Effective Organic Compound Purification

Effective Organic Compound Purification
Effective Organic Compound Purification:

Guidelines and Tactics for Flash Chromatography
Fifth Edition, First Printing
© 2003, 2005, 2008, 2010, 2014, 2015, 2018 Teledyne ISCO. All rights reserved.
Printed in the United States of America
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with respect to accuracy, completeness, or usefulness of the material provided
herein. Teledyne ISCO shall not be liable for any damages that arise from the use of
the information provided herein.
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respective holders.
RediSep Rf® and RediSep Rf Gold® i

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ii Teledyne ISCO
Contents
Contents
Chapter 1
Introduction to Flash Chromatography
Chromatographic Purification in Organic Chemistry . . . . . . . 1
Chapter 2
Flash Chromatography Essentials
Compound Solubility. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Mobile Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Mobile Phase Modifiers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Stationary Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Using TLC to Predict Separation . . . . . . . . . . . . . . . . . . . . . . . . 9
Correlating TLC and Flash. . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Retention Factor and Column Volumes. . . . . . . . . . . . . . . . 9
Method Development Using TLC . . . . . . . . . . . . . . . . . . . . 11
TLC and Mobile Phase Techniques . . . . . . . . . . . . . . . . . . 14
Isocratic Elution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Gradient Elution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Stepped Gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Linear Gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Mixed Gradients. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Loading Capacity of Column . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Column Length Versus Resolution and Purity. . . . . . . . . . . . 27
Flash Column Packings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Particle Shape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Particle Size. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Sample Loading Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Manual Glass Chromatography . . . . . . . . . . . . . . . . . . . . . 29
Automated Chromatography . . . . . . . . . . . . . . . . . . . . . . . 29
Chapter 3
From Traditional Glass Columns to Automated Flash
Chromatography
Manual Glass Column Chromatography. . . . . . . . . . . . . . . . . 37
RediSep Rf® and RediSep Rf Gold® iii

Benefits of Automation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Column Packing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Manually-packed Columns . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Pre-packed Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
TLC Plates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
High Performance Flash Chromatography . . . . . . . . . . . . . . . 46
Column Stacking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Why spherical media?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Higher Resolution with small spherical media . . . . . . . . . 51
Faster purifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Chapter 4
C18 Flash
Chromatography
Overview of Reversed-phase Chromatography . . . . . . . . . . . 57
Normal Phase Silica. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Reversed-phase Silica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
C18 Method Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Thin-layer Chromatography Plates . . . . . . . . . . . . . . . . . . . 60
Using HPLC Systems to Generate Flash Methods . . . . . . . 60
Using the Flash Instrument for Method Development . . . 62
Loading Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Column Care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Solvent Modifiers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Low-solubility Polar Heterocycles . . . . . . . . . . . . . . . . . . . . . . 67
Primary Amines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Carboxylic Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Ionic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
RediSep Rf Gold High Performance C18 Columns . . . . . . . . . 73
RediSep Rf Gold C18 Columns at High pH . . . . . . . . . . . . . . . . 76
Column storage after use in high pH . . . . . . . . . . . . . . . . . 76
RediSep Rf Gold C18Aq for Highly Aqueous Mobile Phases . 78
Use and Care of RediSep Rf Gold C18Aq. . . . . . . . . . . . . . . 79
Water soluble dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Desalting samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Removal of Non-volatile Solvents . . . . . . . . . . . . . . . . . . . . 83
Non-aqueous Reversed-phase. . . . . . . . . . . . . . . . . . . . . . . . . . 85
Changes to the C18 method . . . . . . . . . . . . . . . . . . . . . . . . . 87
iv Teledyne ISCO
Contents
Chapter 5
Advanced Flash Chromatography
Alternative Chromatographic Media . . . . . . . . . . . . . . . . . . . 89
Specialty Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Amine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Basic Alumina. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Neutral Alumina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Cyano . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Trans-anisole oxide purification. . . . . . . . . . . . . . . . . . . . 101
Diol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Purification of trans-anesole oxide . . . . . . . . . . . . . . . . . 112
Chimeric Diol Column Behavior
using Aqueous Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Ion Exchange Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
SCX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Purification of Alkaloids with
RedSep Rf SCX Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Xanthine Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Nicotine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
Harmine and Harmaline. . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Green Tea Extract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
SAX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
RedSep Rf Strong Anion Exchange
Column Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
Gallic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Phenolic Flavanoid Compounds. . . . . . . . . . . . . . . . . . . . 134
Anthocyanin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Green Tea Extract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
Advanced Solvent Strategies . . . . . . . . . . . . . . . . . . . . . . . . . 137
HILIC Purification Strategies . . . . . . . . . . . . . . . . . . . . . . . . . 144
Erioglaucine dye on diol . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Xanthine alkaloids on an Amine column . . . . . . . . . . . . . 148
RedSep Rf Gold Silica and Highly Polar Solvents . . . . . . . . 148
Chapter 6
Natural Products
Cytotoxic Constituents from Butea superba . . . . . . . . . . 155
Alkaloids of Banisteria caapi . . . . . . . . . . . . . . . . . . . . . . . 156
Wide Polarity Range Flash Purifications. . . . . . . . . . . . . . . . 158
Reversed-phase C18 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
Normal Phase Diol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Ion Exchange Columns for Natural Products . . . . . . . . . 162
SCX Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
SAX Columns. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Techniques in Extract Column Screening . . . . . . . . . . . . . . 170
RediSep Rf® and RediSep Rf Gold® v

Silica Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

Alumina Screening. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Diol Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
C18 Screening. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Strong Anion Exchange (SAX) . . . . . . . . . . . . . . . . . . . . . . 177
Strong Cation Exchange (SCX). . . . . . . . . . . . . . . . . . . . . . 180
Chapter 7
Detection Techniques
UV Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Detection with UV-vis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
All-Wavelength Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
Solvent spectrum overlaps compound. . . . . . . . . . . . . . . 187
Sample overloads detector . . . . . . . . . . . . . . . . . . . . . . . . 188
All-wavelength collection on the
CombiFlash Rf and Torrent systems. . . . . . . . . . . . . . . . . 189
Example with a compound mixture . . . . . . . . . . . . . . . . . 189
Example of unknown spectrum . . . . . . . . . . . . . . . . . . . . . 190
Collect related compounds within a UV-vis range . . . . . 192
Other Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Evaporative Light Scattering Detection (ELSD) . . . . . . . . . . 194
Compound Weakly Absorbs UV Light. . . . . . . . . . . . . . . . 194
Compound UV Absorbance is Obscured by Elution Solvent195
Purification of Carbohydrates . . . . . . . . . . . . . . . . . . . . . . 196
Evaporative Light Scattering Detection of
2,3-O-isopropylidene-D-ribofuranose . . . . . . . . . . . . . . . . 196
Mass-directed Fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Single Ion Current (SIC) . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
Extracted Ion Current (XIC) . . . . . . . . . . . . . . . . . . . . . . . . 200
Purification of crude benzidine by XIC . . . . . . . . . . . . . . . 201
Termination upon target mass detection . . . . . . . . . . . . . . . 201
Purification using only UV detection . . . . . . . . . . . . . . . . 203
Terminate after peak with m/z 180 collected . . . . . . . . . 203
Terminate run after collecting m/z 185 . . . . . . . . . . . . . . 204
Terminate run after collecting both m/z 180 and 185. . . 205
Mass-directed purification of steroids with APCI and
CombiFlash Rf+ PurIon . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
Chapter 8
High Performance Liquid Chromatography
Capsaicin compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
vi Teledyne ISCO
Contents
Appendix A
Column Media Selection
Media Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Appendix B
Solvent and UV-vis Wavelength Selection Guide
Solvent Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
Wavelength Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
Appendix C
Theory & Application of Flash Chromatography
Elementary theory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
Appendix D
Troubleshooting
LC Systems
Basic checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
Appendix E
CombiFlash Systems
CombiFlash NextGen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
CombiFlash Rf+ Lumen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
CombiFlash Rf+ PurIon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
CombiFlash Rf+ Torrent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
CombiFlash EZ Prep . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
RediSep Rf® and RediSep Rf Gold® vii

List of Figures
1 Illustration of basic elements in a traditional Flash
column chromatography apparatus . . . . . . . . . . . . . . . . . . . . 2
2 Photo of TLC plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3 Table of common solvents in liquid chromatography . . . . . 6
4 Illustration of mobile phase modifier . . . . . . . . . . . . . . . . . . . 7
5 Table of Rf to CV conversions . . . . . . . . . . . . . . . . . . . . . . . . . 9
6 Illustration of solvent strength optimization . . . . . . . . . . . . 10
7 Illustration of solvent system selectivity optimization. . . . 11
8 Illustration of a solvent system optimized for compound
selectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
9 Table of suggested loading of RediSep Rf silica gel
columns based on Rf differences from TLC plates. . . . . . . . 12
10 Illustration of mobile phase techniques . . . . . . . . . . . . . . . . 13
11 Illustration of isocratic 20% EtOAc in hexane . . . . . . . . . . . 15
16 Illustration of a stepped gradient and chromatogram . . . . 19
17 Illustration of a linear gradient and chromatogram . . . . . . 20
18 Chromatograms resulting from various gradient slopes . . 21
19 Chromatograms of catechol and resorcinol separations . . 22
20 Illustration of CombiFlash Rf Gradient Optimizer and
resulting chromatogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
21 Chromatogram indicating column loading capacity
near limit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
22 Chromatogram indicating column loading capacity
exceeded . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
23 Illustration of gradient mobile phase . . . . . . . . . . . . . . . . . . 25
24 Photos of particle shapes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
25 Illustration of sample injection on glass columns . . . . . . . . 29
26 Photo of syringe injection. . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
27 Photo of a solid sample load cartridge . . . . . . . . . . . . . . . . . 30
28 Photo of solid load cartridge connected to column . . . . . . 31
29 Chromatograms of syringe injection and dried solid
load cartridge techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
30 Photo of pre-packed cartridges . . . . . . . . . . . . . . . . . . . . . . . 33
31 Photo of dry loading sample onto the column. . . . . . . . . . . 33
32 Table of RediSep Rf solid load cartridges . . . . . . . . . . . . . . . 34
33 Photo of a manual Flash system in use . . . . . . . . . . . . . . . . . 35
34 Photo of Teledyne Isco’s CombiFlash Rf system . . . . . . . . . 37
35 Photo of glass column preparation . . . . . . . . . . . . . . . . . . . . 39
36 Chromatograms of compounds separated on a
hand-packed column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
viii Teledyne ISCO

Contents
37 Photo of pre-packed columns . . . . . . . . . . . . . . . . . . . . . . . . 41

38 Photo of matching TLC media. . . . . . . . . . . . . . . . . . . . . . . . 42
39 Table of RediSep Rf TLC plates . . . . . . . . . . . . . . . . . . . . . . . 42
40 Table of solvent migration and plate development time
for RediSep C18 TLC plates . . . . . . . . . . . . . . . . . . . . . . . . . . 43
for RediSep Basic and Neutral Alumina TLC plates . . . . . . 43
42 Chromatograms showing improved resolution . . . . . . . . . 45
43 Chromatograms comparing a single column . . . . . . . . . . . 46
44 Chromatogram of two 24 g stacked columns compared . . 47
45 Chromatogram of bromotoluenes purification. . . . . . . . . . 48
46 Chromatogram of minor compound separation . . . . . . . . . 49
47 Diagram of sample load comparison . . . . . . . . . . . . . . . . . . 50
48 Chromatograms of 3-(2-nitrophenyl amino) propionitrile
purifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
49 Table of RediSep Rf Gold Silica Gel. . . . . . . . . . . . . . . . . . . . 53
50 Diagram of normal phase silica. . . . . . . . . . . . . . . . . . . . . . . 54
51 Diagram of reversed phase silica . . . . . . . . . . . . . . . . . . . . . 55
52 Table of RediSep Rf C18 Reversed Phase columns. . . . . . . 56
for RediSep C18 TLC plates . . . . . . . . . . . . . . . . . . . . . . . . . . 57
54 Illustration of gallic acid and pyrogallol method . . . . . . . . 58
55 Illustration of methyl- and propyl-paraben method. . . . . . 60
56 Diagram of interconversion of diphenyl acetic acid . . . . . 63
57 Illustration of esculin/diphenyl acetic acid purifications . 63
58 Chromatogram of quinoxaline mixture purification . . . . . 64
59 Chromatogram of primary amine mixture purification . . . 65
60 Chromatogram of carbohydrate mixture purification . . . . 66
61 Chromatogram of peptide mixture purification . . . . . . . . . 67
62 Chromatogram of carboxylic acid mixture purification . . 68
63 Chromatogram of ionic mixture purification . . . . . . . . . . . 69
64 Chromatogram of 10 mg compound A purification . . . . . . 71
65 Chromatogram of 46 mg compound A purification . . . . . . 71
66 Chromatogram of 10 mg compound A purification on
a Waters DeltaPrep 4000 system. . . . . . . . . . . . . . . . . . . . . . 72
67 Illustration of analytical HPLC. . . . . . . . . . . . . . . . . . . . . . . . 72
68 Table of Reusable RediSep Rf Gold C18 Reversed Phase
columns, 20–40 microns. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
69 Diagram of functionalized media . . . . . . . . . . . . . . . . . . . . . 76
70 Diagram of amine structure. . . . . . . . . . . . . . . . . . . . . . . . . . 76
71 Chromatogram of normal phase column elution with
hexane/ethyl acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
72 Chromatogram of amine functionalized column with
hexane/ethyl acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
73 Table of Reusable RediSep Rf Amine Columns . . . . . . . . . . 79
RediSep Rf® and RediSep Rf Gold® ix

74 Table of Reusable RediSep Rf Gold Amine Columns . . . . . . 80

75 Chromatogram of normal phase silica column . . . . . . . . . . 81
76 Chromatogram of basic alumina column . . . . . . . . . . . . . . . 82
for RediSep Basic Alumina TLC plates . . . . . . . . . . . . . . . . . 82
78 Table of RediSep Rf Alumina Basic Columns . . . . . . . . . . . . 83
79 Chromatogram of normal phase silica column . . . . . . . . . . 84
80 Chromatogram of neutral alumina column . . . . . . . . . . . . . 85
81 Table of RediSep Rf Alumina Neutral Columns . . . . . . . . . . 86
82 Table of RediSep Rf Alumina Acidic Columns . . . . . . . . . . . 86
83 Diagram of cyano structure . . . . . . . . . . . . . . . . . . . . . . . . . . 87
84 Table of Reusable RediSep Rf Gold Cyano Columns . . . . . . 88
85 Diagram of diol structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
86 Purification of oleyl glycerate . . . . . . . . . . . . . . . . . . . . . . . . 90
87 Purification of tocopherols from corn oil . . . . . . . . . . . . . . . 91
88 Purification of green tea extract . . . . . . . . . . . . . . . . . . . . . . 91
89 Table of Reusable RediSep Rf Gold Diol Columns . . . . . . . . 92
90 Diagram of SCX structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
91 Chromatogram of normal phase column . . . . . . . . . . . . . . . 94
92 Chromatogram of SCX column . . . . . . . . . . . . . . . . . . . . . . . . 95
93 Table of Reusable RediSep Rf SCX Columns. . . . . . . . . . . . . 95
94 Diagram of SAX structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
95 Chromatogram of normal phase column . . . . . . . . . . . . . . . 97
96 Chromatogram of SAX column . . . . . . . . . . . . . . . . . . . . . . . . 97
97 Table of Reusable RediSep Rf SAX Columns. . . . . . . . . . . . . 99
98 Photo of column mount. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
99 Diagram of compounds extracted from Butea superba . . 102
100 Illustration of harmine and harmaline separation on a
silica gel column. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
101 Illustration of harmine and harmaline separation on a
RediSep amine column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
102 Diagram of solvent selectivity . . . . . . . . . . . . . . . . . . . . . . . 105
103 Chart of UV spectra of ethyl acetate and acetone. . . . . . . 107
104 Chart of UV absorbance of 3-(2-nitrophenylamino)
propionitrile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
105 Chromatogram of 3-(2-nitrophenylamino) propionitrile
purification in hexane/ethyl acetate . . . . . . . . . . . . . . . . . . 109
106 Chromatogram of 3-(2-nitrophenylamino) propionitrile
purification in hexane/acetone . . . . . . . . . . . . . . . . . . . . . . 109
107 Chart of stigmasterol absorbance . . . . . . . . . . . . . . . . . . . . 110
108 Chromatogram of stigmasterol purification. . . . . . . . . . . . 110
109 Chart of UV absorbance of catechin . . . . . . . . . . . . . . . . . . 112
110 Chromatogram of hair dye compounds purification. . . . . 113
111 Chromatogram showing detection of chlorophyll,
catechins and caffeine, and tannins . . . . . . . . . . . . . . . . . . 114
x Teledyne ISCO
Contents
112 Chromatogram showing detection of catechin. . . . . . . . . 115

113 Diagram showing UV absorption of various compounds 115
114 Chromatogram of glucose pentaacetate purification . . . 116
115 Chromatogram showing purification of closely-eluting,
saturated peaks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
116 Chromatogram showing ELSD detection of
2,3-O-isopropylidene-D-ribofuranose . . . . . . . . . . . . . . . . . 118
117 Chart for column media selection . . . . . . . . . . . . . . . . . . . 120
118 Table of RediSep Rf Gold Silica Gel Disposable Flash
Columns, 20–40 microns . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
119 Table of RediSep Rf Silica Gel Disposable Flash
Columns, 40–60 microns . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
120 Table of Reusable RediSep Rf Gold C18 Reversed Phase
columns, 20–40 microns. . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
121 Table of Reusable RediSep Rf C18 Reversed Phase
columns, 40–60 microns. . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
122 Table of Reusable RediSep Rf Gold Amine Columns,
20–40 microns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
123 Table of Reusable RediSep Rf Amine Columns,
40–60 microns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
124 Table of Reusable RediSep Rf Gold Cyano Columns,
20–40 microns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
125 Table of Reusable RediSep Rf SAX Columns . . . . . . . . . . . 128
126 Table of Reusable RediSep Rf SCX Columns . . . . . . . . . . . 129
127 Table of Reusable RediSep Rf Gold Diol Columns,
20–40 microns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
128 Table of RediSep Rf Alumina Acidic Columns . . . . . . . . . . 131
129 Table of RediSep Rf Alumina Neutral Columns . . . . . . . . . 132
130 Table of RediSep Rf Alumina Basic Columns. . . . . . . . . . . 133
131 Table of liquid chromatography solvents and their
characteristics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
132 Chart of Solvent Miscibility . . . . . . . . . . . . . . . . . . . . . . . . . 137
133 Table of compound absorbance wavelengths . . . . . . . . . 138
134 Table of compound absorbance wavelengths . . . . . . . . . 139
135 Standard resolution charts . . . . . . . . . . . . . . . . . . . . . . . . . 144
136 Table for troubleshooting peak problems. . . . . . . . . . . . . 146
137 Table for troubleshooting baseline problems. . . . . . . . . . 148
138 Table for troubleshooting recovery and retention
problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
139 Table for troubleshooting pressure problems . . . . . . . . . 149
140 Table for troubleshooting leaks . . . . . . . . . . . . . . . . . . . . . 150
RediSep Rf® and RediSep Rf Gold® xi

xii Teledyne ISCO

Chapter 1
Introduction to Flash
Chromatography
Chromatographic Purification in Organic Chemistry

During the course of developing a chemical reaction to produce a
desired product, the synthetic organic chemist typically goes
through the repeated sequence of reaction setup, workup,
purification, and final product analysis.
When the chemist reaches the purification step, there are several
purification techniques to choose from, including crystallization,
filtration, distillation, and column chromatography.
Traditional column chromatography applies a crude reaction
mixture on top of a bed of silica gel loaded in a glass column. A
gravity-fed solvent mixture (mobile phase) passes through the
vertical column of silica gel (stationary phase), separating the
individual products of the crude reaction mixture.
The separation of the compounds in the mixture is based on their
different affinity for the mobile and stationary phases, which
causes the compounds to migrate through the column at different
rates and emerge from the bottom of the column at different times.
The stationary phase and mobile phase are chosen to achieve the
best possible separation of components, based on the nature of
the sample mixture.
The separated products are collected in test tubes positioned
below the column outlet. Then, identical fractions are gathered
and concentrated.
RediSep Rf® and RediSep Rf Gold® 1

Chapter 1
Compressed air
Solvent
(mobile phase)
Sand
Column media
Separated (stationary phase)
products
Frit
Tap
Empty Separated
collection fractions
tubes
Figure 1: Illustration of basic elements in a traditional Flash column

chromatography apparatus
2 Teledyne Isco, Inc.

The term Flash chromatography was coined in 1978 by W. Clark Still

and coworkers at Columbia University to describe separations in
which a gas-pressurized solvent reservoir is used to accelerate
solvent flow and achieve superior chemical separations in less
time than traditional gravity-based column chromatography.
Today, Flash chromatography is a completely automated
preparative technique influenced by the CombiFlash equipment
designed by Teledyne ISCO. The advantages of using automated
Flash chromatography are many. It’s easy, fast, inexpensive,
requires minimal development time, and has high resolution. For a
complete list of Teledyne ISCO automated Flash systems, refer to
Appendix E - CombiFlash Systems, on page 255.
Flash chromatography is currently one of the most popular
techniques for purifying pharmaceutical intermediates, as well as
final organic products. It is also widely used in natural products
research.
Although silica gel was the media first employed in Flash
chromatography, the introduction of automated systems by
Teledyne ISCO has extended the technique to include other media
such as reversed-phase C18 and other bonded phases, alumina,
and ion exchange resins. This has greatly expanded the application
base of Flash chromatography.

Chapter 1
4 Teledyne Isco, Inc.

Chapter 2
Flash Chromatography
Essentials
Flash chromatography is an easy and simple purification

technique that requires minimal method development. Even
though there are only a few factors to consider when preparing for
a Flash chromatography purification, they all need to be selected
thoughtfully in order to achieve a successful separation. Mobile
phase, stationary phase, type of gradient elution, column loading
capacity, and sample loading technique are some of these factors.
The following describes in detail their influence on the final result
and how they should be approached and selected.
Compound Solubility
The solubility of the crude products mixture to be separated is a
factor the organic chemist should consider when choosing the
solvent system mixture, or mobile phase.
A mobile phase with low polarity properties may precipitate oily
crude mixture products in the flask during dissolution prior to
loading the sample on the column, or after being loaded on top of
the column when the low polarity solvent mixture progression
starts.
To avoid having the sample precipitate unintentionally (or crash),
it is important to choose a solvent system polar enough to cover
both the solubility issue upon sample loading on column and the
maximized separation conditions obtained from Thin-layer
Chromatography (TLC) (see Using TLC to Predict Separation, on
page 9).
Should the sample precipitate in the flask prior to column loading
or be in an initial solid state, the solid loading technique is
recommended (see Solid sample loading, on page 29).

Chapter 2
In the event the sample precipitates after being loaded onto the
column, increasing the polarity of the solvent system through
gradient solvent elution (see Gradient Elution, on page 18) would
eventually reach a solvent system mixture polar enough to
solubilize it. However, precipitated samples often raise the system
pressure thereby reducing the solvent flow. Higher pressure Flash
systems, such as the CombiFlash Rf, is better able to push the
solvent through, making it easier to increase the polarity. Once
solubilized, the sample moves through the stationary phase.For a
Mobile Phase
The solvent system or mobile phase choice for Flash
chromatography is dependent on the polarity of the product(s) to
be isolated and the type of stationary phase to be used.
Typically, the organic chemist will first proceed with a few TLC
analytical trials to determine which solvent system will provide
the optimal separation conditions with respect to the polarity of
the desired product(s) and the selected stationary phase.
The retention distance, Rf , on a TLC plate represents the distance a
given compound migrates from the origin with respect to the
solvent front on the plate. (see Method Development Using TLC, on
page 11.)
Figure 2: Photo of TLC plate Annotations include baseline, sample starting

point, separated compounds, and final solvent front
6 Teledyne ISCO
Figure 3: Table of common solvents and their characteristics in liquid

chromatography (by increasing polarity)
Viscosity Boiling UV Cutoff
Solvent Polarity (cp 20°) Point (°C) (nm)
Hexane 0.06 0.33 69 210
n-Heptane 0.20 0.41 98 200
Toluene 2.40 0.59 111 285
Methylene chloride (DCM) 3.40 0.44 40 245
Tetrahydrofuran 4.20 0.55 66 220
Ethanol 4.30 1.20 79 210
Ethyl acetate 4.30 0.45 77 260
i-Propanol 4.30 2.37 82 210
Acetonitrile 6.20 0.37 82 210
Methanol 6.60 0.60 65 210
Water 10.20 1.00 100 —
During the TLC analytical trials, the chemist will seek the solvent
system that moves the desired product to Rf =0.25±0.05 and keeps
other undesired products to a distance of at least Rf =0.2. These
TLC parameters constitute the ideal Flash chromatography
conditions because of high compound stationary phase contact
time predisposing to high compound resolution during the column
separation.
Many organic solvents are available. Figure 3 lists commonly used
solvents. Table of liquid chromatography solvents and their
characteristics, on page 238 lists additional solvents that may be
more suitable for specialized purifications.
The solvent system strength and selectivity refer respectively to
the solvent system’s ability to migrate all compounds
simultaneously on the column (i.e., purification duration) and to
migrate one specific compound differently from the others (i.e.,
separation resolution).
Typically, the solvent system is a binary mixture of a higher and a
lower strength (polarity) solvent. For instance, organic chemists
commonly initiate their solvent system evaluation and selection

Chapter 2
with hexane/ethyl acetate 1:1 and/or dichloromethane/methanol

95:5 for a normal phase silica gel stationary phase. The different
strength and selectivities of these two solvent mixtures provide
information useful in identifying an appropriate solvent system for
purification of the reaction mixture.
The mobile phase selection is a function of the stationary phase
chosen for the purification. Normal and reversed-phase silica gels
are the most common stationary phases used by organic chemists.
Typically, the solvent system selected for a normal phase silica gel
will have lower protic properties (e.g., hexane/ethyl acetate,
hexane/ether, or dichloromethane/methanol), whereas
reversed-phase silica gel will have higher protic properties (e.g.,
water/acetonitrile, water/isopropanol).
Mobile Phase Modifiers

Acidic and basic organic compounds interact with residual surface
silanol groups on a chromatographic support and cause peak
tailing. The addition of a mobile phase modifier (typically one
percent or less concentration) reduces peak tailing and sharpens
peaks, improving the resolution in separations of basic or acidic
compounds.
Triethylamine, ammonium hydroxide, acetic acid, and
trifluoroacetic acid are common mobile phase modifiers.
Without With
Modifier Modifier
Solvent
Front
Base
Line
Figure 4: Illustration of mobile phase modifier reducing peak tailing on TLC

plates
8 Teledyne ISCO
Stationary Phase
Stationary phase selection is driven by the nature of the products
to be separated. Factors such as compound polarity and
functionality greatly influence the media selection.
The majority of reaction products organic chemists need to isolate
can be purified using a normal phase or reversed-phase silica gel
as the stationary phase.
For some specific types of compounds, however, it is difficult to
achieve an overall satisfactory degree of separation using these
common Flash chromatography stationary phases. The silica gel
suppliers have designed and marketed functionalized silica gel to
provide chemists additional purification media options. Thus,
organic chemists now have a wide range of purification tools
available, which facilitates isolation of compounds with very
different physicochemical properties.
Column Media Selection, on page 221 provides a stationary phase
selection guide and more information on media types.
Using TLC to Predict Separation

Thin-layer Chromatography (TLC) is a simple and practical
chromatography technique organic chemists use to monitor the
evolution of chemical reactions. TLC is also used to optimize Flash
chromatography conditions for purification of crude reaction
mixtures.
Correlating TLC and Flash

The strength with which a compound binds to the stationary
phase is called retention. Provided that the stationary phase is
identical, a correlation can be made between compound retention
in TLC and Flash chromatography.
Retention Factor and Column Volumes

Retention (Rf) of a compound in TLC is measured by the distance it
moves relative to the naturally moving solvent front. This differs
from Flash chromatography, in which the solvent is pumped
through the stationary phase. Instead of relative distances,
retention in Flash chromatography is generally defined in terms of
the volume of solvent necessary to move the components through
the column. This volume, expressed in column volume (CV), is the

Chapter 2
amount of solvent the column can hold in the interstitial space

between the media particles.
Although the measures of retention differ, methods developed
using TLC are generally transferable to Flash chromatography
because of the relationship between Rf and CV:
1-
R f = -------
CV
Figure 5 illustrates this relationship between Rf and CV. A

compound with low retention that moves easily through TLC, (e.g.,
Rf =0.80), can be expected to elute quickly (1.25 CV). Conversely, a
highly retentive compound (e.g., Rf =0.10), binds more strongly to
the stationary phase media and can be expected to elute much
later (10.0 CV).
Figure 5: Table of Rf to CV conversions

Column Volume
Retention (Rf) (CV)
0.90 1.10
0.85 1.17
0.80 1.25
0.75 1.33
0.70 1.40
0.65 1.54
0.60 1.65
0.55 1.81
0.50 2.00
0.45 2.22
0.40 2.50
0.35 2.86
0.30 3.33
0.25 4.00
0.20 5.00
0.15 6.67
0.10 10.00
10 Teledyne ISCO
Method Development Using TLC

Since CV is the measure of compound retention, then CV is the
measure of compound resolution, or the degree to which the
desired product can be isolated from other components in the
mixture.
Chemists perform multiple analytical TLCs to attempt to identify a
solvent system that migrates the desired product spot to
Rf =0.25±0.05 (optimal retention), while migrating all other spots as
far as possible from the desired product (optimal selectivity).
The following figures illustrate this process. Figure 6 shows
progressive attempts to optimize a solvent system to move the
desired compound to optimal retention conditions.
Figure 7 shows the sequential solvent selection attempts to reach
optimal selectivity for a given mixture.
After identifying a solvent system that performs well under the
system conditions, maximum CV for Flash chromatography is
achieved, reflected in the column volume chromatogram of
Figure 8.
EtOAc Hexane/EtOAc Hexane/EtOAc

1:5 1:1
Solvent
front
Rf 0.95
Rf 0.50
Rf 0.25
Base line
Optimal Rf
Figure 6: Illustration of solvent strength optimization

Chapter 2
Hexane/EtOAc CH2Cl2/MeOH CH2Cl2/MeOH

1:1 99:1 95:5
Solvent
front
Rf
Base line
Optimal
selectivity
Figure 7: Illustration of solvent system selectivity optimization
CV
Solvent
front 3
1 2
4
2 1
3
Base line 4
0 3 6 9 12
Column Volumes
Figure 8: Illustration of a solvent system optimized for compound

selectivity and its reflection on the column volume chromatogram
The selectivity obtained will determine the sample loading

capacity on the column. The lower the retention time (Rf) and the
higher the selectivity (Rf) between product spots on the TLC
plate, the higher the amount of sample can be loaded.
12 Teledyne ISCO
Figure 9: Table of suggested loading RediSep Rf silica gel columns based on

Rf differences from TLC plates
The 125 g column is designed for high loads of easily separated
compounds.
Loading
Light Moderate Significant Heavy
Column size
(g silica) Rf < 0.2 0.2 – 0.4 0.4 – 0.6 > 0.6
4g
(69-2203-304) 0.0004 – 0.004 0.004 – 0.16 0.16 – 0.28 0.28 – 0.4
12 g
(69-2203-312) 0.0012 – 0.012 0.012 – 0.48 0.48 – 0.84 0.84 – 1.2
24 g
(69-2203-324) 0.0024 – 0.024 0.024 – 0.96 0.96 – 1.68 1.68 – 2.4
40 g
(69-2203-340) 0.004 – 0.04 0.04 – 1.6 1.6 – 2.8 2.8 – 4
80 g
(60-2203-380) 0.008 – 0.08 0.08 – 3.2 3.2 – 5.6 5.6 – 8
120 g
(69-2203-320) 0.012 – 0.12 0.12 – 4.8 4.8 – 8.4 8.4 – 12
125 g
(69-2203-314) — — 5 – 8.75 8.75 – 12.5
220 g
(69-2203-422) 0.022 – 0.22 0.22 – 8.8 8.8 – 15.4 15.4 – 22
330 g
(69-2203-330) 0.033 – 0.33 0.33 – 13.2 13.2 – 23.1 23.1 – 33
750 g
(69-2203-275) 0.075 – 0.75 0.75 – 30 30 – 52.5 52.5 – 75
1500 g
(69-2203-277) 0.15 – 1.5 1.5 – 60 60 – 105 105 – 150

Chapter 2
Typically, a crude reaction mixture amount corresponding to

1–10% weight of the normal phase silica gel quantity will be loaded
on the column for low selectivity conditions. An amount up to 10%
weight of the normal phase silica gel quantity will be loaded on the
column with high selectivity conditions.
To summarize, when developing a method for Flash
chromatography purification with TLC plates, it is recommended
to:
• Use identical stationary phase for related TLC experiments
and subsequent column runs since the sorbent quality
varies from one manufacturer to another.
• Choose a solvent system that moves the desired product to
Rf =0.25±0.05 and keeps other products in the mixture at a
distance of at least Rf =0.2.
TLC and Mobile Phase Techniques

Because TLC separations closely mimic the behavior of
compounds in a silica gel column and mobile phase combination,
chemists have come to rely upon TLC to scout for optimal
separation conditions.
Isocratic Stepped
Linear Linear with

isocratic hold
Figure 10: Illustration of mobile phase techniques plotted as solvent strength

(Y-axis) over time or column volumes (X-axis)
The separation conditions found while scouting with TLC easily

translate to columns if separations are isocratic. Similarly, a
chemist can perform a series of TLCs to determine the ideal mobile
phase concentrations and translate the conditions to a stepped
gradient separation on a column.
14 Teledyne ISCO
When using linear gradient Flash chromatography to purify organic

compounds, TLC data is less useful because the TLC mobile phase
cannot be dynamically varied.
Given this limitation, TLC is still a practical starting point for
developing effective separation methods using a linear gradient
mobile phase. TLC verifies that the selected solvent system has
the appropriate solvent strength, and that the selected stationary
phase will separate the compounds while ensuring that the
compound of interest will not be permanently retained.
What linear gradient Flash chromatography does is provide the
ideal solvent blend for the separation. This is because the gradient
solvent systems changes infinitesimally from one extreme to
another—at some point the ideal solvent blend is provided for
purification. Testing one point or even several using TLC does little
to help the chemist empirically determine the ideal solvent blend
and gradient curve. The need for analytical TLC prior to
purification is greatly reduced.
Isocratic Elution
Most classical Flash chromatography uses an isocratic mobile
phase to separate compounds. In an isocratic separation, the
mobile phase may be a single solvent or a mixture, but the mobile
phase composition is the same throughout the separation.
TLC is an isocratic technique. Therefore, it can closely correlate to
isocratic separations scaled up to column chromatography.
An isocratic mobile phase can be optimized to purify nearly any
compound of interest. To ensure the separation is selective, the
chemist must control the isocratic conditions beyond just the
right solvent blend. Sample loading and column capacity also must
be closely controlled. But in the end, these efforts yield a
specialized method that will not separate a wide variety of
compounds.
Column capacity is typically limited when using isocratic mobile
phases. If the sample size is increased too much, the mixture’s
compounds will contaminate each other.

