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.
The Importance of Contact Time for Either Desalting or Trapping of Polar Solutes on Trap Columns.
HILIC Webinars and Podcast Training Registration.
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." 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." Selecting buffers for use in partially aqueous mobile phases.
"Mobile-Phase Buffers, Part III Preparation of Buffers." 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

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.
Volume Dependency of Retention with Partition Columns:
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.

HPLC Column Care

HIC for Proteins:
(ALKYL) Aspartamide™ Hydrophobic Intereaction Technical Sheet (Mfg. Instruction Sheet)

Trouble shooting HYDROCELL™ polymeric HIC columns.
Buffer Tables for HIC and IEX.

HILIC for Polar Differences in Molecules:
PolyHYDROXYETHYL™ Aspartamide Technical Sheet (Mfg. Instruction Sheet)
PolyGLYCOPLEX™ (for Complex Carbohydrates) Technical Sheet (Mfg. Instruction Sheet)

ZIC®-HILIC & ZIC®-pHILIC General Operational Parameters Before Getting Started:

ZIC®-HILIC Columns Technical Sheet
ZIC®-cHILIC Columns Technical Sheet
ZIC®-pHILIC Polymeric Columns Technical Sheet
ZIC®-HILIC Capillary Columns Technical Sheet
An Introduction to HILIC Chromatography, A Tutorial and Applications Manual: "A Practical Guide to HILIC"

Ion Exchange:
PolySULFOETHYL™ Aspartamide Strong Cation Exchange Technical Sheet (Mfg. Instruction Sheet)
PolyCAT A™ Weak Cation Exchange Technical Sheet (Mfg. Instruction Sheet)
PolyWAX LP™ Weak Anion Exchange, Technical Sheet (Mfg. Instruction Sheet)
PolyCAT A - PolyWAX LP, Mixed-Bed Ion Exchange Technical Sheet (Mfg. Instruction Sheet)
Trouble shooting HYDROCELL™ polymeric IEX columns.
Buffer Tables for IEX and HIC.

Nano-flow Capillary :
How to Increase Capillary Life with Higgins Caπllary™ columns.
How to Polish Capillary Ends to reduce nano-flow chromatographic system dead volume in tubing connections

Reversed Phase:
General Column Care Information
Higgins Caπllary™ column Care & Maintenance
Higgins & Vydac Column Care & Maintenance
Vydac Comparison of the All-Guard™ Cartridges to the Vydac "...GD54" Guard Cartridges

Size Exclusion (SEC):
SEC Column PolyHYDROXYETHYL Aspartamide Technical Sheet (Mfg. Instruction Sheet)

SPE Column Operating Instructions:


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Small Molecule and Combi-Chem SPE Separations

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

Cerex System-48 or Cerex System-96 for positive pressure SPE filtration or drying
View Demo Cerex System-48
View Demo Cerex System-96

Cerex SPE Cartridges and Plates for high performance (small particle) SPE. Small particle SPE means higher speed, higher loading, and smaller elution volume SPE.

TLC to Flash: Optimizing Your TLC
TLC to Flash: Estimation of Load
TLC to Flash: Flash Solvent Selction
TLC to Flash: Gradient Techniques
Cerex Part Numbers and Prices




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.
MiniSpin & 96-Well Spin Part Numbers and Prices
Operating Instructions for ERLIC, HILIC, RPC, SCX, SEC, WAX, or WCX MiniSpin products.
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
• Ultra MicroTip Part Numbers and Prices
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.

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

Effect of excessive loading volume on SPE Tips or Trap Columns. Use TARGA C18 to increase retention of polar molecules for quantitative experiments, or change loading solvent composition to increase retention.
Six-Step Method Development for Robust Solid Phase Extraction Results.
Ultra-Micro Spin RP clean-up for salt or HILIC for detergent removal from protein digests. Useful for MS applications, due to higher capacity than ZipTip® C18.
• Ultra MicroTip Part Numbers and Prices
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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
0.075 - 0.15mmID Nano-Capillaries (Caπllary)
0.32 - 0.5mmID microCapillaries (Capellini)
Short, Direct Connect, 2.1mmID Narrowbore (Sprite)
0.3-1.0mmID Microbore Guard/Trap (Piccolo)
Cartridge (HAIPEEK) Products
(Type A Silica) HPLC Columns
HAISIL 100™ products
HAISIL 300™ products
Kromasil® 100 small pore columns.
Comparison of Kromasil® to PHALANX™ for pharmaceutical method equivalency.



(Type B Silica) HPLC Columns
HAISIL RS, PROTO™ 200 C4 & C18 for Peptide applications.
Comparison of PROTO™ 200 to Grace/Vydac® C18 Peptide Columns.
Comparison of PROTO 200 for Synthetic Peptides.

