A Look at Column Choices

Does one-size-fit-all?
 Seminar Outline

•Silica Surface Chemistry & Physical Characteristics

•Silica Bonding Procedures
–Special Column Chemistries
•Column Choices

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 Silica Column Characteristics

Suface Chemistry Surface Area

Silanols Bonding Pore Particle

Size Distribution Size Distribution

NOTE: SILICA IS STABLE AT 1.0 > pH < 11.0*

* range depends on manufacturer’s bonding

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 ZORBAX Porous Silica
Particles
Particle Size

Silica Sol 1.8 um

+
Urea (pH-2)
 - or -
3.5 µm
+ - or -
O2 5 µm
CH2O
- or -
7 µm

ZORBAX Rx
Pore
80Å, 95Å, or 300Å

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5
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 History of HPLC Particle Development
Year(s) of Particle Size Most Popular Plates / 15cm
Acceptance Nominal Size
1950’s 100µm 200

1967 57µm (pellicular) 1,000

1972 10µm 6,000

1985 5µm 12,000

1992 3.5µm 22,000

2003 ☻ <2µm* >30,000

*Zorbax "Rapid Resolution HT”, product launch 5/1/03

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Columns Packed with Smaller Particles
Provide Higher Efficiency

Sub 2 micron

3 micron

5 micron

10 micron
FLOW

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 Pore Size Recommendations

Use 60 - 80Å pore size column packings to separate small

molecules equal to or less than 4000MW to maximize
loading capacity and retention.
Use 95 or 300Å pore size columns for larger molecules
like polypeptides ad proteins (from 4000 to 500,000 MW)
to maintain high efficiency.
Increase column diameter to increase loading capacity.

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 Chemistry Variations
Are All C-18s the Same?

1. Type of bonding
2. Completeness of bonding (Carbon Load)
3. Silica Chemistry
4. Bonding Chemistries
• End-capping

NO!
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Monomeric vs. Polymeric Bonding
Highly reproducible
Monomeric bonding
Typical ZORBAX Bonding
•Eclipse Plus
•Eclipse XDB
•StableBond
•Bonus-RP
•Agilent HC/TC

Polymeric Bonding
Eclipse PAH

Trifunctional silane

figure courtesy of Vydac

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2. Carbon Load

Carbon Load refers to the % carbon content of

the silica bonded stationary phase.

Generally speaking, a high carbon load

(example 18-25%) results in a more
hydrophobic surface. The surface is also more
resistant to high pH.

A high carbon load does not necessarily

provide the best resolution.

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3. The Surface of Silica Supports

OH HO OH OH OH

Si Si Si Si

Free Geminol Associated

Silanols Silanols Silanols

decreasing acidity

OH
+ +
M M Si
Surface Metal Internal Metal
(activated silanol)
(most acidic)

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 Chromatographic Improvement
CH2 -OH.
Using Highly Purified
Zorbax Rx-Sil

Original ZORBAX, 1973 and other type A silicas

(basic compound can tail)

Conditions: Flow Rate: 2.0 mL / min.

Mobile Phase: 5% 2-Propanol in Heptane
CH2 -OH

ZORBAX Rx-Sil, 1987 and other Type B silicas

(basic compounds have less tailing; lower
effective silanol pKa)

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9
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Potential Ion Exchange and Hydrogen Bonding Secondary Interactions

Ion-exchange
_ _
SiO Na+ + R3 NH+ SiO N+R3 + Na+
1. Ionized silanols (SiO-) will ion-exchange with protonated bases
(R3NH+) which can cause tailing and method variability. This occurs
most often at mid pH where silanols are ionized.

Hydrogen Bonding
_ _ + _
-SiOH + RCOO -SiO . . . H . . . OOCR
2. Unprotonated acids can compete for H+ with protonated silanols.
This can occur at low pH.

