HPLC Analysis of Biomolecules Technical Guide

1 Successful Separations of Peptides, Proteins and Other BiomoleculesHPLC Analysis of BiomoleculesTechnical GuidechromatographyPart of Thermo Fisher ScientificHPLC Analysis of Biomolecules2 Resources forChromatographersAvailable now, the Thermo Scientific ChromatographyColumns and Consumables 2010/2011 catalog. Beyond this catalog, our Chromatography team shares its extensive expertise through our web-basedChromatography Resource Center and the Separatedby Experience Chromatography Resource Center, accessedat provides Technical support, applications, technicaltips and literature to help you move your separationsforward, quickly and bi-monthly Separated by Experienceenewsletter keeps you up-to-date on the latest Technical and product information of interest to chromatographers.

2 Valuable information developed bychromatographers for chromatographers. Subscribetoday at to Life ScienceMajor advances in proteomics, biochemistry, andmolecular biology will enable new therapeutic treatments and increase our understanding of fundamental life processes. Thermo Scientific hplc and LC/MS systems, software, hplc modularcomponents, hplc particles, columns , and other consumables provide life science researchers thetools, technologies, and integrated solutions neededto tackle the formidable analytical challenges theyface Modes for Biomolecule SeparationsIntroduction5 Reversed Phase ChromatographyReversed Phase hplc of Biomolecules6 Mechanism of RP- hplc Separation6 Particle Size6 Pore Size7 Column Length8 Flow Rate8 Bonded Phase Functionality and Alkyl Chain Length9 Porous Graphitic Carbon (PGC)

3 10 Guidelines for Choosing an RP- hplc Column12 Mobile Phases for RP-HPLC12 Effect of pH on RP- hplc Analyses13 Effect of Ionic Modifier on RP- hplc Analyses13 Effect of Organic Modifier Type and Concentration on RP- hplc Retention14 Effect of Temperature on RP- hplc Retention15 Gradient vs. Isocratic Elution15RP-LC/MS of Biomolecules16 column selection for RP-LC/MS: Column ID16 Maintaining Constant Linear Velocity with Changing Column ID17 Electrospray Ionization (ESI)17 Column Packing Materials for RP-LC/MS18 Column Stability19 Mobile Phase for RP-LC/MS20 Ionic Additives for RP-LC/MS20 Organic/Aqueous Solvent Ratio for RP-LC/MS21 Effect of Connecting Tubing on LC/MS Efficiency21 Ion Exchange ChromatographyBasic Principles of Ion Exchange Chromatography23 Particles for Ion Exchange24 Particle size , Pore size , and Column Length24 Flow Rate24 Temperature24 Ion Exchange Bonded Phases25 Measuring Ion Exchange Capacity25 Mobile Phases for Ion Exchange HPLC26 Mobile Phase pH26 Ionic Strength (Salt Concentration)

4 26 Controlling Retention with Salt and pH Gradients27 Ion Exchange for LC/MS of Biomolecules27 Table of ContentsHPLC Analysis of Biomolecules4 size exclusion ChromatographyBasic Principles of size exclusion Chromatography28 Particles for size Exclusion28 Pore size and Vi/VoRatio29 Molecular Weight Calibration Curves29 Choosing a size exclusion Column Based on Molecular Weight29 Particle size and Column Length30 Mobile Phases for size exclusion Chromatography30 Effect of Mobile Phase pH30 Effect of Mobile Phase Ionic Strength (Salt Concentration)30 Flow Rate and Temperature30 size exclusion Chromatography for LC/MS of Biomolecules31 SEC and Sample Preparation or Fractionation31 Direct Serum Injection31 Two-Dimensional (2-D) Sample Clean-Up32 ProteomicsProteomics and LC/MSn33 Top-Down vs Bottom-Up Approaches33 hplc in Proteomics33 Capillary LC/MS33 Online 2-D HPLC34 Microscale Solid Phase Extraction35 Column Care and MaintenanceGeneral Recommendations for Usage36 Column Protection36 Column Cleaning37 Column Storage37 AppendicesAppendix 1.

