An Efficient Approach to Column Selection in

HPLC Method Development

Let’s join to Pharmatalks

Introduction

 Common Mistakes in Method Development:

 • Inadequate Formulation of Method Goals
 • Little Knowledge of Chemistry of Analyte Mixture
 • Use of the First Reversed Phase C18 Column Available
 • Trial and Error with Different Columns and Mobile
Phases

 These Mistakes Result In:

 • Laborious, Time-consuming Development Projects
 • Methods that Fail to Meet the Needs of the Analyst
 HPLC Method Development - A Proposed
Procedure
 At Your Desk
 • Define your knowledge of the sample
 • Define your goals for the separation method
 • Choose the columns to be considered

 In the Laboratory
 • Choose the initial mobile phase chemistry
 • Choose the detector type and starting parameters
 • Evaluate the potential columns for the sample
 • Optimize the separation conditions (isocratic or gradient) for the
chosen column
 • Validate the method for release to routine laboratories
Choosing the Appropriate HPLC
Column Should Be Based Both Upon
Knowledge of the Sample and Goals
for the Separation

 Benefits of this Approach Include:

 • Small initial time investment
 • Big time savings in the HPLC laboratory
 • More “informed” approach to column selection
 • More efficient than “trial and error” approach
Knowledge of the Sample Influences the
Choice of Column Bonded Phase
Characteristics
 Knowledge of the Sample

 • Structure of sample components? Column Chemistry

(bonded phase, bonding type,
 • Number of compounds present? endcapping, carbon load)

 • Sample matrix?
 • pKa values of sample components?
 • Concentration range?
 • Molecular weight range?
 • Solubility?
 • Other pertinent data?
Goals for the Separation Influence the
Choice of Column Particle Physical
Characteristics

 Goals for the Separation

 • Max. resolution of all components?
 • Partial resolution?
Column Physics
 • Fast analysis? (particle bed dimensions,
particle shape, particle
 • Economy (low solvent usage)? size, surface area, pore
 • Column stability and lifetime? size)

 • Preparative method?
 • High sensitivity?
 • Other goals?
Column Selection Chart

Fast Eqilibration
Suitable for MW
High Resolution
Default Column

High Sensitivity
(Good for most

High Efficiency

Stability at pH
High Capacity

Backpressure

High Stability

Consumption
Applications)

Fast Analysis
High Sample
Method

Low Mobile
Loadability
Goals

Extremes
>2000

Phase
Low
Particle Size
small (3µm) • •
medium (5µm) •
large (10µm) •

Column Length
short (30mm) • • • • •
medium (150mm) •
long (300mm) •

Column ID
narrow (2.1mm) • •
medium (4.6mm) •
wide (22.5mm) •

Surface Area
low (200m2/g) • • •
high (300m2/g) • • •

Pore Size
small (60Å) • •
medium (100Å) •
large (300Å) •

Carbon Load
low (3%) •
medium (10%) •
high (20%) • • •

Bonding Type
monomeric • •
polymeric • • • •

Particle Shape
spherical • • • •
irregular •
 Choosing the Bonded Phase

 Draw the molecular structures for all known components of the

mixture. Identify the two compounds whose structures are the most
similar.
HO HO

 e.g.: O O
OH O OH
HO

O O

Prednisolone Prednisone
 Choosing the Bonded Phase
 For these two molecules, circle the structural features that differ. It is
these differences that should be exploited to optimize the separation.

 e.g.: HO HO

O O
OH O OH
HO

O O

Prednisolone Prednisone
 Choosing the Bonded Phase
 Use the results of the structural comparison to select a bonded
phase showing optimal selectivity for these two molecules. In
this case consider using a silica column (no bonded phase) for its
ability to retain polar solutes through hydrogen bonding.
HO HO

O O
OH O OH
HO

O O
Prednisolone Prednisone
Functional Group Polarity Comparisons
Polarity Functional Group Structure Bonding Types Intermolecular Forces Displayed
Low Methylene R (CH2)2 s London

