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Protocols

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Training

    Basics of Dialysis and Ion Exchange
    Designing a purification protocol using ion exchange resins
    "Basics of HPLC," an on-line book by Dr. Yuri Kazakevich and Dr. Harold McNair
    "Buffer Solubility in Aqueous-Organic Solvents" Adam P. Schellinger and Peter W. Carr, LCGC, JUNE (2004) 22(6): 544-48. Useful for RPC and/or HILIC mobile phase composition selection.
    The Importance of Contact Time for Either Desalting or Trapping of Polar Solutes on Trap Columns.

    Measuring pH in aqueous/organic mixtures:
      Use of the s(w)pH scale method. [Canals I, JA Portal, E Bosch, and M Roses Retention of ionizable compounds on HPLC. 4. Mobile-phase pH measurement in methanol/water Anal Chem, April 15, 2000; 72(8): 1802-9.]
        Three procedures are evaluated:
          - measurement of the pH of the aqueous HPLC buffer before mixing it with the organic modifier,
          - measurement of the pH of the HPLC buffer after mixing it with the organic modifier using a pH electrode system calibrated with aqueous buffers, and

          - measurement of the pH of the HPLC buffer after mixing it with the organic modifier but calibrating the electrode system with reference buffers prepared in the same mixed solvent used as mobile phase.
        Following IUPAC definitions and recommendations, the three pH values are related to the pH scales: w(w)pH, s(w)pH, and s(s)pH, respectively. The relationships between these three pH scales are also presented. The retention of several compounds with acid/base behavior in a C-18 and a polymeric column with buffered methanol/water as mobile phase is related to the mobile phase pH value measured in the three pH scales. They demonstrate that the s(w)pH and s(s)pH scales give better relationships than the w(w)pH scale (pH measured in the aqueous buffer before mixing it with the organic modifier), commonly used on HPLC. The s(w)pH scale is especially recommended because of its simplicity of measurement: the pH is measured after mixing the aqueous buffer with the organic modifier, but the pH calibration is performed with the common aqueous reference buffers.





    Retention Time Predictor Tool for ZIC-HILIC columns.

    Sample Loading and Gradient Modification Data Tables for Column Dimension Changes.

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HPLC Column Care

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Small Molecule and Combi-Chem SPE Separations

Cerex Filtration/Drying Manifolds & SPE Cartridges for Purificaton and Recovery

MiniSpin and MicroTip micro-SPE & Desalting Kits:

    MiniSpin columns and 96-Well Spin Plates  for microliter sample prep. Concentrate, desalt or fractionate 2 - 400µl of chemical or biological materials on over 20 RPC, IEX, and Polar micro-SPE chemistries or 2 - 150µl on SEC materials. UltraMicroTip columns, the tips equivalent to the UltraMicroSpin columns for microliter sample prep. Concentrate, desalt or fractionate 5 - 100µl (3-30 µg) of chemical or biological materials on over 20 RPC, IEX, and Polar chemistries Micro filter Page*Eraser™ empty columns remove the particulates from in-gel digests to prolong capillary LC-MS column life.
    Empty µ-Reactors™ with caps to remove solid reactants from micro quantities of samples. See: Xia B., et al. Anal Biochem. 2009 Apr 15;387(2):162-70. Epub 2009 Feb 10. Glycan reductive isotope labeling (GRIL) for quantitative glycomics.

Ultra-Micro PrepTip™ MS µ-SPE Tips:

    Pipette tips to concentrate, desalt or fractionate 1 - 10µL and 10 - 100µL of biological materials.
    Ultra-Micro PrepTip™ RP clean-up for detergent or salt removal from protein digests. Useful for MS applications, due to lower back pressure and higher capacity than ZipTip® C18.

