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Table of Contents

Applications:

Desalting Columns

Dialysis

Dye Removal

Electrophoresis

e-HILIC

Glycomics

HIC

HILIC

Ion Chromatography

Organic Acids

Peptides & Proteins

Process Columns

Small Molecules

Company

Archives

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Training

    Basics of Dialysis and Ion Exchange
    Brief Overview of Polypeptide Purification Techniques
    Designing a purification protocol using ion exchange resins
    "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.
    Buffers for LCMS or ELSD systems.
    Buffer Preparation Calculator (by Professor Robert J Beynon).
      Choose the buffer species you want to use. Enter parameters for volume, pH, and concentration of buffer species, with an option to modify the ionic strength by addition of neutral salt. Finally, enter the temperature at which you'll use the buffer, and the temperature at which you'll make it up. All in all and excellent tool you'll find useful.
    Buffer Preparation Misconception: "Addressing a Common Misconception: Ammonium Acetate as Neutral pH "Buffer" for Native Electrospray Mass Spectrometry." Konermann, L., J Am Soc Mass Spectrom. 2017 Sep;28(9):1827-1835. doi: 10.1007/s13361-017-1739-3. Epub 2017 Jul 14.
    Detergents & Buffers for HIC or IEX separations.
    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.
    The Importance of Detector Response Time when using Superficially Porous Packings: If not fast enough, at low concentrations, narrow peaks appear to give non-linear responses.
    Improving LC–MS Sensitivity S. Lupo, LCGC North America, Volume 36, 652–660, (2018).
    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.
      "Mobile-Phase Buffers, Part I: The Interpretation of pH in Partially Aqueous Mobile Phases." G. William Tindall, LCGC 2002 20 (11), 1028-1032. A pH change occurs when organic solvent is present and how silica columns can be used at a higher pH.
      "Mobile-Phase Buffers, Part II Buffer Selection and Capacity." G. William Tindall, LCGC 2002 20 (12), 1114-1118. Selecting buffers for use in partially aqueous mobile phases.
      "Mobile-Phase Buffers, Part III Preparation of Buffers." G. William Tindall, LCGC 2003 21 (1), 28-32. The importance of using the weights-and-volumes format.
    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

    Relative Strength of Salts on Retention in HILIC Separations. A.J. Alpert, "Effect of salts on retention in hydrophilic interaction chromatography", J. Chromatogr. A, 1538 (2018) 45–53.
    Retention Time Predictor Tool for ZIC-HILIC columns.
    Scale-up Tables for Sample Loading and Gradient Modification 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 SPE Separations

TLC to Flash: Optimizing Your TLC
TLC to Flash: Estimation of Load
TLC to Flash: Flash Solvent Selction
TLC to Flash: Gradient Techniques

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.&pren;  
    Complete Catalog (9 MB pdf) 0.075 - 50mmID columns.
    HAISIL® HPLC Column configurations Cartridge Column Products
    (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 ® Fast Equilibrating 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:
        Amino acids on HAISIL Columns:
          TARGA (low organic, 100% water wettable RPC) for DABSYL or AccQ-Tag® derivatives
          High Load for fast PTH analyses by conventional HPLC
          PTH columns for an ABI® PTH amino acid sequence analyzer
          HAISIL PTC (ABI 711-0204) for PTC analyses
          HAISIL 100 for Pico-Tag® derivatives
        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.
        Aromatic Organic Compound analyses without buffer
        Combi-Chem analyses of small molecules on Heavily Loaded HAISIL columns

        Metabolomics & Proteomics:

        Fast equilibrating & 100% aqueous loading TARGA, allows extremely wide polarity range separations of sugars, metabolites & polar peptides, or peptide trapping for LC/MS/MS or trapping.
        PROTO™ 200 C4 & C18 Peptide Columns
          Comparison of PROTO™ 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.
      Higgins Columns Part Numbers and Prices

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    Grace/Vydac® Small Molecule HPLC Columns

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    Protein & Peptide Analysis Applications:

    Antibody Concentration and Desalting

      ConSep™ Antibody Concentration and Desalting Columns (115µL --> 20µL without membrane concentration nor spinning).
      HisSep™ spin column optimized for rapid and gentle removal of histidine from antibody formulation buffers.
      mAbSep™ An easy-to-use, high recovery, totally non-denaturing method to buffer exchange, remove salts & small molecules from monoclonal antibodies, antibody drug conjugates (ADC), or high molecular weight protein solutions.

      HIC HPLC Analysis Antibody Drug Conjugates

    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 Columns

      Complete Catalog (7.8 MB pdf) 0.075 - 22mmID columns.
      HAISIL® Product Sheets
      Applications for HAISIL HILIC & Peptide and Protein Columns:
      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).

      Metabolomics & Proteomics:

      Reduce LC-MS Equilibration Time Costs!

