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
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Structure of sample components?
Number of compounds present?
Sample matrix?
pKa values of sample components?
Concentration range?
Molecular weight range?
Solubility?
Other pertinent data?
Column Chemistry
(bonded phase, bonding type,
endcapping, carbon load)
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?
• Economy (low solvent usage)?
• Column stability and lifetime?
• Preparative method?
• High sensitivity?
• Other goals?
Column Physics
(particle bed dimensions,
particle shape, particle
size, surface area, pore
size)
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%)
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Fast Eqilibration
Stability at pH
Extremes
Low Mobile
Phase
Consumption
Fast Analysis
High Sensitivity
High Stability
Suitable for MW
>2000
High Sample
Loadability
High Resolution
Low
Backpressure
High Capacity
High Efficiency
Default Column
(Good for most
Applications)
Method
Goals
Column Selection Chart
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Bonding Type
monomeric
polymeric
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Particle Shape
spherical
irregular
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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
HO
OH
O
O
O
Prednisolone
Prednisone
OH
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.
HO
HO
e.g.:
O
O
HO
OH
O
O
O
Prednisolone
Prednisone
OH
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
HO
OH
O
O
O
Prednisolone
Prednisone
OH
Functional Group Polarity Comparisons
Polarity
Low
Functional Group
Methylene
Structure
R (CH2)2
Phenyl
Bonding Types
Intermolecular Forces Displayed
s
London
s,p
London
R
Halide
Ether
Nitro
Ester
R
F, Cl, Br, I
R
O
-
N O
O
R
O R
O
R
London, Dipole-Dipole
s
London, Dipole-Dipole, H-bonding
s,p
London, Dipole-Dipole, H-bonding
s,p
London, Dipole-Dipole, H-bonding
s,p
London, Dipole-Dipole, H-bonding
s,p
London, Dipole-Dipole, H-bonding
s,p
London, Dipole-Dipole, H-bonding, Acid-base chemistry
R
+
R
Aldehyde
Ketone
O
s
H
O
R
R
Amino
R
NH2
R
OH
Hydroxyl
High
s
O
Carboxylic Acid
R
OH
s,p
London, Dipole-Dipole, H-bonding
London, Dipole-Dipole, H-bonding, Acid-base chemistry
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
R
H
C
C
Si (CH2)3 C
C
C
R
H
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
R
N
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
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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
Column Bed Dimensions
Particle Size
Surface Area
Pore Size
Carbon Load
Bonding Type
Base Material
Particle Shape
Default Column
Optimum Column†
150 x 4.6mm
5µm
200m2/g
100Å
10%
Monomeric
Silica
Spherical
250 x 4.6mm
3* or 5µm
>200m2/g
100Å
16 - 20%
Mono- or Polymeric
Silica
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, multicomponent 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
OH
+
X
Si (CH2)17CH3
R
monomeric
CH3
OH
OH
R
O
CH3
+
Si (CH2)17CH3
bonding
CH3
polymeric
X
Si (CH2)17 CH3
X
Si
bonding
O
(CH2)17 CH3
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. Nonendcapped 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
Sample matrix
pKa values of compounds?
UV spectral information about compounds?
Concentration range of compounds
Molecular weight range of compounds




4
--UV -254
-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.
Anthracene
3-Hexylanthracene
(CH2)5CH3
Note: The structural difference
between these two compounds is the
hydrophobic hexyl side chain. This
suggests a non-polar C18 or C8
column would interact with this area of
difference to help provide separation
of these two compounds.
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
Column bed dimensions (mm)
Particle Size (µm)
Surface area (m2/g)
Pore Size (Å)
Carbon Load (%)
Bonding type
Particle shape
Default Column
150 x 4.6
5
200
100
10
Monomeric
spherical
Ideal Column
100 x 2.1
5
<200
100
10
Monomeric
spherical
Column Selection Example #1
Available packing alternatives meeting the above criteria:
Packing
Base
ParticleParticle Carbon
Material Shape
Size
Load
(µm)
(%)
Pore Surface Bonding
Size Area
Type
(Å)
(m2/g)
Endcap’d
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
Best Peak Shape
Increased Sensitivity,
Low Solvent Consumption,
Fast Analysis
Quick Equilibration
Column of choice: Brava BDS C18, 100x2.1, 5µm (Spherical , 185m2/g,
monomeric)
Good balance of efficiency
& backpressure
Reduced
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
Sample matrix
pKa values of compounds?
UV spectral information about compounds?
Concentration range of compounds
Molecular weight range of compounds



6+
--UV -254
-349 - 645
Column Selection Example #2
Structures of Compounds
N N
O N
H2N
O
H H
NH
S
S
S
O N
O
H H
NH
N
N
H2N
O
HO
O
H H
NH
N
N
N
H H
S
NH
N
O
O
N
O
O
HO
O
S
O
O
O
HO
HO
O
O
S
N
N
S
N
O
N
O
NH
O
O
H
NH
H
N
O
HO
O
S
HO
S
O
N N
N N
H
NH
S
H
S
O
N
O
O
HO
O
O
O
O
NH2
Column Selection Example #2
Which two sample components have the most similar structures?
Draw them, then circle the structural differences between them.
O
H2N
O
H H
NH
H
NH
S
S
H
O
N
O
O
N
O
HO
HO
S
O
O
Notes: both structures very
polar, with amine and pi bond
functions--a RP CN column may
give good separation by mixedmode retention of hydrophobic,
CN---H---NR2 hydrogen bonding
and p-p interactions with double
bonds.
Recommended bonded phase (silica based materials only) – mark one
Normal phase
Reversed phase
silica
C18
NH2
C8
CN
Ph
CN
Column Selection Example #2
Column physical characteristics – use Column Selection Chart and
Method Goals
Column bed dimensions (mm)
Particle Size (µm)
Surface area (m2/g)
Pore Size (Å)
Carbon Load (%)
Bonding type
Particle shape
Default Column
150 x 4.6
5
200
100
10
Monomeric
spherical
Ideal Column
250 x 2.1
5
200 +
Not critical
-Polymeric
spherical
Column Selection Example #2
Available packing alternatives meeting the above criteria:
Packing
Base Particle Particle Carbon
Material Shape
Size
Load
(µm)
(%)
Pore
Size
(Å)
Surface Bonding
Area
Type
2
(m /g)
Endcap’d
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 sensitivity
High res.
Column of choice: Alltima CN, 250 x 2.1 , 5µm ( Spherical , 350 m2/g ,
polymeric)
Good balance of efficiency
& backpressure
Reduced
backpressure
Robust