Some mobile phase additives can be added to the mobile phase to reduce these
interactions and this will be discussed in the mobile phase section.
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 Zorbax StableBond with Rx-SIL
Improves Peak Shape
Mobile Phase: 75% 50 mM KH2PO4, pH 4.4 : 25% ACN Flow Rate: 1.5 mL/min

Silica Type – More Acidic Silica Type – High Purity, Rx-Sil

Column: ODS, 4.6 x 250 mm, 5 m Column: SB-C18, 4.6 x 150 mm, 5 m
Plates: 92 Plates: 6371
USP Tf (5%): 2.90 USP Tf (5%): 1.09

Propranolol OH
pKa 9.5 OCH CHCH NHCH(CH )
2 2 3 2

0 5 10 15 0 5 10
Time (min) Time (min)

• The high purity Rx-SIL improves the peak shape dramatically on a C18 column
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So What Bonded-Phase Do I Choose?*

C18 offers the most retention, maybe too much for some
samples
C8 less retentive than C18, but with similar selectivity in most
cases
Phenyl significantly less retentive for non-polar compounds but
retains polar compounds, so reduces analysis time for
mixture if polar/non polar
CN least retentive, changes in selectivity, good for reducing
analysis time with late eluters and avoiding gradient
separations

* Greater than 20% Organic

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001761S1.PPT
So What Bonded-Phases Do I Choose?
High Aqueous* Mobile Phases

C18 rarely offers retention advantages, and selectivity may

not be optimum with little retention
C8 often provides a little more retention than C18 with similar
selectivity
Phenyl most retentive with changes in selectivity
C3 similar to Phenyl in retention but some changes in
selectivity from C18, C8 and Phenyl
CN least retentive, definite changes in selectivity, good
resolution

* Below 20% Organic

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001762S1.PPT
4. ZORBAX Bonding Technology

All Purpose Rx-Silica Support Application

Phases Targeted Phases

• StableBond • Bonus-RP
• Eclipse XDB • Extend
•Eclipse Plus •Ultra-pure
•Spherical, consistent
pore size
•Fully-hydroxylated
•Sol gel maximizes
strength and lifetime
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 Match Method pH and Column Choice
Choose the Best Bonded-Phase for Each pH Range
StableBond, pH 1-6 Eclipse Plus & XDB, pH 2-9 Bonus-RP, pH 2-8 Extend-C18, pH 2-11.5
1. Uses bulky silanes 1. Proprietary double-endcapping 1. polar alkyl phase 1. unique bidentate structure
2. Non-endcapped 2. triple endcapped 2. double endcapped
3. uses bulky silanes

* R
CH3

O Si
*R1 O Si
R1
C18 C18
O Si PG R
CH3 Si Si
R CH3 R1 O O
OH O Si CH3 CH3
R CH3 Silica Support
Si CH3
CH3
CH3
O Si
O Si R1 R1
CH3
R CH3 O Si PG R
O Si CH3
OH R1
CH3
R CH3 CH3
O Si Si CH3
O Si R1
CH3 CH3

R R1

O Si PG R

R1

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001241P2.PPT
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ZORBAX StableBond Bonding

Superior low pH stability – down to pH 1 R

O Si
Non-endcapped for selectivity and R
lifetime OH
R

Patented sterically protecting bonding O Si

R
OH
5 different selectivities - C18, C8, CN, R
Phenyl, C3 O Si

R
Ideal for most sample types at low pH

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StableBond Reaction to Make a
Sterically-Protected Surface

H3C H CH 3 H 3C H CH 3
C C
Si
OH + X
Si
R
Si
O
Si
R
C C
H 3C H CH 3 H 3C H CH 3
X = Cl, OEt , etc .
R = CN, C8, etc .

