An Efficient Approach to Column Selection in HPLC Method Development

1
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

2
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

3
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

4
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

5
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?
• pKa values of sample components?
• Concentration range?
• Molecular weight range?
• Solubility?
• Other pertinent data?

Column Chemistry (bonded phase, bonding type,
endcapping, carbon load)
6
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)
7
Column Selection Chart
Default Column (Good for most Applications)
Low Mobile Phase Consumption
Method Goals
High Sample Loadability
Suitable for MW gt2000
Stability at pH Extremes
High Efficiency
High Capacity
High Resolution
High Stability
Fast Analysis
Fast Eqilibration
Low Backpressure
High Sensitivity
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 poly
meric Particle
Shape spherical irregular
8
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.

Prednisolone
Prednisone
9
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.

Prednisolone
Prednisone
10
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.

Prednisolone
Prednisone
11
Functional Group Polarity Comparisons
Polarity Functional Group Structure Bonding
Types Intermolecular Forces Displayed
Low Methylene s London Phenyl s ,
p London Halide s London, Dipole-Dipole Ethe
r s London, Dipole-Dipole, H-bonding Nitro
s , p London, Dipole-Dipole, H-bonding Ester
s , p London, Dipole-Dipole, H-bonding Aldehyde
s , p London, Dipole-Dipole,
H-bonding Ketone s , p London,
Dipole-Dipole, H-bonding Amino s , p London,
Dipole-Dipole, H-bonding, Acid-base
chemistry Hydroxyl s London, Dipole-Dipole,
H-bonding High Carboxylic Acid s , p London,
Dipole-Dipole, H-bonding, Acid-base chemistry
12
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

13
Choosing the Bonded Phase

• Phenyl
• Nonpolar - Retention is a mixed mechanism of
  hydrophobic and p - p interactions.
• Example Alltech Phase Platinum Phenyl

14
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

15
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
16
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

17
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 gt200m2/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.
18
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

19
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

20
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

21
Particle Shape

• Effect on chromatography
• Spherical particles offer reduced back pressures
  and longer column life when using viscous mobile
  phases like 5050 MeOHH2O.

22
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.

23
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.

24
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 gt
  2000.

25
Bonding Type

• Effect on chromatography
• Monomeric bonding offers increased mass transfer
  rates, higher column efficiency, and faster
  column equilibration.

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.
26
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.

27
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.

28
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.

29
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)
? 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
What do I know about the sample?
30
Column Selection Example 1
Structures of Compounds
31
Column Selection Example 1
Which two sample components have the most similar
structures? Draw them, then circle the
structural differences between them.
Normal phase silica NH2 CN Reversed
phase C18 C8 Ph CN
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.
Anthracene
3-Hexylanthracene
Recommended bonded phase (silica based materials
only) mark one
32
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
lt200 Pore Size (Å) 100 100 Carbon Load
() 10 10 Bonding type Monomeric
Monomeric Particle shape spherical
spherical
33
Column Selection Example 1
Available packing alternatives meeting the above
criteria Packing Base
Particle Particle Carbon Pore
Surface Bonding End- Material Shape Size
Load Size Area Type capd (µm)
() (Å) (m2/g) silica Sph. 3, 5,
10 12 80 220 Mono. Yes silica Sph. 3,
5 8.5 145 185 Mono. Yes silica Sph. 3, 5,
10 10 80 200 Mono. Yes silica Sph. 3, 5,
10 6 100 200 Mono. Yes Column of choice
Brava BDS C18, 100x2.1, 5µm (Spherical ,
185m2/g, monomeric)
Allsphere ODS-2 Brava BDS C18 Econosphere
C18 Platinum C18
Increased Sensitivity, Low Solvent
Consumption, Fast Analysis
Best Peak Shape
Quick Equilibration
Reduced backpressure
Good balance of efficiency backpressure
34
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 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
What do I know about the sample?
35
Column Selection Example 2
Structures of Compounds
36
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 column may
give good separation by mixed- mode
retention of hydrophobic, CN---H---NR2
hydrogen bonding and ?-? interactions with
double bonds. Normal phase silica NH2 C
N Reversed phase C18 C8 Ph CN
Recommended bonded phase (silica based materials
only) mark one
37
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 2.1Particle Size
(µm) 5 5 Surface area (m2/g) 200 200
Pore Size (Å) 100 Not critical Carbon
Load () 10 -- Bonding type Monomeric Polym
eric Particle shape spherical spherical
38
Column Selection Example 2
Available packing alternatives meeting the above
criteria Packing Base Particle Particle Carbon P
ore Surface Bonding End- Material Shape Size Load
Size Area Type capd (µm) () (Å)
(m2/g) 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 Platin
um 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 res.
High resolution, High sensitivity
Good balance of efficiency backpressure
Robust
Reduced backpressure