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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.
    Effect of excessive loading volume on SPE Tips or Trap Columns: Use of 100% water with TARGA C18 increases retention of polar molecules for quantitative experiments.
    Eliminate tailing due to metals chelating sample on frits or MS emitter tips with a 50mM disodium EDTA treatment (an off-line, non-volatile treatment), or more conveniently, with acetylacetonate in the mobile phase.
    The Importance of Contact Time for Either Desalting or Trapping of Polar Solutes on Trap Columns.
    An Introduction to HILIC Chromatography, A Tutorial and Applications Manual: "A Practical Guide to HILIC"
    An Introduction to Ion Chromatography and trouble shooting manual: "A Practical Guide to Ion Chromatography"
    Measuring pH in aqueous/organic mixtures [(H-0): Bosch et al., Anal. Chem. 68 (1996) 3651-3657]:
      Use of the s(w)pH scale method. [(H-00): 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.
    Monitoring the effects of pH and coulombic interaction on selectivity for ionic HILIC phases with organic acids.

    Molarity of Concentrated Reagents
      Table of Dilutions to make 1 Molar Solutions of Acids & Bases for LC-MS or Ion Exchange

    Retention Time Predictor Tool for ZIC-HILIC columns.
    Sample Loading and Gradient Modification Data Tables for Column Dimension Changes.
    Size Exclusion: A theoretical basis for SEC. Barth, H.G. and Saunders, G.D., "Fundamentals and Properties of Size-Exclusion Chromatography Packings and Columns", Supplement to LCGC, Recent Developments in LC Column Technology, April 2012, 46-53.
    Solid Phase Extraction: Six-Step Method Development (adapted, with thanks to Prof. Michael Burke, Univ. AZ) for robust results.
    Volatile Buffers for LCMS or ELSD systems.
    Volume Dependency of Retention with Partition Columns:
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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.
    Removal of phospholipids using HILIC on silica SPE cartridges with acetone as eluent for eliminating matrix effects in the analysis of biological fluids when using RPC LC-MS.

Volume Dependent Results for Retention with Tip Columns for MS µ-SPE:

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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 (3.67 MB pdf) 0.075 - 22mmID columns.
    Catalogs of HAISIL® HPLC Columns (Type A Silica) HPLC Columns (Type B Silica) HPLC Columns
      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.




Applications for HAISIL 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

Grace/Vydac® Small Molecule HPLC Columns

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Monolithic Columns for Small Molecules & Combi-Chem

EMD Chemicals®

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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


Complex Carbohydrate Separations

Activated Charcoal SPE

C18 Desalting and Purification of Oligosaccharides

Glycan Reductive Isotope Labeling (GRIL)

Using HILIC's Electrostatic (e-HILIC) and Hydrogen Donor Mechanisms for Carbohydrate Separations.

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Glycomics and Proteomics Applications:

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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®
    ERLIC separation using a PolySULFOETHYL™ A, SCX column and phosphate buffer conditions.
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.
TSK-GEL® Go back to menu.

Dialysis:

Simple Dialysis

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Equilibrium Dialysis:

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  • Membranes
  • Dialyzer and Equilibrium Dialyzer Go back to menu.

    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) Reaction Delay & Mixing Coils

    Process Columns

    Adjustable, Empty Glass Process Columns. An overview of properties and capabilities.
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    Last Updated: 09/26/14

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