• Diisopropyl silanes or diisobutyl silanes (C18)

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Bonded Phase Selectivity Differences in 30% ACN

mAU
RRHT SB-CN  5 Different bonded phases
4.6 x 50 mm, 1.8 m
100

0
compared
1  Analysis time of each run is
RRHT SB-Phenyl
mAU
only 2 minutes
100 4.6 x 50 mm, 1.8 m  Comparison done in optimum
0
1 2
% organic
RRHT SB-AQ
mAU
 The fast runs mean a
100 4.6 x 50 mm, 1.8 m comparison can be done even if
0 you have a good separation on
1 2
mAU the C18
RRHT Eclipse Plus C18  More chances to optimize!
4.6 x 50 mm, 1.8 m
100

0
1 2
mAU

RRHT SB-C18
4.6 x 50 mm, 1.8 m
100

0
1 2

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Traditional Stationary Phase Bonding and
Endcapping Reaction

CH 3
CH 3
O Si R
OH O Si R CH 3
CH 3 CH 3 OH
OH OH CH 3 CH 3
+ Cl Si R
OH CH 3
+ Cl Si CH 3 O Si CH 3
OH
CH 3 CH 3 CH 3
OH O Si R CH 3
R = C8, C18, etc . (TMS)
CH 3 O Si R

• Dimethyl silanes
• Endcapped with TMS

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ZORBAX Eclipse Plus & XDB Columns

Improved peak shape for basic

CH3
compounds – especially at Si
O
intermediate pH CH3
Improved
ilica
CH3
e S O Si CH 3 double
Good for all sample types – Bas roved CH 3 endcapping
acid, base, neutral Imp CH3
O Si
CH3
Wide useable pH range, pH 2-9 CH3
O Si CH3

Double end-capped CH 3
CH3

Pore size: O Si
CH3
XDB 80Å, Plus 95Å

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Selectivity of Polar Phases Provides Optimum
Separation of Steroids Versus Non-Polar C18/C8
Column: ZORBAX Eclipse XDB-CN
Column Dimensions: 4.6 x 150 mm, 5 μm
1 2 Mobile Phase: 35:65 ACN:Water Flow Rate: 2.000 ml/min
4 5 Injection Volume: 2.00 μl
3
6 7 Column Temperature: 25 °C
Detector: UV, 210 nm
0 5 10 15 20 min
4,5 Column: ZORBAX Eclipse XDB-Phenyl
1
2 Mobile Phase: 48:52 ACN:Water Sample:
1. Estriol (0.00130 μg/μl),
3
6 7 2. β-Estradiol (0.00130 μg/μl),
3. Ethinyl Estradiol (0.00147 μg/μl),
0 5 10 15 20 4. Dienestrol (0.00123 μg/μl),
5. Diethylstilbestrol (0.00128 μg/μl)
1 2 5 Column: ZORBAX Eclipse XDB-C8 6. Ethynylestradiol 3-methyl ether (0.00103 μg/μl)
3
4 Mobile Phase: 54:46 ACN:Water 7. Ethynodiol Diacetate (0.00139 μg/μl)
6 7

0 5 10 15 20 ¾ Eclipse XDB-CN is the

4
optimum bonded phase – the
3 5
12 Column: ZORBAX Eclipse XDB-C18
Mobile Phase: 60:40 ACN:Water only one that can resolve all
6 7 the components in under 20
0 5 10 15 20 minutes.
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 ZORBAX Bonus-RP
R1
Good peak shape of basic O Si PG R
compounds R1
CH3
Si CH3
Polar alkyl-amide bonded phase CH3
R1

O Si PG R
Unique selectivity
R1
CH3

Enhanced low pH stability with Si

CH3
CH3

sterically protecting bonding R1

O Si PG R

Triple endcapped R1

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Improved Peak Shape of Basic Compounds
Separation of Small Molecule Anorectics on
ZORBAX Bonus-RP and Traditional Alkyl Phase
Columns: 4.6 x 150 mm, 5 μm
1 2
Mobile Phase: 45% 25 mM K2HPO4, pH 7.2 :
55% (MeOH:ACN, 50:50)

Bonus-RP
Flow Rate: 1.0 mL/min.
Detection: 254 nm
Injection vol: 5 µL
Sample: Anorectics (“Fen-phen”)
3
1. Phentermine pKa10.1
2. Fenfluramine pKa 9.1
3. Impurity