Chapter 2
Figures 11 through 15 illustrate tests performed to optimize an

isocratic mobile phase. In this example, Sample A, a blend of
acetophenone (1), methyl paraben (2), and 4-aminobenzoic acid
(3) is separated using 20, 30, 40, 50, and 70% EtOAc and Hexane.
When reviewing the results of this TLC series, we learn that for the
purification of acetophenone 20% EtOAc is best. For methyl
paraben 20 to 30% EtOAc is best, and for the purification of
4-aminobenzoic acid, 40 to 50% of EtOAc is best.
Solvent
front 1
Rf ~.7 1
2
2
3
Baseline
0 1 2 3 4 5 6 7 8 9 10
Column Volumes
Figure 11: Illustration of isocratic 20% EtOAc in hexane

4-aminobenzoic acid does not move from the TLC baseline, nor does it
come off the column.
Rf ~.9 1 1
Rf ~.1 3 3
0 1 2 3 4 5 6 7 8 9 10
Column Volumes

4-aminobenzoic acid has low Rf. Peak 3 is very spread out.
16 Teledyne ISCO
1
1
2
3
2
0 1 2 3 4 5 6 7 8 9 10
Column Volumes

Acetophenone and methyl paraben are no longer pure.
4-aminobenzoic acid is pure.
1
1
2
3
2
0 1 2 3 4 5 6 7 8 9 10
Column Volumes
Figure 14: Illustration of isocratic 50% EtOAc in hexane 4-aminobenzoic

acid remains pure. It comes off the column sooner while maintaining
sufficient resolution.

Chapter 2
0 1 2 3 4 5 6 7 8 9 10
Column Volumes
Nothing is resolved at 70%.
Gradient Elution
Gradient elution describes techniques for decreasing overall
purification time, increasing resolution, and increasing column
capacity by varying the mobile phase composition during the
chromatographic separation. Gradient methods include both
stepwise and continuous changes in the solvent blend, with linear
or straight-line gradients being the most common form of
continuous gradients. A binary gradient is one in which the ratio of
two solvents (or solvent mixtures) is varied during the separation.
Ternary (3-solvent) and quaternary (4-solvent) gradients are also
used in some cases.
It is extremely powerful to have fully programmable control over
the mobile phase components during the course of a separation.
This capability allows you to tailor the resolving power for a
particular set of species that need to be separated on a
chromatographic column.
Until the recent development of automated Flash chromatography
systems, the power of programmable gradients was not readily
available to organic chemists. Gradients are a means of controlling
resolution. By adroit use of gradients, closely eluting compounds
may be separated while compounds with long retention times
(they may be thought of having highly excess resolution) can be
run with reduced time and solvent.
18 Teledyne ISCO
When gradients are applied to Flash purification of small organic

molecules there are several key benefits that a chemist can take
advantage of:
• shorter elution times
• less dependence on pre-determining optimal
separation conditions
• higher purity
• fewer fractions to deal with
• greater sample loading capacity
• greater repeatability
The benefits of gradient chromatography are apparent when

compared to isocratic and stepped solvent systems, and how TLC
relates to each method.
Stepped Gradient
Stepped gradients are a classical technique used in Flash
chromatography. The solvent system is a blend of solvents.
Several different blends are prepared at increasingly polar solvent
strengths.
In the same way an optimal solvent is chosen for an isocratic
separation, optimal solvent blends for stepped gradients are
identified through TLC trials. The goal of the TLC trials is to
determine a blend that moves the compound of interest.
These blends are introduced onto the Flash column in turn. The
solvent strength is increased only after the previous compound
has separated, greatly improving selectivity. As a result, column
capacity can be increased.
Referring to the example separating Sample A, Figure 16 illustrates
a stepped gradient developed from the analytical TLC trials. A
stepped gradient starting at 20% EtOAc and moving to 40% after 4
column volumes will allow the separation of the three compounds
in a single run.

Chapter 2
80% 1
60%
Gradient
40%
2
20% 3
0 1 2 3 4 5 6 7 8 9 10
Column Volumes
Figure 16: Illustration of a stepped gradient and chromatogram
Linear Gradient
Linear gradients begin with a low-strength solvent blend. The
gradient is advanced in infinitesimal steps (limited by the
resolution of the solvent pumping system) until the separation
ends at a high-strength solvent blend.
With isocratic and stepped gradients, it is very important that a
sufficient number of TLCs be performed to determine just the right
solvent blend. In the case of linear gradients, this requirement is
reduced because the gradient that is best for separation of the
compounds is by default provided to the column. This is because
at one point along the gradient profile, the best solvent blend for
separation of the compounds is delivered to the column.
To continue the example, TLC determined that the ideal
concentration of the solvents is between 20 and 40%. To determine
this with confidence it required that 20, 30, 40, 50, and possibly
70% blends be prepared and evaluated for separation
effectiveness. This is because it is difficult to know at the outset
what concentrations will bracket ideal conditions.
However, when a linear gradient is used, since it starts at a
concentration that is lower than the optimal and increases to a
concentration that exceeds the optimal level, it is not necessary to
perform as many TLCs.
20 Teledyne ISCO
Because of this fact, a chemist has only to perform sufficient TLC

work to determine that the solvent system and stationary phase
combination that is to be applied to the separation will separate
the desired compound from the contaminates.
Figure 17 illustrates a linear gradient used to separate the same
mixture, Sample A, used in the previous examples. Note that
because the 20 and 40% blends are part of the linear gradient
profile, it is not necessary to determine the ideal conditions before
you begin the separation.
Optimizing resolution of a column is a function of gradient slope
and column configuration.
file
80% P ro
1
nt
die
60% ra
G
Gradient
40%
2
20% 3
0 1 2 3 4 5 6 7 8 9 10
Column Volumes
Figure 17: Illustration of a linear gradient and chromatogram
Several adjustments can be made to the slope of the gradient.

These include changing the starting and finishing solvent
concentrations, and the duration of the separation. Complex
gradient curves can also be created. The gradient curve may
include points that hold the solvent at a fixed concentration to
prevent contamination by a closely eluting compound, or add
points to sharply increase the concentration to move
highly-retentive compounds.
The slope of the gradient can greatly affect the resolving power of
the media/solvent combination.

Chapter 2
1 CV
2.5 CV
5 CV
10 CV
20 CV
0 5 10 15
Minutes
Figure 18: Chromatograms resulting from various gradient slopes The
duration of the gradient can be manipulated to optimize the purity
required while minimizing the time needed to complete the procedure.
By decreasing the slope of the gradient, the separation of the

peaks and the broadness of the peaks increase. The trick is to find
the ideal gradient slope so that a compromise is struck between
purity and time to get the compound off the column. This is most
often done by trial and error. A good starting point is to begin with
a gradient that extends over ten column volumes.
Mixed Gradients
Mixed gradients are a combination of step and linear gradients.
These gradients are used to reduce run time while maintaining a
separation between closely eluting components. A linear gradient
is started and an isocratic hold is employed during the gradient to
maintain the resolution between closely eluting compounds.
Figure 19 shows catechol and resorcinol purified under isocratic
conditions and a linear gradient.
22 Teledyne ISCO
80%
B Solvent Strength
60% Isocratic
40%
20%
0 2 4 6 8 10 12 14 16 18 20
Column Volumes
80%
B Solvent Strength
60% Linear
40%
20%
0 5 10 15 20 25 30 35 40 45 50 55
Column Volumes
Figure 19: Chromatograms of catechol and resorcinol separations using
isocratic and linear mobile phases
Under isocratic conditions, the peaks are broad and run together.
The linear gradient, while sharpening the peaks, also causes
overlap. Reducing the slope of the gradient would separate the
peaks but they would also be broadened so there is still overlap
between the peaks. Combining a linear gradient with an isocratic
hold generates the chromatogram in Figure 20 where nearly
complete resolution is achieved between the two diols.
PeakTrak software on CombiFlash systems makes it very easy to
create these gradients with just two TLC plates. The retention
factors of the compound of interest and the closest impurity are
entered into the PeakTrak’s Gradient Optimizer window which
then calculates the optimal combination of linear gradient and
isocratic hold prior to elution of the compounds to give the best
separation.

Chapter 2
80%
B Solvent Strength
60%
40%
20%
0 10 20 30 40 50 60 70
Minutes
Figure 20: Illustration of CombiFlash Rf Gradient Optimizer and resulting

chromatogram showing nearly complete resolution of catechol and
resorcinol.
24 Teledyne ISCO
Loading Capacity of Column

Loading capacity of a column is dependent on a number of
variables. Classical Flash chromatography techniques instruct that
for every 1 gram of compound to be purified, 100 grams of silica
gel (1% load) are required. With the addition of gradient
chromatography and on-line UV-vis detection, this loading
capacity is increased to 1 gram of compound requiring as little as
10 grams of silica (10% load) to purify.
If we refer to the chromatogram presented in Figure 21 you will
note that in this isocratic separation at 30% EtOAc the distance
between the first two peaks is quite small. An increase in the
concentration of either the first or second peak will cause overlap
to occur, resulting in loss of purity of both peaks (Figure 22).
In the case where a gradient is used, the capacity of the column is
increased because the separation of the peaks is greater and the
sharpness of the peaks is greater (Figure 23).
0 1 2 3 4 5 6 7 8 9 10
Column Volumes
Figure 21: Chromatogram indicating column loading capacity near limit At

an isocratic mobile phase of 30% EtOAc, there is little distance
between peaks 1 and 2.

Chapter 2
0 1 2 3 4 5 6 7 8 9 10
Column Volumes
Figure 22: Chromatogram indicating column loading capacity exceeded

Increasing the concentration of methyl paraben (peak 2) causes peaks
to overlap under the same conditions.
80% 1
60%
Gradient
40%
2
20% 3
0 1 2 3 4 5 6 7 8 9 10
Column Volumes
Figure 23: Illustration of gradient mobile phase

The gradient slope can be adjusted to improve selectivity and column
loading capacity.
26 Teledyne ISCO
Column Length Versus Resolution and Purity

Column length is another factor that determines resolution and
purity. A longer column increases the distance that each
compound must travel through the stationary phase. Given the
different rates at which the compounds travel, the resolution
increases relative to the increase in distance. However, the
separation time and back pressure increase along with resolution
and purity. Column stacking (see Column Stacking, on page 46) is a
way to achieve longer column lengths.
Flash Column Packings

Flash chromatography columns typically utilize hard, silica based
packing. This silica may be bare or bonded with various functional
groups to offer differing binding characteristics for separation of
different compounds. The silica particle is also available in
different shapes, sizes and porosities which offer unique
separation characteristics.
Particle Shape
Silica is manufactured in either irregular or spherical particles
(Figure 24).
Irregular silica is produced as a large block of amorphous silica
which is ground and then sieved to produce different particle size
ranges.
Figure 24: Photos of particle shapes

Microscopic views or spherical (left) and irregular (right) silica

Chapter 2
Spherical silica is produced by either spraying a neutral silicate

solution and drying the droplets, or by dispersing the solution in
an emulsion and allowing the droplets to gel. Conditions are highly
controlled under the process to give a well defined size
distribution and porosity. This results in a higher manufacturing
cost than irregular silica.
Spherical shaped silica packs into a column body more densely
and uniformly, resulting in very level and narrow separation bands,
thus being the choice for HPLC columns. Irregular shaped particles
have edges that catch on each other during the packing process
which either break, creating finer particles, or create greater
interstitial space and less active surface area in a given column
volume. Spherical particles will pack more silica more densely into
the same amount of space with less interstitial space and the
resulting greater surface area will offer better separation. Teledyne
ISCO recently added spherical silica in the high performance
RediSep Rf Gold columns for greater resolution.
Particle Size
In liquid chromatography, the smaller the particle size of the
column packing leads to greater plate count1. However, as particle
size decreases the back pressure increases. Typical Flash grade,
irregular silica is classified as 40–63 µm or 230 to 400 mesh which
refers to the sieves sizes used to produce that particle
distribution. This particle size provided adequate resolution while
creating low back pressure so gravity and air pressure could
produce a separation with glass columns.
Reducing the particle size generates greater back pressure due to
the viscosity of the solvent. Reversed-phase solvents generally
have a higher viscosity, further increasing the back pressure.
Irregular particles of the same specified size range typically have
more fine particles (>10 µm) in the mixture. Because of the
manufacturing and handling processes, spherical media has fewer
fine particles than irregular of the same particle distribution,
resulting in lower back pressure than irregular particles. Typical
Flash columns of 40–63 µm will create back pressure of around 15–
20 psi with normal phase solvents and 40–60 psi with
reversed-phase solvents (without consideration of sample
interaction.)
1. Introduction to Modern Liquid Chromatography, Snyder, L.R.;

Kirkland, J.J. 2nd edition, John Wiley and Sons,
28 Teledyne ISCO
New, high resolution Flash columns have been introduced in the

market with finer packings such as 20–40 µm, irregular, and 15 µm
packing. These offer improved resolution, but run at higher
pressures. Newer Flash systems, such as the CombiFlash Rf,
operate at up to 200 psi to accommodate greater back pressures.
Teledyne ISCO introduced RediSep Rf Gold high performance
columns which combine a smaller 20–40 µm particle size and
spherical shape to achieve improved resolution without an
increase in back pressure.
Sample Loading Techniques

One of the challenges to producing pure compounds is to load the
compound and reaction by-products onto the column. This can be
accomplished in a number of ways for both manual and automated
chromatography equipment.
Manual Glass Chromatography

Liquid sample loading — The sample as an oil or liquid is slowly
pipetted inside the glass column preloaded with silica gel topped
by a sand layer (Figure 25, left). Then, the solvent mixture selected
for optimal resolution is slowly introduced so to preserve the
stationary phase packing, causing separation to occur.
Solid sample loading — The sample as an oil or solid is dissolved in a
minimal amount of polar solvent and powder silica gel is added.
The solvent is then removed with a rotary evaporator, leaving the
sample coated on the silica gel. This sample on silica is then
introduced in the glass column on top of the sand layer (Figure 25,
right) covering the packed preloaded silica gel. Then, solvent
progression can be initiated to separate the products.
It should be noted that if there is little resolution between
compounds, the loading capacity is correspondingly reduced to
prevent adjacent peaks mixing with each other.
Automated Chromatography
Syringe injection — Syringe injection (Figure 26) is a very common
technique as it is very simple and convenient. It also allows
equilibration of the column for improved separation.
Syringe injection requires that the compounds are soluble in
mobile phase at beginning of gradient.

Chapter 2
Liquid Solid
sample sample
Sample coated
Sand on silica gel
Silica gel
Frit
Figure 25: Illustration of sample injection on glass columns

A liquid sample is applied evenly to the top of the column with a
pipette (left). A solid sample is first adsorbed onto silica gel and then
placed on top of the sand (right).
Figure 26: Photo of syringe injection

to a CombiFlash Rf system
30 Teledyne ISCO
Empty solid load cartridges — These cartridges provide a practical

method for introducing low-solubility samples onto the column.
This method allows equilibration of the column for improved
separation.
Solid load cartridges (Figure 27) can be used with a variety of
adsorbent materials. The most popular material is silica gel, used
by adding powder silica gel onto the neat or dissolved sample to
be purified. Then the solvent is removed with a rotary evaporator
providing the sample coated on a silica bed. This sample on silica
is then poured into an empty cartridge, topped with a frit, and
loaded onto the system.
Adsorbents are not limited to silica: Celite2, diatomaceous earth,
boiling chips, and even cotton balls and Kimwipes3 have been
used. Sometimes, chemists insert boiling chips and cotton balls
into the rotary evaporator flask to reduce the risk of bumping.
These can be removed along with the compound and placed into
the solid load cartridge to easily transfer the sample to the
purification step. Kimwipes can be used if a flask is inadvertently
broken and the contents spill onto the lab bench. This spilled
material can be soaked up onto Kimwipes and placed into the solid
load cartridge for introduction into the column.
Functionalized media, such as C18, can be used in the solid load
cartridge as a guard column or as a scavenger.
2. Celite is a registered trademark of Johns-Manville Corpora-

tion.
3. Kimwipes is a registered trademark of Kimberly-Clark World-
wide, Inc.

Chapter 2
Figure 27: Photo of a solid sample load cartridge
Solid load cartridge techniques do not require that samples be

placed on the column bed. However, these solid load cartridges
can be directly connected to the columns (Figure 28) when
samples that are difficult to keep in solution are being purified.
Direct connection to the column eliminates any possibility of
contamination of the valves in the automated instrument and also
make an easy funnel introduction of the sample onto the column.
32 Teledyne ISCO
Figure 28: Photo of solid load cartridge connected to column

to bypass valves in the instrument
Pre-packed solid load cartridges — These cartridges represent a

faster solid sample loading technique compared with the use of an
empty solid load cartridge. The dissolved sample is introduced
into a cartridge pre-packed with an adsorbent. After absorption of
dissolved sample by the absorbent, the wet pre-packed cartridge
is thoroughly dried with a high vacuum pump and finally loaded
onto the system.
Solid load cartridges that have been completely dried often
perform better than liquid injections. Figure 29 compares two
sample loading techniques in the purification of 260 mg mixtures of
a phenolic ester and pyridine derivative on 12 gram RediSep silica
gel columns. The dry solid load cartridge shows better resolution
than the liquid injection.

Chapter 2
O
Cl
O
HO N NH2
methyl-4-hydroxybenzoate (A) 2-amino-5-chloropyridine (B)
Syringe Injection
1.0 100%
B Solvent Strength
Absorbance
=280nm
0.5 (A) 50%

(B)
0%
0 2 4 6 8 10
Minutes
Sample dissolved in 2mL of acetone loaded injected onto a
silica column and followed by a 1mL acetone chase. Acetone
used as a strong solvent to completely dissolve the sample.
Solvent system hexane/ethyl acetate.
Solid Load Cartridge

(dried) 1.0 100%
B Solvent Strength
(A)
Absorbance
=280nm
0.5 (B) 50%
0%
0 2 4 6 8 10
Minutes
Sample dissolved in 2mL of acetone and loaded in a 5g
pre-packed normal phase silica cartridge. Acetone removed
with a cartridge dryer.
Figure 29: Chromatograms of syringe injection and dried solid load cartridge
techniques Acetone used to dissolve, inject, and chase over-dilutes
the syringe-injected mixture, causing band broadening and
overlapping peaks. The solid load cartridge allows the acetone to be
removed, thus retaining the compounds until the increasing solvent
strength releases them.
34 Teledyne ISCO
Teledyne ISCO manufactures a cartridge dryer, part number

60-2200-010, that dries up to four cartridges at a time (Figure 30).
Figure 30: Photo of pre-packed cartridges on Teledyne ISCO’s cartridge

dryer (Part number 60-2200-010)
Dry loading column — This technique is ideal for quick cleaning of

samples and removing baseline materials. After clean-up, the
sample can be moved to the next synthesis step.
When dry loading a column, bypass column equilibration (Figure
31).
Figure 31: Photo of dry loading sample onto the column

Chapter 2
Figure 32: Table of RediSep Rf solid load cartridges

Part number Description
Prepacked Cartridges
69-3873-238 Sample load prepacked silica gel Rf cartridges, 2.5 gram, pkg. of 20.
69-3873-236 Sample load prepacked silica gel Rf cartridges, 5 gram, pkg. of 20.
69-3873-311 Sample load, prepacked silica gel cartridges 125 grams, pkg. of 4.
68-3873-202 Sample load, prepacked silica gel cartridges 260 grams, pkg. of 4.
Sample load, prepacked silica gel cartridge. For use on the CombiFlash
69-3873-254 Torrent system only.
Sample load, prepacked silica gel cartridge. For use on the CombiFlash
69-3873-255 Torrent system only.
69-3873-247 Sample load, prepacked, C18 Rf cartridges, 2.5 gram, pkg. of 5.
69-3873-237 Sample load, prepacked, C18 Rf cartridges, 5 gram, pkg. of 5.
69-3873-312 Sample load, prepacked, Celite Rf cartridges, 2.5 gram, pkg. of 20.
69-3873-313 Sample load, prepacked, Celite Rf cartridges, 5 gram, pkg. of 20.
Empty Cartridges
69-3873-235 Sample load, empty Rf cartridges (holds up to 5 gram), package of 30.
69-3873-240 Sample load, empty Rf cartridges (holds up to 25 gram) pkg. of 30.
69-3873-225 Sample load, empty Rf cartridges (holds up to 65 gram) pkg. of 12.
69-3873-201 Sample load, empty cartridges (holds up to 260 grams), pkg. of 6.
36 Teledyne ISCO
From Traditional Glass Columns to Automated Flash Chromatography
Chapter 3
From Traditional Glass Columns

to Automated Flash
Chromatography
Manual Glass Column Chromatography

Since its inception, manual glass-column Flash chromatography
has been one of the purification methods most used by several
generations of organic chemists.
Figure 33: Photo of a manual Flash system in use

Chapter 3
Manual glass-column Flash chromatography is a practical method

offering several advantages:
• Low-cost simple material (glass column and silica gel, TLC
plates and UV lamp, test tubes, etc.).
• Stationary phase introduction and packing modes fully
controlled by chemist.
• Either isocratic or step gradient solvent elution
can be used.
• Reasonable separation results for simple crude
reaction mixtures.
• Familiar technique for organic chemists.
There are however important drawbacks associated with it:

• Time consuming overall process, especially at the
collection stage.
• Low resolution.
• Separation of complex crude reaction mixtures is difficult
and time consuming.
• Results not reproducible since silica gel column loading
and packing parameters are not standardized.
• Only isocratic or step gradient solvent elution are available
for use.
• Absence of on-line detection means every tube must be
tested by TLC.
Departments with organic compounds preparation as their core

activity are demanding increasing efficiency and productivity from
synthetic organic chemists. These requirements are conducive to
the search of improved working habits in order to maximize their
typical reaction sequence. Since the purification step often is the
time consuming step in this sequence, chemists are looking for
avenues which would significantly improve ease-of-use, reliability,
product purity, and overall productivity of this step.
Teledyne ISCO has a history of producing automated Flash
chromatography instruments meeting these performance criteria.
For a complete list of Teledyne ISCO automated Flash systems,
refer to Appendix E - CombiFlash Systems, on page 255.
38 Teledyne ISCO
Benefits of Automation
For instance, the CombiFlash Rf system offered by Teledyne ISCO
provides automated Flash chromatography that is user-friendly
and reliable, without the downsides of manual glass Flash
chromatography. For a complete list of Teledyne ISCO automated
Flash systems, refer to Appendix E - CombiFlash Systems, on page
255.
Figure 34: Photo of Teledyne ISCO’s CombiFlash Rf system

with 4x automated sequential purification option
Some of the CombiFlash Rf system’s clear advantages over glass

Flash chromatography columns are:

Chapter 3
• Fully automated system from solvent injection to product

collection. Purification time is dramatically shortened.
Chemists can concentrate on other projects.
• Easy-to-use software.
• Purifies wide range of sample sizes.
• Uses linear gradient elution power for superior peak
separation.
• Gradient Optimizer minimizes solvent usage and
purification time while preserving resolution.
• Higher productivity. There is no need to pack columns. The
pumping system delivers optimal flows and gradients for
fast purifications.
• Reliability.
• Safety features such as pressure-limited solvent delivery,
solvent vapor sensing to detect spills, and conductive
tubing to prevent a static discharge.
• Radio Frequency Identification (RFID) technology speeds
setup while minimizing errors.
• Flow rates to 200 mL/minute allow fast purifications and
better use of reversed-phase columns.
• 200 psi pressure limit allows fast runs and efficient use of
C18 and other bonded phases.
• Active solvent sensing prevents columns running dry and
resulting loss of resolution. Solvent sensing also protects
expensive C18 columns.
• Active waste sensing prevents messy and potentially
dangerous solvent spills. Active waste sensing works with
most types and sizes of waste containers.
• All-wavelength collection triggers collection on any
wavelength for improved sample collection.
Column Packing
Manually-packed Columns
Manual column packing can be used to load specialized column
media. However, commonly-used media are readily available as
pre-packed columns in a variety of sizes. For a complete list of
column media, refer to Appendix A - Column Media Selection, on
page 221.
40 Teledyne ISCO
Figure 35: Photo of glass column preparation Manual column packing allows
the chemist to load stationary phase media as needed for each
purification. Column packing that produces repeatable separation
results can be a labor-intensive process.

Chapter 3
Pre-packed Columns
Pre-packed columns improve the efficiency of compound
purification, offering greater productivity and reproducibility.
By using pre-packed columns, scientists can purify compounds
more quickly because they save the time required to pack the
column. Pre-packed columns also show higher purification
efficiency since the silica is more tightly packed allowing the
compounds more interaction with the stationary phase
(Figure 36).
1.0
0.8 Hand-packed
Absorbance
0.6
0.4
0.2
0.0
0 2 4 6 8 10 12 14 16
Minutes
2.0
1.6 Pre-packed
Absorbance
1.2
0.8
0.4
0.0
0 2 4 6 8 10 12 14 16
Minutes
Figure 36: Chromatograms of compounds separated on a hand-packed
column (top) and pre-packed RediSep Rf column (bottom). The
prepacked column shows near baseline separation and would result in
pure fractions. The hand-packed column gives mixed fractions that
would need to be run again at additional time to improve product
yield.
42 Teledyne ISCO
The use of pre-packed columns is safer than hand packed columns.

The user is not exposed to silica dust from packing the column or
during disposal of the used silica. The CombiFlash Rf will blow out
the solvent to waste from silica columns at the end of a run
allowing easy disposal of the column. The user is not exposed to
the silica or any residual compounds left on the column at the end
of the run.
Figure 37: Photo of pre-packed columns Reusable and disposable Flash

chromatography columns are a cost-effective alternative to manual
column packing, nearly eliminating column setup time. Teledyne
ISCO’s RediSep Rf columns are easy to load, allowing fast
changeover between purifications.

Chapter 3
TLC Plates
RediSep TLC plates are useful to run compounds to determine
optimal purification conditions or to confirm purity at the end of a
run. The plates are small enough to run quickly yet long enough to
allow accurate measurement of Rf differences for programming the
Gradient Optimizer. RediSep TLC plates are available in a variety of
materials to match your purification needs.
Figure 38: Photo of matching TLC media TLC media can be prepared to
match the performance characteristics of a column. Matched media
ensures that favorable analytical TLC results can be developed into
effective column purification methods.
Figure 39: Table of RediSep Rf TLC plates
Part Number TLC Plate description

69-2203-400 TLC plates, Silica Gel, package of 200, 5x10 cm each.
69-2203-401 TLC plates, C18 Reversed Phase, package of 30, 5x10 cm each.
69-2203-403 TLC plates, Alumina Basic, package of 30, 5x10 cm each.
69-2203-569 TLC plates, Alumina Neutral, package of 30, 5x10 cm each.
44 Teledyne ISCO
Figure 40: Table of solvent migration and plate development time for RediSep
C18 TLC plates (69-2203-401)
Solvent Migration
Isopropanol Full, 45 min
Water Plate degrades
Methanol Full, 15 min
Water/Methanol 1:1 Full, 45 min
Acetonitrile (ACN) Full, 15 min
Water/ACN 1:1 Full, 45 min
Water/ACN 6:1 75%, 45 min
Figure 41: Table of solvent migration and plate development time for RediSep
Basic and Neutral Alumina TLC plates
(69-2203-403 and 69-2203-569)
Solvent Migration
Hexane Full, 15 min
Ethyl acetate Full, 15 min
Hexane/Ethyl acetate 1:1 Full, 15 min
Hexane/Isopropanol 5:1 Full, 30 min
Dichloromethane Full, 15 min
Dichloromethane/Methanol 9:1 Full, 15 min

Chapter 3
High Performance Flash Chromatography

Column Stacking
One way to improve resolution is by column stacking, placing one
column on top of another of the same size. If needed, more than
one column may be stacked. Column stacking essentially creates a
long, thin column (Figure 42).
To develop a method using column stacking, the method should be
changed to column volumes. Multiply the time for each segment in
the gradient table for a single by the number of columns being
stacked, and enter this new time for each segment. The flow rate is
kept the same as for a single column since the linear velocity of the
solvent is optimized for each column in the CombiFlash system.
After entering the new gradient segment times into the method,
the method may be displayed in time, if desired, rather than
column volumes. The change to column volumes is required since
the solvent and compound needs to run through two (or more)
columns worth of media.
Column stacking works best with compounds that are weakly
retained on the column as these benefit most from the increased
interaction with the media provided by column stacking. Although
column stacking can improve resolution, it does lead to increased
back pressure due to the increased column length. The run time is
also increased by the number of columns stacked. Figure 43 shows
increases in resolution from column stacking compared to using a
regular column of a similar size, but note that the peaks are slightly
wider. The increased peak width is due to diffusion of the
compounds in the mobile phase. This diffusion is more noticeable
in compounds that have a stronger interaction with the column
media. This diffusion leads to diminishing returns from stacking
more columns in terms of improving resolution.
46 Teledyne ISCO
0.6 min
1 column
1.2 min
2 columns
1.8 min
3 columns
2.3 min
4 columns
3.0 min
5 columns
3.5 min
6 columns
4.1 min
7 columns
4.9 min
8 columns
5.7 min
9 columns
0 2 4 6 8 10 12 14 16
Run Time (min)
Figure 42: Chromatograms showing improved resolution

by stacking columns

Chapter 3
Three 4 g Columns, Stacked Single 12 g Column
1.8 min 1.2 min
2 4 6 8 10 12 14 16 2 4 6 8 10 12 14 16
Run Time (min) Run Time (min)
Figure 43: Chromatograms comparing a single column and three stacked

columns containing equivalent amounts of silica.
An alternative to stacking columns is using a higher resolution

column as it will save time and solvent. In Figure 44, the RediSep Rf
Gold column run needed only 19 minutes to purify the compounds
compared to 30 minutes for the stacked column. The Gold column
used 760 mL solvent compared to 1050 mL for the stacked
columns. In addition to the direct time and solvent saved, the
peaks from the Gold column were sharper reducing the number of
fractions collected resulting in further time savings after the
fractions were collected.
48 Teledyne ISCO
0.5 Two 24 g Columns, Stacked
0.4
Absorbance
0.3
0.2
0.1
0.0
0 5 10 15 20 25 30
Minutes
0.5 One 40 g RediSep Rf Gold Column
0.4
Absorbance
0.3
0.2
0.1
0.0
0 5 10 15
Minutes
Figure 44: Chromatogram of two 24 g stacked columns compared to a
chromatogram of a single 40 g RediSep Rf Gold column

Chapter 3
CHBr2 RediSep Rf silica

Peak 1 2.0 100
Absorbance
%B Solvent
R CH3
Peak 2
R
CH2Br
Peak 3 0.0 0
Run Conditions:
Column size: 40 g RediSep Rf Gold silica
Load: 500 mg (on 3.0 100
5 g cartridge)
%B Solvent
Solvents: Hexane and
Ethyl Acetate
Gradient: 0–20%
Flow rate: 40 mL/min
Run time: 15 min.
0.0 0
Wavelength: 254 nm
0 5 10 15
Minutes
Figure 45: Chromatogram of bromotoluenes purification A 1% sample load
of a bromotoluene mixture on an irregular shaped, larger particle
media (RediSep column, top) and a spherical shaped, smaller particle
media (RediSep Rf Gold, bottom)
50 Teledyne ISCO
Why spherical media?

RediSep Rf Gold spherical media has a smaller particle size than
standard Flash media so it provides higher resolution. The
spherical media reduces back pressure allowing high flow rates
For more information on spherical media, refer to Flash
Chromatography Essentials, on page 5.
Higher Resolution with small spherical media

RediSep Rf Gold silica columns have a particle size range of 20–
40 µm compared to 40–60 µm for standard silica. The smaller
particle size allows higher resolution so closely eluting peaks can
be resolved (Figure 45).
RediSep Rf silica
2.5 100
Absorbance
%B Solvent
0.0 0
Run Conditions:
Column size: 40 g RediSep Rf Gold silica
Load: 500 mg 2.5 100
(1.1% load)
%B Solvent
Solvents: Hexane and

Ethyl Acetate
Gradient: 0–20%
Run time: 19 min.
0.0 0
Wavelength: 254 nm
0 5 10 15 18
Minutes
Figure 46: Chromatogram of minor compound separation
of a proprietary sample using an irregular shaped, larger particle
media (RediSep column, top) and a spherical shaped, smaller particle
media (RediSep Rf Gold, bottom).

Chapter 3
This increased resolution is useful when purifying minor

components which are chemically similar to the major compound.
These minor compounds are collected for the purposes of
screening and patent protection. The minor compounds may show
activity similar to the desired material and may be of further
interest.
Collection of minor compounds is also important to demonstrate
that these materials do not affect the efficacy or toxicity of the
pharmaceutical product. Minor compounds are often difficult to
resolve from the main compounds since they are generally an
isomer of the main compound The sharper peaks allow the minor
compounds to be better resolved from the major peak (Figure 46).
50
Irregular silica, peak 1
Sample Load (mL)
40
Spherical silica, peak 1
30
20
10
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Peak Width (min)
50
Run Conditions:
Column size: 12 g Irregular silica, peak 2
Sample Load (mL)
40
Solvent: Isocratic Spherical silica, peak 2
EtOC:hexane 30
(10:90)
Flow rate: 30 mL/min 20
Run time: 9 min.

