HPLC Analysis of Biomolecules Technical Guide - Interscience

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4 HPLC Analysis of Biomolecules Table of Contents Size Exclusion Chromatography Basic Principles of Size Exclusion Chromatography 28 Particles for Size Exclusion 28 Pore Size and V i /V o Ratio 29 Molecular Weight Calibration Curves 29 Choosing a Size Exclusion Column Based on Molecular Weight 29 Particle Size and Column Length 30 Mobile Phases for Size Exclusion Chromatography 30 Effect of Mobile Phase pH 30 Effect of Mobile Phase Ionic Strength (Salt Concentration) 30 Flow Rate and Temperature 30 Size Exclusion Chromatography for LC/MS of Biomolecules 31 SEC and Sample Preparation or Fractionation 31 Direct Serum Injection 31 Two-Dimensional (2-D) Sample Clean-Up 32 Proteomics Proteomics and LC/MS n 33 Top-Down vs Bottom-Up Approaches 33 HPLC in Proteomics 33 Capillary LC/MS 33 Online 2-D HPLC 34 Microscale Solid Phase Extraction 35 Column Care and Maintenance General Recommendations for Usage 36 Column Protection 36 Column Cleaning 37 Column Storage 37 Appendices Appendix 1: Quick Look-Up Tables Protein Structure Defined 38 Molecular Weight of Some Common Biomolecules 38 Common Amino Acids, Abbreviations, MW and pKa 38 Properties of Common HPLC Solvents 38 Common Buffer Systems Used in HPLC 39 Typical Flow Rates and Injection Volumes Versus HPLC Column ID 39 Preparative Scale-Up Calculations 39 Tubing Internal Diameters and Volumes 39 Appendix 2: Columns for HPLC of Biomolecules 40 Appendix 3: Instruments for HPLC of Biomolecules 44 Appendix 4: Index 46 Appendix 5: References 47
HPLC Modes for Biomolecule Separations Introduction Proteins, peptides, carbohydrates, lipids, amino acids, vitamins, coenzymes, and nucleic acids are a few of the members of the diverse collection of compounds involved in biochemical processes, or biomolecules. The structural and chemical diversity of this group along with the need to consider biological activity and complex matrices demands an arsenal of sample preparation, separation modes, column chemistries, column configurations, and detection techniques to effectively characterize the biomolecule under study. There are dozens of techniques or separation modes bioanalytical chemists use to analyze biomolecules. Typically, in any given separation multiple modes exist; it is rare for a separation to be purely ion exchange or purely size exclusion, for example. This is especially true for large biomolecules that may possess many different functional groups and exist in multiple conformations. The focus of this guide is on three of the most commonly encountered separation modes: reversed phase, ion exchange, and size exclusion. Reversed Phase (page 6) • separation is based on hydrophobicity Ion Exchange (page 23) • separation is based on molecular charge Size Exclusion (page 28) • separation is based on molecular weight and molecular shape Regardless of the separation mode, consistent particle chemistry and efficient column packing are the keys to achieving reliable, reproducible separations of biomolecules. Several of the products that Thermo offers for HPLC of biomolecules appear in Table 1. Separation Modes Thermo HPLC Columns Reversed phase BetaBasic C18, C8, C4, CN, Phenyl BioBasic C18, C8, C4, CN, Phenyl Hypercarb Ion exchange BioBasic AX and SCX Size exclusion BioBasic SEC 60, 120, 300, and 1000 Table 1: Overview of Thermo columns for biomolecule separation BioBasic Columns: 300Å silica-based particles with superb efficiency, reproducibility, and stability for HPLC and LC/MS separations of proteins, peptides, and nucleic acids. BetaBasic Columns: 150Å silica-based particles in a wide array of reversed phase bonded phase chemistry with superb efficiency, reproducibility, and stability for separations of small molecules, nucleotides, nucleosides, peptides, and protein digests. Hypercarb Columns: pH-stable carbon particles provide separations based on both hydrophobicity and molecular geometry. Truly unique selectivity compared to silica-based reversed phases with enhanced retention of polar compounds. 5
Page 1 and 2: c h r o m a t o g r a p h y HPLC An
Page 3: Table of Contents HPLC Modes for Bi
Page 7 and 8: efficiency is observed as the large
Page 9 and 10: Figure 7: Effect of flow rate on gr
Page 11 and 12: Figure 11: Retention of nucleosides
Page 13 and 14: Figure 15: Effect of pH on RP-HPLC
Page 15 and 16: Effect of Temperature on RP-HPLC Re
Page 17 and 18: Maintaining Constant Linear Velocit
Page 19 and 20: Hypercarb, 5 µm, 30 x 3 mm Part No
Page 21 and 22: Organic/Aqueous Solvent Ratio for R
Page 23 and 24: Ion Exchange Chromatography Basic P
Page 25 and 26: Column Type of Ion exchange Particl
Page 27 and 28: Figure 46: Effect of decreasing ion
Page 29 and 30: Figure 51: Molecular weight calibra
Page 31 and 32: Figure 55 shows how temperature con
Page 33 and 34: Proteomics Proteomics and LC/MS n P
Page 35 and 36: Figure 61: 2-D LC/LC-MS 2 tryptic d
Page 37 and 38: Column Cleaning If some components
Page 39 and 40: Common Buffer Systems Used in HPLC
Page 41 and 42: 5 µm BioBasic PEEK Columns Descri
Page 43 and 44: 3 µm & 5 µm Hypercarb Columns Des
Page 45 and 46: Finnigan Surveyor HPLC Systems The
Page 47 and 48: Proteomics Capillary LC/MS ........

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