Alkyl C8 2

0 1 2 3 4 5 6 7
Time (min)

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ZORBAX Extend-C18

Superior high pH stability – up to O Si C18

pH 11.5 with silica particles
O Si C18
Excellent reproducibility

Patented bidentate, C18 O Si C18

bonding
O Si C18
Double endcapping

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Improved Retention of Basic
Antihistamines
on ZORBAX Extend-C18
pH 11 pH 7
30% 20 mM TEA 30% 20 mM Na2HPO4
70% MeOH 70% MeOH
7 7
4
4 Column: 4.6 x 150 mm, 5 μm
Mobile Phase: See Above 2,3
Flow Rate: 1.0 mL/min
3 Temperature: RT 1
Detection: UV 254 nm
5 Sample: 1. Maleate
1 2. Pseudoephedrine
3. Scopolamine 5
4. Doxylamine
2 5. Chlorpheniramine 6
6 6. Triprolidine
7. Diphenhydramine

0 5 10 0 5
Time (min)
Time (min)

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 HPLC Columns
Within the Column is where separation occurs.
Proper choice of column is critical for success in HPLC
Column dimensions in HPLC:

• Analytical [internal diameter (i.d.) 1.0 - 4.6-mm; lengths

15 – 250 mm]
• Preparative (i.d. > 4.6 mm; lengths 50 – 250 mm)
• Capillary (i.d. 0.1 – 0.5 mm; various lengths)
LC Columns - analytical
• Nano (i.d. < 0.1 mm, or sometimes stated as < 100 µm)
Column Particle Sizes:
• 7, 5, 3.5 (RR), & 1.8 um (RRHT)

Materials of construction for the tubing

• Stainless Steel (the most popular; gives high pressure capabilities)
• Glass (mostly for biomolecules)
• PEEK polymer (biocompatible and chemically inert to most solvents)

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Choose Column Configuration for Application
Particle
Column I.D. Lengths Flow Rate Sensitivity
Sizes Applications
Type (mm) (mm) Ranges Increase**
(um)
Proteomics
Nano 0.1, 0.075 150* 3.5 100 – 600 nL/min 2000
LC/MS

Peptide Mapping
Capillary 0.3, 0.5 35 – 250 3.5, 5 1 – 10 μL/min 100
LC/MS
High sensitivity
MicroBore 1.0 30 – 150 3.5, 5 30 – 60 μL/min 20
LC/MS
Sample limited,
Narrow Bore 2.1 15 – 150 1.8, 3.5, 5 0.1 – 0.3 mL/min 5
LC/MS
Solvent
3.0 150, 250 1.8, 3.5, 5 0.3 – 1.0 mL/min Analytical 2
Saver
Analytical 4.6 15 – 250 1.8, 3.5, 5 1 – 4 mL/min Analytical 1
Small scale
Semi-prep 9.4 50 – 250 5 4 – 10 mL/min --
Protein purification
CombiChem
Preparative 21.2 50 – 250 5, 7 20 – 60 mL/min --
purification

** Based on 4.6 mm id columns

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 Resolving Power

Column Resolving Resolving Resolving Typical Analysis

Length Power Power Power Pressure Time*
(mm) N(5 µm) N(3.5 µm) N(1.8 µm) Bar (1.8 µm)

150 12,500 21,000 32,500 N.A.