Phenyl s,p London

R

Halide s London, Dipole-Dipole

R F, Cl, Br, I

Ether R O s London, Dipole-Dipole, H-bonding

R
-
O
Nitro + s,p London, Dipole-Dipole, H-bonding
R N O
O
Ester R s,p London, Dipole-Dipole, H-bonding
O R
O
Aldehyde s,p London, Dipole-Dipole, H-bonding
R H
Ketone
O s,p London, Dipole-Dipole, H-bonding
R
R
Amino s,p London, Dipole-Dipole, H-bonding, Acid-base chemistry
R NH2

Hydroxyl s London, Dipole-Dipole, H-bonding

R OH

High Carboxylic Acid O s,p London, Dipole-Dipole, H-bonding, Acid-base chemistry

R
OH
Choosing the Bonded Phase
Examples of bonded phases used for HPLC packing media:

 C18 or Octadecylsilane (ODS)

 Very nonpolar - Retention is based on London (dispersion) interactions with hydrophobic compounds.
 Example Alltech Phase: Alltima ™ C18

R
Si (CH2)17CH3
R
Choosing the Bonded Phase
 Phenyl
 Nonpolar - Retention is a mixed mechanism of hydrophobic and p - p
interactions.
 Example Alltech Phase: Platinum ™ Phenyl

H H
R C C
Si (CH2)3 C C H
R C C
H H
Choosing the Bonded Phase
 Cyanopropyl
 Intermediate polarity - Retention is a mixed mechanism of
hydrophobic, dipole-dipole, and p - p interactions.
 Example Alltech Phase: Alltima™ CN

R
Si (CH2)3 C N
R
Choosing the Bonded Phase
 Each bonded phase has unique selectivity for certain sample types.

 As a practical example, to separate toluene and ethyl benzene:

 • Note a difference of one -CH2- unit
 • Choose a C18 bonded phase for retention by hydrophobicity
 • Maximize hydrophobic selectivity with a high silica surface area,
high carbon load material like Alltima C18

Toluene Ethyl Benzene

Choosing the Particle Physical
Characteristics
 Use the Column Selection Chart
 • Use “default” column as starting point
 • Match up method goals with individual particle physical
characteristics
 • Change only those particle parameters that affect the method goals
 • Recognize the “optimum” column as a possible compromise


 Example:
 Sample Type: hydrophobic compounds
 Method Goal: highest resolution

Choosing the Particle Physical
Characteristics
Example:
Sample Type: hydrophobic compounds
Method Goal: highest resolution
Column Selection Chart
Default Column Optimum Column†
Column Bed Dimensions 150 x 4.6mm 250 x 4.6mm
Particle Size 5µm 3* or 5µm
Surface Area 200m2/g >200m2/g
Pore Size 100Å 100Å
Carbon Load 10% 16 - 20%
Bonding Type Monomeric Mono- or Polymeric
Base Material Silica Silica
Particle Shape Spherical Spherical
* mobile phase backpressure may be excessive

† Optimum Column: Alltima C18 ™, 5µm, 250 x 4.6mm (Part No. 88056)
*Note that the choice may represent a compromise. Here, the “optimum” column for resolution sacrifices speed.
Choosing the Particle Physical
Characteristics

 Column Dimensions
 • Length and internal diameter of packing bed

 Particle Shape
 • Spherical or irregular

 Particle Size
 • The average particle diameter, typically 3-20µm

 Surface Area
 • Sum of particle outer surface and interior pore surface, in
m2/gram
Choosing the Particle Physical Characteristics
 Pore Size
 • Average size of pores or cavities in particles, ranging from
60-10,000Å

 Bonding Type
 • Monomeric - single-point attachment of bonded phase molecule
 • Polymeric - multi-point attachment of bonded phase molecule

 Carbon Load
 • Amount of bonded phase attached to base material, expressed as
%C

 Endcapping
 • “Capping” of exposed silanols with short hydrocarbon chains after
the primary bonding step
Column Dimensions
 Effect on chromatography