      Fast equilibrating & 100% aqueous loadable TARGA® C18, allows extremely wide polarity range separations of sugars, metabolites or peptides. Packed or custom packed in Caπllary™ 0.075 - 0.15mmID capillaries for LC/MS/MS or trapping.

        Combinatorial Library Screening:
        10nM Protein-Conjugate Screening using SEC-RPC on narrowbore 50mmID columns.
        ∼12K/wk, SEC-RPC 10nM Protein-Conjugate Screening.
        SEC-Native MS Protein-Conjugate Affinity Screening.
        RNA-ALIS: Methodology for screening soluble RNAs as small molecule targets.
        Strategies for the Discovery and Optimization of Small Molecule Ligands for PCSK9 . One hit out of > 200,000 compounds screened was good enough to permit making analogues even more effective for the purpose. Petrilli et al., 2019, Cell Chemical Biology 26, 1–9, December 19, 2019 © 2019 Elsevier Ltd.
        SEC Column PolyHYDROXYETHYL Aspartamide Technical Sheet

        Comparison of TARGA to BetaBasic™ for Polar Peptides.
        Sirt5 deacylase activity: Using short TARGA, Sprite columns to monitor a NAD-Dependent Protein Lysine Demalonylase and Desuccinylase.
      PHALANX Water-wettable, Ultra-high density coating for a wider pH range for peptide applications.


      PROTO™ 200 C4 & C18, Peptide columns .
        Comparison of PROTEO 200 to Grace/Vydac® Peptide Columns.
        Comparison of PROTO 200 for Synthetic Peptides.
        Greater capacity of PROTO 200 C4, 200Å compared to 300Å, C18 for peptide retention.
      PROTO™ 300 Protein columns.
      TARGA, a water-wettable RPC chemistry , allows 10x faster equilibration using 100% aqueous conditions, whereas other AQ columns need 2-5% ACN. Great for LC-MS/MS "no TFA" proteomic separations of peptides using formic acid as a modifier.

      Higgins Columns Part Numbers and Prices

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    IEX, HIC, RPC & SEC Columns for Proteins & Antibody Drug Conjugates


    PolyLC®


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    Sepax-Technologies®

    IEX, RPC & SEC Columns for Biomolecules & Antibody Drug Conjugates Go back to menu.

    Vydac®

    • Application Notes for Vydac columns
    • Vydac Column Care
    • Vydac TP (polymeric bonded phase) columns for peptides
        • Comparison of 218TP to PROTO ™ 200 C18 Peptide Columns for 20-30 amino acid peptides.
        Comparison of PROTO300™ C4 to Vydac C4 for proteins.
    • Everest® C18 (monomeric) Ultra High Resolution Protein & Peptide Tryptic Mapping Columns.
        • Comparison using formic acid to 100Å (LC Packings) column.
        Comparison to PROTO™ 200 C18 Peptide Columns for Phosphopeptides.
    • Importance of TFA Concentration for a reproducible peptide separation.
    • Mobile Phase modifier effects on bonded phase selectivity and on Low TFA MS bonded silica columns
    • Proteomic applications on "low TFA" MS media in prepacked capillary columns from 0.075mmID - 4.6mmID.
    • Validation of Reverse Phase HPLC separations (TFA, PO4, HOAc, HCl) Spec. Sheet
        • Vydac Part Numbers and Prices
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    Amino Acid Specific Peptide & Protein Isolation Spin Columns

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

      Amino Acids (underivatized) from blood for tissue transplant rejection monitoring
      Amino Acids (underivatized) from plant extracts: neutral HILIC column LC-MS/MS
      Amino Acids (underivatized) from plant extracts: Ionic HILIC column LC-MS
      Amino Acids (underivatized) metabolomics

    Dropbox of ERLIC Publications

      ERLIC-MS Peptide Deamidation detection: P. Hao, et al. "Enhanced Separation and Characterization of Deamidated Peptides with RP-ERLIC-Based Multidimensional Chromatography Coupled with Tandem Mass Spectrometry", dx.doi.org/10.1021/pr201048c | J. Proteome Res. 2012, 11, 1804-1811.

    Folic Acid Metabolites
    Gentamycin (aminoglycoside) antibiotics with LC-MS Detection.
    Glucosinolates
    Glycopeptides
    Glycopeptide Tryptic Mapping , LC-MS
    Melamine analysis in food
    Metabolomics Profiling
    Metabolomics HILIC Comparison to RPC (Tolstikov paper).
    Nucleoside Analysis with Volatile Buffers.
    Nucleotide Analysis using Electrostatic Repulsion Hydrophilic Interaction (ERLIC) [alt. eHILIC].
    Organic Acids pH Effects in HILIC application note
    Organic Acids in foods
    Phospholipid Class Separation on iHILIC® Fusion Columns with LC-MS detection
    Phospholipid eHILIC assay with LC-MS or ELSD detection
    Phospholipid removal 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.
    Phosphorylation Variants of Histone H1.1 HILIC-CEX
    Phosphopeptide Isolation using BioPureSPN™ Graphite SPE
    Phosphopeptide Isolation using Electrostatic Repulsion Hydrophilic Interaction (ERLIC) [alt. e-HILIC].
    Polar Molecules HILIC
    Polar Molecules HILIC LC-MS/MS
    Small Molecule (urea, allantoin, lysidone, amino adipic acid, pipicolic acid) applications
    ERLIC vs. HILIC Column Comparisons
    SPE HILIC
    PolyHYDROXYETHYL™ Aspartamide HILIC Technical Sheet