Analysis
100 8,500 14,000 24,000 420 Time -33%
75 6000 10,500 17,000 320 -50%
Peak
50 4,200 7,000 12,000 210 Volume -67%
30 N.A. 4,200 6,500 126 -80%
Solvent
15 N.A. 2,100 2,500 55 Usage -90%

* Reduction in analysis time compared to 150 mm column

• pressure determined with 60:40 MeOH/water, 1ml/min, 4.6mm ID

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Pick the column and
particle size to meet your needs
1
2

Rs(1,2) = 4.8 3 4.6 x 250 mm, 5 μm

29.65
N=21848 4 N=22680

0 1 5 10 15 20 25 30 min
2
3
Rs(1,2) = 3.5 4.6 x 100 mm, 3.5 μm
12.71
4 N=11691

. 0 1 5 10 15 20 25 30 min
2
3 N=6568 4.6 x 30 mm, 1.8 μm
4.15
Rs(1,2) = 3.3 1 mL/min
4
N=6104
0 5 10 15 20 25 30 min

1 2
N=6463 3
Rs(1,2) = 3.1 4.6 x 30 mm, 1.8 μm
4
2.09 2 mL/min
N=6460 0.5 1 1.5 2 2.5

0 5 10 15 20 25 30 min
Columns: ZORBAX SB-C18 Mobile Phase: 50% 20 mM NaH2PO4, pH 2.8: 50% ACN Flow Rate: 1 mL/min Temperature: RT
Detection: UV 230 nm Sample: 1. Estradiol 2. Ethynylestradiol 3. Dienestrol 4. Norethindrone

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 Smaller Particles Maintain Efficiency Over Wider
Flow Rate Ranges
Column: ZORBAX Eclipse XDB-C18
H = A + B/u + Cu
0.0030 Dimensions: 4.6 x 50/30mm
Eluent: 85:15 ACN:Water
Flow Rates: 0.05 – 5.0 mL/min
0.0025
Temp: 20°C
Sample: 1.0μL Octanophenone in Eluent
HETP (cm)

0.0020

5.0μm
3.5μm
0.0015

1.8μm
0.0010

0.0005

0.0000
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

Volumetric Flow Rate (mL/min)

Smaller particle sizes yield flatter curves, minima shift to higher flow rates

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 Different C18 Bonded Phases for Max Selectivity

1st choice 1 2 4 Eclipse Plus C18

Best Resolution
& Peak Shape 3
Mobile phase: (69:31) ACN: water
Flow 1.5 mL/min.
1 2 3 4 5 Temp: 30 °C
2nd choice Detector: Single Quad ESI
Good alternate selectivity due 1 2 4 StableBond positive mode scan
3 Columns: RRHT
to non-endcapped SB-C18 4.6 x 50 mm 1.8 um

1 2 3 4 5 min Sample:
1. anandamide (AEA)
3rd choice 1 2 4 Eclipse 2. Palmitoylethanolamide (PEA)
Good efficiency & peak shape 3 XDB-C18 3. 2-arachinoylglycerol (2-AG)
Resolution could be achieved 4. Oleoylethanolamide (OEA)

4th choice 1 2 3 4 5
Multiple bonded phases
Resolution not likely,
1 4 Extend-C18 for most effective method
Other choices better, for this 2,3 development.
separation. Match to one you are
currently using.
1 2 3 4 5

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# 1 – Analytical 4.6x250mm, 5.0µm PAH Column –
Separation of 16 PAH’s in EPA 610
mAU 2
1 1 = Toluene
1400 Conditions:
2 = Naphthalene
3 = Acenaphthylene Det. 220,4nm No Ref.;
4 = Acenaphthene Flow 2.00 ml/min
5 = Fluorene Mobile Phase A = Water; B = Acetonitrile
1200
6 = Phenanthrene Initial %B = 40
7 = Anthracene Gradient: Time (Min) %B
8 = Fluoranthene 0.00 45
9 = Pyrene 17.5 100
1000
10 = Benzo(a)anthracene 24.0 100
11 = Chrysene 25.5 40
12 = Benzo(b)fluoranthene 27.5 40
800 13 = Benzo(k)fluoeanthene Stop Time = 25.0
14 = Benzo(a)pyrene Temp. = 25° C
15 = Dibenzo(a,h)anthracene 500 nanogm on Col for each Component
Rs = 2.2
16 = Benzo(g,h,i)perylene
600 17 = indeno(1,2,3-c,d)pyrene