10
0
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4
Peak Width (min)
Figure 47: Diagram of sample load comparison by purifying a mixture of

acetophenone (Peak 1) and 4'-methoxyacetophenone (Peak 2) in
hexane using an irregular silica (non-RediSep) column and a
spherical silica RediSep Rf Gold column.
52 Teledyne ISCO
NO 2 NO 2
+ CN
CN
NH 2 N
H
Standard Flash Column
2.0 100
19 minutes
Run Conditions: 1.1 L solvent used
UV Absorbance
Solvent %B
Column size: 40 g
Load: 400 mg
Solvent: Hexane:Ace-
tone
Run time: 19 min. 0 0
Wavelength: 229 nm
0 5 10 15
Run Time (min)
0.4
B
A
AU
Analytical HPLC
A: Acetone
B: peak 2 compound 0
40
RediSep Rf Gold Column

2.5 5 minutes 100
Run Conditions:
670 mL solvent used
Column size: 40 g
UV Absorbance
Solvent %B
Load: 400 mg
Solvent: Hexane:Ace-
tone
Run time: 5 min.
Wavelength: 229 nm 0 0
0 1 2 3 4 5
Run Time (min)
0.4
A
B
Analytical HPLC
A: Acetone
B: peak 2 compound 0
40
Figure 48: Chromatograms of 3-(2-nitrophenyl amino) propionitrile
purifications compared to standard Flash grade silica, the RediSep
Rf Gold column demonstrated 60% time savings, 30% solvent savings,
with no loss in purity.

Chapter 3
Faster purifications
The higher resolution of RediSep Rf gold columns can be used to
more quickly purify compounds saving time and solvent. This is
accomplished by increasing the gradient; in some cases the flow
rate can be increased as well. Using CombiFlash Rf systems, the
fast parameters are optimized as a “Gold Speed” method.
To use Gold Speed, the retention factor difference between the two
compounds of interest should be greater than 0.1.
The resolution of the spherical column under Gold Speed
conditions is equivalent to that of a standard Flash column run
under standard conditions, but less solvent was used (Figure 48).
Since the peaks are much narrower, there is less solvent collected
with the peaks reducing the time required to dry the fractions.
Gold Speed also allows compounds sensitive to silica gel to be
purified since the compound has less contact time on the silica.
54 Teledyne ISCO
Figure 49: Table of RediSep Rf Gold Silica Gel Disposable Flash Columns,
spherical, 20–40 microns
Column Teledyne
Sample Size Size ISCO Part Description
(g) Number
20 mg - 0.4 g 4 69-2203-344 4 gram RediSep Rf Gold Silica Gel Disposable
columns, pkg. of 14.
60 mg - 1.2 g 12 69-2203-345 12 gram RediSep Rf Gold Silica Gel
Disposable columns, pkg. of 14.
120 mg - 2.4 g 24 69-2203-346 24 gram RediSep Rf Gold Silica Gel
200 mg - 4 g 40 69-2203-347 40 gram RediSep Rf Gold Silica Gel
400 mg - 8 g 80 69-2203-348 80 gram, RediSep Rf Gold Silica Gel
600 mg - 12 g 120 69-2203-349 120 gram, RediSep Rf Gold Silica Gel
1.1 - 22 g 220 69-2203-359 220 gram, RediSep Rf Gold Silica Gel
15 - 300g 3000 69-2203-529 3000 gram, RediSep Rf Gold Silica Gel
Disposable column, pkg. of 1.

Chapter 3
56 Teledyne ISCO
C18 Flash Chromatography
Chapter 4
C18 Flash
Chromatography
Overview of Reversed-phase Chromatography

Normal Phase Silica
“Bare” or unfunctionalized silica gel consists of a tetrahedral
crosslinked polymer of [SiO4] (Figure 50). In aqueous media, the
free valences of the exposed Si atoms adsorb hydroxyl groups
form polar silanol groups on the surface. These silanol groups
produce the polar adsorption properties that make bare silica
such a useful medium for normal phase Flash chromatography.
O O
Si O Si OH
O O
Si O Si OH
O O
Si O Si OH
O O
Figure 50: Diagram of normal phase silica Bare silica allows silanol groups to
form on the exposed surfaces.

Chapter 4
Reversed-phase Silica
In reversed-phase silica stationary phases, silanol groups are
replaced with less polar or nonpolar functional groups such as C18
(Figure 51) or phenyl.
O O
Si O Si ( )n CH3
O O
Si O Si ( )n CH3
O O
Si O Si ( )n CH3
O O
Figure 51: Diagram of reversed-phase silica
The resulting lipophilic properties obtained are useful for

purification of compounds with high polar and high protic
characteristics.
The C18 reversed-phase usefulness is illustrated in the purification
of the following classes of compounds that surveyed organic
chemists have pointed out as typically difficult to purify. C18 is the
second most common media used for Flash chromatography after
silica because it is easy to use and can purify a wide range of
compounds. Because the C18 groups protect the silica from
hydrolysis, C18 columns are reusable if treated with care. Flash
chromatography allows the use of less expensive equipment to
purify compounds in reversed-phase. The PeakTrak software on
CombiFlash systems is often easier to use than traditional prep
HPLC systems. Acid sensitive compounds can often be run on C18
because the reactive silanol groups are bound to the C18. For a
C18 columns are generally run at a pH of 8 or less because high pH
causes hydrolysis of the siloxane bond causing loss of the C18.
58 Teledyne ISCO
RediSep Rf Gold C18 columns may be run at higher pH for longer

periods of time before any performance degradation occurs.
C18 columns are often ranked by “carbon load” which runs
between 16 and 22%. This is determined by elemental analysis.
Often a higher carbon load is considered better because it
suggests more C18 material bonded onto the silica and a greater
interaction between the column and compounds to be purified.
In contrast to silica gel, 100% water can be used on C18 columns
after conditioning with organic solvent. If more than 95% water is
kept in the column, the long, oil-like, alkyl chains forming the C18
will self-associate; this phenomenon is known as “phase collapse”.
This also called “dewetting”, “chain folding”, or “matting”. Phase
collapse symptoms include retention loss, retention
irreproducibility, increased tailing, and long gradient regeneration
times. There are some C18 media, such as Teledyne ISCO C18Aq,
designed to run under aqueous conditions that exhibit reduced
phase collapse.
Figure 52: Table of RediSep Rf C18 Reversed-phase columns sample loading

capacities
Teledyne
Column ISCO Part Reversed-phase (C18) RediSep Rf
Sample Size Size (g) Number Column Description
4.3 - 86 mg 4.3 69-2203-410 4.3 gram columns, pkg. of 2.
13 - 260 mg 13 69-2203-411 13 gram column, pkg. of 1.
86 mg - 1.72 g 86 69-2203-416 86 gram column, pkg. of 1.
The typical loading on a C18 column is reduced to about 1%

(depending on the separation between compounds) compared to
loading up to 20% on silica gel.
Other types of reversed-phase chromatography include C1, C4,
and C8. These are similar to C18 but with shorter chains. These
columns have similar characteristics to C18 but show decreased

Chapter 4
retention for strongly hydrophobic compounds. Peptides and

proteins are often purified on reversed-phase columns with short
chain alkyl groups.
C18 Method Development

Thin-layer Chromatography Plates
C18 TLC plates can be used for method development if the
compound is relatively nonpolar. C18 TLC plates generally can’t be
used in highly aqueous solutions because the stationary phase
degrades. Compounds are spotted on C18 TLC plates and
developed in a manner similar to silica TLC plates. The PeakTrak
software Gradient Optimizer can also be used to develop the
gradient. Development times for C18 TLC plates range from 15 to
45 minutes.
Figure 53: Table of solvent migration and plate development time or
RediSep C18 TLC plates
Solvent Migration
Water Plate degrades
Water/Methanol 1:1 Full, 45 min
Acetonitrile (ACN) Full, 15 min
Water/ACN 1:1 Full, 45 min
Using HPLC Systems to Generate Flash Methods

Analytical HPLC systems are fairly common in labs. These units
produce very accurate gradients and require little sample to
develop a method. The steps for creating a method for Flash
chromatography are very similar to that for creating an analytical
method.
60 Teledyne ISCO
The first step is to run a gradient and determine where the

compound of interest elutes. Adjust the gradient so the desired
compound is resolved from contaminants. The next step is to
transfer the method to the Flash system. Figure 54 shows gallic
acid and pyrogallol developed on an HPLC system and transferred
to Flash. The only change was to lengthen the run time, which
“flattened” the gradient to allow better resolution.
CO2H
Gallic Acid Pyrogallol
(A) (B)
HO OH HO OH
OH OH
Analytical HPLC Instrument: Agilent 1100 HPLC

Column: Polaris C18, 3µm, 3x100mm
Gradient: 1- 50% acetonitrile in 10 min
(A)
(B) Flow Rate: 0.5 mL/min
Flow Rate: 1 mL/min
Detection Wavelength: 215 nm
Instrument: CombiFlash Rf
Flash System Column: 4.3 g RediSep C18, 40-63µm
Sample Load: 10 mg
0.50 Gradient: 0-50% acetonitrile over 14100min
0.45 Flow Rate: 16 mL/min 90
0.40 Detection Wavelength: 215 nm 80
0.35 70
Gradient % Solvent B
0.30 60
Absorbance
0.25 50
(B)
0.20 40
0.15 30
0.10
(A) 20
0.05 10
0.00 0
0 2 4 6 8 10 12 14 16
Minutes
Figure 54: Illustration of gallic acid and pyrogallol method

developed on an analytical HPLC and transferred to a Flash
reversed-phase system

Chapter 4
The compounds eluted at three to four minutes in the analytical

method. When this method was transferred to the Flash system,
the compounds eluted at the same time as the HPLC. Starting the
gradient at 0% acetonitrile gave baseline separation while allowing
a higher loading of 10 mg. Using an analytical HPLC allows
methods to be easily developed with minimal compound use.
Using the Flash Instrument for Method Development

Because C18 columns are reusable, one also can develop the
method on the Flash instrument. The use of a 4.3 g C18 column
reduces run time, solvent, and compound required to develop the
method. It is easiest to start with the default method on the
instrument and load only a few milligrams to determine where the
compounds elute. Increase or reduce the gradient slope to achieve
the desired resolution. After the method is developed, save it and
change to the column size to be used for your separation. Load the
method you saved—the CombiFlash system will automatically
scale the method for the larger column.
62 Teledyne ISCO
Methyl paraben Propyl paraben

O
(A) O
(B)
O O
OH OH
Analytical HPLC (B) Instrument: Agilent 1100 HPLC

Column: Adsorbosphere C18, 5µm, 4.6x250mm
Mobile Phase: A: H2O, B: CH3CN
(A) Gradient: 60 to 75%B in 7 minutes
Flow Rate: 1.0 mL/min
Flash System Instrument: CombiFlash Rf

Column: 4.3 g RediSep C18, 40-63µm
Sample Load: 10 mg
Mobile Phase: A: H2O, B: CH3CN
Gradient: 0 to 100% B in 13 minutes
Flow Rate: 16 mL/min
0.50 100
(A)
0.45 90
0.40 80
0.35 70
(B)
0.30 60
Absorbance
0.25 50
0.20 40
0.15 30
0.10 20
0.50 0.05 100

10
0.00 0
0.45 90
0 2 4 6 8 10 12 14 16
Minutes 80
0.40
0.35 70
0.30 60
(A)
Absorbance
0.25 (B) 50
0.20 40
0.15 30
0.10 20
0.05 10
0.00 0
0 2 4 6 8 10 12 14 16
Minutes
Figure 55: Illustration of methyl- and propyl-paraben method developed on

an analytical HPLC instrument and transferred to a Flash
reversed-phase system

Chapter 4
Loading Compounds
Loading techniques for C18 are similar to those for silica. The
compound can be liquid-loaded onto the column. Alternatively,
compounds may be adsorbed onto an inert material such as Celite.
The compound may also be loaded onto C18 solid load cartridges.
If C18 cartridges are used they should be conditioned by washing
them with the “B” solvent to be used in the separation (typically
methanol or acetonitrile) followed by the “A” solvent. This
double-wash step “raises” the C18 chain from the silica bed so it
can interact with the sample. The compound can be loaded in the
same fashion as done with a Solid Phase Extraction (SPE)
cartridge.
If loose C18 is used, the compound should be dissolved in an
organic solvent prior to adding the C18 and drying. This will
activate the C18. The compound adsorbed on the C18 can be
added to an empty RediSep Rf solid load cartridge.
Using C18 as an adsorbent (either loose or in a pre-packed
cartridge) also acts as a guard column that protects the main
column.
Column Care
With proper care, RediSep Rf C18 columns may be used for over 20
purifications. RediSep Rf C18 columns are shipped dry-packed;
before its first run, the C18 column must be washed with at least 6
column volumes of water: organic solvent prior to use. The organic
solvent is typically the “B” solvent that will be used for the
separation. The minimum concentration of organic solvent is 1:1;
higher concentrations of organic solvent are generally better. Once
wetted, a C18 column should never be allowed to dry out or
channels will form that will adversely affect future separations.
After initial conditioning, use three column volumes of the initial
solvent conditions prior to the run to equilibrate the column. After
the run is complete, make sure the system does not purge the
solvent from the column (change the method to turn off “Air
Purge” prior to the run if needed). If using a CombiFlash Rf system
with RediSep Rf columns, the column’s RFID tag will tell the system
not to purge the solvent.
The C18 methods preprogrammed into CombiFlash systems
reduce the solvent concentration to 80% B solvent for column
64 Teledyne ISCO
storage. Any modifiers (such as TFA) should be washed from the

column before storage. Store the column with the end caps in
place. The end caps that were shipped with the columns should be
retained for this purpose.
For best results, a column run with a particular organic solvent
should continue to be run in that solvent.
Solvent Modifiers
The most common solvent modifier used for reversed-phase is
trifluoroacetic acid (TFA). TFA is compatible with the stainless
steel fittings used in HPLC and CombiFlash systems. Use of a pH
above 7.5 to 8 causes the silica gel supporting the C18 to dissolve;
TFA keeps the solvent system at a low pH. TFA can also be easily
removed by lyophilization. RediSep Rf Gold columns have
demonstrated the ability to perform multiple runs at pH 10 (see
Column storage after use in high pH, on page 76).
At neutral pH, acids and bases may form their conjugates and
appear as two peaks or as a broad peak. Figures 56 and 57
illustrate this effect and how the use of TFA forces these
compounds into a single peak.
When diphenyl acetic acid is purified from esculin (Figure 57)
without TFA, the diphenyl acetic acid shows as two peaks at 3.5
and 9 column volumes. The chromatogram appears as if it has
contains three compounds with the first tailing into the esculin
peak. One hint that the first peak is diphenyl acetic acid is that it
tails back while the peak at 6 column volumes tails forward due to
the conversion between diphenyl acetic acid and its conjugate
base. In more extreme examples, the two peaks will appear to be
joined together by the tailing or “bridging”.
Solvent modifiers are not required when purifying neutral
compounds.
O O
OH O
Figure 56: Diagram of interconversion of diphenyl acetic acid and its

conjugate base

Chapter 4
Without TFA
2.00 100
1.80 90
1.60 80
1.40 70
1.20 60
Absorbance
1.00 50
0.80 40
0.60 30
0.40 20
0.20 10
0.00 0
0 2 4 6 8 10 12 14 16
Run Length 16.0 CV (28.3 min)
With TFA
2.00 100
1.80 90
1.60 80
1.40 70
1.20 60
Absorbance
1.00 50
0.80 40
0.60 30
0.40 20
0.20 10
0.00 0
0 2 4 6 8 10 12 14 16
Figure 57: Illustration of esculin/diphenyl acetic acid purification without

and with TFA modifier
66 Teledyne ISCO
Low-solubility Polar Heterocycles

The separation of a mixture of quinoxalines was investigated:
CN CN
N N
(A) NH2
(B)
NH2
N N N
N
O
CN
N
NH2
N N
(C) H2N
Flash chromatography of the quinoxalines mixture on a

CombiFlash Rf system with a C18 reversed-phase 4.3 g RediSep
column fully separated the products with water + 0.1%
TFA/acetonitrile + 0.1% TFA as the mobile phase (Figure 58).
The sample was loaded using the solid sample technique by
dissolving the sample in a necessarily high amount of methanol.
C18 reversed-phase silica was then added and the solvent was
removed in vacuo. The resulting pre-coated sample was placed in
an empty RediSep solid load cartridge.
0.50 100
0.45 90
0.40 80
0.35 70
0.30 60
Absorbance
0.25 50
0.20 (C) 40
0.15 30
0.10 (B) 20
(A)
0.05 10
0.00 0
0 10 20 30 40 50
Column Volumes
Figure 58: Chromatogram of quinoxaline mixture purification on C18

reversed-phase RediSep column with water + 0.1% TFA/acetonitrile +
0.1% TFA

Chapter 4
In addition to C18 reversed-phase RediSep Rf columns, amine or

basic alumina RediSep Rf columns are also options to consider for
the purification of low-solubility polar heterocycles. Method
development would indicate which column and solvents to select.
Primary Amines
The separation of a mixture of primary amines was investigated:
(A) (B) (C) NH2
N
N
N
N NH2 N NH2
(D)
N NH2
N
Flash chromatography of the primary amines mixture on a

CombiFlash Rf 4x system with C18 reversed-phase 4.3g RediSep
column separated the products (Figure 59).
1.00 100
0.90 90
0.80
(C) 80
0.70 70
0.60 60
Absorbance
0.50 50
(B)
0.40 40
(A) 30
0.30
0.20 (D) 20
0.10 10
0.00 0
0 20 40 60 80 100
Column Volumes
Figure 59: Chromatogram of primary amine mixture purification on C18

reversed-phase RediSep column with water/acetonitrile
In addition to C18 reversed-phase RediSep Rf columns, amine or

basic alumina RediSep Rf columns are also options to consider for
the purification of primary amines. Method development would
indicate which column and solvents to select.
68 Teledyne ISCO
Carbohydrates
The separation of a mixture of carbohydrates was investigated:
OH
(A)
O
HO
HO
OH
(B) O
OH
HO O O
O
HO
HO
OH
O
NO2
Flash chromatography of the carbohydrates mixture on Teledyne

ISCO CombiFlash Rf 4x system with C18 reversed-phase 13 g
RediSep column fully separated the products with
water/acetonitrile as the mobile phase (Figure 60). The use of a
high performance C18 column is discussed later in this chapter.
0.4 (A)
0.3
Absorbance (214 nm)
(B)
0.2
0.1
0.00
0 10 20 30
Run Time (min)
Figure 60: Chromatogram of carbohydrate mixture purification on C18

reversed-phase RediSep column with water/acetonitrile
Evaporative Light Scattering Detection (ELSD) is useful when the

compound possesses no chromophores. CombiFlash Rf and
Torrent systems can cut peaks based on a peak signal from an
external ELSD detector.

Chapter 4
Peptides
The separation of a mixture of peptides was investigated:
Gly-Pro-Ala (A) Val-Tyr-Val (B)
Flash chromatography of the peptides mixture on a Teledyne ISCO

CombiFlash Rf 4x system with C18 reversed-phase 13 g RediSep
column fully separated the products with water + 0.1%
TFA/acetonitrile + 0.1% TFA as the mobile phase (Figure 61).
1.00 100
0.90 90
0.80 80
0.70 (A) 70
0.60 60
Absorbance
0.50 50
0.40 40
0.30 (B) 30
0.20 20
0.10 10
0.00 0
0 5 10 15 20 25
Column Volumes
Figure 61: Chromatogram of peptide mixture purification

on C18 reversed-phase RediSep column with water + 0.1%
TFA/acetonitrile + 0.1% TFA
70 Teledyne ISCO
Carboxylic Acids
The separation of a mixture of shikimic acid (A) and maleic acid
(B) was investigated:
COOH
H2OC CO2H
(B)
HO OH
OH
(A)
Flash chromatography of the carboxylic acids mixture on a

Teledyne ISCO CombiFlash Rf 4x with C18 reversed-phase 4.3 g
RediSep column fully separated the products with water + 0.1%
TFA/acetonitrile+ 0.1% TFA as the mobile phase (Figure 62).
1.00 100
0.90 90
0.80 (B) 80
0.70 70
0.60 60
Absorbance
0.50 50
0.40
(A) 40
0.30 30
0.20 20
0.10 10
0.00 0
0 5 10 15 20 25 30 35 40 45 50
Column Volumes
Figure 62: Chromatogram of carboxylic acid mixture purification on C18

reversed-phase RediSep column with water + 0.1% TFA/acetonitrile +
0.1% TFA

Chapter 4
Ionic Compounds
The separation of a mixture of pyrrolidinium (A) and piperidinium
chlorides (B) was investigated:
Cl Cl
N N
N
Cl
NO2 NO2
NO2
(A) (B)
Flash chromatography of the ionic mixture on a Teledyne ISCO

CombiFlash Rf 4x system with C18 reversed-phase 13 g RediSep
column separated the products with water + 0.1% TFA/acetonitrile
+ 0.1% TFA as the mobile phase (Figure 63).
0.50 100
0.45 90
(A)
0.40 80
0.35 70 Gradient % Solvent B
0.30 60
Absorbance
0.25 50
0.20
(B) 40
0.15 30
0.10 20
0.05 10
0.00 0
0 5 10 15 20 25 30
Column Volumes
Figure 63: Chromatogram of ionic mixture purification

on C18 reversed-phase RediSep column with water + 0.1%
TFA/acetonitrile + 0.1% TFA
72 Teledyne ISCO
RediSep Rf Gold High Performance C18 Columns

Although the standard RediSep Rf C18 columns purify most
compounds well, there are times when greater resolution is
required. RediSep Rf Gold C18 columns can be used for these
purifications. This material provides near-HPLC performance at
lower pressure and cost. RediSep Rf Gold C18 columns utilize the
benefits provided by spherical silica in a smaller 30 µm average
particle size. Due to their spherical shape, back pressures are
100–150 psi at flow rates providing optimal separation—well
within the range of modern Flash equipment.
The purification of a mixture containing 5-benzyl-N--N-im-di-
t-Boc-L-His (Compound A) was investigated4:
O O
H
N
OH
O
N
N
O
Compound A was purified on a CombiFlash Rf system using

RediSep Rf Gold C18 and standard RediSep Rf C18 columns (Figure
64). The solvent system was 5–95% ACN:H2O) both containing
0.1% TFA. The RediSep Rf Gold C18 column (Figure 65) allowed
much higher loading and provided a sharper peak.
For comparison, the same sample was purified using a Waters
DeltaPrep 4000 using a Vydac 10250 mm column, 5  particle size
(Figure 66). Analytical HPLC showed no difference in purity
between the compound eluted from the HPLC or that from the
CombiFlash Rf system using a RediSep Rf Gold C18 column
(Figure 67).
4. The collaboration of Dr. David Smith at Creighton University,

School of Pharmacy is gratefully acknowledged.

Chapter 4
100
0.80
Absorbance
%B Solvent
0 0
0 10 20 30 40 50 CV
Figure 64: Chromatogram of 10 mg compound A purification on a 13 g
RediSep Rf C18 column
1.50 100
Absorbance
%B Solvent
0.75
0 0
0 10 20 30 40 50 CV
Figure 65: Chromatogram of 46 mg compound A purification on a 15.5 g
RediSep Rf Gold C18 column
74 Teledyne ISCO
100
Absorbance
%B Solvent
0
Time
Figure 66: Chromatogram of 10 mg compound A purification on a Waters
DeltaPrep 4000 system using a Vydac 10x250 mm column, 5 μ
particle size
23.186
CombiFlash Rf system with

RediSep Rf Gold C18 column
29.239
3.150
START
STOP
23.005
DeltaPrep 4000 system

with Vydac column
29.266
3.361
START
STOP
Figure 67: Illustration of analytical HPLC to compare purity of compound A

separated on two systems

Chapter 4
RediSep Rf Gold C18 Columns at High pH

Many compounds generate multiple ionic species during the
course of C18 purification that cause broadened peaks which
reduce resolution. If the interconversion of the compounds is slow,
each ionic compound may appear as an individual peak.
Trifluoroacetic acid (TFA) often is used for ion pairing (with basic
compounds) or ion suppression (acidic compounds) since C18
columns are compatible with low pH.
There is a need to purify compounds on reversed-phase columns
at higher pH. Many compounds are acid sensitive yet form
peak-broadening ionic species at neutral pH. Purifying the
compounds at basic pH would improve their purity and yield. C18
columns typically are not stable at pH greater than 7.5 because the
basic solution dissolves the silica underlying the C18 chains,
eventually washing the C18 away and causing a decrease in column
performance. These changes take the form of reduced plate counts
or changes in retention time. RediSep Rf Gold C18 columns are
end-capped to resist attack of the silica by the mobile phase.
RediSep Rf Gold C18 columns can be used for purifying
compounds up to pH 10 with only minimal change over time.
Compared to steel C18 prep HPLC columns, RediSep Rf Gold C18
columns are inexpensive and useful for milligram to gram scale
purifications.
RediSep Rf Gold columns have been run for several hours exposed
to pH 10 mobile phases. Like other silica-based C18 columns, the
RediSep Rf Gold columns will degrade over time. However, the
degradation is slow enough that the column is still usable for many
runs. The degradation is reduced as the pH is lowered or exposure
time is decreased.
Column storage after use in high pH

After use, wash the column with 5 column volumes of the organic
solvent. Run the column with a standard gradient method from 5 to
95% organic solvent in unbuffered water. The column was stored in
80% organic solvent in water as described earlier (Column Care, on
page 64). The extended wash is intended to remove as much of the
ammonium hydroxide from the column as possible. After washing,
store the column in 80% methanol or acetonitrile. The column can
be stored indefinitely without further degradation as long as it
does not dry out.
76 Teledyne ISCO
Figure 68: Table of Reusable RediSep Rf Gold C18 Reversed-phase

columns 20–40 microns sample loading capacities
Column Teledyne
Size ISCO Part
Sample Size (g) Number Description
5.5 - 110 mg 5.5 69-2203-328 5.5 gram RediSep Rf Gold C18 columns,
pkg. of 2.
15.5 - 310 mg 15.5 69-2203-334 15.5 gram RediSep Rf Gold C18 column,
pkg. of 1.
30 - 600 mg 30 69-2203-335 30 gram RediSep Rf Gold C18 column,
pkg. of 1.
50 mg - 1.0 g 50 69-2203-336 50 gram RediSep Rf Gold C18 column,
pkg. of 1.
100 mg - 2 g 100 69-2203-337 100 gram RediSep Rf Gold C18 column,
pkg. of 1.
150 mg - 3 g 150 69-2203-338 150 gram RediSep Rf GoldC18 column,
pkg. of 1.
pkg. of 1.
pkg. of 1.
0.95 - 19 g 950 69-2203-492 950 gram RediSep Rf Gold C18 column,
pkg. of 1.
1.9 - 38 g 1900 69-2203-493 1.9 kg RediSep Rf Gold C18 column,
pkg. of 1.
3.8 - 76 g 3800 69-2203-528 3.8 kg RediSep Rf Gold C18 column,
pkg. of 1.

Chapter 4
RediSep Rf Gold C18Aq for Highly Aqueous Mobile Phases

RediSep Rf Gold C18Aq media consists of C18 bonded to silica with
modifications to show improved performance under conditions
where the solvent contains a high concentration (>50%) of water. If
compounds elute with more than 50% organic solvent, the RediSep
Rf Gold C18 media shows improved performance compared to
C18Aq media. Purifications of water-soluble organic compounds
are demonstrated in Figure 69 where the polar compound exhibits
improved retention.
Standard C18 columns are designed to reduce the interactions of
unreacted surface silanols by end-capping with a methyl group or
another nonpolar entity. This limits their selectivity towards highly
polar compounds due to “phase collapse” (Figure 69). Phase
collapse causes reduced retention due to decreased interaction of
the sample with the C18 chains. Irreproducible runs are also
another symptom of phase collapse.
H2 O
H2O
Silica Surface
H2O
H2O H2O
H2O
R H 2O
H2O
R H2O H2O
H2O
R H 2O
H2O H2O
Figure 69: C18 chains are fully extended when organic solvent is present but
undergo “phase collapse” (left) under highly aqueous
conditions. Hydrophilic groups (R) reduce phase collapse (right).
RediSep Rf Gold C18Aq media, with its hydrophilic groups, reduces

tendencies toward phase collapse and allows better retention of
highly polar samples.
78 Teledyne ISCO
Compounds that require more than 50% water in the mobile phase
for elution are candidates for purification with RediSep Rf Gold
C18Aq. Examples include water soluble dyes, glycosylated
compounds, and other materials containing multiple polar groups.
RediSep Rf Gold C18Aq columns are a preparative alternative to
analytical separations currently performed with hydrophilic C18
columns.
Although RediSep Rf Gold C18Aq columns work well under all
percentages of organic solvent, improvements in resolution are
seen using RediSep Rf Gold C18 for compounds that elute using
more than 50% organic solvent.
Use and Care of RediSep Rf Gold C18Aq

RediSep Rf Gold C18Aq columns are dry packed and may be stored
indefinitely under these conditions.
Prior to use, the column should be activated with the organic
solvent used in the purification. Activate the column by washing
with ~6 column volumes of solvent containing ~80% of the organic
solvent used in the mobile phase. After the initial wash, the
column should be stored with end caps, to prevent it from drying.
Additionally, the columns should be stored in at least 50% organic
solvent.
RediSep Rf Gold C18Aq can be run with acetic acid, trifluoroacetic
acid, or other modifiers to improve peak shape. After use, these
modifiers should be washed from the column to improve column
life.

Chapter 4
Water soluble dyes

50 mg each of tetrazine and brilliant blue were dissolved in 2.0 mL
water. Of this solution, a 1.0 mL sample was injected onto a 30 g
RediSep Rf Gold C18 and a RediSep Rf Gold C18Aq column. The
columns were run according to the conditions listed in Figure 70.
O O– +
S O Na–
S O
O
O
Brilliant Blue Na+ N O
N+ –O N
Compound 2
N OH
N
O
OH
S
HN N O Tetrazine
O Compound 1
S
O –O O
Na+
The RediSep Rf Gold C18Aq column displayed increased retention

of both compounds with better resolution between compounds.
100
1.6 1
2
2
UV Absorbance, 214 nm
1.2
50
0.8 1
0.4 RediSep Rf Gold C18Aq

0 %B
RediSep Rf Gold C18

0.0
0 6 12 18
Run Time, Minutes
Figure 70: Comparison of RediSep Rf Gold C18Aq and standard RediSep Rf

Gold C18 with sample requiring highly aqueous mobile phase. The
mobile phase for both runs was water (A) and methanol (B) at 35
mL/min. Compound 1 is not retained on the RediSep Rf Gold C18
column under low organic solvent conditions.
80 Teledyne ISCO
Desalting samples
Compounds are often purified with solvents that contain salts or
buffers that need to be removed prior to subsequent studies with
the compound. The RediSep Rf Gold C18Aq columns have the
ability to adsorb compounds under highly aqueous conditions and
are ideal for removing the salt from a sample. By following the
procedures for desalting samples you will remove a large portion
of water from samples dissolved in aqueous solvents facilitating
evaporation. The procedure is similar to solid phase extraction,
but on a larger prep scale.
Compounds run on ion exchange media are often eluted with
buffers that are difficult to remove from the sample after
purification. Buffers, such as phosphate salts, are often used to
improve resolution during purification on C18 by maintaining the
pH of the solvent at a value such that the eluted compounds
remain in a consistent ionization state.
The RediSep Rf Gold C18Aq columns are well-suited to desalting
applications because they resist phase collapse. Samples requiring
desalting are generally dilute and the large volume of water
eventually causes phase collapse with other types of C18.
The general procedure for desalting samples is as follows:
1. Remove any organic solvents by rotary evaporation.
Organic solvents tend to reduce the binding of compounds
to C18. Stop evaporation if the sample appears to be
precipitating.
2. Condition the RediSep Rf Gold C18Aq column by washing
with an organic solvent such as methanol or acetonitrile
for 5 column volumes (CV) followed by water for 5 CV.
3. Load the sample. For desalting purposes, load up to 5% by
weight on the column because the desired compound is
captured from the salt solution and released. For example,
a 5.5 g RediSep Rf Gold C18Aq column can be used to
capture up to ~250 mg compound. If further purification is
desired after washing off the salt, use a 1% load. Larger
volumes can be loaded with a CombiFlash sample load
pump. Since the sample is being loaded in a weak, polar
solvent the total volume injected may exceed the column
volume without sample loss.