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) columns.
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:
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



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.

Comparison of TARGA to BetaBasic™ for Polar Peptide Separations
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.


HAISIL PEEK and HAIPEEK HPLC Cartridge Columns.

Higgins Columns Part Numbers and Prices

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
  Spheribond™ ODS1: Selectivity is similar under a number of comparative conditions.
  Spheribond™ ODS2: Selectivity is very close under a number of comparative conditions.
  
• 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

Denali® C18 (monomeric) Ultra High Peak Symmetry Small Molecule Columns.
Vydac Application Notes

Vydac Column Care

Part Numbers & Prices "238DE™ Series".

Monolithic Columns for Small Molecules & Combi-Chem

EMD Chemicals®

Chromolith® columns catalog. Low back pressure, faster assays, longer life columns.
Chromolith® Part Numbers

HPLC Columns for Protein & Peptide Analysis Applications:

Detergent Removal

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 (3,468K pdf) 0.075 - 22mmID columns.
Catalogs of HAISIL® HPLC Columns
0.075 - 0.15mmID Nano-Capillaries (Caπllary)
0.32 - 0.5mmID microCapillaries (Capellini)
Short, Direct Connect, 2.1mmID Narrowbore (Sprite)
0.3-1.0mmID Microbore Guard/Trap (Piccolo)
HILIC HAISIL® 100 Silica Columns
Cartridge (HAIPEEK) Products.


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:

Fast equilibrating & 100% aqueous loading TARGA, allows extremely wide polarity range separations of sugars, metabolites & polar peptides, or peptide trapping for LC/MS/MS.

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.
Comparison of PROTO300 C4 for hemoglobin analyses.
Comparison of PROTO300 C4 to Vydac C4 for proteins.
Comparison of PROTO300 C4 to Vydac C8 and Zorbax® C8 for protein digests.

PROTO300 C4 for Polyvalent, Unimolecular Glycopeptide Separations.
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:

(In 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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IEX, HIC & SEC Columns for Proteins




PolyLC®

• Ion Exchange columns, Operation and Care.
• Mixed-Bed Ion Exchange for Complete Protein Capture and Fractionation
• PolyCAT A™ Weak Cation Exchange Technical Sheet
Decreasing pH Gradients for Weak Cation Exchange of Peptides
Effect of Organic Solvent on WCX of Proteins
Hemoglobin Analyses by WCX. Comparison of 3µm to 5µm columns for FACS & A1c.
Intact Protein Isolation for Proteomics Bulletin 2006
Monoclonal Antibody Variant Analysis Application Sheet
Protein Variant Analysis Application Sheet


• PolySULFOETHYL® AspartamideSCX Strong Cation Exchange Spec Sheet
SCX post ICAT™ and 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.
HILIC-SCX of Lung Surfactant Proteins from excess lipids.




• PolyWAX LP® Weak Anion Exchange, Spec Sheet
  Dropbox of ERLIC-WAX Publications
  ERLIC-WAX, Tryptic Phosphopeptide Enrichment (ASMS poster)
  ERLIC-WAX SPE Phosphopeptide SPE Instructions
  SDS Removal


• HIC (Hydrophobic Interaction)
  The Hofmeister Effect and its implications for HIC. A discussion of the ranking of various ions toward their ability to retain (precipitate) a mixture of proteins. See also, Douglas J. Tobias and John C. Hemminger, Science, 319, 1197-98 (2008) for a discussion of Specific Ion Effects regarding the hydration of ions on surfaces.
  (ALKYL) Aspartamide™ HIC Spec Sheet
  HIC Operation and Methods Development.
• PolyGLYCOPLEX™ for Complex Carbohydrate Separations, Spec Sheet
• Rapid, Sensitive Combinatorial Library Screening: 10nM Protein-Conjugate Screening by SEC
• SEC Column PolyHYDROXYETHYL Aspartamide Technical Sheet

Bibliography of PolyLC column references.

PolyLC Part Numbers and Prices

Sepax-Technologies® IEX & SEC Columns for Proteins & Biomolecules

• 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. A theoretical basis for SEC, which helps explain why Sepax SEC columns are so effective.
• Comparison of Non-Porous to Porous IEX Resins for Intact Proteins
• Fast Mab Aggregate Analysis Using Zenix® SEC Columns
• Heavy Chain & Light Chain Mab Analysis Using Zenix® SEC-300 Columns
• Mab Analyses in the Presence of Tween Detergents Using Sepax SEC Columns
• Sepax 3µm Zenix SEC Columns for Biomolecules.
• Sepax 5µm SRT SEC Columns for Biomolecules.
• Sepax SRT-C and Zenix-C (lay-down) SEC Phases Provide a Wider SEC Range of Separation for Hydrophobic Biomolecules.
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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) HILIC LC-MS/MS from plant extracts
Folic Acid Metabolites
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.