400 3

8 10 12
6 11
200 5 15
13 16
7 9 14
17

5 10 15 20 25 min

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# 4 – Rapid Resolution – 4.6x150mm, 3.5µm PAH
Column – Separation of 16 PAHs in EPA 610
mAU 1 2
1 = Toluene Conditions:
2 = Naphthalene Det. 220,4nm No Ref.; Stop time = 20.0min
3 = Acenaphthylene Flow 2.00 ml/min
1400
4 = Acenaphthene Mobile Phase A = Water; B = Acetonitrile
5 = Fluorene Gradient: Time (Min) %B
6 = Phenanthrene 0.00 40
1200 7 = Anthracene 1.13 40
8 = Fluoranthene 15.00 100
9 = Pyrene 18.5 100
10 = Benzo(a)anthracene 20.0 40
1000
11 = Chrysene 21.5 40
12 = Benzo(b)fluoranthene Stop Time = 23.5
13 = Benzo(k)fluoeanthene Temp. = 25° C
800 Rs = 2.6 14 = Benzo(a)pyrene 500 nanogm on Col for each Component
15 = Dibenzo(a,h)anthracene
16 = Benzo(g,h,i)perylene
4 17 = indeno(1,2,3-c,d)pyrene
600

3
400
8 10
11 12
6 15
5 13 16
200 7 9 14
17

2 4 6 8 10 12 14 16 18 min

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#6- Rapid Resolution Eclipse PAH 4.6x50mm,
3.5µm Column – Separation of 16 PAHs in EPA 610
mAU 1
1 = Toluene
1750 2 2 = Naphthalene Conditions:
3 = Acenaphthylene Det. 220,4nm No Ref.
4 = Acenaphthene Flow 2.00 ml/min
1500 5 = Fluorene Mobile Phase A = Water; B = Acetonitrile
6 = Phenanthrene Gradient: Time (Min) %B
7 = Anthracene 0.00 42
8 = Fluoranthene 0.33 42
1250 9 = Pyrene 10.00 100
10 = Benzo(a)anthracene 12.5 100
11 = Chrysene 13.5 42
12 = Benzo(b)fluoranthene 15 42
1000 13 = Benzo(k)fluoeanthene Stop Time = 15.1
14 = Benzo(a)pyrene Temp. = 25° C
Rs = 2.0 15 = Dibenzo(a,h)anthracene 500 nanogm on Col for each Component
16 = Benzo(g,h,i)perylene
750
17 = indeno(1,2,3-c,d)pyrene
4

500 3

8 10 15
6 11 12 16
250 5 13 17
7 9 14

2 4 6 8 10 min

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#7- Rapid Resolution HT Eclipse PAH 4.6x100mm,
1.8 µm Column - Separation of 16 PAHs in EPA 610

1 = Toluene
mAU 2 = Naphthalene
Conditions:
100 3 = Acenaphthylene
Agilent 1200SL
4 = Acenaphthene
DAD 220,4nm No Ref. DAD
5 = Fluorene
Flow 2.0 ml/min
6 = Phenanthrene
Mobile Phase A = Water; B = Acetonitrile
7 = Anthracene
Gradient: Time (Min) %B
80 8 = Fluoranthene
0.00 40
9 = Pyrene
0.9 40
10 = Benzo(a)anthracene
12 100
11 = Chrysene
14.5 100
12 = Benzo(b)fluoranthene Rs = 3.63
15 40
60 13 = Benzo(k)fluoeanthene
Stop Time = 16
14 = Benzo(a)pyrene
Temp. = 25° C
15 = Dibenzo(a,h)anthracene
50 nanogm on Col for each Component
16 = Benzo(g,h,i)perylene
no Mixer & no Pulse Dampener
17 = indeno(1,2,3-c,d)pyrene
40