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Small Molecule and Combi-Chem HPLC Separations

Higgins Analytical® HPLC columns

(Exceptionally Good Performance, A Wide Range of Configurations.) Our "Best Value" Product Line.
    Complete Catalog (3,408K pdf)
    Catalogs of HAISIL® HPLC Columns (0.3 - 22mmID), or Short, Direct Connect, 2.1mmID Narrowbore (Sprite), 0.3-1.0mmID Microbore Guard/Trap (Piccolo), 0.075 - 0.15mmID Nano-Capillaries (Capellini), and/or Cartridge (HAIPEEK) Products:
      (Type A Silica) (Type B Silica)
        HAISIL RS, PROTO™ 200 C4 & C18 for Peptide applications. HAISIL RS, PROTO™ 300 C4 & C18 for Protein applications.
        HAISIL TS, TARGA ® C8 & C18 for Polar Analytes & Proteomics.
        HAISIL CS, CLIPEUS™ Silica, CN, Phenyl, C8, & C18 phases, excellent for Small Molecule applications.
      Heavily Coated (Type B Silica) Phases
      :
        HAISIL VS, HEAVY™ Ultra-high density coating for wide pH range applications.
        HAISIL HL, High Load High density coating for Combi-Chem & Fast LC Analyses.
        HAISIL PS, PHALANX™ Water-wettable, Ultra-high density coating for wide pH range applications.





      Polymeric RPC and Ion Exchange (Capillary - < 2mmID column configurations.)
        PL-Gel® PLRP-S, 3µm, 100Å pores, for Small Molecule, high pH applications, such as negative ion mode LC/MS; or 300Å pores for Peptide applications.
        PLRP-S, RP1K & RP4K, 5µm, with 1000Å or 4000Å pores for Protein & Antibody RPC.
        PL-SAX, PL-SCX, PL-WAX & PL-WCX, 5µm, with 1000Å or 4000Å pores for Protein and Antibody ion exchange applications.


    Applications for HAISIL Columns:
      Amines (without tailing), or for LC-MS/MS, "no TFA" peptide separations: CLIPEUS with WOW! selectivity in C8, C18, CN or Phenyl phases that do not require base deactivation.
      Combi-Chem analyses of small molecules on Heavily Loaded HAISIL columns

      PROTO™ 200 C4 & C18 Peptide Columns
        Comparison of PROTEO™ C18 to Other Columns for Phosphopeptides.
      PROTO™ 300 C4 & C18 Protein Columns
        Comparison of PROTO300™ C4 to Vydac C4 for proteins.
        Comparison of PROTO™300 C4 to Vydac C8 and Zorbax® C8 for protein digests.
      Proteomics: Fast equilibration & 100% aqueous loading on TARGA, allows an extremely wide polarity range for LC/MS/MS "no TFA" peptide separations.

      HAISIL PEEK and HAIPEEK HPLC Cartridge Columns.

    Higgins Columns Part Numbers and Prices
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MACCEL™ HPLC Columns

  • Enhanced polar selectivity on MACCEL AQPS™, a water wettable (low organic RPC) phase, with great selectivity for all polar molecules.
      Engelhard Test: Polar Selectivity of unbuffered acids and bases on AQPS phases.
      Polar Selectivity of acids and bases on AQPS phases under acidic buffered, ACN conditions.
      Optimum Conditions for acids and bases on AQPS phases: neutral buffered, MeOH conditions.

  • Compare selectivity on MACCEL Spheribond™ ODS1 and ODS2 to Waters™ Spherisorb® ODS1 and ODS2
  • Polar molecule selectivity on MACCEL Hypersorb™ ODS is very close to Thermo™ Hypersil® ODS
      Hypersorb™ ODS: Selectivity under a number of comparative conditions is very similar.
  • Maccel Analytical and Microbore HPLC columns

    MACCEL Small Molecule HPLC Application Notes

    MACCEL Part Numbers and Prices


Grace/Vydac® Small Molecule HPLC Columns

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Complex Carbohydrate Separations

Glycan Reductive Isotope Labeling (GRIL)

Using HILIC's Electrostatic and Hydrogen Donor Mechanisms for Carbohydrate Separations.

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HPLC Columns for Protein & Peptide Analysis Applications:

Detergent Removal

Technical Sheet for HILIC microSPE Detergent Removal Cartridges or SDS Removal Guard columns.

Peptide Retention Time Calculations

    Oleg Krokhin's RT Calculator. New sequence-specific correction factors for prediction of peptide retention in RP-HPLC: application to protein identification by off-line HPLC-MALDI-MS. This version has been developed for the same set of ~2000 tryptic peptides and features new correction factors related to a peptide's propensity to form helical structures. Correlation about 0.98 (R-squared value) was obtained for the training set of 2000 peptides compared to 0.96 for version 2.

Higgins Analytical HILIC and RPC Peptide Columns

    Complete Catalog (3,408K pdf)
    HILIC HAISIL® 100 Silica Columns and Cartridge (HAIPEEK) Products.

