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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.


    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.

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.
    Catalogs of HAISIL® HPLC Columns
      Short, Direct Connect Narrowbore (Sprite), Microbore Guard/Trap (Piccolo), and/or Cartridge (HAIPEEK) Products.
      Complete Catalog or individual sections:
      (Type A Silica) (Type B Silica)
        HAISIL RS PROTEO™ 200 C4 & C18 peptide columns or in LC-MS/MS Capillary (Capellini™), Guard/Trap 0.3 - 1mmID (Piccolo™) or Direct Connect Ballistic Gradient, 2mmID (Sprite™) configurations.
          Comparison of PROTO™ 200 to Grace/Vydac® C18 Peptide Columns.

        HAISIL RS PROTO™ 300 protein columns or in LC-MS/MS Capillary (Capellini™), Guard/Trap 0.3 - 1mmID (Piccolo™) or Direct Connect Ballistic Gradient, 2mmID (Sprite™) configurations.
        HAISIL TS Targa ® polar analyte columns or in LC-MS/MS Capillary (Capellini™), Guard/Trap 0.3 - 1mmID (Piccolo™) or Direct Connect Ballistic Gradient, 2mmID (Sprite™) configurations.
        HAISIL CS Clipeus® columns or in LC-MS/MS Capillary (Capellini™), Guard/Trap 0.3 - 1mmID (Piccolo™) or Direct Connect Ballistic Gradient, 2mmID (Sprite™) configurations.
        HAISIL VS HEAVY® Ultra-high density coating for wide pH range applications as columns or in LC-MS/MS Capillary (Capellini™), Guard/Trap 0.3 - 1mmID (Piccolo™) or Direct Connect Ballistic Gradient, 2mmID (Sprite™) configurations.
        HAISIL HL High Load High density coating as columns or in LC-MS/MS Capillary (Capellini™), Guard/Trap 0.3 - 1mmID (Piccolo™) or Direct Connect Ballistic Gradient, 2mmID (Sprite™) configurations.
        HAISIL PS Phalanx Water-wettable, Ultra-high density coating for wide pH range applications as columns or in LC-MS/MS Capillary (Capellini™), Guard/Trap 0.3 - 1mmID (Piccolo™) or Direct Connect Ballistic Gradient, 2mmID (Sprite™) configurations.





      (Polymers: RPC and Ion Exchange)
        PL Gel, 3µm, PLRP-S with 100Å or 300Å pores for high pH applications, such as negative ion mode LC-MS/MS in Capillary (Capellini™), Guard/Trap 0.3 - 1mmID (Piccolo™) or Direct Connect Ballistic Gradient, 2mmID (Sprite™) configurations.
        PL Gel, 5µm, PL-SCX and PL-SAX for protein ion exchange applications for LC-MS/MS Capillary (Capellini™), Guard/Trap 0.3 - 1mmID (Piccolo™) or Direct Connect Ballistic Gradient, 2mmID (Sprite™) configurations.


  • Applications for HAISIL Columns
  • Amino acids on HAISIL Columns:
  • Clipeus (WOW! selectivity) in C8, C18, CN or Phenyl phases that do not require base deactivation to chromatograph small polar amines without tailing; or for LC-MS/MS "no TFA" peptide separations.
  • Combi-Chem analyses of basic molecules on HAISIL HL "High Load" columns
  • PROTO™ 200 C4 & C18 Peptide Columns or in LC-MS/MS Capillary (Capellini™), Guard/Trap 0.3 - 1mmID (Piccolo™) or Direct Connect Ballistic Gradient, 2mmID (Sprite™) configurations.
      • Comparison of PROTEO™ C18 to Other Columns for Phosphopeptides.
  • PROTO™ 300 C4 & C18 Protein columns or in LC-MS/MS Capillary (Capellini™), Guard/Trap 0.3 - 1mmID (Piccolo™) or Direct Connect Ballistic Gradient, 2mmID (Sprite™) configurations.
      • Comparison of PROTO300™ C4 to Vydac C4 for proteins.
  • Targa. for fast equilibration using 100% aqueous conditions for an extremely wide polarity range of compounds; or for LC-MS/MS "no TFA" peptide separations.
  • HAISIL PEEK and HAIPEEK HPLC Columns and Cartridges.

    • 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

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

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Protein, Peptide, & Multi-Ring Cpd. HPLC and Capillary Column 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

  • HILIC HAISIL® 100 (Type A Silica) Silica Columns and Cartridge (HAIPEEK) Products.
  • Clipeus (Type B Silica) RPC columns are excellent for the smaller range of LC-MS/MS "no TFA" tryptic map, peptide separations.
  • PROTO™ 200 C4 & C18 peptide columns (Type B Silica) or in LC-MS/MS Capillary (Capellini™), Guard/Trap 0.3 - 1mmID (Piccolo™) or Short, Direct Connect, Ballistic Gradient, 2mmID (Sprite™) configurations.
      • Comparison of PROTEO 200 to Grace/Vydac® Peptide Columns.
  • PROTO™ 300 protein columns (Type B Silica) or in LC-MS/MS Capillary (Capellini™), Guard/Trap 0.3 - 1mmID (Piccolo™) or Short, Direct Connect, Ballistic Gradient, 2mmID (Sprite™) configurations.
      • Comparison of PROTO300™ C4 to Vydac C4 for proteins.
  • Targa. (Type B Silica) for 10x faster RPC equilibration with 100% aqueous conditions where other AQ columns have been applied are also great for LC-MS/MS "no TFA" peptide separations of peptides using formic acid.
  • Phalanx (Type B Silica) Ultra-high density coating for wide pH range applications and for 10x faster RPC equilibration with 100% aqueous conditions. Available as columns or in LC-MS/MS Capillary (Capellini™), Guard/Trap 0.3 - 1mmID (Piccolo™) or Short, Direct Connect, Ballistic Gradient, 2mmID (Sprite™) configurations.
  • "Wide pore" HAISIL® Reversed Phase (Type A Silica) HPLC Columns and Cartridge Products have been used for virus particles and protein, peptide separations.

    • 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).
    ZIC-HILIC Publications
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Grace/Vydac®

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Proteomics Fractionation Applications:

    Amino Acid Specific Peptide & Protein Isolation Spin Columns

    HPLC & SPE 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
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DNA & Oligonucleotide Applications:

Higgins Analytical®
    Nucleotide Analysis on Targa C18 5µm 250x4.6mm column using 15mM ammonium acetate, pH 6.0 with a gradient to 10% MeOH.
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 Chamber and FAST 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 & SDS Removal

    • ElectroPrep Gel Extractor, Protein Concentrator, and SDS removal device

    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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