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An Efficient Approach to Column Selection in HPLC Method Development

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:

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An Efficient Approach to Column Selection in HPLC Method Development

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  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 >2000 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 • • polymeric • • • • 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 Ether 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 • C18 or Octadecylsilane (ODS) • Very nonpolar - Retention is based on London (dispersion) interactions with hydrophobic compounds. • Example Alltech Phase: Alltima™ C18 • Examples of bonded phases used for HPLC packing media:

  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 >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.

  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 50:50 MeOH:H2O.

  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 Alltech’s Alltima™, Adsorbospherel® HS, and Adsorbosphere® UHS packings. Low surface area silicas are used in Alltech’s 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 > 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 Alltech’s 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. • Alltech’s 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 <200 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 cap’d (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 CN 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 Polymeric Particle shape spherical spherical

  38. 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) (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 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 res. High resolution, High sensitivity Good balance of efficiency & backpressure Robust Reduced backpressure

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