Chapter 4
a. Place the Rf+ in manual control, and select Liquid

Injection (Pumps Disabled) from the Valve Position
control. Place the column on the instrument and load
the sample.
b. For an Rf 150, place the column on the system and
load the sample.
c. Verify the column has been conditioned according to
the manufacturer’s instructions.
4. Wash the salt from the column using manual control. On
the Rf+, select Through column and flow cell from the Valve
Position control. On an Rf 150, connect the solvent line to
the column shuttle. Set the flow rate to the value listed on
the column label and press the Prime A button. Wash the
column for at least one, and preferably five, column
volumes.
5. Elute the compound. Change the method to a step gradient
after 1 CV. The step should go to 100% B. If the compound
requires further purification, use a gradient suitable for the
purification. From the Run Requirement screen, select
None-On Column from the Sample Loading Technique
control because the column was equilibrated and the
sample is on the column.
Always run a small sample to verify the compound will adsorb on
the column before running the entire sample mixture.
The example in Figure 71 is brilliant blue purified from a WAX
column eluted with 1M NaH2PO4 in water. As there was no organic
solvent involved, the mixture was not evaporated prior to loading
on the column.
The step gradient provides the following features:
• Run time and solvent usage is reduced.
• The compound elutes in a sharper band reducing the
volume of solvent to evaporate, saving time.
There is relatively little water in the sample, so drying time is
reduced because organic solvents, such as methanol, are easier to
evaporate.
If desired, a regular gradient can also be run if the compound
requires further purification instead of the step gradient.
82 Teledyne ISCO
2.0 100
O
O–
S
80 O
1.5
Absorbance (214 nm)
N+
60
Methanol (%)
1.0
40
O
OH
S
0.5 HN N
O
20
0.0 0 O
0 5 10 15 20
Time (CV)
Figure 71: Desalting of brilliant blue with a RediSep Rf Gold C18Aq

column
Removal of Non-volatile Solvents

Samples are often dissolved in non-volatile solvents, such as
dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), for
experiments such as nuclear magnetic resonance (NMR) studies.
Recovery of the sample from these solvents is difficult because of
the solvent’s high boiling point. RediSep Rf Gold C18Aq columns
are useful for retaining the desired compound while removing the
non-volatile solvent. The compound is then released using a
solvent with a lower boiling point that is more easily evaporated.
This method allows the recovery of valuable material for further
experiments.
DMSO-d6 is often used to dissolve polar compounds for NMR
structure determination because it has a simple NMR spectrum
itself and freshly-opened ampoules do not contain hydrogen that
can exchange with labile protons such as those on amines,
alcohols, or phenols. DMSO boils at 189° C and DMF, another
common NMR solvent, boils at 153° C under atmospheric pressure.
These solvents are difficult to evaporate under vacuum and
heating the mixture to speed evaporation may damage the
compound of interest.
Many of these non-volatile solvents are soluble in water allowing
compounds to be captured on a reversed-phase resin, such as
RediSep Rf Gold C18Aq, and released into a volatile solvent, such

Chapter 4
as methanol, after washing the solvent used for the NMR

experiment off the column. As NMR studies generally use purified
compounds, there is no material left on the column after washing
and the column can be used for this purpose several times.
The RediSep Rf Gold C18Aq columns are well suited to this
application because they resist phase collapse. NMR samples are
generally dilute and the large volume of water used to dissolve the
sample and non-volatile solvent can eventually cause phase
collapse with other types of C18.
2.5 100
OH
2.0 80
OH
Absorbance (214 nm)
1.5 60 HO O
MeOH (%)
OH
1.0 40
O
OH OH
0.5 20 O
OH
0.0 0
0 5 10 15 OH
Time (CV)
Figure 72: Elution of epigallocatechin gallate after removal of DMSO-d6
A sample (95 mg) of epigallocatechin gallate was isolated5 and

dissolved in DMSO-d6 for proton and carbon NMR analysis. A 5.5 g
RediSep Rf Gold C18Aq column was conditioned by washing with 2
column volumes (CV) methanol, followed by 5 CV water. The 1 mL
sample was removed from the NMR tube and the mixture dissolved
in 5 mL water which was loaded on the column. The CombiFlash Rf
system was run in manual control mode and the column was
washed with 5 CV water to remove the DMSO. The column was
then run with a step gradient to 100% methanol (Figure 72). The
peak was monitored at 214 nm. The chromatogram shows the
compound eluted in a single sharp peak. A linear gradient could be
run if the compound required further purification. The sample load
5. Purification of Phenolic Flavonoids with Flash

Chromatography, Silver, J.E.; Drooby, M.; Lewis, R.L.
presented at the International Congress on Natural Products
Research 31 July 2012
84 Teledyne ISCO
should be limited to 5% of the column weight to avoid sample

“breakthrough”. For example, a 5.5 g RediSep Rf Gold C18Aq
column would desolvate up to ~250 mg sample. The column
injection and wash should be captured if there is any question
whether the desired compound will adsorb on a C18 column so as
to avoid possible sample loss.
Using a RediSep Rf Gold C18Aq column allows facile recovery of a
compound from a non-volatile solvent. The example compound
elutes from a C18 column at 30% methanol when run as a gradient.
It dissolves in water, but it still adsorbed well onto the C18Aq
column to allow removal of the DMSO-d6. This represents a general
method for isolating compounds from such solvents for further
studies.
Non-aqueous Reversed-phase
Non-aqueous reversed-phase is a useful method to purify nonpolar
compounds allowing resolution of materials that are difficult to
purify on silica gel. The method is suitable for long-chain,
non-polar compounds such as lycopenes or acyl glycerides. Some
symmetric molecules such as crown ethers may also be purified
with this method. The purification of lycopenes is described.
Lycopenes were used as a model of other antioxidants of similar
polarity to determine the best method to purify this class of
compounds. Lycopene, xanthophylls, and carotenes are long chain
pigment compounds that act as antioxidants and are common in
nutraceuticals and natural-based dyes. These compounds are
characterized as long conjugated chains that may be terminated
with ring systems. The rings may contain ketones or hydroxyl
groups.
Nonpolar compounds containing long hydrocarbon chains are
difficult to purify using normal phase chromatography due to their
weak interaction with the stationary phase. These hydrophobic
compounds are also strongly retained under reversed-phase
conditions. Non-aqueous reversed-phase is a useful means of
purifying these compounds. Non-aqueous reversed-phase is not
commonly used because the solvents typically used reduces UV
detection of compounds6. All-wavelength collection, unique to
CombiFlash systems, suppresses the solvent absorbance and
6. Snyder, L.R.; Kirkland, J.J.; Glajch, J.L.; Practical HPLC

Method Development, 2nd Ed, John Wiley & Sons, 1997, p.264

Chapter 4
allows collection of compounds with UV spectra that overlap the

solvent absorbance.
In addition, the low concentration of most compounds of interest
require large medium pressure liquid chromatography systems to
obtain compound from a large volume of starting material,
typically tens to hundreds of grams. The large scale system is
useful for purifying minor components as well. In this example, a
reversed-phase chromatography system is used to purify
carotenoid compounds as a model for other natural products.
Method optimization was performed on a small scale with a
CombiFlash Rf system equipped with a UV-vis detector with a
15.5 g RediSep Rf Gold C18 column using methanol and ethyl
acetate, acetone, or methylene chloride. Samples were adsorbed
on 5 g C18 Solid Load Cartridges or adsorbed onto Celite and
placed into empty 5 g solid load cartridges. Lycopene compounds
were detected at 473 or 360 nm. The standard method for the C18
column was changed to start with 100% methanol (Solvent A), and
end with 100% of the “B” solvent.
The non-aqueous reversed-phase method was able to purify the
mixture into three main groups of peaks. Silica gel and the other
media failed to purify the mixture. Method development on silica
gel for these compounds is difficult because a small amount of
strong “B” solvent causes the mixture to move together on a TLC
plate.
Non-aqueous reversed-phase can be used with a variety of
solvents. Methanol/methylene chloride can be used as can
methanol/ethyl acetate. Acetonitrile is a substitute for methanol.
The C18 is chemically bonded to the silica so using strong organic
solvents will not harm the column and it can be used for 20 to 30
runs. The column should be stored in methanol, acetonitrile, or
isopropanol.
86 Teledyne ISCO
Changes to the C18 method

The standard C18 method was modified as follows for non-aqueous
reversed-phase:
• The initial segment was changed to 0% B.
• The gradient was ramped to 100%
• The final segment was changed to 0% B. By returning to 0%
B, the column is ready for the next run.
The column should be stored in alcohol or acetonitrile which may
be used as the starting solvent.
Using C18 TLC plates, it was determined that the carotenoids did
not elute with 100% methanol. Using 1:1 methanol:
dichloromethane, the carotenoids had an Rf of 0.72. Using 1:1
methanol: ethyl acetate, the carotenoids had an Rf of 0.81;
indicating that a gradient of ethyl acetate or dichloromethane
would elute the compounds.
The use of a CombiFlash Rf system allowed the method to be
quickly fine tuned. The “scale-up” feature in the PeakTrak software
was used to generate the method for the CombiFlash Torrent
module.
100
0.8 90
All-Wavelength
Detection 200-360 nm 80
70
Percent Dichloromethane
0.6
60
AU 473 nm
50
0.4
40
30
0.2
20
10
0.0 0
1 2 3 4 567 8
-0.1
0 2 4 6 8 10 12 14 16
Run Time (Column Volumes)
Figure 73: Purification of carotenoids with methanol/dichloromethane

using all-wavelength collection and collection at 473 nm. Fractions
collected identified by bands and numbers.

Chapter 4
Figure 74: Mass and yield of fractions collected

Fraction 1 2 3 4 5 6 7 8 Total
Fraction 1.8742 5.4886 0.271 0.0582 0.1594 0.1577 0.2757 0.8827 9.1675
Mass (g)
% of Crude 16.9% 49.4% 2.4% 0.5% 1.4% 1.4% 2.5% 8.0% 82.6%
Extract
1.4 100
large scale MPLC
90
1.2
80
1.0 70
Percent Dichloromethane
60
0.8
AU
50
0.6
40
0.4 30
20
0.2
10
0.0 0
0 10 20 30 40
Run Time (Minutes)
Figure 75: Analytical HPLC for fraction number 8
88 Teledyne ISCO
Chapter 5
Advanced Flash
Chromatography
Alternative Chromatographic Media

Chemists generally proceed with normal phase silica gel for
purification of medium to low polarity products. Reversed-phase
silica gel is used for high polarity products.
When a mixture cannot be separated on normal or reversed-phase
silica gel, or when the use of solvent modifiers becomes
impractical, the alternative is to use specialty stationary phase
media. Specialty media offers different adsorptive properties and
selectivities, such as by acidic or basic moiety, or by ionic charge.
Media options include:
• Normal-phase silica • C18 reversed-phase silica

• Amine • Cyano
• Alumina-basic • Diol
• Alumina-neutral • Strong anion exchange
• Alumina-acidic • Strong cation exchange

Chapter 5
Specialty Media
Some specialty media “end cap” the carbon tether chain with a
functional group conferring the stationary phase specific
properties. This allows for different selectivity than silica or C18,
and is useful for purification of delicate compounds.
O O
Si O Si ( )n FG
O O
Si O Si ( )n FG
O O
Si O Si ( )n FG FG = NH2
= CN
O O =...
Figure 76: Diagram of functionalized media Binding chains of functional

groups to the tetrahedral silica can create media with an affinity for a
specialized characteristic of a compound.
Some examples of specialty columns and their applications are

listed in the following paragraphs.
Amine
Compounds that have an acidic or basic moiety may streak or tail
with normal or reversed-phase silica. Streaking or tailing will
ultimately cause overlapping fractions.
Typically, when using silica, chemists spike their solvents with
either triethylamine (TEA) if they have a basic component, or
acetic acid (AcOH) if they have an acidic component in their target
compound. The problem here is that solvents have to be swapped
and primed before compounds can be separated, then purged from
the system after the run.
90 Teledyne ISCO
With an acid or base moiety covalently bound to the stationary

phase, the need to switch solvents is eliminated. Additionally, TEA
or AcOH are not used so they don’t need to be removed after the
purification is complete.
Si NH2
Figure 77: Diagram of amine structure
To illustrate how an amine media can be of assistance, the

separation of a mixture of nicotine (A) and 1-methylbenzimidazole
(B) was investigated.
(A) N
N
N (B)
Separation of the mixture on normal phase and amine

functionalized silica were examined.
Flash chromatography of the heterocycles mixture with an amine
functionalized RedSep column fully separated the products with
hexane/ethyl acetate as the mobile phase (Figures 78 and 79). The
normal phase column did not separate them.
The amine functionalized silica can be used in either normal or
reversed-phase conditions. It is useful in purification of
compounds holding basic properties. This is particularly true if
spiking with TEA is required for purification with normal phase
silica. Although amine columns can be used with reversed-phase
solvents, it is easiest to think of amine as a normal phase column,
where a more polar solvent causes faster elution of the
compounds.
The use of an amine column can eliminate the need to spike with
TEA, and as a result, reduce time required to remove the TEA
mixed with the solvent from purified fractions.
Amine columns are reusable because the amine group is bonded to
the silica. After the first use, do not allow the column to dry out
since drying the column will adversely affect future purifications.

Chapter 5
Turn off the air purge on your Flash system’s method. The
CombiFlash system will turn off the air purge by reading the
column RFID tag. Remove all organic modifiers by flushing with
three column volumes of 80% acetonitrile in water or 100%
isopropanol and store the column in the wash solvent. If the
storage solvents are immiscible with the solvents used for the
separation, you may need to wash the column with an
intermediate solvent prior to storage. For a complete list of
Teledyne ISCO automated Flash systems, refer to Appendix E -
CombiFlash Systems, on page 255.
2 3 45
0.50 100
0.45 90
0.40 80
Absorbance (AU)
0.35 70
0.30 60
0.25 50
0.20 40
0.15 (A+B) 30
0.10 20
0.05 10
0.00 0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Column Volumes
Figure 78: Chromatogram of normal phase column elution with hexane/ethyl

acetate Heterocycles mixture did not separate.
0.50 6 7 8 9 10 11 100
0.45 90
0.40 80
0.35
(A) 70
Absorbance (AU)
0.30 60
0.25 50
0.20 40
(B)
0.15 30
0.10 20
0.05 10
0.00 0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Column Volumes
Figure 79: Chromatogram of amine functionalized column with hexane/ethyl

acetate Heterocycles mixture successfully separated.
Ketones and aldehydes may react with the aminopropyl stationary

phase to form imines so care should be taken when purifying these
92 Teledyne ISCO
materials. A small amount should be purified first on a small

column, and scaled up if the purification is successful.
Method development for amine columns can be done using a
sample with a small column.
Figure 80: Table of Reusable RedSep Rf Gold Amine Columns
20–40 microns
Column Teledyne
Size ISCO Part
5.5 - 110 mg 5.5 69-2203-504 5.5 gram Amine RedSep Rf Gold columns,
pkg. of 2.
15.5 - 310 mg 15.5 69-2203-505 15.5 gram Amine RedSep Rf Gold column,
pkg. of 1.
30 - 600 mg 30 69-2203-506 30 gram Amine RedSep Rf Gold column,
pkg. of 1.
50 mg - 1.0 g 50 69-2203-507 50 gram Amine RedSep Rf Gold column,
pkg. of 1.
100 mg - 2 g 100 69-2203-508 100 gram Amine RedSep Rf Gold column,
pkg. of 1.
150 mg - 3 g 150 69-2203-509 150 gram Amine RedSep Rf Gold column,
pkg. of 1.
pkg. of 1.
pkg. of 1.
0.9 - 19 g 950 69-2203-512 950 gram Amine RedSep Rf Gold column,
pkg. of 1.
1.9 - 38 g 1900 69-2203-513 1.9 kg Amine RedSep Rf Gold column,
pkg. of 1.
3.8 - 76 g 3800 69-2203-534 3.8 kg Amine RedSep Rf Gold column,
pkg. of 1.

Chapter 5
Basic Alumina
Basic alumina is a mixture of different aluminum oxides that are
partially dehydrated. The intrinsic basicity of this media gives it
similar applications to the amine media.
To illustrate how a basic alumina media can be of assistance, the
separation of a mixture of quinazolinone and benzimidazole
derivatives was investigated.
(A) 3-(1-piperdinylmethyl)-4(3H)-quinazolinone
O
N N
(B) 5,6-dimethyl-1-(piperdinomethyl)benzimidazole
N
Flash chromatography of the heterocycles mixture with a normal

phase silica gel did not separate the two products successfully
(Figure 81).
0.50 100
0.45
0.40
75
0.35
Absorbance (AU)
0.30
0.25 50
0.20
(A+B)
0.15
25
0.10
0.05
0.00 0
2 4 6 8 10 12 14 16 18 20
Time (minutes)
Figure 81: Chromatogram of normal phase silica column

Elution with hexane/ethyl acetate
94 Teledyne ISCO
However, the use of basic alumina RedSep column successfully

resolved the two nitrogen-containing heterocycles (Figure 82).
Although the two product peaks on the chromatogram show
incomplete baseline resolution, analytical examination of resulting
fractions has shown limited cross-contamination.
0.50 100
0.45
0.40
75
0.35
Absorbance (AU)
0.30
0.25 50
0.20
(A) (B)
0.15
25
0.10
0.05
0.00 0
2 4 6 8 10 12 14
Time (minutes)
Figure 82: Chromatogram of basic alumina column

Alumina columns are not multiple use columns, so there is no need

for cleaning these columns.
Methods can be developed on basic alumina plates.
Figure 83: Table of solvent migration and plate development time for RedSep
Basic Alumina TLC plates (69-2203-403)
Solvent Migration
Hexane Full, 15 min
Ethyl acetate Full, 15 min
Hexane/Ethyl acetate 1:1 Full, 15 min
Hexane/Isopropanol 5:1 Full, 30 min
Dichloromethane Full, 15 min
Dichloromethane/Methanol 9:1 Full, 15 min

Chapter 5
Figure 84: Table of RedSep Rf Alumina Basic Columns

Column Teledyne
Size ISCO Part
40 - 320 mg 8 69-2203-450 8 gram Alumina Basic RedSep Rf columns,
pkg. of 20.
120 - 960 mg 24 69-2203-451 24 gram Alumina Basic RedSep Rf columns,
pkg. of 20.
240 mg - 1.92 g 48 69-2203-452 48 gram Alumina Basic RedSep Rf columns,
pkg. of 15.
400 mg - 3.2 g 80 69-2203-453 80 gram Alumina Basic RedSep Rf columns,
pkg. of 15.
0.8 - 6.4 g 160 69-2203-456 160 gram Alumina Basic RedSep Rf columns,
pkg. of 12.
pkg. of 10.
pkg. of 6.
pkg. of 4.
96 Teledyne ISCO
Neutral Alumina
Neutral alumina is a useful media in situations when acid-sensitive
products partially or fully degrade during purification due to the
intrinsic slight acidity of normal phase silica gel. The neutral
properties of this media also allows the purification of substances
holding basic properties.
To illustrate this latter possibility, the separation of a mixture of
two pyridine derivatives was investigated.
(A) 2-(2-piperdinoethyl)pyridine
(B) 1-(2-pyridiyl) piperazine
N N NH
Flash chromatography of the heterocycles mixture on a normal

phase RedSep column did not separate the two products
successfully (Figure 85).
0.50 100
0.45
0.40
75
0.35
Absorbance (AU)
0.30
0.25 50
0.20
0.15
25
0.10
0.05
0.00 0
0 5 10 15 20 25 30 35 40 45 50
Column Volumes
Figure 85: Chromatogram of normal phase silica column

Elution with hexane/ethyl acetate – peaks not visible.

Chapter 5
However, the use of neutral alumina RedSep column successfully

separated the two nitrogen-containing heterocycles (Figure 86).
1.00 100
0.90
(B)
0.80
75
0.70
Absorbance (AU)
0.60
0.50 (A) 50
0.40
0.30
25
0.20
0.10
0.00 0
0 10 20 30 40 50
Column Volumes
Figure 86: Chromatogram of neutral alumina column

Alumina columns are not multiple use columns, so there is no

information for cleaning and storing these columns.
98 Teledyne ISCO
Figure 87: Table of RedSep Rf Alumina Neutral Columns

Column Teledyne
Size ISCO Part Alumina Neutral RedSep Rf column
40 - 320 mg 8 69-2203-440 8 gram columns, pkg. of 20.
240 mg - 1.92 g 48 69-2203-442 48 gram columns, pkg. of 15.
1.2 - 9.6 g 240 69-2203-444 240 gram columns, pkg. of 10.
Figure 88: Table of RedSep Rf Alumina Acidic Columns

Column Teledyne
Size ISCO Part Alumina Acidic RedSep Rf column
5.5 - 110 mg 8 69-2203-430 8 gram columns, pkg of 20.
15.5 - 310 mg 24 69-2203-431 24 gram columns, pkg. of 20.
50 mg - 1.0g 80 69-2203-433 80 gram columns, pkg. of 15.
100 mg - 2 g 160 69-2203-436 160 gram columns, pkg. of 12.
150 mg - 3 g 240 69-2203-434 240 gram columns, pkg. of 10.
0.9 - 19 g 660 69-2203-435 660 gram columns, pkg. of 4.

Chapter 5
Cyano
Cyano functionalized silica acts similar to normal phase silica
when using similar solvents.
In reversed-phase conditions, it is similar to C4 reversed-phase
columns, although the elution order and selectivity of compounds
may be different. This allows chemists to purify compounds that
may not be well resolved on C18.
C N
Si
Figure 89: Diagram of cyano structure
Cyano columns are reusable. After the first use, do not allow the
column to dry out since drying the column will adversely affect
future purifications. Turn off the air purge on your Flash system’s
method. The CombiFlash system will turn off the air purge by
reading the column RFID tag. Remove all organic modifiers by
flushing with three column volumes of 80% acetonitrile in water or
100% isopropanol and store the column in the wash solvent. If the
intermediate solvent prior to storage.
Figure 90: Synthesis of trans-anesole oxide
100 Teledyne ISCO

Trans-anisole oxide purification

RedSep Rf Gold Cyano and RedSep Rf Gold Diol columns provide
viable alternative methods for the purification of delicate
compounds, such as epoxides, that often decompose on silica gel.
A reusable cyano solid load cartridge was made by filling a 5 g
“empty” solid load cartridge with 2.5 g RedSep bulk cyano media.
The reaction mixture was dissolved in dichloromethane and
placed in the cyano solid load cartridge. The dichloromethane was
evaporated by placing the solid load cartridge on the CombiFlash
Rf+ system and blowing air through the cartridge with the air-purge
feature via manual control. The cyano solid load cartridge ensures
a compatible adsorption surface and minimizes compound
decomposition. The mixture was eluted with a hexane-ethyl
acetate gradient (Figure 91) using a 50 g RedSep Rf Gold Cyano
column. Fractions were collected at 280 nm and evaporated into
tared flasks at 45° C.
10
2.0x10 0.5 0.5 100
0.4 0.4
10
1.5x10
% B (water)
Absorbance (All-Wavelength)
Ion Current (1-1200 Da)
Absorbance (254 nm)
0.3 0.3
10
1.0x10 50
% B (EtOAc) % B (EtOAc)
0.2 0.2
9
5.0x10
0.1 0.1
0.0
0.0 0.0 0
0 20 40 60
Run Time (CV)
Figure 91: Purification of trans-anisole oxide with RedSep Rf Gold Cyano

column

Chapter 5
Figure 92: Compound Recovery for Cyano

Fraction Recovered Mass (g)
1 0.0575
2 0.3012
3 0.421
4 0.0107
5 0.0076
6 0.0510
7 0.0187
Total: 0.4888
Acidic silanols on the chromatographic media likely open the

epoxide to form an ester that irreversibly binds to the silica. No
material was found to elute from silica. The cyano group is bonded
to the silica media which protects the compound by preventing
reaction of the epoxide with acidic silanol groups while providing a
surface with the required selectivity to purify the mixture running
as normal phase columns. The use of low-boiling normal phase
solvents allows rapid evaporation of fractions at a moderate
temperate compared to reversed-phase solvents, further reducing
the stress on fragile compounds.
102 Teledyne ISCO

Figure 93: Table of Reusable RedSep Rf Gold Cyano Columns

20–40 microns
Column Teledyne
Size ISCO Part
5.5 - 110 mg 5.5 69-2203-494 5.5 gram Cyano RedSep Rf Gold columns,
pkg. of 2.
15.5 - 310 mg 15.5 69-2203-495 15.5 gram Cyano RedSep Rf Gold column,
pkg. of 1.
30 - 600 mg 30 69-2203-496 30 gram Cyano RedSep Rf Gold column, pkg.
of 1.
50 mg - 1.0 g 50 69-2203-497 50 gram Cyano RedSep Rf Gold column, pkg.
of 1.
100 mg - 2 g 100 69-2203-498 100 gram Cyano RedSep Rf Gold column,
pkg. of 1.
150 mg - 3 g 150 69-2203-499 150 gram Cyano RedSep Rf Gold column,
pkg. of 1.
275 mg - 5.5 g 275 69-2203-500 275 gram Cyano RedSep Rf Gold column,
pkg. of 1.
415 mg - 8.3 g 415 69-2203-501 415 gram Cyano RedSep Rf Gold column,
pkg. of 1.
0.9 - 19 g 950 69-2203-502 950 gram Cyano RedSep Rf Gold column,
pkg. of 1.
1.9 - 38 g 1900 69-2203-503 1.9 kg Cyano RedSep Rf Gold column,
pkg. of 1.
3.8 - 76 g 3800 69-2203-533 3.8 kg Cyano RedSep Rf Gold column,
pkg. of 1.

Chapter 5
Diol
Diol columns work well with long-chain compounds. Diol columns
are useful for compounds that are unstable, or irreversibly bind to
silica.
Diol functionalized silica is less polar and has higher retention time
than normal phase bare silica. Diol functionalized silica offers an
interesting alternative to normal phase bare silica for difficult
separations. The OH moieties on diol are less active than those on
silica making this media useful for compounds that decompose or
are difficult to elute with silica. Being normal phase, diol uses
solvents that are easy-to-evaporate compared to solvents used in
reversed-phase.
Si O OH
OH
Figure 94: Diagram of diol structure
Diol columns are reusable. After the first use, do not allow the
reading the column RFID tag. Remove all organic modifiers by
flushing with three column volumes of 80% acetonitrile in water or
100% isopropanol and store the column in the wash solvent. If the
intermediate solvent prior to storage.
Method development with diol columns is similar to that of silica
gel. TLC plates can be used for non-aqueous solvent systems. If
aqueous systems are required, it is faster to develop the method
with a small diol column using a small amount of sample. Although
diol columns are compatible with reversed-phase solvents, it is
easiest to think of the column as always working in normal phase
with the ability to run aqueous solvent systems. The combination
of a less active surface and the ability to elute with a strong water
solvent allows diol to be used with a wide range of compounds.
104 Teledyne ISCO

With a minimal amount of compound, TLC plates can be used to

rapidly scout solvent systems and predict chromatography. The
relationship between the TLC retention factor (Rf) and the elution
time for a compound is Rf=1/CV, where CV= the number of column
volumes (see Retention Factor and Column Volumes, on page 9).
Figures 95 and 96 demonstrate that diol TLC plates provide a
reasonable prediction of how a variety of compounds will behave
in a column. As the polarity of the solvent is increased, the
compounds show higher retention factors demonstrating that the
diol is running as normal phase. This predictability is useful
because the Rf-to-Gradient feature in the PeakTrak software may
be used to optimize the method for improved purification.
Hexane/isopropanol is a useful solvent system for diol columns
because it has a wide polarity range.
Figure 95: TLC of compounds on diol using hexane/ethyl acetate with
comparisons of estimated and actual retention times
Predicted Actual
Hexane Ethyl Acetate Retention Retention
Compound % % Rf (CV) (CV)
Dibutyl phthalate 75 25 0.79 1.3 1.3
0 100 0.89 1.1
Phenol 75 25 0.51 1.9 1.9
0 100 0.81 1.2
Hydroquinone 75 25 0.125 8 5.4
0 100 0.73 1.4
Caffeine 50 50 0.22 4.6 5.4
0 100 0.59 1.7
Theophylline 50 50 0.17 5.8 9.1
0 100 0.48 2.1

Chapter 5
Figure 96: TLC of compounds on diol using hexane/isopropanol with

comparisons estimated and actual retention times
Predicted Actual
Hexane Isopropanol Retention Retention
Phenol 75 25 0.72 1.4 1.4
50 50 0.83 1.2
Hydroquinone 75 25 0.42 2.4 2.4
50 50 0.83 1.2
4-nitro- 75 25 0.18 5.4 6.8
phenl--D-gluco-
pyranoside
50 50 0.5 2
Caffeine 75 25 0.26 3.8 4.8
50 50 0.48 2.1
106 Teledyne ISCO

The following demonstrates the utility of a diol column in normal

phase with a compound readily purified on silica gel. Figure 97
shows the diol generates a a similar chromatogram as silica gel.
Compounds generally elute slightly earlier due to the reduced
polarity of diol compared to silica gel.
Figure 97: Purification of 3-(2-nitrophenyl amino) propionitrile on RedSep

Gold diol and RedSep Gold silica columns using a hexane/ethyl
acetate gradient

Chapter 5
Figure 98: TLC of compounds on amine plates with comparisons of

estimated and actual retention times using various solvents
Predicted Actual
Hexane Ethyl Acetate Retention Retention
caffeine 50 50 0.23 4.4 4.5
nicotine 50 50 0.66 1.5 1.5
Predicted Actual
Hexane Isopropanol Retention Retention
phenol 80 20 0.45 2.2 2.4
60 40 0.65 1.5 1.7
hydroquinine 80 20 0.11 9.1 10.4
60 40 0.33 3.3 3.6
Dichloro Predicted Actual

methane Isopropanol Retention Retention
caffeine 100 0 0.3 3.3 1.5
80 20 0.4 2.6 1
theophylline 80 20 0 No elution 5
Long hydrocarbon chain compounds on diol. Compounds containing

long organic chains are easily purified on diol compared to silica.
Diol is not as polar as silica so compounds with a strong
hydrocarbon character interact better with the stationary phase
and do not elute all at once when a slightly more polar solvent is
run on the column.
108 Teledyne ISCO

Oleyl Glycerate. Oleyl glycerate is made from oleic acid7, which is

available at only 85% purity. The compounds eluted together using
silica. The desired compound can be easily purified with a diol
column (Figure 99).
HO O
HO
0.12
0.10
Absorbance (AU)
0.08
0.06
0.04
0.02
0.00
0 5 10 15 20 25 30
Run Time (min)
Figure 99: Purification of oleyl glycerate with RedSep Rf Gold diol column
7. Fong, C.; Wells, D.; Krodkiewska, I.; Booth, J.; Hartley, P.G.
Synthesis and Mesophases of Glycerate Surfactants J. Phys
Chem B 2007, 111, 1384

Chapter 5
Tocopherols. Tocopherols represent another example of

compounds with a long hydrocarbon chain. Diol columns were
used to isolate the tocopherols from a variety of vegetable oils.
The purification was attempted on silica gel but the tocopherols
eluted immediately upon addition of any amount of “B” solvent.
The diol column provided greater control (Figure 100).
HO
100
1.50
Absorbance (AU)
% Solvent B
0.00 0
0 3 6 9 12 15 18
Column Volumes
Figure 100: Purification of tocopherols from corn oil

with RedSep Rf Gold diol column
110 Teledyne ISCO

Diol Column Versatility. As an example of the versatility of the diol

column, a methanolic extract of green tea was eluted from a diol
column with a gradient starting a hexane/isopropanol gradient and
ending with an isopropanol/water gradient in a single run (Figure
101). The sample was adsorbed onto Celite. Compounds eluted
include chlorophylls (A), xanthine alkaloids (B), catechins (B), and
tannins (C). The tannins would bind to silica.
100
2.50
Gradient– Gradient–
hexane/isopropanol isopropanol/water
Absorbance (AU)
(C)
% Solvent B
(B)
(A)
0.00 0
0 10 20 30 40 50
Column Volumes
Figure 101: Purification of green tea extract using a RedSep Rf Gold diol
column

Chapter 5
Purification of trans-anesole oxide

A reusable diol solid load cartridge was made by filling a 5 g
“empty” solid load cartridge with 2.5 g RedSep bulk diol media.
The reaction mixture was dissolved in dichloromethane and
placed in the diol solid load cartridge. The use of diol media in the
solid load cartridge provided a compatible media for elution for
the chromatography. The dichloromethane was evaporated by
placing the solid load cartridge on the CombiFlash Rf+ instrument
and blowing air through the cartridge with the air-purge feature via
manual control. As illustrated in Chromatography Application
Notes 888 and 899, loading the sample on a solid load cartridge
improves resolution. The diol packing ensures a compatible
adsorption surface and minimized compound decomposition.
The mixture was eluted with a hexane-ethyl acetate gradient
(Figure 102) using a 50 g RedSep Rf Gold Diol column. Fractions
were collected at 280 nm and evaporated into tared flasks at 45 °C.
2.0 100
90
80
1.5
70
Absorbance (280 nm)
Ethyl Acetate (%)
60
1.0 50
40
30
0.5
20
10
0.0 0
0 2 4 6 8 10 12 14 16
Time (CV)
Figure 102: Purification of trans-anisole oxide with RedSep Rf Gold Diol

column
8. http://www.isco.com/WebProductFiles/Applications/101/
Application_Notes/AN88_C18_Flash_Column_Loading.pdf
retrieved 21 June 2014.
9. http://www.isco.com/WebProductFiles/Applications/101/
Application_Notes/AN29_Overview_of_Silica_Column_Sample_
Loading_Techniques.pdf retrieved 21 June 2014.
112 Teledyne ISCO

Figure 103: Table of Compound Recovery for Diol

Fraction Recovered Mass (g)
1 0.0549
2 0.2766
3 0.1030
4 0.0139
5 0.0136
6 0.0543
Total: 0.5163
Care should be taken when purifying ketones, 1°, or 2° amines as

these may react with the diol stationary phase. Care should also be
taken when using acetone (or other ketones) as a solvent as they
may react with the diol side chain, especially if trace amounts of
acids are present. If in doubt, try the purification on a small
column and scale up to a larger column if the purification is
successful.

Chapter 5
Figure 104: Table of Reusable RedSep Rf Gold Diol Columns

20–40 microns
Column Teledyne
Size ISCO Part
5.5 - 110 mg 5.5 69-2203-514 5.5 gram Diol RedSep Rf Gold columns,
pkg. of 2.
15.5 - 310 mg 15.5 69-2203-515 15.5 gram Diol RedSep Rf Gold column,
pkg. of 1.
30 - 600 mg 30 69-2203-516 30 gram Diol RedSep Rf Gold column,
pkg. of 1.
50 mg - 1.0 g 50 69-2203-517 50 gram Diol RedSep Rf Gold column,
pkg. of 1.
100 mg - 2 g 100 69-2203-518 100 gram Diol RedSep Rf Gold column,
pkg. of 1.
150 mg - 3 g 150 69-2203-519 150 gram Diol RedSep Rf Gold column,
pkg. of 1.
pkg. of 1.
pkg. of 1.
0.9 - 19 g 950 69-2203-522 950 gram Diol RedSep Rf Gold column,
pkg. of 1.
1.9 - 38 g 1900 69-2203-523 1900 gram Diol RedSep Rf Gold column,
pkg. of 1.
3.8 - 76 g 3800 69-2203-535 3.8 kg Diol RedSep Rf Gold column,
pkg. of 1.
114 Teledyne ISCO

Chimeric Diol Column Behavior

using Aqueous Solvents
Diol columns have been marketed as being “useful in normal and
reversed-phase”10 with little indication about how to determine
the conditions for “normal” and “reverse” phase. For organic
solvents, diol columns act as “normal phase”11.
A column acts as “normal phase” if a compound exhibits reduced
retention as the solvent polarity is increased. Conversely, a column
showing reduced compound retention with reduced solvent
polarity is acting as “reversed-phase”. It is possible for a column to
exhibit both normal and reversed-phase behavior at the same time
with a mixture of compounds.
FD&C Blue #1 (1.0 g) was dissolved in water and mixed with Celite
541 (18.0 g); butyl paraben (1.0 g) was dissolved in methanol and
added to the dye/Celite mixture. The sample mixture was dried
under vacuum and was used for all experiments; 200 mg of this
mixture was used for each run.
10.Diol Phase.
http://www.justchromatograhy.com/wiki/diol-phase, 7 October
2011
11.Diol Columns – pretend they're normal phase.
http://www.isco.com/WebProductFiles/Applica
ions/101/Poster_and_Paper_Reprints/
Diol_Columns-Pretend_They're_Normal_Phase.pdf (accessed
Feb 2012).