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    HILIC Articles (alt: "Aqueous Normal Phase" (ANP)) - HILIC Is Partition Chromatography with Electrostatic (e-HILIC) and Hydrogen Donor Mechanisms Utilized for Separations.

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    iHILIC® Fusion & iHILIC®-(P) Classic™

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    SeQuant® ZIC®-HILIC Columns

      ZIC-cHILIC, cholinate stationary phase for proteomic 2-D identification of more than 20000 unique peptides corresponding to over 3500 proteins.
      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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    Complex Carbohydrate Separations

    Activated Charcoal SPE

    C18 Desalting and Purification of Oligosaccharides

    Glycan Reductive Isotope Labeling (GRIL)

    HILIC Electrostatic and Hydrogen Donor Mechanisms for Carbohydrate Separations.

      - Isolation of O-Linked Glycans using iSPE® HILIC (Anal. Chem. 2020, 92, 3758−3768)
      - Nature Protocols, (2020) Glycomic Chacterization. Recommends iSPE® HILIC for (Steps 100–117) Glycoproteomic analysis: protein digestion, enrichment, and MS analysis.
      - Desalting and Purification of N-glycan Derivatives by HILIC SPE (Courtesy of New England BioLabs).
      - Comparison of 100Å to 200Å Pores for the separation of monosaccharides and neutral oligosaccharides.
      - Dramatic effect of a 200Å or greater Pore Size on the HILIC separation of derivatized sialylated complex glycans.
      - ERLIC conditions for Sialylated or sulfated carbohydrates.
      - iSPE® HILIC SPE Glycopeptide Enrichment shows favorable results: "Using this technique, we observed multifold increases in the number and abundance of glycopeptides identified and more than 90% enrichment of glycopeptides over non-glycosylated peptides in the cell membrane fraction. In addition, iSPE®-HILIC resolved glycosites previously unidentified using conventional zwitterionic ZIC®-HILIC." Diane Dayoung Park, et al., Chem. Sci., 2018, 9, 6271 "Membrane glycomics reveal heterogeneity and quantitative distribution of cell surface sialylation" DOI: 10.1039/c8sc01875h .
      - 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.
      - PolyGLYCOPLEX™, PolyWAX HipH™, & PolySAX™ HipH for Carbohydrate HPLC.

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    Glycomics and Proteomics Applications:

    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.
    • Comparison of eHILIC Solvent Conditions Using PolyWAX LP™ (column or SPE) for "Simultaneous Identification of Unmodified Tryptic Peptides, Phosphopeptides, Glycopeptides, and Deamidated Peptides" (2011 ASMS, Siu Kwan Sze Poster, Nanyang Technological University, Singapore).
    • Comprehensive Phosphopeptide Enrichment Strategy
    • eHILIC (ERLIC) 2-D fractionation of peptides vs. SCX prior to RPC for proteomics
    • ERLIC for shotgun proteomics: "[sic] comprehensive profiling of rat kidney proteome", P. Hao, et al., Journal of Proteome Research (impact factor: 5.11). 06/2010; 9(7):3520-6. DOI:10.1021/pr100037h.
    • 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.
    • - iSPE® HILIC SPE Glycopeptide Enrichment shows favorable results: "Using this technique, we observed multifold increases in the number and abundance of glycopeptides identified and more than 90% enrichment of glycopeptides over non-glycosylated peptides in the cell membrane fraction. In addition, iSPE®-HILIC resolved glycosites previously unidentified using conventional zwitterionic ZIC®-HILIC." Diane Dayoung Park, et al., Chem. Sci., 2018, 9, 6271 "Membrane glycomics reveal heterogeneity and quantitative distribution of cell surface sialylation" DOI: 10.1039/c8sc01875h .
    • SPE BioPureSPN Spin columns and 96-Well Plates for microliter sample prep. For the hardware challenged: Use BioPureSPN HIL-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).
    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).
    HILICON® Fusion & Fusion(P)
      Mono-, di- and tri-phosphate Nucleotides on iHILIC® Fusion ERLIC columns.
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    Dialysis:

    Simple Dialysis

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    Equilibrium Dialysis:

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    Membranes


  • Dialyzer and Equilibrium Dialyzer Go back to menu.

    Electrophoresis 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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