20

2 4 6 8 10 12 14 min

Group/Presentation Title
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Month ##, 200X
# 8 – Rapid Resolution HT Eclipse PAH 4.6x50mm,
1.8µm Column – Separation of 16 PAHs in EPA 610
mAU 2
Conditions:
1 = Toluene Agilent 1200SL
160 2 = Naphthalene DAD 220,4nm No Ref. DAD
1 3 = Acenaphthylene Stop Time = 5.60min
4 = Acenaphthene Flow 2.00 ml/min
140 5 = Fluorene Mobile Phase A = Water; B = Acetonitrile
6 = Phenanthrene Gradient: Time (Min) %B
7 = Anthracene 0.00 45
120 8 = Fluoranthene 3.5 100
9 = Pyrene 4.9 100
10 = Benzo(a)anthracene 5.2 45
Rs = 2.0 11 = Chrysene
100 Stop Time = 5.6
12 = Benzo(b)fluoranthene Temp. = 25° C
13 = Benzo(k)fluoeanthene 50 nanogm on Col for each Component
4 14 = Benzo(a)pyrene no Mixer & no Pulse Dampener
80
15 = Dibenzo(a,h)anthracene
16 = Benzo(g,h,i)perylene
17 = indeno(1,2,3-c,d)pyrene
60
1
3
10 12
40 11
8 13 15
6 16
5 14
17
20
7 9

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 min

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Month ##, 200X
#10 Solvent Saver Eclipse PAH 3.0x250 mm, 5 µm
Column - Separation of 16 PAHs in EPA 610
1 = Toluene
2 = Naphthalene Conditions:
mAU
3 = Acenaphthylene Agilent 1200SL
140
4 = Acenaphthene DAD 220,4nm No Ref. DAD
5 = Fluorene
6 = Phenanthrene Flow 0.85 ml/min
120 7 = Anthracene Mobile Phase A = Water; B = Acetonitrile
8 = Fluoranthene Gradient: Time (Min) %B
9 = Pyrene 0.00 40
10 = Benzo(a)anthracene 2 40
100 11 = Chrysene 17 100
12 = Benzo(b)fluoranthene 24 100
13 = Benzo(k)fluoeanthene 24.1 40
80 14 = Benzo(a)pyrene Stop Time = 27
15 = Dibenzo(a,h)anthracene Temp. = 25° C
16 = Benzo(g,h,i)perylene 50 nanogm on Col for each Component
17 = indeno(1,2,3-c,d)pyrene no Mixer & no Pulse Dampener
60

Rs = 2.15
40

20

5 10 15 20 25 min

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#11 Narrow Bore Eclipse PAH 2.1x250 mm, 5 µm
Column - Separation of 16 PAHs in EPA 610
1 = Toluene
2 = Naphthalene Conditions:
mAU
3 = Acenaphthylene Agilent 1200SL
4 = Acenaphthene DAD 220,4nm No Ref. DAD
5 = Fluorene Flow 0.417 ml/min
6 = Phenanthrene Mobile Phase A = Water; B = Acetonitrile
7 = Anthracene Gradient: Time (Min) %B
200 8 = Fluoranthene 0.00 40
9 = Pyrene 2 40
10 = Benzo(a)anthracene 17 100
11 = Chrysene 24 100
12 = Benzo(b)fluoranthene 24.1 40
150 13 = Benzo(k)fluoeanthene Stop Time = 27
14 = Benzo(a)pyrene Temp. = 25° C
15 = Dibenzo(a,h)anthracene 50 nanogm on Col for each Component
16 = Benzo(g,h,i)perylene no Mixer & no Pulse Dampener
17 = indeno(1,2,3-c,d)pyrene
100

Rs = 2.00

50

5 10 15 20 25 min

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#13 Narrow Bore Rapid Resolution Eclipse PAH
2.1x100mm, 3.5 µm Column - Separation of 16 PAHs in EPA 610