Chapter 5
% water
(Isocratic) Absorbance, 628 nm % ACN
0 100
10 90
Normal Phase
20 80
30 70
40 60
50 50
60 40
Reversed Phase
70 30
80 20
90 10
100 0
0 10 20 30
Time (Column Volumes)
Figure 105: FD&C Blue #1 exhibits both normal and reversed-phase behavior
on a diol column. Shaded area denotes “normal phase” behavior
for this column and compound.
A 15.5 g diol column was used for all experiments. The column was
run isocratically for 20 column volumes (see details of Figures 105
and 106 for solvent concentrations) using water and acetonitrile as
solvents. The column was washed after 20 column volumes. Butyl
paraben was detected at 254 nm while the dye was monitored at
628 nm. The chromatograms are plotted separately for each
compound.
Figure 105 shows that FD&C Blue #1 exhibits minimum retention at
50% acetonitrile in water. As the concentration of water increases
from 0 to 50%, the retention time of the dye decreases consistent
with normal phase behavior. As the solvent polarity is decreased
(0 to 50% acetonitrile) the retention time again decreases,
consistent with reversed-phase.
116 Teledyne ISCO

Butyl paraben (Figure 106) is a non-polar compound that exhibits

purely reverse behavior on a diol column under aqueous
conditions. Note that using solely organic solvents, the diol
column will act as a normal phase column.
% ACN
(Isocratic) Absorbance, 254 nm
100
90
80
70
60
50
40
30
20
10
0
0 10 20 30
Time (CV)
Figure 106: Butyl paraben exhibits reversed-phase behavior on a diol column

under aqueous conditions

Chapter 5
Diol columns can exhibit both normal and reversed-phase

behavior under aqueous conditions. Diol behavior can be
summarized as:
• Diol columns run as normal phase when using only organic
solvents.
• Non-polar compounds will run as reversed-phase under
aqueous conditions and will not work well in HILIC
purifications.
• Water soluble compounds can show both normal phase
and reversed-phase behavior. They will generally show
normal phase behavior from 0 to 40 or 50% water, which
corresponds to the commonly used range for aqueous
normal phase.
• More polar organic solvents (such as methanol) are likely
to show a reduced range of normal phase behavior with
water soluble compounds.
Ion Exchange Media

Various strong and weak anion or cation exchange bonded silica
gel are available and can be used for the separation of almost any
type of charged molecule, from large proteins to small nucleotides
and amino acids.
SCX
The SCX (Strong Cation Exchange) media is a silica-bound sulfonic
acid.
O
O H
S
Si O
Figure 107: Diagram of SCX structure
The strong acidity of this media induces the full retention of any
compounds with basic properties subjected through it. This
intrinsic media property can be exploited several ways.
SCX media can be used as a practical and efficient tool for the
selective isolation of either basic or non-basic compounds from a
crude reaction mixture containing both.
118 Teledyne ISCO

To illustrate how SCX media can be of assistance, the separation of

a mixture of chromone and a benzimidazole derivative was
investigated.
(A) Chromone
O
(B) 1-(1-piperidinylmethyl)-1H-benzimidazole
N
N
Flash chromatography of the mixture on a normal phase silica

RedSep column showed release of the two products (Figure 108).
The use of a RedSep SCX column showed total retention of the
benzimidazole derivative onto the column and release of the
chromone (Figure 109).
This isolation of the basic compound, while allowing one or more
organic compounds holding neutral properties to migrate freely
through the column, demonstrates that the column can be
effective as a clean up tool.
Conversely, the SCX media also represents a practical tool for the
isolation of desired compounds holding basic properties. In this
case, the contaminants would be the neutral compounds which
would be immediately released then discarded by the SCX column
run. The compounds holding basic properties retained by the SCX
column are liberated by injecting a solution of ammonia in
methanol. In this case, the SCX media works as a catch and release
process.
SCX columns may be reused. After the first use, do not allow the
reading the column RFID tag. Regenerate the column with 10
column volumes of 1M acetic acid and store in 80% methanol in
water or 100% isopropanol.

Chapter 5
2.00 (A) 100
1.75
1.50 75
Absorbance (AU)
1.25
1.00 50
0.75
0.50 25
(B)
0.25
0.00 0
2 4 6 8 10 12 14 16 18 20 22 24 26 28
Time (minutes)
Figure 108: Chromatogram of normal phase column Elution with

dichloromethane/methanol
2.00 (A only) 100
1.75
1.50 75
Absorbance (AU)
1.25
1.00 50
0.75
0.50 25
0.25
0.00 0
2 4 6 8 10 12 14 16 18 20 22 24 26 28
Time (minutes)
Figure 109: Chromatogram of SCX column Elution with

dichloromethane/methanol
120 Teledyne ISCO

Figure 110: Table of Reusable RedSep Rf SCX Columns

Column Teledyne
Sample Size ISCO Part
Loada (g) Number Description
1.8 mMol 5 69-2203-390 5 gram Strong Cation Exchange RedSep Rf
columns, pkg. of 2.
column, pkg. of 1.
column, pkg. of 1.
column, pkg. of 1.
35 mMol 100 69-2203-396 100 gram Strong Cation Exchange RedSep Rf
column, pkg. of 1.
column, pkg. of 1.
column, pkg. of 1.
column, pkg. of 1.
a. Sample load = mMol  compound molecular weight/1000

Chapter 5
Purification of Alkaloids with

RedSep Rf SCX Columns
Ion exchange media separate molecules by net charge. Because
ions have differing affinities for the ion exchange media, it is
possible to selectively remove ions from solutions and release
them later. SCX columns can be used in a “catch-and-release”
mechanism where the basic compounds are removed from the
crude mixture and released after the impurities are washed away.
Alternatively, the ionic strength and pH of the solvent system can
be altered to purify a collection of bases. Both methods were
combined to isolate alkaloids from green tea extract.
Teledyne ISCO RedSep Rf SCX columns consist of a sulfonic acid
moiety bound to silica. Because the ion exchanger is chemically
bound to silica, the media will not swell when organic solvents are
used; RedSep Rf SCX columns can be used with solvents such as
dichloromethane.
Many small molecules of interest are weak bases and are difficult
to adsorb onto SCX columns. Salts of these compounds are often
very soluble in water, reducing interaction with the ion-exchange
column when the sample is loaded. To overcome these issues, the
samples can be dissolved in acidic solutions of organic solvent
such as methanol. The presence of acid forces the alkaloids to
have a positive charge, allowing interaction with the column.
The load limit for ion exchange columns is determined by the
number of ion exchange sites on the column, generally expressed
in millimoles. To determine the sample load, use the following
equation:
Sample load (g) = mmol × Compound Molecular Wt.
1000
RedSep Rf ion exchange columns can be reused. After the run is
complete, regenerate the column with ten column volumes of 0.1 N
strong mineral acid (sulfuric or phosphoric acid), followed by
washing with ten column volumes of water. Store the column in
80% methanol in water or 100% isopropyl alcohol.
122 Teledyne ISCO

Xanthine Alkaloids
Separate solutions of caffeine and theophylline were prepared by
dissolving 200 mg of alkaloid in 20 mL methanol containing 5%
glacial acetic acid. The mixture run on the column was made by
mixing 2.0 mL of each solution. This mixture (40 mg alkaloids) was
injected onto a 15 g RedSep Rf SCX column.
Detection was at 210 and 270 nm. Solvent A was methanol; Solvent
B was water containing 1% glacial acetic acid. The column was
washed with 10 column volumes (CV) of methanol before initiating
a linear gradient to 30% Solvent B over 20 CV.
The resulting chromatogram is illustrated in Figure 111.
2.0 100
Absorbance: 210 nm, 270 nm
80
1.5
% Solvent B
60
1.0
40
0.5
20
0.0 0
0 20 40
Time (CV)
Figure 111: Catch and release of xanthine alkaloids Caffeine is the first peak
eluted.

Chapter 5
Nicotine
Nicotine was run in a similar fashion as the xanthine alkaloids.
Nicotine (100 mg) was dissolved in 1.0 mL methanol containing 5%
acetic acid and injected onto a 15 g RedSep Rf SCX column. A
gradient of 5% acetic acid was followed by a gradient of 5%
ammonium hydroxide. The column was regenerated with 5% acetic
acid. Detection was 260 nm.
The resulting chromatogram is illustrated in Figure 112.
% Solvent B: Water/5% HOAc, Water/5% NH4OH

0.5 100
0.4 80
Absorbance 260 nm
0.3 60
0.2 40
0.1 20
0.0 0
0 20 40 60 80
Time (CV)
Figure 112: Catch and release of nicotine on a RedSep Rf SCX column
Unlike the xanthine alkaloids, nicotine is not displaced by

hydrogen ions but requires the “stronger” ammonium ions for
elution.
124 Teledyne ISCO

Harmine and Harmaline

These compounds were run similar to nicotine. The alkaloid (50
mg) was dissolved in 1 mL methanol containing 5% acetic acid
followed by injection onto a 15 g RedSep Rf SCX column. A gradient
of 5% acetic acid was followed by a gradient of 5% ammonium
hydroxide. The column was regenerated with 5% acetic acid.
Detection was 245 nm (harmine) or 360 nm (harmaline). Both
compounds were also detected with all-wavelength detection
(range 220–360 nm).
The resulting chromatograms are illustrated in Figures and 114.
% Solvent B: Water/5% HOAc, Water/ 5% NH4OH

4 100
Absorbance: 245 nm, All Wavelength
80
3
60
2
40
1
20
0 0
0 20 40 60 80
Time (CV)
Figure 113: Catch and release of harmine on a RedSep Rf SCX column

Chapter 5
2.0 100
% Solvent B: Water/5% HOAc, Water/ 5% NH4OH

Absorbance: 360 nm, All Wavelength 80
1.5
60
1.0
40
0.5
20
0.0 0
0 20 40 60 80
Time (CV)
Figure 114: Catch and release of harmaline on a RedSep Rf SCX column
126 Teledyne ISCO

Green Tea Extract

Green tea extract is used as a model to demonstrate the technique
using an actual plant extract. Green tea was extracted in methanol
and the extract dried. A portion of this extract (0.524 g) was
dissolved in 5.0 mL methanol containing 5% glacial acetic acid; the
entire sample was injected onto a 15 g RedSep Rf SCX column.
Detection was at 210 and 270 nm. A gradient using 5% acetic acid in
water followed by a gradient containing 5% ammonium hydroxide
in water was used to isolate the alkaloids.
3 100
Water/5% HOAc, Water/5% NH4OH

% Solvent B:
50
0 0
0 20 40 60 80
Time (CV)
Figure 115: Purification of alkaloids from green tea extract. The time window
(shaded region) excludes collection during the column
wash/regeneration.
The non-polar and acidic compounds in the plant extract are

washed off the column early in Figure 115. The alkaloids elute as
the acetic acid gradient is run. Other basic compounds elute
during the second gradient utilizing ammonium hydroxide. The
column is regenerated in the last gradient for a subsequent
purification. A time window was utilized so that fractions were
only collected during the first two gradients and not during the
column regeneration.
Acetic acid and ammonium hydroxide are used because they are
relatively easily removed from the sample after purification
compared to other acids or bases. Other useful acids include
formic and trifluoroacetic acid.

Chapter 5
SAX
The SAX (Strong Anion Exchange) media is a silica-bound
quaternary amine.
CH3
Cl -
Si N+
CH3
HC3
Figure 116: Diagram of SAX structure
Any compounds with acidic properties including weakly acidic

compounds subjected through SAX media will be fully retained.
SAX media can be used as a practical and efficient tool for the
selective isolation of either acidic or non-acidic compounds from a
crude reaction mixture containing both.
To illustrate how SAX media can be of assistance, the separation of
a mixture of chromone and 2,4-dihydroxybenzoic acid was
investigated.
O COOH
OH
(A)
Chromone OH
(B)
2,4-Dihydroxybenzoic acid
Flash chromatography of the mixture on a normal phase RedSep

column showed release of the two products (Figure 117).
The use of a RedSep SAX column showed total retention of the
aromatic carboxylic acid compound onto the column and release
of the chromone (Figure 118).
128 Teledyne ISCO

(B)
1.50 100
1.25
75
(A)
1.00
Absorbance (AU)
0.75 50
0.50
25
0.25
0.00 0
0 2 4 6 8 10 12 14
Column Volumes
Figure 117: Chromatogram of normal phase column

1.00 100
(A only)
0.90
0.80
75
0.70
Absorbance (AU)
0.60
0.50 50
0.40
0.30
25
0.20
0.10
0.00 0
0 2 4 6 8 10 12 14
Column Volumes
Figure 118: Chromatogram of SAX column


Chapter 5
This isolation of the acidic compound, while allowing one or more

organic compounds holding neutral properties to migrate freely
through the column, demonstrates that the SAX media can be
effective as a clean up tool.
Conversely, the SAX media also represents a practical tool for the
isolation of desired compounds holding acidic properties. In this
case, the contaminants would be the neutral compounds which
would be immediately released then discarded by the SAX column
run. The compounds holding acidic properties retained by the SAX
column are liberated by injecting a solution of acetic acid in
methanol. This solution can be directly injected through the
column with a syringe flush. In this case, the SAX column works as
a catch and release process.
SAX columns may be reused. After the first use, do not allow the
reading the column RFID tag. Regenerate the column with 10
column volumes of 5% NH4OH in methanol and store in 80%
methanol in water, or 100% isopropanol.
130 Teledyne ISCO

Figure 119: Table of Reusable RedSep Rf SAX Columns

Column Teledyne
Size ISCO Part
Sample Sizea (g) Number Description
3.8 mMol 5.7 69-2203-381 5.7 gram Strong Anion Exchange RedSep Rf
columns, pkg. of 2.
11.2 mMol 17 69-2203-382 17 gram Strong Anion Exchange RedSep Rf
column, pkg. of 1.
column, pkg. of 1.
column, pkg. of 1.
column, pkg. of 1.
column, pkg. of 1.
column, pkg. of 1.
column, pkg. of 1.

Chapter 5
RedSep Rf Strong Anion Exchange

Column Applications
The compounds of interest to synthetic and natural product
chemists are often weak acids such as phenols or carboxylic acids.
Such compounds include antioxidants, pesticides, natural colors,
and pharmaceuticals. The use of the SAX column allows the acidic
compound to be captured while the basic and neutral compounds
are washed from the column. The purified acids can be washed
from the column with at least partial resolution. The RedSep Rf
SAX column is reusable, therefore the same column was used for
all experiments in this application note.
Figure 120: Table of Loading Capacity of RediSep Rf SAX columns
Column Size Maximum Sample Load Sample Weight

(g) (mmol) (MW=200 AMU, grams+)
5.7 3.8 0.7524
17 11.2 2.244
34 22.4 4.488
57 37.6 7.524
114 75.2 15.048
170 112.2 22.44
313 206.6 41.316
470 310.2 62.04
+Maximum sample load = mmol * compound molecular weight / 1000 where mmol is the value listed on the
column. For a compound with a molecular weight of 200g/mol purified on a column with a maximum capacity
of 11.2 mmol, the maximum load for this compound is 11.2 * 200/1000 = 2.24 g
Column Conditioning — The column was first washed with five

column volumes of methanol. RedSep Rf SAX columns are packed
with a chloride counterion. The conjugate bases of the desired
compounds fail to displace the chloride ion leading to poor
binding. Compounds would reliably bind to the column after the
second wash, because the column was completely converted to
the acetate counterion.
132 Teledyne ISCO

Gallic Acid
Gallic Acid (16 mg, Sigma Aldrich, St. Louis, MO) was dissolved in
0.5 mL methanol containing 5% ammonia. The detection
wavelength was 270 nm.
0.30
O OH
100
80
Absorbance (270 nm)
HO OH
% B Solvent
OH
20
0.00 0
0 5 10 15 20 25
Time (CV)
Figure 121: Purification of Gallic Acid on a RedSep Rf SAX column

Chapter 5
Phenolic Flavanoid Compounds

Epicatechin (16 mg, Sigma Aldrich) was dissolved in 0.5 mL
methanol containing 5% ammonium hydroxide. All-wavelength
collection was used with a wavelength range of 200 – 250 nm, and a
peak width of 2 minutes. All-wavelength collection is a detection
technique unique to CombiFlash Rf and Torrent systems which
allow detection and collection of compounds with unknown
absorbance. All-wavelength collection is also used for baseline
suppression.
OH
OH
HO O
1.0
OH 100
OH
All-Wavelength (AU)
B Solvent (%)
All Wavelength
Collection Artifact
0.0 0
0 5 10 15 20 25
Time (CV)
Figure 122: Epicatechin purification on a RedSep Rf SAX column
134 Teledyne ISCO

Anthocyanin
An unknown anthocyanin (25 mg) was mixed with 1 mL methanol
containing 5% ammonia. Water was added until the sample was in
solution (~0.5 mL). All-wavelength collection (200 – 250 nm, 2 min
peak width) was used to detect the anthocyanin compound. The
shoulder at 16 CV was determined to be another anthocyanin
compound. There is also a weak peak appearing at ~22 CV.
0.5
100
All-Wavelength (AU)
B Solvent (%)
Anthocyanin
Impurities
0.0 0
0 5 10 15 20 25
Time (CV)
Figure 123: Anthocyanin purification on a RedSep Rf SAX column

Chapter 5
Green Tea Extract

Green Tea was extracted in methanol, and dried of which 0.75 g
was dissolved in 15 mL methanol containing 5% ammonia. The
solvent was limited to 15 mL to prevent sample loss as this volume
is similar to the void volume of the column. The neutral and basic
compounds do not bind to the column and eluted during loading
and column wash. The sample was run similar to the others except
that the gradient was changed from 10 to 30 CV. The fractions were
collected at 275 nm. Even at this high loading, there was no
“breakthrough” of the acidic compounds.
HPLC separation against reference compounds showed the peak
eluted during the gradient contained catechin and other
unidentified compounds. The gradient was lengthened compared
to the other runs to allow these minor components to be better
purified from catechin.
100
Peak A 1.2 HPLC A
Methanol (%)
2.5
AU
100
Absorbance (AU) 275 nm
0.0 0
0 20
B Solvent (%)
catechin
0.5 100
HPLC B
B Methanol (%)
Peak
AU
0.0 0
0.0 0
0 Time (Minutes) 20
0 10 20 30 40
Time (CV) HPLC Runs
Figure 124: Green Tea Extract purified on a RedSep Rf SAX column HPLC A
is from the fraction containing the major peak (A) eluting at 17 CV;
HPLC B is the fraction containing the shoulder peak (B) eluting at 27
CV.
136 Teledyne ISCO

Advanced Solvent Strategies

Running various solvents on TLC plates allows a user to select a
solvent system that would cause a good retention (measured by
Rf), but poor resolution between compounds. Resolution can be
improved by using solvents with different selectivity. Figure 125
shows a “selectivity triangle”. This three-axis chart allows the
scientist to achieve a change in solvent selectivity much more
rapidly than testing various solvents randomly.
H Acceptors
8 6
5
H 7 Large
Donors Dipole
Group 1 Group 3 Acetone (5.40)

i-Propyl ether (2.40) Tetrahydrofuran (4.20) Acetonitrile (6.20)
Ethyl ether (2.90) Dimethyl formamide (6.40) Group 7
Group 2 Group 4 Toluene (2.40)
Isobutyl alcohol (3.00) Acetic acid (6.20) Chlorobenzene (2.70)
n-Butanol (3.90) Ethylene glycol (6.90) Benzene (3.00)
n-Propanol (4.00) Group 5 Group 8
i-Propanol (4.30) Methylene chloride (3.40) Chloroform (4.40)
Ethanol (4.30) Water (10.20)
Methanol (6.60) Group 6
Ethyl acetate (4.30)
Dioxane (4.80)
Figure 125: Diagram of solvent selectivity Solvent polarity is listed in

parentheses. Refer to Figure 210 for additional solvent properties.

Chapter 5
As an example, assume separation trials on silica TLC plates

suggest the correct solvent strength is obtained with a solvent
system of 8:2 hexane:methylene chloride. Using the polarity values
in Figure 116 on page 122, the total solvent system polarity can be
determined using the following equation:
PT = A  PA + B  PB
where A and B are the ratios of solvent A and B;

PA and PB are the polarities.
For our example, the polarity is 0.73 = 0.8  0.06 + 0.2  3.40
To move the compound up the TLC plate in the same fashion, we
need a solvent system with a polarity of 0.73.
Referring to Figure 125, methylene chloride is in solvent group 5.
To significantly change the selectivity, the solvent group most
distant from group 5 in should be chosen. Group 8 is distant from
group 5; chloroform is a choice from this group. To determine the
concentration of chloroform, use the equation:
Bnew = B  PB  Pnew
where Bnew is the ratio of new solvent B;

Pnew is the polarity of the new solvent B.
For our example: 0.16 = 0.2  3.4  4.4, so the new solvent system is
0.84: 0.16 hexane: chloroform.
If this system failed to work well, a solvent from group 2 such as
n-propanol could be used.
Choosing a solvent from the same group will make little difference
to the selectivity. Changing from ethyl acetate to acetone, both in
group 6, makes little difference in the selectivity. This knowledge
can be used to choose a solvent in the same group that may have
physical properties, such as absorbance, more appropriate for the
purification.
For reversed-phase, changing the less polar solvent (solvent B) is
less effective in changing selectivity. Changing from methanol to
acetonitrile generally results in little change to the chromatogram.
A change to tetrahydrofuran sometimes produces good results.
138 Teledyne ISCO

In addition to determining the best solvent system for purifying

compounds, the concept of solvent groups can also be used to find
solvents that allow easier detection of compounds. When using
silica gel, hexane-ethyl acetate is the most common solvent pair
used. These solvents purify a wide range of compounds and are
commonly used for reactions and extractions, making them
plentiful in labs. Many compounds show only end absorption of UV
light, from 200–220 nm (the end of the detector range). Ethyl
acetate adsorbs in this range causing sloping baselines which hide
smaller peaks. Ethyl acetate is in solvent group 6 (Figure 125).
Acetone is also in group 6 and has similar characteristics to ethyl
acetate for chromatography—both solvents have similar polarity
and solvating powers for most organic compounds.
0.75
0.65
Ethyl Acetate
0.55
Absorbance (AU)
0.45
0.35
0.25
Acetone
0.15
0.05
-0.05
200 225 250 275 300 325
Wavelength (nm)
Figure 126: Chart of UV spectra of ethyl acetate and acetone Ethyl acetate
absorbs UV light strongly at short wavelengths which masks
compounds that show only end absorption.
Acetone is slightly more polar (see Figure 210 on page 238) so

peaks may elute slightly earlier. Acetone is also readily available in
most labs because it is commonly used as a solvent. Acetone
absorbs UV light from 225 nm through 300 nm making this solvent
an excellent choice for compounds that only absorb UV light at
shorter wavelengths (Figure 126).

Chapter 5
Acetone does have some drawbacks—it readily absorbs moisture

from the air so containers should be capped when not in use.
Acetone absorbs UV light with wavelengths longer than 220 nm.
For this reason, it is not useful for detectors that operate only at
254 nm. Acetone also should not be used to purify compounds
containing diols, or primary and secondary amines since there is a
possibility of reactions with those compounds.
The synthesis and purification of 3-(2-nitrophenylamino)
propionitrile provides an example of the utility of acetone as an
alternative to ethyl acetate.
NO 2 NO 2
+ CN
CN
NH 2 N
H
3-(2-nitrophenylamino) propionitrile absorbs most strongly at
230 nm, lower than the UV cutoff for ethyl acetate. Setting the
detection wavelength to 230 nm will cause the detector to see the
absorbance of both the ethyl acetate and the desired compound.
0.75
0.65
0.55
Absorbance (AU)
0.45
0.35
0.25
0.15
0.05
-0.05
200 250 300 350 400 450
Wavelength (nm)
Figure 127: Chart of UV absorbance of 3-(2-nitrophenylamino) propionitrile
140 Teledyne ISCO

PeakTrak software’s Gradient Optimizer on the CombiFlash Rf

system was used to create a method using acetone and hexane as
the elution solvents. The desired compound was purified from the
starting compound and other impurities while maintaining the
maximum sensitivity for detecting the compounds on the
instrument.
Figure 128 shows the purification of 3-(2-nitrophenylamino)
propionitrile at 229 nm (dashed absorbance trace) and at 285 nm
(solid absorbance trace) The 285 nm trace is much smaller,
reflecting the reduced absorbance at this wavelength. The
stronger absorbing 229 nm trace is riding on the sloping baseline
from the ethyl acetate.
2.00 100
90
80
1.50
70
Absorbance (AU)
60
1.00 50
40
30
0.50
20
10
0.00 0
0 5.0 10.0 15.0 20.0

Figure 128: Chromatogram of 3-(2-nitrophenylamino) propionitrile

purification in hexane/ethyl acetate The dashed trace is
absorbance at 229 nm; solid trace is absorbance at 285 nm.

Chapter 5
Running the same mixture in hexane/acetone provides the

chromatogram in Figure 129. The baseline in the acetone run is flat
allowing easy observation of minor impurities. The peaks are
rounded at the top due to high loading on the column.
2.00 100
90
80
1.50
70
Absorbance (AU)
60
1.00 50
40
30
0.50
20
10
0.00 0
0 5.0 10.0 15.0 20.0

Figure 129: Chromatogram of 3-(2-nitrophenylamino) propionitrile

purification in hexane/acetone Absorbance measured at 229 nm.
Another example is the purification of stigmasterol, a plant steroid.

This compound, in common with many natural products, shows
only weak end absorption (Figure 130).
The use of the traditional hexane/ethyl acetate solvent system
would make this compound difficult to observe so that the fraction
collector could collect the peak. Using acetone allows the
compound to be observed. The alternative to the use of acetone in
this situation would require the system to be set to “collect all”
and manual TLC evaluation of each fraction by the chemist.
142 Teledyne ISCO

The baseline is still drifting slightly since acetone has a slight

absorbance at 205 nm but the compound and impurities are still
detected very well (Figure 131).
0.45
Absorbance (AU)
0.35
0.25
0.15
0.05
0.00
-0.05
200 210 220 230 240 250
Wavelength (nm)
Figure 130: Chart of stigmasterol absorbance shows absorbance only at

shorter wavelengths
1.50 100
90
80
70
Absorbance (AU)
60
0.75 50
40
30
20
10
0.00 0
0 5.0 10.0 15.0 20.0 25.0

Figure 131: Chromatogram of stigmasterol purification

using hexane/acetone

Chapter 5
HILIC Purification Strategies

Water soluble compounds, such as anthocyanins, dyes,
nucleotides, compounds containing carbohydrates, tannins or
other polyphenols, and alkaloids can be difficult to purify because
they are poorly retained on reversed-phase columns while being
strongly retained on silica. HILIC (Hydrophilic Interaction Liquid
Chromatography) is a technique that uses chromatography media
that are compatible with water and are run as normal phase
columns. The technique involves a gradient starting with an
organic solvent and ending with a solvent mixture containing
water. The use of water gives the technique the alternate name of
Aqueous Normal Phase. The purification of alkaloids on an amine
column and a synthetic dye on a diol column are demonstrated.
Many compounds are difficult to purify due to their polarity and
strong solubility in water. These compounds are insoluble in most
normal phase solvents and irreversibly are retained on silica or
alumina. Under reversed-phase conditions, the compounds are
poorly retained on C18.
The use of water-compatible normal phase media and solid load
cartridges allow these compounds to be purified. Aqueous normal
phase also exhibits different selectivity, allowing purification of
compounds that may otherwise closely elute.
The gradient begins with a water miscible organic solvent and runs
typically to 50% water. In general, less polar solvents are preferred
for the starting solvent in the gradient as they give greater control
over the purification but other solvents may give differences in
selectivity. Because the compounds elute using mostly organic
solvents, there is less water to be removed, allowing faster drying
of the sample.
144 Teledyne ISCO

Figure 132: List of solvents suitable for Aqueous Normal Phase

Chromatography .
acetone ethanol
acetonitrile acetonitrile
isopropanol tetrahydrofuran
Compounds should be adsorbed onto a material such as Celite

prior to the purification. Compounds dissolved in water and
injected onto the column may not interact with the column media
causing poor separation. Celite is useful because it adsorbs water
while retaining the compound poorly. HILIC purifications require at
least 1-2% water. If running a gradient, the gradient should start
with minimum of 1-2% B solvent (water) to allow the stationary
phase to be properly hydrated during equilibration. The
equilibration should be at least 5 column volumes to ensure
development of the hydration layer on the stationary phase.
Erioglaucine dye on diol

Erioglaucine dye was adsorbed onto Celite as described above and
then placed in a solid load cartridge. The sample was run (0.05 g
dye) on a 15.5 g RedSep Rf Gold diol column. Solvent A was
acetonitrile; Solvent B was water containing 0.1% TFA. The
gradient was run from 0 to 50% B. The compound was fractionated
using all-wavelength collection. The dye was resolved from the
impurities.

Chapter 5
– –
O3S – SO3
SO3
N N+
0.6
100
Absorbance
%B Solvent
0.0 0
0 10 20 30
Figure 133: Purification of an erioglaucine dye on a diol column
146 Teledyne ISCO

theophylline caffeine
1.5
100
Absorbance
%B Solvent
0.0 0
0 10 20 30
2.5
100
Absorbance
%B Solvent
0.0
0 10 20
Figure 134: Purification of theophylline and caffeine on an amine column

(top) using aqueous normal phase and silica (bottom) with
adichloromethane/methanol gradient.

Chapter 5
Xanthine alkaloids on an Amine column

Both columns were loaded with 0.15 g total alkaloids and run with
the solvent gradients depicted in Figure 134. The amine column
was a 15.5 g RedSep Rf Gold Amine column; a 12 g RedSep Rf Gold
Silica column was eluted with a dichloromethane/methanol
gradient.
The amine column under HILIC conditions exhibited greater
resolution between the alkaloids compared to silica. In addition,
the purification was achieved without the use of chlorinated
solvents. Both compounds eluted with less than 50% water; greater
resolution could be achieved by reducing the maximum gradient
to 50% water.
RedSep Rf Gold Silica and Highly Polar Solvents

Compounds including nucleotides, dyes, and many basic
compounds are very polar and are difficult to purify without
resorting to correspondingly polar solvents on silica gel. HILIC is
increasingly used to analyze these compounds with HPLC.
Common HILIC media include diol, amine, and bare silica. Many of
these compounds could be purified on silica with high
concentrations of methanol.
It is commonly held that silica gel dissolves in methanol or water.
However Alexander, et al., demonstrated the solubility of silica in
methanol to be 0.0014%12; the same study demonstrated aqueous
silica solubility of 0.01%. Both solubility measurements were
obtained after equilibrating for several months, much longer than
the residence time in a Flash column. This same study also
demonstrated a large increase in silica solubility above pH 7.
Figure 135 shows the purification of adenine from
dichloromethane. Adenine elutes at ~46% methanol. Both peaks
have good peak shape with slight tailing of the adenine peak. This
compares favorably to the reversed-phase elution using a
water/methanol gradient (Figure 136). This run used a 30 g RedSep
Rf Gold C18 column and both solvents contained 0.1% TFA.
12.Alexander, G.B.; Heston, W.M.; Iler, R.K. The Solubility

of Amorphous Silica In Water J. Phys. Chem.
1954, 58(6), 453-455
148 Teledyne ISCO

3.0 100
80
% B Solvent
2.0
60
270 nm (AU)
40
1.0
20
0.0 0
0 2 4 6 8 10 12 14 16
Figure 135: Purification of adenine from caffeine using a

dichloromethane/methanol gradient
2.5 100
80
% B Solvent
60
210 nm (AU)
40
20
0.0 0
0 5 10 15
Time (CV)
Figure 136: Caffeine and adenine elute together on C18

Chapter 5
The ability to use water on RedSep Rf Gold silica columns allows

users to avoid chlorinated solvents. The elution of caffeine and
adenine were similar using dichloromethane or water.
2.0 100
1.5
% B Solvent
210 nm (AU)
1.0
30
0.5
0.0 0
0 4 8 12 16
Time (CV)
Figure 137: Elution of caffeine and adenine using silica HILIC on a RedSep Rf
Gold Silica column
2.0 100
1.5
% B Solvent
210 nm (AU)
1.0
30
0.5
0.0 0
0 4 8 12 16
Time (CV)
Figure 138: Improved adenine peak symmetry with 0.1% TFA in water
150 Teledyne ISCO

Water soluble vitamins were run because they have a range of

functional groups and show poor resolution using a C18 column.
For this experiment, 4-aminobenzoic acid, niacinamide, and
thiamine hydrochloride were separately adsorbed onto silica (20%
loading) and 150 mg of each vitamin/silica mixture were mixed in a
5 g Solid Load Cartridge. Solvent A was water containing 0.1% TFA;
Solvent B was acetonitrile. The gradient in Figure 139 was run on a
12 g RedSep Rf Gold silica column.
1.0 100
Acetonitrile (%)
HO O
N NH2
NH2
3 0.0 0
0 8 16
NH2 CH3
215 nm, 250 nm, All Wavelength
OH 70
N
H3C S
Absorbance (AU)
Water (%)
0 0
0 10 20 28
Figure 139: Water soluble vitamins purified on a RedSep Rf Gold silica

column using an acetonitrile/water gradient. The inset shows the
mixture exhibits poor selectivity on a C18Aq column designed for
purifying polar molecules.