    Amino acids on HAISIL Columns:
      TARGA (low organic, 100% water wettable RPC) for DABSYL or AccQ-Tag® derivatives
      High Load for fast PTH analyses

      HAISIL PTC (ABI 711-0204) for PTC analyses

      HAISIL 100 for Pico-Tag® derivatives
    CLIPEUS RPC columns are excellent for smaller peptide, "no TFA" separations (without tailing).
    PHALANX Water-wettable, Ultra-high density coating for a wider pH range for peptide applications.

    Proteomics: Fast equilibration & 100% aqueous loading on TARGA, allows for an extremely wide polarity range for LC/MS/MS "no TFA" peptide separations.
    PROTO™ 200 C4 & C18, Peptide columns . PROTO™ 300 Protein columns.
      Comparison of PROTO300™ C4 to Vydac C4 for proteins.
      Comparison of PROTO™300 C4 to Vydac C8 and Zorbax® C8 for protein digests.
    TARGA Water-wettable, 10x faster RPC equilibration using 100% aqueous conditions, where other AQ columns need 2-5% ACN, are great for LC-MS/MS "no TFA" proteomic capture and separations of peptides using formic acid.
    "Wide pore" HAISIL® Reversed Phase (Type A Silica) HPLC Columns and Cartridge Products have been used for virus particles and protein, peptide separations.

    Polymeric RPC and Ion Exchange (Capillary - < 2mmID column configurations.)
      PL-Gel® PLRP-S, 3µm, 100Å pores, for Small Molecule, high pH applications, such as negative ion mode LC/MS; or 300Å pores for Peptide applications.
      PLRP-S, RP1K & RP4K, 5µm, with 1000Å or 4000Å pores for Protein & Antibody RPC.
      PL-SAX, PL-SCX, PL-WAX & PL-WCX, 5µm, with 1000Å or 4000Å pores for Protein and Antibody ion exchange applications.

    Higgins Columns Part Numbers and Prices

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PolyLC®

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HILIC Methods for Separating Polar Molecules by Polar Differences - Alt.: "Aqueous Normal Phase" (ANP)

HILIC Articles (alt: "Aqueous Normal Phase" (ANP)) - HILIC Is Actually Partition Chromatography with Electrostatic and Hydrogen Donor Mechanisms to Utilize for a Separation.

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SeQuant® ZIC®-HILIC Columns

    Applications for Zwitterion pHILIC on a Polymer Based Betain Sulfonate Surface
      ZIC®-pHILIC for Adenosine Phosphate Analysis (AMP, cAMP, dAMP, ADP, dATP and ATP) Using HILIC Conditions with Volatile Buffers (70% ACN, 30% Ammonium Carbonate, pH 8.8, 100 mM).
      Adenosine Phosphate Analysis (AMP, ADP, ATP, CoA, Acetyl-CoA, and Pantothenate) Using HILIC Conditions with Volatile Buffers (80% ACN, 20% 10 mM Ammonium Carbonate, 0.2% NH4OH).

    Retention Time Predictor Tool for ZIC-HILIC columns.

    ZIC-HILIC Publications
    ZIC®-HILIC & ZIC®-pHILIC General Operational Parameters Before Getting Started:
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Grace/Vydac®

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Glycomics and Proteomics Applications:

    Amino Acid Specific Peptide & Protein Isolation Spin Columns

    Glycomic Separation Techniques

      IP-NPLC [eHILIC] for high mannose and sialylated glycopeptides. "Identification and Quantification of Glycoproteins Using Ion-Pairing Normal-Phase LC and MS," Wen Ding, Harald Nothaft, Christine M. Szymanski, and John Kelly, MCP Papers in Press, June 12, 2009.