1 = Toluene
mAU 2 = Naphthalene Conditions:
3 = Acenaphthylene Agilent 1200SL
300 4 = Acenaphthene DAD 220,4nm No Ref. DAD
5 = Fluorene Flow 0.417 ml/min
6 = Phenanthrene Mobile Phase A = Water; B = Acetonitrile
7 = Anthracene Gradient: Time (Min) %B
250 8 = Fluoranthene 0.00 40
9 = Pyrene 0.9 40
10 = Benzo(a)anthracene 12 100
11 = Chrysene 14.5 100
200 12 = Benzo(b)fluoranthene 15 40
13 = Benzo(k)fluoeanthene Stop Time = 16
14 = Benzo(a)pyrene Temp. = 25° C
15 = Dibenzo(a,h)anthracene 50 nanogm on Col for each Component
150 16 = Benzo(g,h,i)perylene no Mixer & no Pulse Dampener
17 = indeno(1,2,3-c,d)pyrene

100 Rs = 2.52

50

2 4 6 8 10 12 14 min

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#14 – Narrow Bore RRHT Eclipse PAH 2.1x100mm
1.8 µm – Separation of 16 PAHs in EPA 610

1 = Toluene
mAU Conditions:
2 = Naphthalene
Agilent 1200SL
3 = Acenaphthylene
DAD 220,4nm No Ref. DAD
4 = Acenaphthene
Flow 0.417 ml/min
175 5 = Fluorene
Mobile Phase A = Water; B = Acetonitrile
6 = Phenanthrene
Gradient: Time (Min) %B
7 = Anthracene
0.00 40
150 8 = Fluoranthene
0.9 40
9 = Pyrene
12 100
10 = Benzo(a)anthracene
14.5 100
11 = Chrysene
125 15 40
12 = Benzo(b)fluoranthene Rs = 3.61
Stop Time = 16
13 = Benzo(k)fluoeanthene
Temp. = 25° C
14 = Benzo(a)pyrene
100 50 nanogm on Col for each Component
15 = Dibenzo(a,h)anthracene
no Mixer & no Pulse Dampener
16 = Benzo(g,h,i)perylene
17 = indeno(1,2,3-c,d)pyrene
75

50
toluene

25

2 4 6 8 10 12 14 min

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#15 – Narrow Bore RRHT Eclipse PAH 2.1x50mm,
1.8µm PAH Column - Separation of 16 PAHs in EPA 610
1 = Toluene
mAU 2 = Naphthalene Conditions:
350 3 = Acenaphthylene Agilent 1200SL
4 = Acenaphthene DAD 220,4nm No Ref. DAD
5 = Fluorene Stop Time = 7.0min
6 = Phenanthrene Flow 0.417 ml/min
300 7 = Anthracene Mobile Phase A = Water; B = Acetonitrile
8 = Fluoranthene Gradient: Time (Min) %B
9 = Pyrene 0.00 45
10 = Benzo(a)anthracene 3.5 100
250
11 = Chrysene 4.9 100
12 = Benzo(b)fluoranthene 5.2 45
13 = Benzo(k)fluoeanthene Stop Time = 7
200 14 = Benzo(a)pyrene Temp. = 25° C
15 = Dibenzo(a,h)anthracene 50 nanogm on Col for each Component
16 = Benzo(g,h,i)perylene no Mixer & no Pulse Dampener
17 = indeno(1,2,3-c,d)pyrene
150

Rs = 2.16
100

50

1 2 3 4 5 6 min

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Month ##, 200X
How To “Match” a Column to a ZORBAX RRHT
Column

General Phase Type Starting ZORBAX Choice

Typical “endcapped” C18 or C8

Eclipse Plus C18 or C8
bonded phases, newer columns
Endcapped C18 or C8 columns,
Eclipse XDB-C18 or C8
older generation
Non-endcapped columns StableBond C18

Older types of columns StableBond C18, C8 etc.

Aqueous “type” columns SB-AQ

CN or Phenyl SB-CN, SB-Phenyl

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 Conclusion

There is no one-size-fits all column

Choose the best for your application needs

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