Chapter 5
For the C18 experiment (Figure 139, inset), 100 mg of each vitamin
was dissolved in 1.0 mL 1:1 acetonitrile:water; 0.3 mL of this
mixture (30 mg each vitamin) was run on a 15.5 g RedSep Rf Gold
C18Aq column. Solvent A was acetonitrile, solvent B was water;
both solvents contained 0.1% TFA. The compounds were
determined to elute in the reverse order compared to the silica
column.
For both experiments, the detector simultaneously measured
absorbance at 215, 250 nm, and all-wavelength collection (200–300
nm, 2 min peak width).
The vitamins showed excellent resolution and peak shape on
silica, but failed to resolve on C18.
152 Teledyne ISCO

Natural Products
Chapter 6
Natural Products
Natural products chemistry differs from synthetic chemistry

because the structure of the compound is unknown until after the
material is purified. The desired compounds must be removed
from their matrix (plant, fermentation, or marine organism) and
then isolated from a host of other materials without a priori
knowledge of which compound is active.
In addition to the detectors on the CombiFlash system, the
purification is also directed by biological activity, or “assay guided
fractionation”. Automated Flash chromatography helps the natural
products chemist with well-packed columns that improve
resolution and precise gradient control. Additionally, CombiFlash
systems allow easy scale-up and large sample capacity. UV-vis
detection helps find compounds easily, although this is of greater
use later in the purification. For a complete list of Teledyne ISCO
automated Flash systems, refer to Appendix E - CombiFlash
Systems, on page 255.
Automated Flash chromatography systems can be used with a
wide variety of pre-packed columns that work well on a wide range
of compounds. Columns that are usually hand-packed due to
variable swelling of the stationary phase in different solvents, such
as LH-20 or CHP-20, are easily adapted to CombiFlash systems to
provide the benefits of automation to these purifications as well.
The parameters for a particular sized column can be entered
manually into the system and saved for future use. Automated
gradients avoid the need for pre-mixing solvents.
Purification of natural products is most often started with a
chemical/extraction screening. Compounds that extract into ethyl
acetate are ideal candidates for silica gel using hexane/ethyl
acetate or hexane/acetone gradients. Compounds that adsorb on
to XAD-16 can be purified with silica gel using more polar solvents
systems including methylene chloride/methanol. CombiFlash
systems allow easy changing of solvent systems during the run
with four-solvent inlet lines. This enables single-step purification of

Chapter 6
a wider range of compounds in a single run, and allows “wide

polarity range chromatography” where gradients using different
solvents are “chained” together in a single run. Combining this
technique with the use of different columns allows rapid screening
to determine initial purification parameters such as column type
and solvent systems. The use of detectors, such as the PurIon
mass spectrometer, permit correlation of peaks and molecular
weight with biological activity.
Figure 140: Photo of column mount part number 60-5394-432 (shown below
valve) used to adapt a CHP-20 column to a CombiFlash Rf system
154 Teledyne ISCO

Natural Products
Cytotoxic Constituents from Butea superba

Butea superba13 is a legume with the common name “red Kwao
Krua” in Thailand. This species has shown anti-proliferation effects
on the growth of MCF-7 and HeLa cells.
The dried tubers of Butea superba were extracted with methanol
and the methanolic extract was re-extracted with hexane followed
by extraction with CH3Cl. The chloroform extract was separated
by Flash chromatography on RediSep columns with 100% CH2Cl2
with 1% step gradients of methanol. Compounds A–E (Figure 141)
were obtained from 1–2% MeOH in CH2Cl2. The compounds were
identified by spectral comparison with reported values in the
literature without further purification.
Cytotoxicity was determined by MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)
assay. Compounds B and D showed moderate cytotoxic activity on
KB cell lines.
H O OCH3
HO
3-hydroxy-9-methoxypterocarpan (A)
O H
R’’’ See table below

O
R’
R’’
R R’ R’’ R’’’ Compound

OCH3 H H OH 7-hydroxy-4’-methoxy-isoflavone (B)
OCH3 H H OCH3 7,4’-dimethoxyisoflavone (C)
OH OH H OCH3 5,4’-dihydroxy-7-methoxy-isoflavone (D)
OCH3 H OCH3 OH 7-hydroxy-6,4’-dimethoxyisoflavone (E)
Figure 141: Diagram of compounds extracted from Butea superba
13.Ngamrojanavanich, N.; Loontaisong, A.; Pengpreecha, S.;

Cherdshewasart, W.; Pornpakakul, S.; Pudhom, K.; Roengs-
umran, S.; Petsom, A. J. of Ethnopharmacology 2007, 109, 354

Chapter 6
Alkaloids of Banisteria caapi

The plant Banisteria caapi14 is the source of the alkaloid harmine,
known for its hallucinogenic properties, and the related harmaline.
Harmaline was purified from Florisil using a chloroform-methanol
gradient. The dried vine was refluxed in methanol. The extract was
dried and extracted into chloroform with 5% ammonium
hydroxide. In our labs, we found that harmine would elute readily
from silica gel using methylene chloride, but harmaline would only
elute slowly with a broad peak even with 100% methanol. We then
used a RediSep Amine functionalized column using a gradient from
50 to 100% ethyl acetate in hexane. Although the harmaline still
produces a broad peak, it is much more compact than the run from
silica gel. Further optimization of the gradient could be employed
to sharpen the harmaline peak.
The use of amine functionalized silica in this example illustrates
the ability to purify even difficult samples.
Teledyne ISCO produces a number of pre-packed columns
containing other media such as acidic, basic, and neutral alumina,
cyano, amine, and ion exchangers.
14.Hochstein, F. A. and Paradies, A. M. J. Amer. Chem Soc. 1957,

79(21), 5735.
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N N
O O
N N
H H
Harmine Harmaline
(A) (B)
1.00 100
90
80
0.75
70
Absorbance (AU)
60
0.50 50
40
30
0.25
20
10
0.00 0
0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0
Figure 142: Illustration of harmine and harmaline separation on a silica gel

column with a methylene chloride/methanol gradient solvent system
0.50 100
0.45 90
0.40 80
0.35 70
Absorbance (AU)
0.30 60
0.25 50
0.20 40
0.15 30
0.10 20
0.05 10
0.00 0
0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0
Figure 143: Illustration of harmine and harmaline separation on a RediSep

amine column with hexane/ethyl acetate gradient solvent system

Chapter 6
Wide Polarity Range Flash Purifications

In most reactions and subsequent purifications, compounds
exhibit similar polarity and solubility properties to allow
purification with a single pair of solvents in the gradient. There are
times when compounds of interest span a large polarity range,
such as pharmacognosy applications. By adroit use of gradients
and solvents, chromatograms can be run that span the polarity
range from hexane through water in a single run for a normal
phase column. Reverse phase columns can be run that span the
range from water through methylene chloride in a single run. The
ability to automatically change solvents and gradients saves time
and can extend column life. Solid load cartridges avoid solubility
issues since all compounds are released from the cartridge at the
appropriate time in the purification. Additionally, wide polarity
range chromatography is useful for screening unknown
compounds to determine the best combination of columns and
solvents.
Hexane is a weak solvent for normal phase, while water is a strong
solvent for normal phase. For reversed-phase, water is the weak
solvent and organic solvents such as dichloromethane are strong
solvents. It is possible to “chain” gradients based on solvent
strength and miscibility to create a gradient that covers a wide
range of polarities.
Examples using a normal phase diol and reversed-phase C18
columns are presented.
Reversed-phase C18
A crude tomato paste extract was run with a CombiFlash Rf system
equipped with a UV-vis detector (PN 68-5230-008) with a 43 g
RediSep Rf Gold C18 column (PN 69-2203-334) using a water to
methanol gradient followed by a methanol to methylene chloride
gradient with automatic solvent switching. A sample was adsorbed
onto Celite and placed into empty 25 g Solid Load Cartridges (PN
69-3873-240). Lycopene compounds were detected at 473 nm;
other compounds were fractionated with all-wavelength collection
(200–360 nm range). The column was initially run with a
water/methanol gradient which eluted polar compounds then
automatically switched to a methanol/dichloromethane gradient
to purify the lycopenes. This gradient program, including the
solvent changes, was programmed into the CombiFlash Rf system
(Figure 144).
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2 100
MeOH DCM
%B Gradient
Absorbance
H2O MeOH
0 10 20 30
Run Time (Column
473 nm
All-wavelength
Figure 144: Wide polarity purification of tomato extract A1: H2O; B1: MeOH;
A2: MeOH; B2: DCM
Figure 144 demonstrates the ability of the CombiFlash Rf system to

automatically switch solvents during purification and isolate a
wide range of compounds in a single run. The compounds range
from essentially water-soluble to completely water insoluble.
Normal Phase Diol

Crude tea extract purification was performed on a CombiFlash Rf
system (PN 68-5230-006). One gram of extract was dissolved in
methanol and adsorbed onto Celite 545 (Acros Organics) in a 5 g
RediSep solid load sample cartridge (PN 69-3873-235). The
compound was eluted with a hexane/isopropanol gradient
followed by an isopropanol/water gradient on a 15.5 g RediSep Rf
Gold diol column (PN 69-2203-515). The multiple solvent gradient
was programmed to automatically switch on the CombiFlash Rf
system. The alkaloids and catechin compounds eluted together
while the tannins eluted early in the water gradient (Figure 145).
Fractions were collected using the all-wavelength collection
algorithm. Detection was 254 nm (peak width=1 min) and
All-Wavelength (200–360 nm, peak width=8 min)

Chapter 6
.
2.5 100
IPA H 2O
%B Gradient
Absorbance
hexane IPA
0
0 10 20 30 40 50
Run Time (Column
254 nm
All-wavelength
Figure 145: Initial purification of green tea extract A1: Hexane; B1: IPA; A2:
IPA; B2: H2O
Wide polarity range chromatography works well in normal phase

(Figure 145). In this case, the porphyrin compounds elute first,
followed by catechols and xanthine alkaloids, and finally the
tannins at the start of the water gradient. Silica and alumina can
also be run as a wide polarity range purification.
For these experiments, one solvent was common to all portions of
the gradient. This solvent was defined as B1 and A2 (Figure 146);
solvent lines A2 and B1 were placed in this solvent container.
Figure 146: One arrangement of solvents for wide polarity chromatography on

C18
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Figure 147 demonstrates the use of four solvents for wide polarity
chromatography. The diol column is initially run with a
hexane/ethyl acetate gradient. The second gradient is ethyl
acetate/methanol; the final gradient is methanol/water. This is a
gradient useful for scouting both polar and non-polar compounds
for natural products.
Figure 147: Four-solvent gradient for wide polarity range chromatography

on a diol column starting with hexane, going through 100% ethyl
acetate to 100% methanol and finishing with water

Chapter 6
Wide polarity range chromatography is an easy method to purify

compounds of widely differing solubility with a single run. The use
of solid load cartridges avoid issues of solubility, and avoids
compounds precipitating and clogging the column. The solid load
cartridge allows the full method to be run automatically; the
solvent changes are programmed into the gradient table of the
system. The adroit use of solvent switching allows the use of
immiscible solvents, such as hexane through ethyl acetate, and
then to some concentration of methanol on a silica column.
Ion Exchange Columns for Natural Products

These columns are specific for basic (SCX columns) and acidic
(SAX columns). Compounds that are retained, and eluted from
these columns often require only one more purification step from a
crude extract.
SCX Columns
The use of a Strong Cation Exchange (SCX) resin is demonstrated
to isolate a variety of alkaloids using a “catch-and-release”
mechanism. Automatic solvent switching is used to generate
multiple gradients with cations of increasing strength that elute
the alkaloids using a combination of ionic strength and pH control.
The multiple gradients are used within the same purification run to
isolate various alkaloids from the same crude extract; green tea
extract is used as a model for other plant extracts. Linear gradients
allow purification of related compounds from each other. The use
of multiple gradients permits purification of unknown alkaloids
without prior knowledge of their pKa.
Ion exchange columns represent a powerful method to quickly
purify desired natural products from the rest of the extract. One
common method to isolate alkaloids include extracting them into
an acidic aqueous solution, making the aqueous solution basic,
followed by back extraction into an organic solvent. However,
extraction does not allow families of alkaloids to be purified from
each other. Ion exchange media separate molecules by net charge.
Because ions have differing affinities for the ion exchange media, it
is possible to selectively remove ions from solutions and release
them later. SCX columns can be used in a “catch-and-release”
mechanism where the basic compounds are removed from the
crude mixture and released after the impurities are washed away.
Alternatively, the ionic strength and pH of the solvent system can
be altered to purify a collection of basic compounds. Both
162 Teledyne ISCO

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methods were combined to isolate alkaloids from green tea

extract. RediSep Rf SCX columns consist of a sulfonic acid moiety
bound to silica. Because the ion exchanger is chemically bound to
silica, the media will not swell when organic solvents are used.
RediSep Rf SCX columns can be used with solvents such as
dichloromethane.
Many small molecules of interest are weak bases and are difficult
to adsorb onto SCX columns. Salts of these compounds are often
very soluble in water, reducing interaction with the ion-exchange
column when the sample is loaded. To overcome these issues, the
samples were dissolved in acidic solutions of methanol. The
presence of acid forces the alkaloids to have a positive charge,
allowing interaction with the column. Using methanol caused
better binding than dissolving the sample in water.
Xanthine Alkaloids. Caffeine and theophylline were both displaced
by protons from the acetic acid. The two peaks are partially
resolved showing that, in addition to being captured and released,
the compounds can be partially resolved. Binding of the alkaloids
to the column is demonstrated by their eluting only after the
gradient is run, starting 10 column volumes (CV) into the run.
Substituting water for methanol for Solvent A caused the alkaloids
to elute early during the initial 10 CV column wash.
2.0 100
80
1.5
caffeine
% Solvent B
theophylline 60
1.0
40
0.5
20
0.0 0
0 20 40
Time (CV)
Figure 148: Capture and release of caffeine and theophylline using a SCX
column

Chapter 6
Nicotine. Nicotine eluted only during the ammonia gradient. The

following examples use a double gradient since this has been
found to elute different classes of compounds. The first gradient,
using hydrogen ions (acid), elutes weak bases such as xanthine
alkaloids. Stronger bases are eluted with a second gradient
containing ammonium, a stronger eluting ion then hydrogen ions.
The increasing concentration of ammonium also raises the pH of
the eluting solvent, tending to convert the eluting compounds to a
free base which binds less strongly to the ion exchange column.
This double gradient has the advantage that the chemist does not
need to know the pKa of their basic compound, simplifying the
purification.
0.5 100
% Solvent B: Water/5% HOAc, Water/5% NH4OH

0.4 80
Absorbance 260 nm
0.3 60
0.2 40
0.1 20
0.0 0
0 20 40 60 80
Time (CV)
Figure 149: Capture and release of nicotine using a SCX column
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Harmine and Harmaline. Both harmine and harmaline were captured

by the RediSep Rf SCX column (Figure 150). Both compounds
eluted at the same time during the second ammonia gradient.
Water/5% HOAc, Water/ 5% NH4OH

2.0 100
80
1.5
% Solvent B:
60
1.0
40
0.5
20
0.0 0
0 20 40 60 80
Time (CV)
4.0 100
Water/5% HOAc, Water/ 5% NH4OH

80
3.0
60 % Solvent B:
2.0
40
1.0
20
0 0
0 20 40 60 80
Time (CV)
Figure 150: Capture and release of harmine (top) and harmaline (bottom)

Chapter 6
Green Tea Extract. Green tea extract is used as a model for other
purifications because, in addition to the alkaloids, it also contains
other materials in the plant extract that could possibly interfere
with alkaloid binding.
Figure 151 shows that acidic and neutral compounds eluted from
the column with methanol. Xanthine alkaloids eluted with the
acetic acid gradient while an unknown compound eluted during
the ammonia gradient demonstrating that the technique is useful
for purifying families of alkaloids.
3 100
Water/5% HOAc, Water/5% NH4OH

% Solvent B:
50
0 0
0 20 40 60 80
Time (CV)
Figure 151: Purification of alkaloids in green tea extract The shaded area
denotes a time window used to collect alkaloids and exclude wash
gradient.
SAX Columns
Phenolic flavonoids are common plant secondary metabolites.
They occur as flavonoids, anthocyanidins, and anthocyanins. The
interest in flavonoids is due to their anti-oxidant activity; they are
also studied as natural food colorants that may also possess
nutraceutical benefits. As the compounds are chemically similar to
each other, obtaining pure material can be difficult. Advances in
flash chromatography columns, systems, and detection
techniques allow easier purification of phenolic flavonoids.
Methanolic extract from Camellia sinensis, a rich source of
phenolic flavonoids, is used as a model system for purification of
these compounds.
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Flavonoids are polyphenolic compounds that are ubiquitous in

nature and are categorized by chemical structure into flavonols,
flavones, flavanones, isoflavones, catechins, anthocyanidins and
chalcones. Flavonoids are found in fruits, vegetables, beverages
such as tea, coffee, beer, wine and fruit drinks, and in flowers.
Flavonoids are interesting because of their potential beneficial
effects on human health. Anthocyanidins are of interest because,
besides their potential health benefits, they may replace artificial
colors in food.
Most of the compounds in this class are acidic from the phenolic
groups, lending themselves to purification with ion-exchange
columns.
A RediSep Rf SAX column (17 g, PN 69-2203-382) was conditioned
by washing with methanol followed by a gradient to 100% water
containing 5% glacial acetic acid; the gradient was run twice to
ensure the column possessed an acetate counter-ion15. Green tea
extract (10.0 g) was dissolved in 100 mL methanol and 3 mL
concentrated ammonium hydroxide was added to the mixture
which was immediately filtered and loaded onto the column with a
sample load pump (PN 60-5247-007). The column was washed with
100 mL methanol, and the purification in Figure 152 was run.
The SAX column quickly purified the acidic compounds from the
basic and neutral compounds. Using a SAX column allowed various
compound classes, if present, to be purified at the same time that
the acidic compounds are isolated from the basic and neutral
compounds.
15.RediSep Rf Strong Anion Exchange Column Applications,

http://www.isco.com/WebProductFiles/Applications/101/Applic
ation_Notes/AN87_RediSep%20Rf_SAX_Column_Application
s.pdf, retrieved 19 June 2012

Chapter 6
2.5 100
% B Solvent (5% Glacial Acetic/water)

2.0 80
Absorbance 254 nm (AU)
1.5 60
1.0 40
0.5 20
0.0
0
0 5 10 15 20 25 30
Time (CV)
Figure 152: SAX purification of compounds from green tea extract
100
2.5
% B Solvent (0.1% Formic Acid in MeOH)
80
2.0
60
1.5
40
1.0
20
0.5
0.0 0
0 2 4 6 8 10 12 14 16 18
Time (CV)
Figure 153: Antioxidant purification on a RediSep Rf Gold C18Aq column
168 Teledyne ISCO

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100
2.5
% B Solvent (0.1% Formic Acid in MeOH)

80
2.0
60
1.5
40
1.0
20
0.5
0.0 0
0 2 4 6 8 10 12 14 16 18
Time (CV)
Figure 154: Antioxidant purification on a RediSep Rf Gold C18Aq column 9.33

9.23
9.09
8.94
8.75
8.06
6.82
6.41
5.94
5.94
5.84
5.84
5.37
4.96
3.50
2.96
2.95
2.92
2.92
2.68
2.65
2.50
OH
epigallocatechin
OH
gallate
HO O
OH
OH OH
O
OH
OH
9 8 7 6 5 4 3 2 1 ppm
1.00
1.69
0.97
0.95
1.66
0.79
1.63
1.64
0.86
0.87
0.81
0.81
0.85
0.85
OH
epicatechin OH
gallate
HO O
OH OH
O
OH
OH
Figure 155: ESI+ mass spectroscopy and proton NMR evaluation

Chapter 6
A Flash chromatography system is capable of purifying natural

products beginning with the crude extract through the final,
purified, compound.
CombiFlash chromatography systems can run a variety of columns
beyond silica gel. The columns are not limited to those pre-packed
by the manufacturer, but can include user-packed columns such as
Sephadex LH-20, polyamide, or CHP-20 reversed-phase. Using such
columns with the Flash system allows the use of the fraction
collector and the detector for accurate peak cutting.
Techniques in Extract Column Screening

Wide polarity range chromatography can be run on many different
flash columns. The use of several columns allows a user to quickly
determine the polarity of the compound based on the solvents
which elute the material from the column. The best columns for
initial and secondary purification can also be determined. The use
of UV-vis and mass spectrometer detection allows peak collection
and early identification of previously known compounds. Column
screening is a systematic method to determine a purification
strategy for unknown compounds. Columns useful for a screen are
silica, alumina, diol, C18, SAX, and SCX column. Each column
possesses different selectivity.
Column screening is an integral first step in purifying natural
products. Efficient column screening allows facile and rapid
purification of active components for further purification. The
utilization of automated flash chromatography offers
opportunities to streamline the column screenings of extracts.
Combining pre-packed columns of a variety of chemistries with
wide-polarity range chromatography is the first step in
streamlining the process. Useful column chemistries include silica,
ion-exchange, alumina, C18, and diol. Coincident to streamlining
column screening is the incorporation of simultaneous detection
techniques such as UV-vis, mass spectrometry, and ELSD. Utilizing
this enhanced automated procedure not only allows rapid
purification and early correlation with previously discovered
compounds, but also enables the researcher to focus on
potentially novel active targets. The purification of extracts from
the fruits of Capsicum annuum using silica, alumina, diol, and
reversed-phase C18 columns establishes a model protocol for
other extracts. Using this same example the advantages of
simultaneous multiple detection techniques is illustrated.
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When a natural products purification is begun, the active

compound(s) are generally unknown until they are purified and
identified. As the compounds are unknown, one needs to screen
columns to discover the method that resolves the active
compounds from the inactive material. The use of wide polarity
range chromatography on different media allow determination of
the active compound polarity and an initial separation from
inactive material in a single run. The use of multiple detectors
improves the chances of correlating activity to specific peaks
eluting from a column. The peaks also provide information about
purity as well. A mass spectrometer detector helps to screen
known compounds early in the purification. Capsaicin compounds
are used as a model for plant extracts. These compounds are of
interest for pain relief16, suppression of tumorigenesis17, increased
bladder capacity, and reduced nausea18.
Silica Screening
Crude extract (0.8 g) was dissolved in a mixture of toluene/ethyl
acetate/methanol to make a clear, green, liquid. This was mixed
with 4 g silica (PN 60-3874-091) and evaporated to a dry powder
which was placed in a solid load cartridge (PN 69-3873-235). A 4 g
RediSep Rf Gold silica column (PN 69-2203- 344) was used for the
purification. An initial gradient of hexane/ethyl acetate was run,
followed by an ethyl acetate/methanol gradient, followed by a
methanol/water gradient using the automatic solvent switching on
a CombiFlash Rf+ PurIon system fitted with an integrated ELSD.
The mass spectrometer was set to monitor a range of 100-1200 Da;
the ELSD silica parameters were used. Mass spectra were collected
as ESI+ only.
16.Rusterholz, D.B.; Capsaicin, from Hot to Not. Can New

Pain-Relieving Drugs Be Derived from This Substance Known
To Cause Pain? J. Chem. Educ., 2006, 83 (12), 1809
17.Malagarie-Cazenave, S.; Olea-Herrero, N.;Vara, D.; Morell, C.;
Díaz-Laviada, I. The vanilloid capsaicin induces IL-6 secretion
in prostate PC-3 cancer cells. Cytokine, 2011, 54(3) ,330-337
18.Hayman, M.; C.A. Kam, P. C.A. Capsaicin: A review of its
pharmacology and clinical applications. Current Anaesthesia
& Critical Care, 2008, 19(5-6), 338-343

Chapter 6
0.6 0.15 100
% B (% EtOAc) % B (% water)
1.0x1011
0.10
MS (Ion count 100-1200 Da)
0.4
Absorbance (254 nm)

ELSD
0.05 50
5.0x1010
0.2
% B (% EtOAc)
0.00
0.0 0.0 -0.05 0

0 50 100 150
Run Time (CV)
Figure 156: Silica screen of extract shows capsaicins eluting mixed with other
compounds
The mass spectrometer detector showed capsaicins eluting in ~8

column volumes (CV), but these were mixed with other
compounds. It should be noted that the capsaicin compounds
were detected as the sodium adducts, confirmed by purifying
authentic pure purchased capsaicin. This suggests that when
evaluating mass spectra of eluted compounds, one should
consider the presence of adducts such as sodium, methanol, or
acetonitrile depending on the solvents used.
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Alumina Screening
with 7.25 g neutral alumina and evaporated to a dry powder which
was placed in a solid load cartridge (PN 69-3873-235). A 24 g
RediSep Rf neutral alumina column (PN 69-2203-441) was used for
the purification. An initial gradient of hexane/ethyl acetate was
run, followed by an ethyl acetate/methanol gradient, followed by a
methanol/water gradient using the automatic solvent switching on
a CombiFlash Rf+ PurIon S system. The mass spectrometer was set
to monitor a range of 100-1200 Da. An ESI probe was used with
automatic spectra collection in both positive and negative mode.
10
2.0x10 0.5 0.5 100
0.4 0.4
10
1.5x10
% B (water)
Ion Current (1-1200 Da)
Absorbance (254 nm)
0.3 0.3
1.0x1010 50
% B (EtOAc) % B (EtOAc)
0.2 0.2
9
5.0x10
0.1 0.1
0.0
0.0 0.0 0
0 20 40 60
Run Time (CV)
Figure 157: Alumina screen of extract shows capsaicins elute together, but are
well resolved from other compounds

Chapter 6
The neutral alumina column provided better resolution of the

capsaicin from the impurities as compared to the silica. However,
the mass spectrum shows evidence of impurities within the
capsaicin peak at 24 CV.
Diol Screening
with 4.5 g Celite 545 and evaporated to a dry powder which was
placed in a solid load cartridge (PN 69-3873-235). A 15.5 g RediSep
Rf Gold Diol column (PN 69-2203-515) was used for the purification.
An initial gradient of hexane/2-propanol was run, followed by a
2-propanol/water gradient using the automatic solvent switching
on a CombiFlash Rf+ PurIon L system. The mass spectrometer was
set to monitor a range of 100-2000 Da. An ESI probe was used with
automatic spectra collection in both positive and negative mode.
3x1010 0.3 100
% B (Hexanes) % B (2-propanol)
Absorbance (254 nm, 280 nm, All-Wavelength)
2x1010 0.2
50
0.1
1x1010
0.0
0 0
0 50
Run Time (CV)
Figure 158: Capsaicins not observed to elute from diol column, indicating this
is a poor choice for purifying these compounds.
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C18 Screening
with 4 g Celite 545 and evaporated to a dry powder which was
placed in a solid load cartridge (PN 69-3873-235). A 15.5 g RediSep
Rf Gold C18 column (PN 69-2203-334) was used for the purification.
An initial gradient of water/methanol was run, followed by a
methanol /dichloromethane gradient, using the automatic solvent
switching on a CombiFlash Rf+ PurIon system. The mass
spectrometer was set to monitor a range of 100-1200 Da. Mass
spectra were collected in ESI+ mode.
10
6.0x10 0.25 0.50 100
0.45
0.20 0.40
0.35
10
4.0x10
0.15 0.30
MS (100-1200 Da)
ELSD
0.25
% B (DCM)
50
0.10 0.20
10
2.0x10
Absorbance (254 nm)
0.15
% B (MeOH)
0.05 0.10
0.05
0.00
0.0 0.00
0
0 20 40
Run time (CV)
Figure 159: C18 resolves capsaicins from each other, but are not well resolved
from other impurities

Chapter 6
The C18 column did not resolve the capsaicins cleanly from other
compounds as indicated by the mass spectrometer trace, but
showed that the various capsaicin compounds could be resolved
from each other suggesting C18 as a secondary, orthogonal,
purification method. To demonstrate this, the capsaicin peak from
the alumina column screening was run. The sample (35 mg) was
dissolved in ethyl acetate, loaded onto a 2.5g C18 solid load
cartridge (PN 69-3873-247). The cartridge was dried by using the
air purge on the CombiFlash system. A gradient was run from 50 to
100% methanol in water on a 15.5g RediSep Rf Gold C18 column
(PN 69-2203-334). Mass spectrometer detection was ESI+ mode, run
on a CombiFlash Rf+ PurIon system.
2.0x1010 0.50
0.45
100
0.40
1.5x1010
0.35
Ion Currrent (300-350 Da)
Abrorbance (280 nm)
0.30
% B (MeOH)
1.0x1010 0.25 50
0.20
0.15
5.0x109
0.10
0
0.05
0.0 0.00
0 20
Run Time (CV)
Figure 160: C18 Flash purification of capsaicins after initial purification of

crude extract on neutral alumina
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Strong Anion Exchange (SAX)

Using green tea flavonoids, the following example demonstrates
how a SAX column is employed as a part of a column screening
strategy. A RediSep Rf SAX column (17 g, PN 69-2203-382) was
conditioned by washing with methanol followed by a gradient to
100% water containing 5% glacial acetic acid; the gradient was run
twice to ensure the column possessed an acetate counter-ion19.
Green tea extract (0.5 g) was dissolved in 40 ml methanol and 3 ml
concentrated ammonium hydroxide was added to the mixture
which was immediately filtered and loaded onto the column. The
ammonium hydroxide converted the acetic flavonoids to their
conjugate bases that can interact with the SAX column. The
column was washed with 200 ml methanol, and the purification in
Figure 162 was run. Detection was a combination of mass
spectrometer (ESI+, range 100-2000 Da); All-wavelength collection
(200-400 nm), and detection at 214 and 254 nm.
The SAX column quickly purified the acidic compounds from the
basic and neutral compounds. Using a SAX column allows various
acidic compound classes, if present, to be purified at the same
time that the acidic compounds are isolated from the basic and
neutral compounds. The wash at the end of the run is included
because, during the elution with acid, compounds may be
converted to the less polar free-acids which may not be soluble in
water.
19.RediSep Rf Strong Anion Exchange Column Applications,

ation_Notes/AN87_RediSep%20Rf_SAX_Column_Application
s.pdf, retrieved 19 June 2012

Chapter 6
100
1.5
Mass Spectrometer (100-2000 Da)
1.0x1010 80
Absorbance (214 nm) (AU)

1.0 60
Methanol (%)
5.0x109
40
0.5
20
0.0 0.0
0
0 2 4 6 8 10 12 14 16 18 20 22 24 26
Run Time (min)
OH
ESI+ ESI- OH
HO O
OH
OH OH
O
epigallocatechin OH
gallate OH
Figure 161: C18 column purification of epigallocatechin gallate Mass

spectrum shows both the [M-H]- and its dimer.
178 Teledyne ISCO

Natural Products
4x1010 100
3
Absorbance (254 nm, 214 nm, All-Wavelength 200-400 nm)

Mass Spectrometer (100-2000 Da) (Ion Current)
5% Acetic Acid/water (%)

2
2x1010 50
0 0 0
0 20 40
Run Time (CV)
3.5x108
180 180 8x108 180
3.0x108
184 8.0x108
7x108
6x108
2.5x108
6.0x108 5x108
Ion Count
Ion Count
Ion Count
2.0x108
4x108
1.5x108 4.0x108
3x108
290
1.0x108
308 2.0x10 8 458 2x108
7
5.0x10 1x108
0.0 0.0 0
200 400 600 800 1000 1200 1400 1600 1800 2000 200 400 600 800 1000 1200 1400 1600 1800 2000 200 400 600 800 1000 1200 1400 1600 1800 2000
m/z (Da) m/z (Da) m/z (Da)
8x106
6x107
451 8x107
441
7x106 5x107
441 7x107
6x106 6x107
7
4x10
5x106 5x107
Ion Count
Ion Count
Ion Count
4x106 3x107
4x107
6
3x10 3x107
2x107
2x106 2x107
1x106
1x107 873 898 1x107
0 0 0
200 400 600 800 1000 1200 1400 1600 1800 2000 200 400 600 800 1000 1200 1400 1600 1800 2000 200 400 600 800 1000 1200 1400 1600 1800 2000
m/z (Da) m/z (Da) m/z (Da)
10.5 - 19 CV 19 - 25.1 CV 33.1 - 35 CV

0.0334 g 0.2100 g 0.0063 g
Figure 162: SAX column purification of crude green tea extract

Chapter 6
Strong Cation Exchange (SCX)

Using alkaloids from green tea as an example, a RediSep Rf SCX
column (15 g, PN 69-2203-391) was conditioned by washing with
methanol followed by 200 ml water containing 1N sulfuric acid to
ensure the column possessed a hydrogen counter-ion, followed by
a further wash of 200 ml water. Green tea extract (0.5 g) was
dissolved in 20 ml methanol/ 5% acetic acid, filtered and then
loaded on to the column. The column was washed with 200 ml
methanol, and the purification in Figure 163 was run. Detection
was a combination of mass spectrometer (ESI+, range 100-2000
Da); and detection at 214 and 270 nm.
The SCX column quickly purified the basic compounds from the
acidic and neutral compounds. Using a SCX column allows various
basic compound classes, if present, to be purified at the same time
that the basic compounds are isolated from the acidic and neutral
compounds. The double gradient is used since it resolves different
classes of alkaloids20. The xanthine alkaloids were found to be
displaced by hydrogen ions (acid) under aqueous conditions while
other basic compounds elute during the second gradient using
ammonium hydroxide. The wash at the end of the run is included
because, during the elution with base, compounds may be
converted to the less polar free-bases which may not be soluble in
water. The xanthine alkaloids can be purified on C18 columns. The
peak eluting during the second gradient is as yet an unidentified
compound.
20.Purification of Alkaloids with RediSep Rf SCX columns,

ation_Notes/AN40_Purification_of_Alkaloids_with_RediSep_
Rf_SCX_Columns.pdf, retrieved 17 July 2015
180 Teledyne ISCO

Natural Products
% B Solvent (5% acetic acid/water, 5% ammonium hydroxide/water)

6.0x1010 1.0 100
Mass Spectrometer (100-2000 Da) (Ion Current)

0.8 80
Absorbance (AU, 210 nm, 270 nm)

4.0x1010
0.6 60
0.4 40
2.0x1010
0.2 20
0.0 0.0
0
0 20 40 60
Run Time (CV)
195 195
8
4x10
4x108
3x108 3x108
Ion Count
Ion Count
2x108 153 2x108
1x108 1x108
153
0 0
200 400 200 400
m/z (Da) m/z (Da)
Caffeine (195 Da) Caffeine (195 Da)
195 1.8x108 229

1.0x108
1.6x108
341
7 1.4x108
8.0x10
1.2x108
153 241
Ion Count
Ion Count
6.0x107 1.0x108
8.0x107
4.0x107
181 6.0x107
4.0x107
2.0x107
153 2.0x107
0.0 0.0
200 400 600 800 1000 200 400 600 800
m/z (Da) m/z (Da)
Caffeine (195 Da) and Theophylline Unknown Compound
Figure 163: Ion exchange purification of Xanthine alkaloids and other basic
compounds

Chapter 6
Column screening in combination with a mass spectrometer

detector allows one to determine good column conditions for both
primary and downstream purification. The mass spectrometer
detector used with ELSD and UV detection, allow an initial
assessment of peak purity. The mass spectrometer allows initial
identification of known compounds; however, care has to be taken
to consider adducts. In these experiments, the capsaicin
compounds generally eluted as sodium adducts confusing initial
identification. Common adducts include sodium, methanol, and
acetonititrile.
182 Teledyne ISCO