    HPLC & SPE Proteomic Separation Techniques

    • Capillary Columns for LC-MS/MS Proteomics Fractionation.
    • Comparison of SCX, TiO2, HILIC (HEA) and HILIC (WAX) for Phosphopeptide segregation and/or fractionation.
    • HILIC 2-D fractionation of peptides prior to RPC for proteomics.
    • SCX pre-columns for post ICAT™ and MudPIT (DALPC) fractionation of proteins for proteomics.
    • Use of pH and salt gradients for 2-D Nano-SCX/RPC-LC/MSMS proteomics fractionation of tryptic digests.
    • SPE MiniSpin columns and 96-Well Spin Plates for microliter sample prep. For the hardware challenged: Use MiniSpin Tips or UltraMicroSpin SCX columns to fractionate, then load into an autosampler for RPC LC-MS/MS
    • Micro filter Page*Eraser™ empty columns remove the particulates from in-gel digests to prolong capillary LC-MS column life
    • Mixed-Bed Ion Exchange for Complete Protein Capture and Fractionation
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DNA & RNA Oligonucleotide Separations:

Higgins Analytical®
    Nucleotide Analysis on Targa C18 5µm 250x4.6mm column using 15mM ammonium acetate, pH 6.0 with a gradient to 10% MeOH.
    Post Transcriptional Modification, Small RNA Nucleotide Separations (C. Callie Coombs, University of Cincinnati, Masters Thesis in Chemistry).
Hydrocell®
    Protein and Oligonucleotide separations by HIC or IEX on polymer based columns.
Macherey-Nagel®
    Nucleogen® for DNA, RNA and Oligonucleotides.
    Oligonucleotide separations on NS 1000 and DEAE-60 columns in volatile buffers.
    • DEAE-60 Macherey-Nagel Part Numbers and Prices

PAGE, DEAE technique for Oligonucleotide clean-up for LC-MS applications:
    DEAE MacroSpin column after PAGE or RPC column separation
PolyLC® SeQuant®
    ZIC®-pHILIC for Adenosine Phosphate Analysis (AMP, cAMP, dAMP, ADP, dATP and ATP) Using HILIC Conditions with Volatile Buffers (70% ACN, 30% Ammonium Carbonate, pH 8.8, 100 mM).
    Adenosine Phosphate Analysis (AMP, ADP, ATP, CoA, Acetyl-CoA, and Pantothenate) Using ERILIC Conditions with Volatile Buffers (80% ACN, 20% 10 mM Ammonium Carbonate, 0.2% NH4OH).
Grace Vydac®
    Mono-, di- and tri-phosphate Nucleotides on Vydac 302IC SAX columns.
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Dialysis:

Simple Dialysis

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Equilibrium Dialysis:

    Overview of Reusable dialyzers with replaceable membranes or Disposable Equilibrium Dialysers for Protein or DNA Binding Studies.
    Individual Equilibrium Dispo-Dialyzer®: 75µL disposable kits.
    Modular Plate 96-Well Equilibrium Dialyzer™ with individual membranes on 50-200 µL wells. New! ASME robotic dimensioned plates.
    96-Well Equilibrium Dialyser Comparison: Old vs. New Specifications.
    96-Well Equilibrium Dialyser 8 Plate Rotator picture.
    Reusable Two or Three Chamber MicroEquilibrium and FAST Micro-Equilibrium DIALYZERS™

    MicroEquilibrium and FAST Micro-Equilibrium DIALYZER™ Instructions

    Twenty-cell Multi-Equilibrium Dialyser
    Equilibrium Dialysis Formulas and Protocols (Note: 5.7M file using Harvard Apparatus p/n, not Nest p/n)

    Equilibrium Dialyzer Part Numbers & Prices








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  • Membranes
  • Dialyzer and Equilibrium Dialyzer
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    Electrophoresis Applications

    Agarose for DNA Electrophoresis

    • Agarose General Characteristics & Applications
    • EXCLU-SIEVE® Agarose: Characteristics, Applications, and Technical Data
    • EXCLU-SIEVE™ Part Numbers and Prices

      • Table Cross Referencing Agarose Products and Applications

    Electro-Elution removes a 20-50x molar excess of fluorescent dyes from labelled proteins in 20 minutes

    • ElectroPrep Gel Extractor, Protein Concentrator, Excess Dye or SDS Removal Device
    • Fast ElectroPrep for 20 minute Removal of Excess Dye from Labelled Protein with Minimal Protein Loss.

    Protein Gel Destaining

    • Cozap Removes Coomassie Blue From Protein Gels. Crystal clear gels, no blue desk tops, and less expensive to use than tissues or foam. Zap those Coomassie Blues!
    • Cozap Part Numbers & Prices
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    Knitted Open Tubular (KOT) Reactors

    Process Columns

    Adjustable, Empty Glass Process Columns. An overview of properties and capabilities.
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