An Efficient Approach to Column

Selection in HPLC Method

Development

Craig S. Young and Raymond J. Weigand

Alltech Associates, Inc.

2051 Waukegan Road Deerfield, IL 60015
Phone: 1-800-ALLTECH Web Site: www.alltechweb.com
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?

Number of compounds present?
Sample matrix? Column Chemistry
pKa values of sample components? (bonded phase, bonding type,
endcapping, carbon load)
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?
Fast analysis? Column Physics
(particle bed dimensions,
Economy (low solvent usage)?
particle shape, particle
Column stability and lifetime? size, surface area, pore
Preparative method? size)
High sensitivity?
Other goals?
Column Selection Chart

Low Mobile Phase

Fast Eqilibration
Suitable for MW
High Resolution

High Sensitivity
Default Column
(Good for most

High Efficiency

Stability at pH
High Capacity

High Stability
Backpressure
Applications)

Consumption
Fast Analysis
High Sample
Method

Loadability
Goals

Extremes
>2000
Low
Particle Size
small (3m)
medium (5m)
large (10m)

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 (60)
medium (100)
large (300)

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.

e.g.:

HO HO

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 London

Phenyl , London

R
Halide London, Dipole-Dipole
R F, Cl, Br, I
Ether London, Dipole-Dipole, H-bonding
R O
Nitro R , London, Dipole-Dipole, H-bonding
-
O
+
Ester R N O , London, Dipole-Dipole, H-bonding

O
Aldehyde R , London, Dipole-Dipole, H-bonding
O R
Ketone O , London, Dipole-Dipole, H-bonding

R H
Amino , London, Dipole-Dipole, H-bonding, Acid-base chemistry
O
R
Hydroxyl R London, Dipole-Dipole, H-bonding

High Carboxylic Acid R NH2 , London, Dipole-Dipole, H-bonding, Acid-base chemistry

R OH

O
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
- 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 - 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 5m 3* or 5m
Surface Area 200m2/g >200m2/g
Pore Size 100 100
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, 5m, 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 3-20m

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
60-10,000

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:H 2O.
Particle Size

Effect on chromatography
Smaller particles offer higher efficiency, but also cause higher
backpressure. Choose 3m particles for resolving complex, multi-
component samples. Otherwise, choose 5 or 10m 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 Alltechs Alltima ,
Adsorbospherel HS, and Adsorbosphere UHS packings. Low surface
area silicas are used in Alltechs 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
150 or less for sample MW 2000. Choose a pore size of 300 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 Alltechs 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.
Alltechs 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
Anthracene
hydrophobic hexyl side chain. This
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 C18 C8 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 (m) 5 5
Surface area (m2/g) 200 <200
Pore Size () 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 capd
(m) (%) () (m /g)
2

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

Brava BDS C18 silica Sph. 3, 5 8.5 145 185 Mono. Yes

Econosphere C18 silica Sph. 3, 5, 10 10 80 200 Mono. Yes

Platinum C18 silica Sph. 3, 5, 10 6 100 200 Mono. Yes

Increased Sensitivity,
Best Peak Shape Low Solvent Consumption, Quick Equilibration
Fast Analysis

Column of choice: Brava BDS C18, 100x2.1, 5m (Spherical , 185m2/g,

monomeric)
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

O
O
H H
polar, with amine and pi bond
H2N H H NH
S O
functions--a RP CN column may
NH S
S
O
N O give good separation by mixed-
N
O HO O
mode retention of hydrophobic,
HO O CN---H---NR2 hydrogen bonding
and - interactions with double
bonds.
Recommended bonded phase (silica based materials only) mark one

Normal phase silica NH2 CN

Reversed phase C18 C8 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 (m) 5 5
Surface area (m2/g) 200 200 +
Pore Size () 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 capd
(m) (%) () (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

High resolution,
High res.
High sensitivity

Column of choice: Alltima CN, 250 x 2.1 , 5m ( Spherical , 350 m2/g ,

polymeric)
Good balance of efficiency Reduced
& backpressure Robust
backpressure