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].
Phospholipid eHILIC assay with LC-MS or ELSD detection
Phosphorylation Variants of Histone H1.1 HILIC-CEX
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
HILIC Column Comparisons
• Dropbox of ERLIC Publications.
• General HILIC Information
• Comparison of PolyHYDROXYETHYL A® to Amide-80® for small molecule HILIC LC-MS analysis of fermentation broth.
• HILIC for small polar acids, nucleic acid bases and nucleosides. A Comparison (Y. Guo & S. Gaiki, J. Chromatogr. A, 1074 (2005) 71-80.) of Amide, Amine, Silica and betain sulfonate functional groups.
• HILIC, even on raw silica, Increases LC-MS Sensitivity. LCGC article: " Hydrophilic Interaction Chromatography Using Silica Columns for the Retention of Polar Analytes [small molecules sic] and Enhanced ESI-MS Sensitivity"
• Pore Size Effects for Complex Carbohydrate Analyses

SPE HILIC
High Capacity HILIC micro-SPE tips
Operating Instructions for HILIC MiniSpin products.
UltraMicroTip columns (tips equivalent to the UltraMicroSpin columns for microliter sample prep).

PolyHYDROXYETHYL™ Aspartamide HILIC Technical Sheet



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

Bibliography of PolyLC HILIC column references.
Dropbox of ERLIC Publications.
Complex Carbohydrate ZIC- HILIC References
Evaluation of an inverted zwitterionic, ZIC-cHILIC, cholinate stationary phase for proteomic 2-D identification of more than 20000 unique peptides corresponding to over 3500 proteins.
HILIC Day, June 16, 2011, 8:30 A.M. - 4:00 P.M. at NJ Registration.
HILIC Newsletter-1108. Discusses melamine analysis in food products.
HILIC Newsletter-1208. Discusses methyl malonic acid analysis in serum, plasma and urine plus an ELSD detection method for choline and inositol.
HILIC Newsletter-0109. Discusses diethylene glycol analysis in medicine, and suggestions on how to reduce consumption of acetonitrile.
HILIC Newsletter-0309. Discusses alternatives to ACN for HILIC assays and announces the U.S. HILIC Day prior to the ASMS meeting in Philadelphia.
HILIC Newsletter-1009. Illustrates increased sensitivity for Peptide Mapping by HILIC and references Kato, et al., Underivatized Amino Acid Analysis publication: "Application of amino acid analysis using hydrophilic interaction liquid chromatography coupled with isotope dilution mass spectrometry for peptide and protein quantification."
HILIC Newsletter-0110. Approaches to the analysis of sugars and an invitation to register for HILIC Training webinars and podcasts.
HILIC Review Article: Petrus Hemstrom and Knut Irgum, J. Sep. Sci., 2006, 29, 1784 - 1821. "Hydrophilic Interaction Chromatography."
Hydrophilic interaction liquid chromatography with alcohol as a weak eluent, M. Liu, J. Ostovic, E.X. Chen, N. Cauchon, J. Chromatogr. A, Mar 2009; 1216(12): 2362-70.
Retention Time Predictor Tool for ZIC-HILIC columns.
ZIC-HILIC Publications Search

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SeQuant® ZIC®-HILIC Columns

Applications for Zwitterion HILIC on a Silica Based Betain Sulfonate Surface
"An HPLC Method for the Determination of Pharmaceutical Salts", LCGC AUG-06: Brian W. Pack and Donald S. Risley
Simultaneous Measurement of Anions & Cations: Method Validation
Poster on use of betain sulfonates bound to silica for Zwitterion HILIC separations
ZIC- HILIC Brochure for Polar Separations
Zwitterion HILIC of glucosinylates, an improved method
Zwitterion HILIC analysis of melamine in food
Zwitterion HILIC of morphine and glucuronated metabolites
Zwitterion HILIC of organophosphonate nerve agent metabolites
Zwitterion HILIC orthogonal method to RPC
Zwitterion HILIC of peptides prior to RPC. 2-D fractionation for proteomics.
Zwitterion HILIC of tryptic peptides
Zwitterion HILIC of homocysteine, methylmalonic acid and succinic acid from biological fluids
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 Part Numbers and Prices
ZIC®-HILIC & ZIC®-pHILIC General Operational Parameters Before Getting Started:
ZIC®-HILIC Columns Technical Sheet
ZIC®-pHILIC Polymeric Columns Technical Sheet

ZIC®-HILIC Capillary Columns Technical Sheet

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Grace/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.
• Denali™ C18 (monomeric) C18 for basic and acidic compounds with superior efficiency and reproducibility
  Best for Small Molecules.
  Part Numbers at "238DE™" Prices.
  