 Column Dimension
 • Short (30-50mm) - short run times, low backpressure
 • Long (250-300mm) - higher resolution, long run times
 • Narrow ( 2.1mm) - higher detector sensitivity
 • Wide (10-22mm) - high sample loading
Particle Shape
 Effect on chromatography
 Spherical particles offer reduced back pressures and longer
column life when using viscous mobile phases like 50:50
MeOH:H2O.
Particle Size
 Effect on chromatography
 Smaller particles offer higher efficiency, but also cause higher
backpressure. Choose 3µm particles for resolving complex, multi-
component samples. Otherwise, choose 5 or 10µm packings.
Surface Area
 Effect on chromatography
 High surface area generally provides greater retention, capacity and
resolution for separating complex, multi-component samples. Low
surface area packings generally equilibrate quickly, especially
important in gradient analyses.
 High surface area silicas are used in Alltech’s Alltima ™,
Adsorbospherel® HS, and Adsorbosphere ® UHS packings. Low surface
area silicas are used in Alltech’s Platinum ™, Econosphere™, and Brava™
packings.
Pore Size
 Effect on chromatography
 Larger pores allow larger solute molecules to be retained longer through
maximum exposure to the surface area of the particles. Choose a pore size
of 150Å or less for sample MW  2000. Choose a pore size of 300Å or
greater for sample MW > 2000.
Bonding Type
 Effect on chromatography
 Monomeric bonding offers increased mass transfer rates, higher column
efficiency, and faster column equilibration.

CH3 R
monomeric
OH + X Si (CH2)17CH3 Si (CH2)17CH3
bonding
CH3 R

OH CH3 O CH3
polymeric
+ X Si (CH2)17CH3 Si
bonding
OH X O (CH2)17CH3

Polymeric bonding offers increased column stability, particularly

when highly aqueous mobile phases are used. Polymeric bonding
also enables the column to accept higher sample loading.
Carbon Load
 Effect on chromatography
 Higher carbon loads generally offer greater resolution and longer run
times. Low carbon loads shorten run times and many show a different
selectivity, as in Alltech’s Platinum line of packings.
Endcapping
 Effect on chromatography
 Endcapping reduces peak-tailing of polar solutes that interact
excessively with the otherwise exposed, mostly acidic silanols. Non-
endcapped packings provide a different selectivity than do endcapped
packings, especially for such polar samples.
 Alltech’s Platinum™ EPS packings are non-endcapped to offer enhanced
polar selectivity.
Conclusion
 In this approach to HPLC column selection, the bonded phase
chemistry of the column is chosen on the basis of an analysis of the
sample component structures. The physics of the column is chosen
according to an analysis of the goals for the separation method. This
approach succeeds in predicting unique, optimum bonded phase
chemistries and particle bed physical characteristics that are likely to
meet the goals for the separation method.
Column Selection Example #1
What goals do I have for the method?
Maximum resolution of all components?
Best Peak Shape for difficult samples? ✓
Fast analysis? ✓
Economy (low solvent consumption)? ✓
Column stability-long lifetime?
Purify one or more unknown components for characterization?
High sample loadability?
High sensitivity? …Other (High Sample Throughput-
-Quick Equilibration) ✓

What do I know about the sample?

Number of compounds present 4
Sample matrix --
pKa values of compounds? --
UV spectral information about compounds? UV -254
Concentration range of compounds --
Molecular weight range of compounds 94 - 323
Column Selection Example #1

Structures of Compounds

OH
N

(CH2)3CH3

Phenol 3-Butylpyridine

(CH2)5CH3

Anthracene 3-Hexylanthracene
Column Selection Example #1

Which two sample components have the most similar structures?