Chapter 7
Prior to the introduction of automated Flash chromatography

systems, the user would collect all of the column eluent into
fractions. The fractions would be spotted onto TLC plates and
visualized to determine how to cut the fractions. Most compounds
absorb ultraviolet (UV) light, so Flash systems use UV lamps and
detectors. The detector generates a voltage proportional to the
absorbance which is used by the fraction collector to cut fractions
based on signal intensity and/or slope. Using the detector and
fraction collector can eliminate the need to test the fraction with
TLC to determine which fractions to combine and also saves time
and test tubes because only eluent with an absorbing compound is
collected.
UV Detection
The oldest form of detection in Flash chromatography, UV
detection is also the most common detection technique.
Compounds are detected by absorption of light. Ideally, the
detector should be set to the absorbance of the compound which
may be measured with a spectrophotometer, or referring to Table
of compound absorbance wavelengths, on page 241. Most
compounds can be detected within the range of 200–360 nm used
by UV detectors.
The default wavelength commonly used is 254 nm because many
compounds absorb at this wavelength. Since some compounds
exhibit weak absorbance at this wavelength (Figure 164), they will
only show a small peak on the detector.
For this reason, the absorbance spectrum of the compound should
be known before starting the purification.
RediSep columns can be loaded with enough sample that the
detector becomes saturated as the peak elutes. The detection
wavelength can be moved to a different value during the elution on
CombiFlash systems such as a minor absorbance (280 nm using

Chapter 7
the spectrum in Figure 164), or a shoulder of the major absorbance

if saturation of the detector prevents fractionation of closely
eluting peaks. For a complete list of Teledyne ISCO automated
Flash systems, refer to Appendix E - CombiFlash Systems, on page
255.
0.5
0.4
Absorbance (AU)
0.3
0.2
0.1
0.0
200 250 300 350 400

Wavelength (nm)
Figure 164: Chart of UV absorbance of catechin
184 Teledyne ISCO

Detection with UV-vis

A detector sensitive to ultraviolet and visible (UV-vis) light is
useful for fractionating compounds that absorb in the visible light
range such as pigments and dyes. Since fewer compounds absorb
in the visible range, the chromatogram is simpler allowing
collection only of those compounds of interest. Some compounds
may show their greatest absorbance in the UV range, but the
absorbance of the purifying solvent may overlap the compound
spectrum. An extended wavelength detector allows the detection
of the compound peak at another wavelength. The CombiFlash Rf
and the development-scale CombiFlash Torrent systems
configured with UV-vis option have a working range of 200–
800 nm.
480 nm
630 nm
700 nm
660 nm
360 nm
3.00 100
90
80
70
Absorbance (AU)
60
1.50 50
40
30
20
10
0.00 0
0 2 4 6 8 10 12
Run Length 12.8 minutes
Figure 165: Chromatogram of hair dye compounds purification with UV-vis

detection

Chapter 7
All-Wavelength Detection
All-wavelength collection in CombiFlash systems measures the
average absorbance on all wavelengths detected on a photodiode
array. The signal is processed to remove baseline drift caused by
solvent absorbance. This creates a single voltage that allows the
fraction collection program in the MPLC or Flash chromatography
system to properly cut the peak. All-wavelength detection is useful
when:
• The UV-vis spectrum is unknown, such as compounds
purified from natural products.
• There is a mixture of compounds with various absorbances
such that a single wavelength can’t “see” all the
compounds in the mixture.
• The elution solvent spectrum overlaps that of the desired
compound.
• Compounds with similar spectra overload the detector,
making it difficult to properly fractionate compounds.
• Only compounds within a specified range of absorbance
are desired. This method would exclude some starting
materials, of the products have a different absorbance
spectrum.
All-wavelength detection enhances the ability for a CombiFlash
system to purify compounds in an automated fashion.
186 Teledyne ISCO

Solvent spectrum overlaps compound

Ethyl acetate and dichloromethane are two of the most commonly
used solvents for Flash chromatography. Both of these solvents
absorb UV light below 250 nm which interferes with detection of
compounds that also absorb in this wavelength range when
gradients are used. The constantly changing baseline interferes
with the ability of the fraction collector to properly cut fractions.
Glucose pentaacetate shows only weak end adsorption which is
further suppressed by the absorption of the dichloromethane
(Figure 166). As the dichloromethane concentration is decreased,
the baseline drifts downwards. This drift tends to confuse common
fraction collection programs but is not an issue with all-wavelength
collection. All-wavelength collection filters out the baseline drift to
create a baseline usable by the fraction collector in the
CombiFlash system.
0.08 0.08
AcO
All-Wavelength Absorbance
210 nm Absorbance (AU)
O
AcO
AcO
OAc
OAc
0.00 0.00
-0.08
0 5 10 15
Run Time (min)
Figure 166: Chromatogram of glucose pentaacetate purification

using a dichloromethane/methanol gradient and an all-wavelength
collection range of 200 – 250 nm

Chapter 7
Sample overloads detector

High sample loads that cause the absorbance to saturate the
detector are common in Flash chromatography. If compounds are
closely eluting, the saturation prevents the fraction collector from
separating the compounds properly since the saturated peak is
seen as a single large peak.
All-wavelength collection measures all absorbance within the
range selected by the user and can cut the peaks since the
program detects an absorbance change at non-saturating
wavelengths. In Figure 167 catechol and resorcinol are purified
from an overloaded, overlapping peak with all-wavelength
collection.
2.0 0.7
207 nm Absorbance (AU)
All-Wavelength Absorbance
207 nm
All Wavelength
0.0 0.0
0 20 40 60
Column Volumes
Figure 167: Chromatogram showing purification of closely-eluting, saturated

peaks using all-wavelength collection with a range of 200 – 360 nm
188 Teledyne ISCO

All-wavelength collection on the

CombiFlash Rf and Torrent systems
All-wavelength collection in CombiFlash systems measure the
average absorbance on all wavelengths detected on a photodiode
array. The signal is processed to remove baseline drift caused by
solvent absorbance. This creates a single voltage that allows the
fraction collection program in the MPLC or Flash chromatography
system to properly cut the peak. All-wavelength detection is useful
when:
• The UV-vis spectrum is unknown, such as compounds
purified from natural products.
• There is a mixture of compounds with various absorbances
such that a single wavelength cannot see all the
compounds in the mixture.
• The elution solvent spectrum overlaps that of the desired
compound.
• Compounds with similar spectra overload the detector,
making it difficult to properly fractionate compounds.
All-wavelength detection improves the ability for a CombiFlash
system to purify compounds in an automated fashion. These
improvements are illustrated in the following examples.
Example with a compound mixture
2.5 A1: hexane A2: isopropanol 100

B1: isopropanol B2: water
Absorbance
%B Solvent
(C)
(B)
(A)
0.0 0
0 10 20 30 40 50
Figure 168: Chromatogram shows the purification of chlorophyll (A), caffeine

and catechins (B), and tannins (C) using all-wavelength collection
with a diol column. All of these compounds have differing spectra yet
all were detected with all-wavelength collection.

Chapter 7
Example of unknown spectrum
0.25 100
(B) (C)
Absorbance
%B Solvent
(A)
254 nm
All-wavelength collection
0.0 0
0 20 40 60 80
Figure 169: Detection of catechin (A) along with caffeine (B) and other
catechin compounds (C) using all-wavelength collection
In Figure 169, catechin is not detected with the commonly used 254
nm wavelength since this is a minimum in the spectrum, but the
all-wavelength collection was able to detect and fractionate the
compound. This is an especially useful technique for natural
products where the absorbance of the desired compound
generally is not known until after the final purification.
190 Teledyne ISCO

OH
0.5 OH catechin (A)
OH
AU
HO O
OH
0.0
200 254 300 400
O C H3
1.0 H3 C
N
N caffeine (B)
N N
AU
O
C H3
0.0
200 300 400
1.0
other catechin compounds (C)
AU
0.0
200 300 400
Figure 170: Diagram showing UV absorption of compounds separated in

Figure 169

Chapter 7
Collect related compounds within a UV-vis range

Since all-wavelength collection wavelength range can be changed,
it is possible to purify compounds with a range of wavelengths
different from others. This permits only the collection of
compounds of interest. Although one could choose a single
wavelength to perform this task, all-wavelength collection can be
set to a range that permits collection of a family of related
compounds.
Figure 171 shows three compounds collected at 254 nm. Figure 172
demonstrates selective purification using all-wavelength collection
where peaks 1 and 3 absorb between 295 and 325 nm.
1.0
(1)
Absorbance, 254 nm
(2)
(3)
0.0
0 5 10 15 20
Figure 171: Purification of compounds at 254 nm
192 Teledyne ISCO

0.2
(3)
Absorbance, 254 nm
(1)
0.0
0 5 10 15 20
Figure 172: Selective purification of compounds with all-wavelength

collection between 295 and 325 nm
Other Detectors
Occasionally, other detectors are used to purify compounds such
as refractive index (RI), fluorescence, or evaporative light
scattering detectors (ELSD). External detectors may be connected
to CombiFlash Rf and Torrent systems. These systems will then cut
peaks using an input from the external detector.
1.0
UV Absorbance (AU)
0.0
0 6 12 18
Run Time (min)
Figure 173: Chromatogram showing ELSD detection of

2,3-O-isopropylidene-D-ribofuranose using a CombiFlash Rf
system. ELSD runs as an external detector

Chapter 7
Evaporative Light Scattering Detection (ELSD)

Evaporative Light Scattering Detection (ELSD) is a useful
technique for weakly absorbing compounds. Although these
compounds can be detected with a UV detector, their response is
very weak resulting in low compound recovery. If the absorbance
of the compound overlaps that of the solvent, it may be difficult to
fractionate the compound due to a decreased signal after
subtracting the solvent baseline.
In the following experiments, tocopherols were dissolved in ethyl
acetate and mixed 10% (w/w) with Celite. The mixture was
evaporated to a free flowing powder. The sample (load listed in
each section) was placed in a 5g empty RediSep solid load
cartridge (PN 69-3873-235) and run with the solvents and gradients
as described in each section. Normal phase ELSD parameters were
used on a CombiFlash Rf system.
Compound Weakly Absorbs UV Light
0.5 100
90
0.4 80
Absorbance (280 nm), AU
70
0.3 60
% B Solvent
ELSD (V)
50
0.2 40
30
0.1 20
10
0.0 0
0 2 4 6 8 10 12 14 16
Time (CV)
Figure 174: Purification of tocopherols on a diol column with a hexane/ethyl

acetate gradient Shaded area denotes area of improved recovery
using ELSD compared to UV absorbance.
194 Teledyne ISCO

In Figure 174, a mixture of tocopherols is purified using a 30g

RediSep Rf Gold Diol column (PN 69-2203-516) with a hexane/ethyl
acetate gradient. Tocopherols exhibit a weak absorbance at 280
nm. Using ELSD allows enhanced detection of the compounds and
improved recovery, denoted by the shaded areas. The shaded
areas denote material not detected with the UV detector, but
collected with the ELSD.
Compound UV Absorbance is Obscured by Elution Solvent

0.5 0.5 100
0.4 0.4 80
0.3 0.3 60
Acetone (%)
Absorbance
ELSD
0.2 0.2 40
- 280 nm
0.1 0.1 - ALL-Wavelength 20
- ELSD
0.0 0.0 0
0 2 4 6 8 10 12 14 16
Time (CV)
Figure 175: Tocopherols purified on a diol column using a hexane/acetone

gradient Detection using ELSD, all-wavelength collection, and
absorbance at 280 nm.
The sample in Figure 175 was run with an acetone gradient, which
absorbs at 280 nm. Detection at 280 nm was obscured by the
solvent absorbance. The ELSD detection was unaffected by the
solvent. All Wavelength Collection weakly detected the first two
peaks. The collection at 280 nm was partially obstructed by the
solvent absorbance.

Chapter 7
Purification of Carbohydrates
Carbohydrate containing compounds generally pose a challenge
for MPLC equipment because they show weak or no UV
absorbance. Derivatized carbohydrate compounds may show a
weak absorbance in the same wavelengths as the eluting solvent
making detection and fractionation difficult. Detection of
carbohydrate containing compounds is usually only difficult early
in the synthesis when there are few chromophores that absorb UV
light. As the synthesis proceeds closer to the targeted compound,
UV detection becomes easier.
Evaporative Light Scattering Detection of

2,3-O-isopropylidene-D-ribofuranose
Evaporative Light Scattering Detection (ELSD) is useful when the
compound possesses no chromophores. ELSD is not a universal
detector as some low-melting compounds fail to cause a
response21. ELSD is also a destructive detector since a portion of
the material is lost in the detector.
A sample volume of 0.1mL of crude
2,3-O-isopropylidene-D-ribofuranose was injected onto a 12 g
RediSep Rf silica column on a CombiFlash Rf system. The gradient
was 0 to 100% ethyl acetate in hexane.
The product was easily purified using the ELSD detector. The
active flow splitter allows the purification to be easily scaled to
larger reactions because the optimal sample size is delivered to
the ELSD regardless of flow rate.
21.Webster, G.K.; Jensen, J.S.; Diaz, A.R. Investigation into

Detector Limitations Using Evaporative Light-Scattering
Detectors For Pharmaceutical Applications. J.
Chromatographic Science, V. 42 Oct 2004, p.484
196 Teledyne ISCO

Figure 176: ELSD detection of 2,3-O-isopropylidene-D-ribofuranose with a

CombiFlash Rf system
Mass-directed Fractionation
There is a need for a flash chromatography detector that allows
researchers to identify compounds as they are purified so time is
not wasted concentrating the product of a side reaction. Mass
spectrometers are useful as detectors because the molecular
weight of a synthesized compound is known. Used in conjunction
with UV detection, specific compounds can be collected without
additional confirmation of the compound identity after elution.
Likewise, in natural products, certain species are known to
produce certain compounds. Knowledge of these molecular
weights allows the user to screen potentially interesting
compounds during elution.

Chapter 7
Single Ion Current (SIC)

Single ion current (SIC) is an experimental run where the mass
spectrometer is programmed to generate a data trace from a
narrow range of mass-to-charge ratio (m/z) values, typically 1 or 2
Daltons. This generates a trace specific for a molecular weight and
allows purification of a single compound. As a demonstration,
caffeine and theophylline were dissolved in methanol and
adsorbed onto Celite 545 (1:9 w/w alkaloids: Celite) to make a 10%
sample. To determine the appropriate molecular weight to collect
caffeine, the Mass Spectrometer Method Development function in
PeakTrak was used. The sample was made 0.05 mg/mL and
injected into the system (Figure 177).
The mass spectrum from the method development screen verified
that the [M+H]+ ion for caffeine was seen (as opposed to a major
fragment or an adduct). Since the method development program
allows a user to directly inject a sample into the mass
spectrometer, it is also useful for monitoring the progress of a
reaction.
The alkaloid/Celite mixture 0.3 g of (30 mg alkaloids, 0.12% column
load each compound) was placed in a 5 g solid load cartridge (PN
69-3873-235) and run on a 12 g RediSep Rf silica column (PN
69-2203-312). The mass spectrometer was set to a m/z value of 195
(caffeine [M+H]+) and run as electrospray positive ion mode (ESI+).
The compounds were resolved with a gradient of 2 to 30%
methanol in dichloromethane (Figure 178).
Figure 177: Experimental mass spectrometer detection Method Development

screen
198 Teledyne ISCO

3x109 0.75 30
25
0.60
2x109 Abs 254 nm, 275 nm (AU) 20

0.45
Intensity (cps)
Methanol (%)
15
0.30
1x109 10
0.15
5
0 0.00 0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Time (CV)
Indicates mass directed fractionation
153
10
1x10
Mass spectrum Caffeine

8x109
Measured at 6-CV Note: Colored bars
6x10 9
denote collected
[M=H]+
fractions.
4x109
195
2x109
0
200 400 600
Mass (Daltons)
1x1010 153
10
1x10 Mass spectrum Theophylline
measure at 8 CV
10
1x10
Intensity (cps)
8x109
[M+H]+
6x109
4x109
[M+H+MeOH]+
181
2x109 213
0
200 400 600
Mass (Daltons)
Figure 178: Purification of caffeine from theophylline Although the MS was

programmed for SIM, mass spectra are collected for all compounds.

Chapter 7
Extracted Ion Current (XIC)

The Extracted Ion Current (XIC) was run in the same fashion as the
single ion experiment (on a 12 g RediSep Rf column, PN
69-2203-312) except 4.5 g alkaloids on Celite was run (4% column
load). The mass spectrometer was programmed to collect
compounds within the range of 180-200 Daltons. Even with the
heavy load, the mass spectrometer was able to detect both
compounds (Figure 179).
4 30
25
Absorbance 254 nm, 275 nm (AU)
1x1010
3
20
Intensity (cps)
Methanol (%)
2 15
9
5x10
10
1
0 0 0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Time (CV) Indicates mass directed fractionation
Figure 179: Purification of caffeine and theophylline by FCMS using XIC

range 180-200 Daltons
200 Teledyne ISCO

Purification of crude benzidine by XIC

Crude benzidine was synthesized and adsorbed onto 18.74 g silica
(PN 60-3874-091); 5.0 g of the silica/sample mixture (1.03 g reaction
mixture) was run on a 40 g RediSep Rf silica column with a
hexane/ethyl acetate gradient. The mass spectrometer detection
range was set to 175-300 Daltons; the carrier solvent was methanol
containing 0.1% formic acid. The recovery from the Flash
Chromatography - Mass Spectrometry (FCMS) system was 0.76 g.
FCMS is a useful tool for validating the results of a reaction as well
as ensuring the desired reaction product is collected. In the
benzidine synthesis, the major compound eluted was an oxidation
product and could be ignored due to observation of the mass
spectrum in real-time. The ion trace can be run as a “Single Ion
Current”, allowing a single component to be collected or as a mass
range allowing several compounds of interest to be fractionated.
All purifications were performed with ACS grade solvents with
standard flash columns. Nothing was found from these columns or
solvents that interfered with the purification by mass
spectroscopy. The CombiFlash Rf+ PurIon system acquires a full
range mass spectrum from 50-1200 Dalton even if SIC is run
allowing evaluation of interesting peaks during the purification.
Termination upon target mass detection

Mass directed fractionation using the CombiFlash Rf+ PurIon Flash
chromatography system offers significant time and solvent savings
to the chemist. The mass spectrometer can collect fractions on the
basis of a molecular ion that is unique to the desired or targeted
compound. This capability permits confirmation that the desired
compound is being collected in real time as it is eluting. The need
for post purification fraction verification by either thin layer
chromatography (TLC) or LC-MS is thereby eliminated. The user
has the option to collect only the desired compound, based on its
unique molecular ion and thus minimize the number of test tubes
and solvent volume collected for post purification processing.
When collecting on a target molecular ion using the “Terminate on
Target” feature in PeakTrak, the run can be ended immediately
after the unique ion elutes saving both significant solvent and time.
A series of four purifications were run using the same alkaloid
mixture. The first run collected all peaks, based on UV detector
response, and was allowed to complete its programmed run time.
The second run, utilized the “Terminate on Target” feature where

Chapter 7
collection was driven by both the UV and the mass spectrometer

signal and the target molecular ion corresponded to that of the
first eluting peak. Using the same feature, the third run was
programmed to collect only the molecular ion that corresponded
to that of the second peak and collection was driven only by the
mass spectrometer for this run. The final run was similar to the
second and third, except that the PurIon was programmed to
collect based on molecular ions associated with both peaks one
and two. The two component mixture was purified using 12 g
RediSep Rf Gold silica columns (PN 69-2203-345). The compounds
were eluted with hexane/ ethyl acetate; the standard gradient
method was altered slightly by starting at 2% ethyl acetate to allow
for improved elution of the peak with m/z (mass-to-charge ratio) of
180 Da.
The desired molecular ion peaks were determined by use of the MS
Method Development screen (Figure 180). An aliquot of the
mixture was diluted to an approximate concentration of 1 mg/20
mL, filtered through a 0.22 µm filter, and injected. The desired peak
(185 Da in this example) was selected from the touch screen which
automatically entered the mass into the “Detection Ions” table.
Any masses entered into this table are automatically transferred to
the mass spectrometer parameters in the column run method,
when the MS Method Development window is closed.
Figure 180: MS Method Development screen with the 185 Da ion selected for
detection
202 Teledyne ISCO

Purification using only UV detection
Figure 181: Alkaloids purified using UV detection only
The alkaloids were purified using UV detection only. In this case,

the UV peaks are weak due to a lack of strong UV absorbance for
these compounds. Collection was based on 254 nm.
Terminate after peak with m/z 180 collected
Figure 182: Run terminated after elution of peak with m/z=180 Da

Chapter 7
In this run, the PurIon was run with the target mass set to 180 Da
and programmed to terminate after this peak eluted. Collection
was based on both UV and mass, demonstrating the ability to use
either detector to collect compounds if desired. The peak eluted,
and the run was stopped (Figure 182) automatically based on the
elution of a peak with m/z 180 Da. The user is presented with the
option to continue the run, or terminate the purification at this
point. If the user knew that additional compounds were contained
in the sample with the same mass, such as an isomer, the
purification could be continued until the desired isomer eluted.
Terminate run after collecting m/z 185
Figure 183: Collection of only compounds with m/z 185 Da
The collection in this purification was entirely based on the target

mass signal from the PurIon; the UV signals were only monitored.
The system was programmed to terminate the run after the
compound with m/z 185 eluted. The first eluting compound was
ignored and was not collected, reducing the number of fractions
collected.
204 Teledyne ISCO

Terminate run after collecting both m/z 180 and 185
Figure 184: Termination after elution of compounds with molecular ion peaks
of both 180 and 185
The last example uses two target masses to purify the compounds;
one at 180 Da and the other with a molecular ion peak at 185 Da.
The run does not terminate until both peaks have eluted. The
PurIon system can be programmed to run up to 4 distinct target
masses, or a mass range and up to 3 distinct target masses. The
mass range can include the entire range for the PurIon mass
spectrometer or a subset of the total range. Such a range can be
used to terminate a run for both a molecular ion and an adduct of
that ion, for example. Working off such a range guarantees that the
compound is detected, and the run terminated, regardless of
whether the adduct is observed during a column run as opposed
to the direct injection. The use of both the adduct and the
molecular ion increases the sensitivity for that compound and
increases yield.

Chapter 7
Mass-directed purification of steroids with APCI and

CombiFlash Rf+ PurIon
Steroids are an important class of compounds that include sex
hormones, corticosteroids, anabolic steroids, and
anti-inflammatory compounds such as dexamethasone. This class
of compounds is not generally detected with electrospray
ionization (ESI) but is easily seen with atmospheric pressure
chemical ionization (APCI).
This application note demonstrates the use of APCI for detection
and purification of crude ergosterol as well as the use of
dichloromethane (DCM) as a carrier solvent for very non-polar
compounds in APCI detection.
Crude ergosterol (monoisotopic mass 396.3 Da) was dissolved in
dichloromethane and directly injected into the PurIon L system
using the method development screen; methanol containing 0.1%
formic acid was used as the carrier solvent. No molecular ion peak
or fragment was observed under ESI+ or ESI- (Figure 185).
Figure 185: ESI+ spectrum of crude ergosterol
206 Teledyne ISCO

The ESI probe was changed for an APCI probe and the sample was
injected using the same carrier as for the ESI experiment. APCI+
showed a strong peak at 379 Daltons (Da).
The use of the Ion Finder feature (Figure 186) suggested the base
peak for ergosterol to be 379 Da after loss of water from the [M+H]+
ion. Loss of water is a common fragmentation pathway of sterol
ions under APCI22. The 379 Da peak was selected as the detection
ion in the Ion Finder window and this value was automatically
transferred to the method editor for the column.
Figure 186: Ion Finder used to determine fragments of ergosterol with APCI
22.Lagarda, M.J.; Garcia-Llatas, G.; Farre, R. Analysis of

phytosterols in foods. Journal of Pharmaceutical and
Biomedical Analysis 41 (2006) 1486-1496

Chapter 7
To purify the ergosterol, 0.4943 g crude ergosterol was dissolved in

dichloromethane and mixed with 2.5 g silica gel (PN 60-3874-091);
the mixture was rotary evaporated to dryness and placed in a 5 g
empty solid load cartridge (PN 69-3873-235). The purification was
run on a 40 g RediSep Rf Gold silica column (PN 69-2203-347) using
the standard Gold Resolution method using a hexane/ethyl acetate
gradient. Compounds were collected by mass directed
fractionation using 379 Da (as selected in the Ion Finder).
5x108
379 Tube 1
4x108
3x108
Intensity (cps)
2x108
1x108
0
100 200 300 400 500 600 700 800
m/z (Da)
2.0x108
1.8x108 Tube 2
1.6x108 379
1.4x108
1.2x108
Intensity (cps)
1.0x108
8.0x107
6.0x107
4.0x107
2.0x107
0.0
100 200 300 400 500 600 700 800
m/z (Da)
Figure 187: Purification of crude ergosterol with mass spectrometer and UV

detection Mass spectra show the contents of each collection fraction
are the same.
208 Teledyne ISCO

A peak detected at 379 Da was collected into two tubes. The

Fraction Tube Composite Mass Spectrum feature (Figure 188) in
PeakTrak confirmed that both fractions contained the same
material, while a difference in the mass spectrum would suggest an
impurity in one of the fractions. This feature is useful to determine
which fractions to combine to reduce impurities in the final
product. The fractions were combined and evaporated to yield
0.1274 g (26% yield).
Figure 188: Fraction Tube Composite Mass Spectrum is seen by selecting a

tube on the touchscreen (tube 44 in this example)

Chapter 7
Figure 189: Crude ergosterol (left) and purified (right) The CombiFlash Rf+
PurIon system with an APCI probe is able to detect compounds that
systems only using ESI probes are not able to detect. These are
generally relatively non-polar compounds.
The PeakTrak MS Method Development and Ion Finder windows

allow confirmation that compounds ionize. They also help the
chemist to identify fragments that can be used for detection at
times when the [M+H]+ (or [M-H]- for negative ionization) are not
present. The Ion Finder is also useful for finding adducts of
sodium, potassium, and solvents such as methanol and
acetonitrile, if these are used.
The CombiFlash Rf+ PurIon system can also provide information
about co-eluting peaks, impurities, and confirmation of compound
elution from a column.
210 Teledyne ISCO

Chapter 8
High Performance Liquid

Chromatography
High performance liquid chromatography (HPLC) is on a

continuum of techniques leading from flash chromatography. High
performance chromatography works by using columns with a
smaller particle size.
Figure 190: Methods to improve resolution between compounds
There are several ways to improve resolution between

compounds, depicted in Figure 190. One can increase retention
time by reducing the solvent polarity as described in Flash
Chromatography Essentials, on page 5. However, this increases
solvent usage and run time. One can also change the selectivity,
the relative peak retention, by changing either the solvent or the
column (see Advanced Flash Chromatography, on page 89). One can

Chapter 8
also improve resolution by changing the number of theoretical

plates with the use of improved packing materials. RediSep Rf Gold
silica (see From Traditional Glass Columns to Automated Flash
Chromatography, on page 37) is one example of a more efficient
media; RediSep Prep columns uses finer media (5 µ particles).
Such fine particles tend to create higher back pressure, which is
handled by the CombiFlash EZ Prep system. This system is able to
use both Flash and Preparative (Prep) HPLC columns. For a
complete list of Teledyne ISCO chromatography systems, refer to
Capsaicin compounds
In Natural Products, on page 153, the purification of capsaicin
compounds was discussed as an example of column screening.
The capsaicin compounds were purified with a Flash C18 column
(Figure 191).
Figure 191: C18 Flash purification of capsaicin compounds
The same eluent from an alumina column was run on the

CombiFlash EZ Prep system with a PurIon L mass spectrometer
using a RediSep Prep column to generate the chromatogram in
Figure 192.
212 Teledyne ISCO

Figure 192: Final capsaicin purification on prep HPLC column
Using the increased efficiency of the Prep HPLC column, the

compounds were resolved to near-baseline resolution. In addition,
an additional compound (Figure 193, Compound 1) was detected
with the mass spectrometer as a shoulder of the peak eluting at
~18 minutes.

Chapter 8
Figure 193: Table of mass spectra and assigned structures for purification
Compound Structure and mass spectrum
1
HO
H
N Nordihydroapsaicin
O
Formula Weight : 293.4 ([M+Na]+= 316)

Exact Mass : 293.2
Formula : C17H27NO3
214 Teledyne ISCO


2
HO
H
N
Capsaicin
O
O
Formula Weight : 305.4 ([M+23] = 328)
Exact Mass : 305.20
Formula : C18H27NO3
Composition : C 70.8% H 8.9% N 4.6% O 15.7%

Chapter 8

3
HO
H
N Dihydrocapsaicin
O
O Formula Weight : 307.4 ([M+23] = 330)

Exact Mass : 307.21
Formula : C18H29NO3
216 Teledyne ISCO


4
HO
H
N
O Homodihydrocapsaicin
Formula Weight : 321.4 ([M+23]= 344)

Exact Mass : 321.23
Formula : C19H31NO3
Peptides
Peptide synthesis and purification is becoming increasingly
important. Peptides are used as active site models in drug
discovery and are also increasingly being used as Active
Pharmaceutical Ingredients (APIs). The increased use of peptides
necessitates improved purification techniques. Impurities in
synthesized peptides come from impurities in the protected amino
acid reagents used and incomplete reactions as the peptide chain
is grown.
The peptide HNWYPAAPH was studied as an ACE inhibiter23 and
was synthesized by peptide synthesis and purification lab. The
method development screen of the CombiFlash EZ Prep system
was used to verify the identity of the peptide and to confirm that
the default ionization settings would detect the peptide (Figure
194).

Chapter 8
Figure 194: Verification of purchased HNWYPAAPH crude peptide
23.Lee,S-J; Kim, Y-S; Kim, S-E; Kim, E-K; Hwang, J-W; Park,
T-K; Kim, B.K; Moon, S-H; Jeon, B-T; Jeon, Y-J; Ahn, C-B; Je,
J-Y, Park, P-J. Purification and Characterization of a Novel
Angiotensin I-Converting Enzyme Inhibitory Peptide Derived
from an Enzymatic Hydrolysate of Duck Skin Byproducts. J.
Agric. Food Chem. 2012, 60, 10035o 10040.
218 Teledyne ISCO

The mass was found to compare well with the expected mass
(monoisotopic mass 1091.5 Da). Peaks visible include the [M+H]+
(1092 Da); [M+Na]+ (1114 Da); and the doubly-charged [M+2H]2+
ion (546 Da). The 546 and 1092 Da peaks were selected as the
detection ions in the Method Development window (Figure 194)
and these values were automatically transferred to the run method
for the column (Figure 195). For an initial study, 10.2 mg of the
sample was purified on a 10 mm x 150 mm RediSep Prep C18
column. A water/acetonitrile gradient, both containing 0.1%
trifluoroacetic acid (TFA), at 5.0 mL/min was used to elute the
peptide. Of the sample loaded, the major peak collected between
16.3 and 17.6 minutes yielded 7.8 mg (76.4% yield) at greater than
99% purity.
Figure 195: Purification of HNWYPAAPH on CombiFlash EZ Prep

Chapter 8
220 Teledyne ISCO

Appendix A
Media Selection
The following charts and figures can assist with the selection of
stationary phase media based on sample properties and size.
• Figure 196: Chart for column media selection
• Figure 197: Table of RediSep Rf Gold Silica Gel Disposable
Flash Columns 20–40 microns
• Figure 198: Table of RediSep Rf Silica Gel Disposable Flash
Columns, 40–60 microns
• Figure 199: Table of Reusable RediSep Rf Gold C18
Reversed-phase columns, 20–40 microns
• Figure 200: Table of Reusable RediSep Rf C18
Reversed-phase columns, 40–60 microns
• Figure 201: Table of Reusable RediSep Rf Gold Amine
• Figure 202: Table of Reusable RediSep Rf Amine Columns,
40–60 microns
• Figure 203: Table of Reusable RediSep Rf Gold Cyano
• Figure 204: Table of Reusable RediSep Rf SAX Columns
• Figure 205: Table of Reusable RediSep Rf SCX Columns
• Figure 206: Table of Reusable RediSep Rf Gold Diol Columns,
20–40 microns
• Figure 207: Table of RediSep Rf Alumina Acidic Columns
• Figure 208: Table of RediSep Rf Alumina Neutral Columns
• Figure 209: Table of RediSep Rf Alumina Basic Columns

Appendix A
Sample Stationary Phase (conditions)

Characteristics
Normal Phase
Low or Medium Diol
Polarity Neutral Alumina
Reversed Phase
High C18
Polarity Cyano
C18
Normal Phase
Amine
Basic
Properties Basic Alumina
SCX
Cyano
Neutral Alumina
Sample
Normal Phase
C18
Acidic Acidic Alumina

Properties SAX
Cyano
Neutral Alumina
Diol
Acid
Neutral Alumina
Sensitive
Cyano
C18
Charged
Cyano
Figure 196: Chart for column media selection for purification of small mole-
cules (MW<2000)
222 Teledyne ISCO

Figure 197: Table of RediSep Rf Gold Silica Gel Disposable Flash Columns
20–40 microns Sample loading capacities and ordering information
Column Teledyne
Size ISCO Part
Sample Size
(g) Number Description
20 mg - 0.4 g 4 69-2203-344 4 gram RediSep Rf Gold Rf Gold Silica Gel
1.1 - 22 g 220 69-2203-359 220 gram RediSep Rf Gold Silica Gel
7.5 - 150 g 1500 69-2203-428 1.5 kg RediSep Rf Gold Silica Gel Disposable
columns, pkg. of 2.
15 - 300 g 3000 69-2203-529 3 kg RediSep Rf Gold Silica Gel Disposable
columns, pkg. of 1.