5 Quick Look-Up TablesProtein Structure Defined38 Molecular Weight of Some Common Biomolecules38 Common Amino Acids, Abbreviations, MW and pKa38 Properties of Common hplc Solvents38 Common Buffer Systems Used in HPLC39 Typical Flow Rates and Injection Volumes Versus hplc Column ID39 Preparative Scale-Up Calculations39 Tubing Internal Diameters and Volumes39 Appendix 2: columns for hplc of Biomolecules40 Appendix 3: Sample Preparation Products for Biomolecules44 Appendix 4: Index46 Appendix 5: References47 Table of Contents5 IntroductionProteins, peptides, carbohydrates, lipids, amino acids, vitamins,coenzymes, and nucleic acids are a few of the members of thediverse collection of compounds involved in biochemical processes,or Biomolecules .

6 The structural and chemical diversity of this groupalong with the need to consider biological activity and complexmatrices demands an arsenal of sample preparation, separationmodes, column chemistries, column configurations, and detectiontechniques to effectively characterize the biomolecule under are dozens of techniques or separation modes bioanalyticalchemists use to analyze Biomolecules . Typically, in any given separation multiple modes exist; it is rare for a separation to bepurely ion exchange or purely size exclusion , for example. This isespecially true for large Biomolecules that may possess many different functional groups and exist in multiple conformations.

7 Thefocus of this Guide is on three of the most commonly encounteredseparation modes: reversed phase, ion exchange, and size Phase (page 6) separation is based on hydrophobicityIon Exchange (page 23) separation is based on molecular chargeSize exclusion (page 28) separation is based on molecular weight and molecular shapeRegardless of the separation mode, consistent particle chemistry and efficient column packing are the keys to achievingreliable, reproducible separations of Biomolecules . Table 1 presentsseveral Thermo Scientific hplc products for biomolecular ModesThermo Scientific hplc ColumnsReversed phaseBetaBasic C18, C8, C4, CN, PhenylBioBasic C18, C8, C4, CN, PhenylHypercarb Ion exchangeBioBasic AX and SCXSize exclusionBioBasic SEC 60, 120, 300, and 1000 Table 1: Overview of Thermo Scientific columns for biomolecule separationBioBasic columns :300 silica-based particles with superb efficiency, reproducibility,and stability for hplc and LC/MS separations of proteins, peptides,and nucleic acids.

8 BetaBasic columns :150 silica-based particles in a wide array of reversed phase bonded phase chemistry with superb efficiency, reproducibility, andstability for separations of small molecules, nucleotides, nucleosides,peptides, and protein columns :pH-stable carbon particles provide separations based on bothhydrophobicity and molecular geometry. Truly unique selectivitycompared to silica-based reversed phases with enhanced retentionof polar Modes for Biomolecule SeparationsHPLC Analysis of Biomolecules6 Reversed Phase hplc of BiomoleculesReversed phase hplc (RP- hplc ) is a very powerful and widely-usedtechnique for separating Biomolecules , both large and small.

9 Its primary advantages over other separation modes are high efficiencyand the ability to distinguish between compounds that are chemicallyvery similar. For example, RP- hplc can separate large peptides whoseprimary sequences differ by only one amino acid residue. For analystsusing LC/MS in proteomics applications, it is an indispensable tech-nique for separating the peptides in enzymatic digests of of RP- hplc SeparationThe term reversed phase is applied because the first hplc separa-tions employed a polar support particle and a non-polar eluent. In atypical RP- hplc separation, these are reversed.

10 The support particleis non-polar; either a resin or carbon particle or a silica particlewhich has been bonded with a non-polar molecule such as alkylchains or phenyl rings. Common RP- hplc eluents are polar solventssuch as water, methanol, or acetonitrile. Retention of proteins and peptides by RP- hplc usually occursthrough a combination of adsorption/desorption and a partitioning typemechanism, compared to small molecules which usually separatestrictly by a partitioning type mechanism. The hydrophobic footprint of the protein reversibly adsorbs to the bonded phase at the head orinlet of the column.