Draw them, then circle the structural differences between them.
Note: The structural difference
between these two compounds is the
hydrophobic hexyl side chain. This
Anthracene
suggests a non-polar C18 or C8
column would interact with this area of
difference to help provide separation
(CH2)5CH3
of these two compounds.
3-Hexylanthracene

Recommended bonded phase (silica based materials only) – mark one

Normal phase silica NH2 CN
Reversed phase C18C8 Ph CN
Column Selection Example #1

Column physical characteristics – use Column Selection Chart and

Method Goals

Default Column Ideal Column Column bed

dimensions (mm)150 x 4.6 100 x 2.1
Particle Size (µm) 5 5
Surface area (m2/g) 200 <200
Pore Size (Å) 100 100
Carbon Load (%) 10 10
Bonding type Monomeric Monomeric
Particle shape spherical spherical
 Column Selection Example #1

Available packing alternatives meeting the above criteria:

Packing Base ParticleParticle Carbon Pore Surface Bonding End-

Material Shape Size Load Size Area Type cap’d
(µm) (%) (Å) (m /g)
2

Allsphere ODS-2 silica Sph. 3, 5, 10 12 80 220 Mono. Yes

silica Sph. 3, 5 8.5 145 185 Mono. Yes
Brava BDS C18 silica Sph. 3, 5, 10 10 80 200 Mono. Yes
silica Sph. 3, 5, 10 6 100 200 Mono. Yes
Econosphere C18

Platinum C18

Column of choice: Brava BDS C18, 100x2.1,

Increased 5µm (Spherical , 185m2/g,
Sensitivity,
monomeric)Best Peak Shape Low Solvent Consumption,
Fast Analysis
Quick Equilibration

Reduced
Good balance of efficiency backpressure
& backpressure
Column Selection Example #2
What goals do I have for the method?
Maximum resolution of all components? ✓
Partial resolution, resolving only select components?
Fast analysis?
Economy (low solvent consumption)?
Column stability-long lifetime? ✓
Purify one or more unknown components for characterization?
High sample loadability?
High sensitivity? ✓
…Other

What do I know about the sample?

Number of compounds present 6+
Sample matrix --
pKa values of compounds? --
UV spectral information about compounds? UV -254
Concentration range of compounds --
Molecular weight range of compounds 349 - 645
Column Selection Example #2

Structures of Compounds

N N
N O N
O N O
N
O O O H H
S
H H H H H H NH
H2N S NH S S
NH S NH O N O O
N
O
N N N
H2N O O NH2
O HO O
O S N
HO O HO O HO O N
O S

N
O N O S O
NH H H H
H N N S O
O NH
O NH N S
O N N S N O
O HO O
HO O
HO
Column Selection Example #2

Which two sample components have the most similar structures?

Draw them, then circle the structural differences between them.

Notes: both structures very

polar, with amine and pi bond functions--a RP
CN columnO
may O
H H
give good separation by mixed-
H2N H H mode retention
NH
S
of hydrophobic,
O
CN---
S
H---NR2 hydrogen bonding
NH S
O
N O and p-p interactions with
N
double O bonds.
HO O
HO O

Normal phase silica NH2 CN

Recommended bonded phase (silica based materials only) – mark one
Reversed phase C18C8 Ph CN
Column Selection Example #2

Column physical characteristics – use Column Selection Chart and

Method Goals

Default Column Ideal Column Column bed

dimensions (mm)150 x 4.6 250 x 2.1
Particle Size (µm) 5 5
Surface area (m2/g) 200 200 +
Pore Size (Å) 100 Not critical
Carbon Load (%) 10 --
Bonding type Monomeric Polymeric
Particle shape spherical spherical
Column Selection Example #2

Available packing alternatives meeting the above criteria:

Packing Base Particle Particle Carbon Pore Surface Bonding End-

Material Shape Size Load Size Area Type cap’d
(µm) (%) (Å) (m /g)
2

Adsorbosil CN silica Irreg. 5, 10 -- 60 450 Poly. Yes

Alltima CN silica Sph. 3, 5 -- 100 350 Poly. Yes
Allsphere CN silica Sph. 3, 5, 10 -- 80 220 Mono. No
Platinum CN silica Sph. 3, 5, 10 -- 100 200 Mono. No

Column of choice: Alltima CN, 250 x 2.1 , 5µm ( Spherical , 350 m2/g ,
polymeric) High resolution,
High res.
High sensitivity

Good balance of efficiency Reduced

& backpressure Robust
backpressure