Appendix A
Figure 198: Table of RediSep Rf Silica Gel Disposable Flash Columns, 40–60
microns Sample loading capacities and ordering information
Column Teledyne
Size ISCO Part
Sample Size
20 mg - 0.4 g 4 69-2203-304 4 gram RediSep Rf Disposable Flash columns,
pkg. of 20.
60 mg - 1.2 g 12 69-2203-312 12 gram RediSep Rf Disposable Flash
120 mg - 2.4 g 24 69-2203-324 24 gram RediSep Rf Disposable Flash
200 mg - 4 g 40 69-2203-340 40 gram RediSep Rf Disposable Flash
columns, pkg of 15.
400 mg - 8 g 80 69-2203-380 80 gram RediSep Rf Disposable Flash
600 mg - 1 2g 120 69-2203-320 120 gram RediSep Rf Disposable Flash
N/A 125 69-2203-314 125 gram RediSep Rf Disposable Filter
column, pkg. of 6.
1.1 - 22 g 220 69-2203-422 220 gram RediSep Rf Disposable Flash
column, pkg. of 6.
1.65 - 33 g 330 69-2203-330 330 gram RediSep Rf Disposable Flash
columns, pkg. of 4.
3.8 - 75 g 750 69-2203-275 750 gram RediSep Disposable Flash columns,
pkg. of 4.
7.5 - 150 g 1500 69-2203-277 1.5 kg RediSep Disposable Flash columns,
pkg. of 3.
15 - 300 g 3000 69-2203-527 3 kg RediSep Silica Gel Disposable columns,
pkg. of 1.
224 Teledyne ISCO

Figure 199: Table of Reusable RediSep Rf Gold C18 Reversed-phase columns,

20–40 microns Sample loading capacities and ordering information
Column Teledyne
Size ISCO Part
Sample Size
5.5 - 110 mg 5.5 69-2203-328 5.5 gram RediSep Rf Gold C18 columns, pkg.
of 2.
15.5 - 310 mg 15.5 69-2203-334 15.5 gram RediSep Rf Gold C18 column, pkg.
of 1.
30 - 600 mg 30 69-2203-335 30 gram RediSep Rf Gold C18 column, pkg.
of 1.
50 mg - 1.0 g 50 69-2203-336 50 gram RediSep Rf Gold C18 column, pkg.
of 1.
100 mg - 2 g 100 69-2203-337 100 gram RediSep Rf Gold C18 column, pkg.
of 1.
150 mg - 3 g 150 69-2203-338 150 gram RediSep Rf Gold C18 column, pkg.
of 1.
of 1.
of 1.
0.95 - 19 g 950 69-2203-492 950 gram RediSep Rf Gold C18 column, pkg.
of 1.
1.9 - 38 g 1900 69-2203-493 1.9 kg RediSep Rf Gold C18 column, pkg. of
1.
3.8 - 76 g 3800 69-2203-528 3.8 kg RediSep Rf Gold C18 column, pkg. of
1.

Appendix A
Figure 200: Table of Reusable RediSep Rf C18 Reversed-phase columns, 40–

60 microns Sample loading capacities and ordering information
Column Teledyne
Size ISCO Part
Sample Size
4.3 - 86 mg 4.3 69-2203-410 4.3 gram Reverse Phase (C18) RediSep Rf
columns, pkg. of 2.
13 - 260 mg 13 69-2203-411 13 gram Reverse Phase (C18) RediSep Rf
column, pkg. of 1.
column, pkg. of 1.
column, pkg. of 1.
86 mg - 1.72 g 86 69-2203-416 86 gram Reverse Phase (C18) RediSep Rf
column, pkg. of 1.
column, pkg. of 1.
column, pkg. of 1.
column, pkg. of 1.
226 Teledyne ISCO

Figure 201: Table of Reusable RediSep Rf Gold Amine Columns, 20–40

Column Teledyne
Size ISCO Part
Sample Size
5.5 - 110 mg 5.5 69-2203-504 5.5 gram Amine RediSep Rf Gold columns,
pkg. of 2.
15.5 - 310 mg 15.5 69-2203-505 15.5 gram Amine RediSep Rf Gold column,
pkg. of 1.
30 - 600 mg 30 69-2203-506 30 gram Amine RediSep Rf Gold column, pkg.
of 1.
50 mg - 1.0 g 50 69-2203-507 50 gram Amine RediSep Rf Gold column, pkg.
of 1.
100 mg - 2 g 100 69-2203-508 100 gram Amine RediSep Rf Gold column,
pkg. of 1.
150 mg - 3 g 150 69-2203-509 150 gram Amine RediSep Rf Gold column,
pkg. of 1.
275 mg - 5.5 g 275 69-2203-510 275 gram Amine RediSep Rf Gold column,
pkg. of 1.
415 mg - 8.3 g 415 69-2203-511 415 gram Amine RediSep Rf Gold column,
pkg. of 1.
0.9 - 19 g 950 69-2203-512 950 gram Amine RediSep Rf Gold column,
pkg. of 1.
1.9 - 38 g 1900 69-2203-513 1.9 kg Amine RediSep Rf Gold column, pkg.
of 1.
3.8 - 76 g 3800 69-2203-534 3.8 kg Amine RediSep Rf Gold column, pkg.
of 1.

Appendix A
Figure 202: Table of Reusable RediSep Rf Amine Columns, 40–60 microns

Sample loading capacities and ordering information
Column Teledyne
Size ISCO Part
Sample Size
23 - 235 mg 4.7 69-2203-350 4.7 gram Amine RediSep Rf columns, pkg. of
2.
70 mg - 0.7 g 14 69-2203-351 14 gram Amine RediSep Rf column, pkg. of 1.
0.7 - 7 g 140 69-2203-354 140 gram Amine RediSep Rf column, pkg of 1.
1.3 - 12.8 g 260 69-2203-358 260 gram Amine RediSep Rf column, pkg. of
1.
1.92 - 19.25 g 385 69-2203-355 385 gram Amine RediSep Rf column, pkg. of
1.
228 Teledyne ISCO

Figure 203: Table of Reusable RediSep Rf Gold Cyano Columns, 20–40

Column Teledyne
Size ISCO Part
Sample Size
5.5 - 110 mg 5.5 69-2203-494 5.5 gram Cyano RediSep Rf Gold columns,
pkg. of 2.
15.5 - 310 mg 15.5 69-2203-495 15.5 gram Cyano RediSep Rf Gold column,
pkg. of 1.
30 - 600 mg 30 69-2203-496 30 gram Cyano RediSep Rf Gold column, pkg.
of 1.
50 mg - 1.0 g 50 69-2203-497 50 gram Cyano RediSep Rf Gold column, pkg.
of 1.
100 mg - 2 g 100 69-2203-498 100 gram Cyano RediSep Rf Gold column,
pkg. of 1.
150 mg - 3 g 150 69-2203-499 150 gram Cyano RediSep Rf Gold column,
pkg. of 1.
275 mg - 5.5 g 275 69-2203-500 275 gram Cyano RediSep Rf Gold column,
pkg. of 1.
415 mg - 8.3 g 415 69-2203-501 415 gram Cyano RediSep Rf Gold column,
pkg. of 1.
0.9 - 19 g 950 69-2203-502 950 gram Cyano RediSep Rf Gold column,
pkg. of 1.
1.9 - 38 g 1900 69-2203-503 1.9 kg Cyano RediSep Rf Gold column, pkg.
of 1.
3.8 - 76 g 3800 69-2203-533 3.8 kg Cyano RediSep Rf Gold column, pkg.
of 1.

Appendix A
Figure 204: Table of Reusable RediSep Rf SAX Columns Sample loading

capacities and ordering information
Column Teledyne
Size ISCO Part
6.27 mMol 5.7 69-2203-381 5.7 gram Strong Anion Exchange RediSep Rf
columns, pkg. of 2.
18.7 mMol 17 69-2203-382 17 gram Strong Anion Exchange RediSep Rf
column, pkg. of 1.
column, pkg. of 1.
column, pkg. of 1.
column, pkg. of 1.
187 mMol 170 69-2203-385 170 gram Strong Anion Exchange RediSep Rf
column, pkg. of 1.
column, pkg. of 1.
517 mMol 470 69-2203-386 470 gram Strong Anion Exchange RediSep Rf
column, pkg. of 1.
230 Teledyne ISCO

Figure 205: Table of Reusable RediSep Rf SCX Columns Sample loading

Column Teledyne
Size ISCO Part
3.5 mMol 5 69-2203-390 5 gram Strong Cation Exchange RediSep Rf
columns, pkg. of 2.
10.5 mMol 15 69-2203-391 15 gram Strong Cation Exchange RediSep Rf
column, pkg. of 1.
21 mMol 30 69-2203-392 30 gram Strong Cation Exchange RediSep Rf
column, pkg. of 1.
column, pkg. of 1.
column, pkg. of 1.
column, pkg. of 1.
column, pkg. of 1.
column, pkg. of 1.

Appendix A
Figure 206: Table of Reusable RediSep Rf Gold Diol Columns, 20–40

Column Teledyne
Size ISCO Part
Sample Size
5.5 - 110 mg 5.5 69-2203-514 5.5 gram Diol RediSep Rf Gold columns, pkg.
of 2.
15.5 - 310 mg 15.5 69-2203-515 15.5 gram Diol RediSep Rf Gold column, pkg.
of 1.
30 - 600 mg 30 69-2203-516 30 gram Diol RediSep Rf Gold column, pkg.
of 1.
50 mg - 1.0 g 50 69-2203-517 50 gram Diol RediSep Rf Gold column, pkg.
of 1.
100 mg - 2 g 100 69-2203-518 100 gram Diol RediSep Rf Gold column, pkg.
of 1.
150 mg - 3 g 150 69-2203-519 150 gram Diol RediSep Rf Gold column, pkg.
of 1.
of 1.
of 1.
0.9 - 19 g 950 69-2203-522 950 gram Diol RediSep Rf Gold column, pkg.
of 1.
1.9 - 38 g 1900 69-2203-523 1.9 kg Diol RediSep Rf Gold column, pkg. of
1.
3.8 - 76 g 3800 69-2203-535 3.8 kg Diol RediSep Rf Gold column, pkg. of
1.
232 Teledyne ISCO

Figure 207: Table of RediSep Rf Alumina Acidic Columns Sample loading

Column Teledyne
Size ISCO Part
Sample Size
5.5 - 110 mg 8 69-2203-430 8 gram Alumina Acidic RediSep Rf columns,
pkg of 20.
15.5 - 310 mg 24 69-2203-431 24 gram Alumina Acidic RediSep Rf columns,
pkg. of 20.
30 - 600 mg 48 69-2203-432 48 gram Alumina Acidic RediSep Rf columns,
pkg. of 15.
50 mg - 1.0g 80 69-2203-433 80 gram Alumina Acidic RediSep Rf columns,
pkg. of 15.
100 mg - 2 g 160 69-2203-436 160 gram Alumina Acidic RediSep Rf
150 mg - 3 g 240 69-2203-434 240 gram Alumina Acidic RediSep Rf
415 mg - 8.3 g 440 69-2203-438 440 gram Alumina Acidic RediSep Rf
columns, pkg. of 6.
0.9 - 19 g 660 69-2203-435 660 gram Alumina Acidic RediSep Rf
columns, pkg. of 4.

Appendix A
Figure 208: Table of RediSep Rf Alumina Neutral Columns Sample loading

Column Teledyne
Size ISCO Part
Sample Size
40 - 320 mg 8 69-2203-440 8 gram Alumina Neutral RediSep Rf columns,
pkg. of 20.
120 - 960 mg 24 69-2203-441 24 gram Alumina Neutral RediSep Rf
240 mg - 1.92 g 48 69-2203-442 48 gram Alumina Neutral RediSep Rf
1.2 - 9.6 g 240 69-2203-444 240 gram Alumina Neutral RediSep Rf
columns, pkg. of 6.
columns, pkg. of 4.
234 Teledyne ISCO

Figure 209: Table of RediSep Rf Alumina Basic Columns Sample loading

Column Teledyne
Size ISCO Part
Sample Size
40 - 320 mg 8 69-2203-450 8 gram Alumina Basic RediSep Rf columns,
pkg. of 20.
120 - 960 mg 24 69-2203-451 24 gram Alumina Basic RediSep Rf columns,
pkg. of 20.
240 mg - 1.92 g 48 69-2203-452 48 gram Alumina Basic RediSep Rf columns,
pkg. of 15.
400 mg - 3.2 g 80 69-2203-453 80 gram Alumina Basic RediSep Rf columns,
pkg. of 15.
0.8 - 6.4 g 160 69-2203-456 160 gram Alumina Basic RediSep Rf columns,
pkg. of 12.
pkg. of 10.
pkg. of 6.
pkg. of 4.

Appendix A
236 Teledyne ISCO

Appendix B
Solvent and UV-vis Wavelength

Selection Guide
Solvent Selection
Figure 210 lists typical chromatography solvents and their
properties by increasing polarity. Figure 211 may be used to select
miscible solvents.
Wavelength Selection
Figure 212 lists substances and the wavelength at which they
typically may be detected. Figure 212 lists wavelengths that are
available with Teledyne ISCO optical detection units and
substances that have good absorbance at these wavelengths.

Appendix B
Figure 210: Table of liquid chromatography solvents and their

characteristics (by increasing polarity)
Boiling Point (°C)

Viscosity (cp 20°)
Selectivity Group
UV Cutoff (nm)
(Fig. 125)
Polarity
SOLVENT
Pentane 0.00 0.23 36 210 —
Petroleum ether 0.01 0.30 30—60 210 —
Hexane 0.06 0.33 69 210 —
Cyclohexane 0.10 1.00 81 210 —
Isooctane 0.10 0.53 99 210 —
Trimethylpentane 0.10 0.47 99 215 —
Cyclopentane 0.20 0.47 49 210 —
n-Heptane 0.20 0.41 98 200 —
Trichloroethylene 1.00 0.57 87 273 —
Carbon tetrachloride 1.60 0.97 77 265 —
i-Propyl ether 2.40 0.37 68 220 1
Toluene 2.40 0.59 111 285 7
Chlorobenzene 2.70 0.80 132 — 7
o-Dichlorobenzene 2.70 1.33 180 295 —
Ethyl ether 2.90 0.23 35 220 1
Benzene 3.00 0.65 80 280 7
Isobutyl alcohol 3.00 4.70 108 220 2
238 Teledyne ISCO

Boiling Point (°C)

Viscosity (cp 20°)
Selectivity Group
UV Cutoff (nm)
(Fig. 125)
Polarity
SOLVENT
Methylene chloride 3.40 0.44 40 245 5
Ethylene dichloride 3.50 0.79 84 228 —
n-Butanol 3.90 2.95 117 210 2
n-Butyl acetate 4.00 — 126 254 —
n-Propanol 4.00 2.27 98 210 2
Tetrahydrofuran 4.20 0.55 66 220 3
Ethanol 4.30 1.20 79 210 2
Ethyl acetate 4.30 0.45 77 260 6
i-Propanol 4.30 2.37 82 210 2
Chloroform 4.40 0.57 61 245 8
Dioxane 4.80 1.54 102 220 6
Acetone 5.40 0.32 57 205, 6
225 – 300
Acetic acid 6.20 1.28 118 230 4
Acetonitrile 6.20 0.37 82 210 6
Dimethyl formamide 6.40 0.92 153 270 3
Methanol 6.60 0.60 65 210 2
Ethylene glycol 6.90 19.90 197 210 4
Dimethyl sulfoxide 7.20 2.24 189 268 —
Water 10.20 1.00 100 — 8

100
240
Ac
eti
100 A ce
cA
to cid
100 Ac
eto
ne
Figure 211:
0.18 Be
nz
nit
rile
en
0.43 n-B e
uta
7.81 Bu
tyl
no
l
Appendix B
0.08 Ca Ac
rb eta
0.815 Ch on te
lor Te
0.01 C o for
tra
c
ycl
m hlo
oh rid
0.81 1,2
-D ex
an e
1.6 D i c hlo e
ich
lor ro
100 Di
me om eth
e an
e
100 Di
me
thy
lfo
tha
ne
D t r m
100 iox hy
lS a mi
an ulf de
100 e
Chart of Solvent Miscibility
Eth ox
an
ol id e
8.7 Eth
yl A
6.89 Di
-et ce
tat
0.0003 He hy
l e
pta Ethe
H ne r
Miscible
0.001 ex
Immiscible
an
100 Me e
tha
M n
Values provided are solubility in water, %w/w
4.8 eth ol
Pe yl-
0.004 nta t-B
ne uty
100 n-P
ro lE
100 I so p an
the
r
-pr ol
1.71 Di op
-is an
Te o-P ol
100 tra ro
T hyd py
0.051 olu lE
en ro the
W e fur
an r
ate
r
Teledyne ISCO
Figure 212: Table of compound absorbance wavelengths

(by compound)
Compound Wavelength Compound Wavelength

(nm) (nm)
acridine 365 napthalenes 365
amino acids 214, 280a ninhydrin 435
aromatic amino acids 254 ninhydrin-amino acid reaction 580
product (DYDA)
amino acids, ninhydrin-primary 365 non-aromatic peptides 214
and secondary
antibiotics 310 nucleic acids 254
aromatics 254 nucleoproteins 254
benzophenones, substituted 310 nucleotides 214
carotenoids 435, 546 o-napthaquinone 365
chlorophylls and derivatives 546, 580 porphyrin derivatives 365
enzymes; NADH, NADPH 340 porphyrins 435, 546, 580
ferretin 310 proline 435
ferricyanide 365 proteins 254, 280
ferroproteins 365 pterins 254
flavoproteins 470 pyr-heme a2 hemachrome 620
hemachrome 620 rubredoxin, oxidized 326
heme proteins, oxidized 636 steroids 214, 365
hydroxyproline 435 tropolene derivatives 365
lactase 620 tropolene, natural 310
lipids 214 vitamins 254, 310
a. Amino acids which have greater absorbance at 280 nm are too dependent on pH for ad-
equate accuracy at 254 nm.

Appendix B
242 Teledyne ISCO

Appendix C
Theory & Application of Flash

Chromatography
Elementary theory
Given a compound X in a Flash column:
 X  s = concentration of X in stationary phase (g/mL or g/g)
 X  m = concentration of X in mobile phase (g/mL)
K =  X  s   X  m where K is the distribution coefficient between

the stationary and mobile phases
The larger value of K, the longer the retention time. Differences in

K values for different compounds lead to differential migration
through the column.
Compound X is characterized by two parameters:
t r = retention time (in units of time)
t w = baseline bandwidth (same units as t r 
t r = V r  F tw = Vw  F
where: V r = retention volume of band
V w = baseline bandwidth (in mL)
F = solvent flow rate in mL/sec
The relationship between tr (or Vr) and K can be obtained as

follows.
Vs  X s V
- = -------s- K
k = --------------------
Vm  X m Vm

Appendix C
where: k = capacity factor

V s = volume of stationary phase in column
V m = volume of mobile phase in column
The velocity of compound X in the column is given as vx

v x = v s   1 + k 
where: v s = velocity of solvent in column (cm/sec)
Since the distance traveled by X through the column is constant

and since the velocity of a non-retained band is equal to that of the
mobile phase,
L = tr vx = to vs
where:
L = column length
t o = retention time of non-retained band
Therefore:
 tr – to 
t r = t o  1 + k  and k = -----------------
-
to
and V r = V m  1 + k  = V m + V s K
The extent of broadening (tw or Vw) of the band of compound X is

defined in terms of the number of theoretical plate (N) of the
column, which is a measure of column performance.
2 2 2
N = 16  t r  t w  = 16  V r  V w  = 5.54  t r  t 0.5w 
where:
t0.5w = bandwidth at one-half the peak height.
244 Teledyne ISCO

Application
The basic function of LC is to separate a mixture of two or more
substances. Given two compounds, X and Y, in a column, their
relative separation or resolution is defined as:
t r y – t r x
R s = ----------------------------------------
-
1  2  t w y + t w x 
The resolution can also be related to the conditions of separation:

R s = 1  4  k   1 + k   N   – 1 
where:
k is the capacity factor of either compound
N is the number of theoretical plates

k
 is the separation factor, and is equal to ------y-
k x
The efficiency with which a column resolves or separates
compounds X and Y is a function of N. It is useful to determine this
efficiency as a proportionality constant (HETP) which is the height
equivalent to a theoretical plate.
HETP = L  N
The first attempt to separate a mixture by liquid chromatography
rarely gives the desired results. However, these results can give
some clues as to what changes must be made to achieve an
adequate separation. The Standard Resolution Curves (Figure 213)
give the Rs values for band ratios of 1:1, 4:1, and 16:1. These charts
give some idea how much Rs has been achieved and how much it
must be increased to give the desired resolution.
Generally a large increase in Rs or decrease in tr is most readily
accomplished by varying k. Small values of k give low Rs values
and tr values near to. Large k values give long tr and large tw (or
Vw) values. If exclusion chromatography is being employed, k
values are altered by changing the column packing. For other
methods, the solvent strength is varied. Stronger solvents give
smaller k values and weaker solvents give larger k values.
Following this discussion is a list of various solvents arranged in
their order of eluting strength. For separations involving several

Appendix C
components, k must be varied during separation. In this case

gradient elution techniques are employed.
Where a change in k does not result in the desired change in Rs,
resolution can be improved by increasing N. this involves changing
the column length, solvent velocity, and/or temperature of the
column.
The easiest method of increasing N is to reduce the mobile phase
flow rate. This however, increases tr.
Once it is understood how various parameters affect each other,
experimental can be predicted for a desired change in Rs or tr.
Following this discussion is a table showing how the resolution can
be changed by varying time, pressure, and/or column length. This
table was derived from:
n
H = D
where D is a column efficiency parameter, and n=0.4. Lower and
higher values are sometimes encountered and tables for n=0.3 and
n=0.5 are available.
In separations where Rs for bands of components X and Y is <0.5
and k >2, an increase in N may not be practical. Resolution in this
case can be improved by using gradient elution.
The foregoing discussion has been oversimplified as it is only our
purpose to provide a quick reference to the basic theory of liquid
chromatography and some data for its application. More detailed
information may be obtained from the following references:
Snyder, L.R.; Kirkland, J.J.; Introduction to Modern Liquid
Chromatography, Wiley-Interscience, N.Y. (1979)
Runser, D.J., Maintaining and Troubleshooting HPLC Systems,
Wiley-Interscience, N.Y. (1981)
Krstulovic, A.M.; Brown, P.R.; Reversed-phase High Performance
Liquid Chromatography, Wiley-Interscience, N.Y. (1982)
246 Teledyne ISCO

1:1 ratio of two bands
0.4 0.5 0.6 0.7
84
88
92
0.8 1.0 1.25
98 99.4
95

0.4 0.5 0.6 0.7
90 93
0.8 1.0 1.25
95 98 99.5

0.4 0.5 0.6 0.7
96
0.8 1.0 1.25
97 99 99.6
Figure 213: Standard resolution charts

Numbers above the bands refer to Rs. The arrows indicate the cut point which will yield
bands of equal purity while the number above or below refers to the purity of each of the
two resulting bands.
Charts adapted by permission of L.R. Snyder and Preston Technical Abstracts Co., from
Journal of Chromatographic Science 10, 364 (1972)

Appendix C
248 Teledyne ISCO

Troubleshooting LC Systems
Appendix D
Troubleshooting
LC Systems
Basic checklist
The list below summarizes common problems that can be quickly
checked and remedied.
• Instrument(s) not plugged in
• Instrument(s) not turned on
• Fuse(s) blown
• No mobile phase
• Air lock in pump lines
• Leaks
• No sample being introduced
• Temperature gradients across system
• Contaminated or plugged column
• Wrong column type or size
• Flow through column reversed
• Wrong detector setting
• Dirty detector cell
• Indicator light or gauge malfunction
• Sample chemistry misinterpreted
Troubleshooting
The following figures list common LC problems and solutions.
• Figure 214: Table for troubleshooting peak problems
• Figure 215: Table for troubleshooting baseline problems
• Figure 216: Table for troubleshooting recovery and retention
problems
• Figure 217: Table for troubleshooting pressure problems

Appendix D
• Figure 218: Table for troubleshooting leaks
Figure 214: Table for troubleshooting peak problems
Symptom Cause Remedy

Broad peaks Retention time too long Use stronger mobile phase, increase flow rate,
select different column type.
Too much sample injected Decrease injection volume.
Compounds eluted too early Use larger-volume column, smaller injection
due to sample overloading. volumes, or dilute the sample.
Late elution from previous Extend run times, finish runs with stronger
injection percentage of mobile phase B, use longer col-
umn equilibration times before run.
Peak tailing High pH Change to reversed-phase column, change to
column with a better pH stability.
High temperature Maintain temperatures below 40° C.
Interactions with silica station- Use reversed-phase column, decrease mobile
ary phase phase pH, strengthen mobile phase, add trieth-
ylamine to mobile phase, or buffer sample.
Interfering peak Improve sample purification before injection,
alter mobile phase, select an affinity column.
Peak fronting Poor column packing Replace column.
Column overloading Use larger-capacity column or smaller sample
volumes.
250 Teledyne ISCO


Peak doubling Interfering peak Improve sample purification before injection,
alter mobile phase, select an affinity column.
interfering compound from pre- Extend run times, finish runs with stronger
vious run percentage of modifier solvent, use longer col-
Column overloading Use larger-capacity column or smaller sample
volumes.
Poor column packing Replace column and decrease mobile phase
solvent strength.
Injection solvent too strong Prepare sample with weaker solvent, then
adjust mobile phase strength.
Sample and mobile phase Use miscible solvents.
incompatible
Inverted peaks Mobile phase absorbance Select mobile phase with lower absorbance,
greater than that of elute. verify purity of mobile phase.
Air injected with sample Ensure solid sample loop or liquid injection is
free of air.
Ghost peaks Impure mobile phase Verify purity of mobile phase or use HPLC
grade solvents.
Sample preparation Improve sample purification before injection.
Spikes Bubbles in mobile phase Degas solvents, check for bad fluid line fit-
tings.
Faulty UV detector Test detector operation, replace lamp.

Appendix D
Figure 215: Table for troubleshooting baseline problems

Drifting baseline Column contaminated Purge column with strong solvent, replace col-
umn.
Temperature effects Control environment, allow system to warm
up and stabilize.
Leak in system Locate and repair.
Rising baseline Absorbance of mobile phase B Change to low- or non-absorbing mobile
phase, add absorbing compound to mobile
phase A, select a different wavelength.
Sample preparation Improve sample purification before injection.
Falling baseline Absorbance of mobile phase A Change to low- or non-absorbing mobile

phase, add absorbing compound to mobile
phase B, select a different wavelength.
Noisy baseline Residual compounds in system Purge system with strong solvent, replace col-
umn.
Mobile phase contamination Use HPLC grade solvents.
252 Teledyne ISCO

Figure 216: Table for troubleshooting recovery and retention problems

Poor recovery of sample Low yield (less than 90%) Acidify the mobile phase for acidic com-
pounds, add competing base for basic com-
pounds, change to ion-exchange column.
Interaction with stationary Change to ion exchange or affinity column,
phase eliminate possible reactive groups.
Adsorption on column media Increase mobile solvent strength.
Varying retention times Equilibration volume too small Increase column equilibration before run.
Column contaminated Purge column with strong solvent, replace col-
umn.
Interaction with active sites in Manage interaction by modifying the mobile
stationary phase phase, inject large volume of sample onto col-
umn to condition it before use.
Figure 217: Table for troubleshooting pressure problems

Increasing or high pressure Column blocked, sample Improve sample purification before injection,
purge column with strong solvent, replace col-
umn.
Column blocked, microbial Add organic solvent or growth inhibitor to
growth mobile phase. If column is installed but not in
use, purge regularly. Store column filled with
sufficient organic solvent and sealed end-caps.
Tubing blocked Refer to flow diagram and disconnect compo-
nents to locate obstruction.
Precipitation Use miscible solvents and buffers, use
less-ionic mobile phase, avoid rapid changes
to characteristics of mobile phase.
Low pressure Leaks Inspect and correct system tubing and fittings.
Low flow rate Check pump and tubing from pump.
Pump inlet Check solvent containers and pump inlet tub-
ing, degas solvent.
Figure 218: Table for troubleshooting leaks

Appendix D
Leak Location Cause Remedy

Injection Loose fitting Re-seat sample cartridge or injection needle in
fitting, inspect fitting for damage or wear,
replace fitting.
Injection valve damaged Replace injection valve, prepare sample and
solvents to avoid precipitation or formation of
salts.
Column Loose fitting Re-seat column in fitting, inspect fitting for
damage or wear, replace fitting.
System tubing and compo- Loose fitting or seal Tighten or replace.
nents Damaged tubing Replace tubing
Leak from system component Service or replace the component.
such as damper, detector, or
pump.
254 Teledyne ISCO

CombiFlash Systems
Appendix E
CombiFlash Systems
CombiFlash NextGen
The CombiFlash NextGen system is offered in three different

models: NextGen, NextGen 300, and NextGen300+. The NextGen
offers flow rates up to 300 mL/min at up to 300 psi (20 bar)
depending on the model. UV, UV-Vis ELSD, or MS options are
available and this system is customizable to meet application
needs. The following are additional features of the NextGen
system:
• Complete control on one touch screen
• Configurable to meet specific application parameters
• New default methods speed up the flow without sacrificing
performance
• Greener option as it conserves solvent with optimized
gradient
• Designed to take up minimal lab space without sacrificing
performance

Appendix E
CombiFlash Rf+ Lumen
The CombiFlash Rf+ Lumen automated Flash system with

integrated Evaporative Light Scattering Detector (ELSD)
compliments UV or UV-vis detection to deliver the widest
compound detection range. This system detects compounds with
minimal or no UV absorption such as carbohydrates, steroids, and
lipids which require more than just UV absorbance detection. The
following are additional features of the Rf+ Lumen system:
• No increase in footprint with addition of ELSD
• Light-sensitive compounds detected when UV
detection is off
256 Teledyne ISCO

CombiFlash Systems
CombiFlash Rf+ PurIon
The CombiFlash Rf+ PurIon automated Flash system with

integrated mass spectrometer offers mass directed fractionation
by collecting only the peaks of interest. Detecting a wide range of
compounds with minimal or no UV absorption using up to 2000
Dalton mass range, the PurIon can easily change electrospray
ionization (ESI) and atmospheric-pressure chemical ionization
(APCI) interfaces. The following are additional features of the Rf+
PurIon system:
• Integrated mass spectrometer control to minimize
learning curve
• Ion finder to verify ionization and identify possible adducts
• “Terminate on Target” stops separation once peaks of
interest are collected
• Mass spectral data display locates compound in real-time
and post-run
• Positive/negative switching

Appendix E
CombiFlash Rf+ Torrent
The CombiFlash Torrent automated Flash system can adapt to a

variety of large scale purification needs. The following are
additional features of the Torrent system:
• Up to 1000 ml/min gradient delivery
• Hundreds of grams purified in a few hours
• Large fraction collection up to 72 bottles or 6-port fraction
valve with unlimited volume
• Active solvent and waste level sensing to avoid spills
• Multiple fraction collector options
258 Teledyne ISCO

CombiFlash Systems
CombiFlash EZ Prep
The CombiFlash EZ Prep dual function purification system

combines Flash and Preparative (Prep) purifications on the same
instrument. Up to 200 mL/min binary gradient allows diverse types
of Flash and Prep HPLC columns. The following are additional
features of the EZ Prep system:
• Minimal bench space required
• RFID tags on rack to ensure proper tube fill, enable rack
exchange during separation and viewing last run on
detected rack
• PeakTrak software automatically loads proper method and
solvents with RFID Flash columns
• “Press and Play” functionality using PeakTrak software
• Color-coded screen displays whether system is in Flash or
Prep HPLC mode
• Active solvent and waste level sensing to avoid spills
• Up to 3500 PSI when running Prep HPLC columns
• Up to 200 PSI for Flash applications
• Automatic phase change when switching from
reversed-phase Prep HPLC to normal phase Flash

Appendix E
260 Teledyne ISCO

Effective Organic Compound Purification

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Effective Organic Compound Purification

Effective Organic Compound Purification:

CombiFlash®, Companion®, CombiFlash Torrent™, RediSep®, RediSep Rf

RediSep Rf® and RediSep Rf Gold® i

Need more copies?

We welcome your input!

RediSep Rf® and RediSep Rf Gold® iii

RediSep Rf® and RediSep Rf Gold® v

Silica Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

RediSep Rf® and RediSep Rf Gold® vii

viii Teledyne ISCO

37 Photo of pre-packed columns . . . . . . . . . . . . . . . . . . . . . . . . 41

RediSep Rf® and RediSep Rf Gold® ix

74 Table of Reusable RediSep Rf Gold Amine Columns . . . . . . 80

112 Chromatogram showing detection of catechin. . . . . . . . . 115

RediSep Rf® and RediSep Rf Gold® xi

xii Teledyne ISCO

Chromatographic Purification in Organic Chemistry

RediSep Rf® and RediSep Rf Gold® 1

Figure 1: Illustration of basic elements in a traditional Flash column

2 Teledyne Isco, Inc.

The term Flash chromatography was coined in 1978 by W. Clark Still

RediSep Rf® and RediSep Rf Gold® 3

4 Teledyne Isco, Inc.

Flash chromatography is an easy and simple purification

RediSep Rf® and RediSep Rf Gold® 5

Figure 2: Photo of TLC plate Annotations include baseline, sample starting

Figure 3: Table of common solvents and their characteristics in liquid

RediSep Rf® and RediSep Rf Gold® 7

with hexane/ethyl acetate 1:1 and/or dichloromethane/methanol

Mobile Phase Modifiers

Figure 4: Illustration of mobile phase modifier reducing peak tailing on TLC

Using TLC to Predict Separation

Correlating TLC and Flash

Retention Factor and Column Volumes

RediSep Rf® and RediSep Rf Gold® 9

amount of solvent the column can hold in the interstitial space

Figure 5 illustrates this relationship between Rf and CV. A

Figure 5: Table of Rf to CV conversions

Method Development Using TLC

EtOAc Hexane/EtOAc Hexane/EtOAc

Figure 6: Illustration of solvent strength optimization

RediSep Rf® and RediSep Rf Gold® 11

Hexane/EtOAc CH2Cl2/MeOH CH2Cl2/MeOH

Figure 7: Illustration of solvent system selectivity optimization

Figure 8: Illustration of a solvent system optimized for compound

The selectivity obtained will determine the sample loading

Figure 9: Table of suggested loading RediSep Rf silica gel columns based on

Light Moderate Significant Heavy

RediSep Rf® and RediSep Rf Gold® 13

Typically, a crude reaction mixture amount corresponding to

TLC and Mobile Phase Techniques

Linear Linear with

Figure 10: Illustration of mobile phase techniques plotted as solvent strength

The separation conditions found while scouting with TLC easily

When using linear gradient Flash chromatography to purify organic

RediSep Rf® and RediSep Rf Gold® 15

Figures 11 through 15 illustrate tests performed to optimize an