• 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.

Part Numbers at "238EV™" Prices.

• Importance of TFA Concentration for a reproducible peptide separation.
• New! Grace Vydac 0.075 and 0.150mmID Capillary Columns for "Low TFA" LC-MS operation enhanced detection of peptides.
• Mobile Phase modifier effects on bonded phase selectivity and on Low TFA MS bonded silica columns
• Monomeric C18 , 238TP RPC chemistry gives unique selectivities for hydrophobic peptides which elute in the 40-60% MeCN region of the gradient
• Process scale bulk TP and 259VHP medias
• 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
• New! Denali™ C18 (monomeric) C18 provides excellent peak symmetry for basic and acidic compounds with superior efficiency and reproducibility for Small Molecules. Prices at "238DE™ Series" Prices.
• Vydac Cross Reference Guide
• Vydac Column Care
Vydac Part Numbers and Prices
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Complex Carbohydrate Separations

Activated Charcoal SPE

Desalting and Purification of Oligosaccharides and Their Derivatives on MiniSpin columns.

C18 Desalting and Purification of Oligosaccharides

TARGA C18 SPE
Volume Limits for SPE

Glycan Reductive Isotope Labeling (GRIL)

Empty µ-Reactors™ with caps 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.

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

Approaches to the analysis of sugars by ZIC®-HILIC.
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.
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:

Amino Acid Specific Peptide & Protein Isolation Spin Columns

BioMolecular Technologies®
Pi³ Methionine
Pi³ Tryptophan
Pi³ Tyrosine Phosphate

Glycomic Separation Techniques

Activated Charcoal SPE: Desalting and Purification of Oligosaccharides and Their Derivatives on MiniSpin columns.
"Simultaneous Identification of Unmodified Tryptic Peptides, Phosphopeptides, Glycopeptides, and Deamidated Peptides" (2011 ASMS, Siu Kwan Sze Poster, Nanyang Technological University, Singapore).
Effect of Pore Size On The HILIC Separation of Glycans
IP-NPLC [e-HILIC] 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.
Comparison of Hydrazide Covalent Chromatography and ERLIC for Glycopeptide Isolation.
Polyvalent, Unimolecular Glycopeptide Separation Using PROTO300 C4 RPC.

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.
• 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.
Hydrocell Part Numbers and Prices
Trouble shooting guide.

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®
PolyHYDROXYETHYL Aspartamide™ for DeOxy RiboNucleoside Analysis Using HILIC Conditions with Volatile Buffers.
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®
Most common TSK-GEL® SEC column and ToyoPearl® media questions and answers.
Amide-80® Comparison to PolyHYDROXYETHYL A™ for small molecule HILIC LC-MS analysis of fermentation broth

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Dialysis:

Simple Dialysis

Ultra-filtration, Dialysis, or 96 Well Plates for Salt Removal & Buffer Exchange
Reusable Teflon® DIALYZER™ for fast, small volume, salt removal (10µl to 5ml)
Micro DispoDIALYZER® for fast Small Volume (10µl to 100µl) salt removal. Disposable Dialysers at a fraction of the cost of slide or cassette products.
Micro Dispo-DIALYZER (1-100µL unit) Instructions.
DispoDIALYZER™ Moderate Volume (1-10ml) Disposable Dialysers.
96-Well DispoDIALYZER™ plates.
SpinCon™ Ultra-Micro DispoDIALYZER for ultra-small volume (1-5µL) ultra-filtration of samples.
SpinCon Operating Instructions.
Flow Through Macro DIALYZER for larger volume dialysis used in conjunction with the ElectroPREP™ system.
On-line, HPLC effluent buffer exchange & micro dialysis
.
Removal of Excess Dye from Labelled Proteins in 20 minutes with Minimal Protein Loss.

Dialyzer Part Numbers & Prices

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Equilibrium Dialysis:

Overview of Reusable dialyzers with replaceable membranes or disposable equilibrium dialysers for Protein or DNA Binding Studies.
Individual DispoEquilibrium DIALYZER™: 75µL disposable kits.
Modular Plate 96-Well DispoEquilibrium DIALYZER™ with individual membranes on 50-200 µL wells. ASME robotic dimensioned plates.
96-Well DispoEquilibrium DIALYZER 8 Plate Rotator picture.
Reusable Fast Micro-Equilibrium DIALYZER™

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
  Dialysis Membrane Conditioning and storage
  Dialysis Membrane MSDS data
  Dialysis Membrane Overview
  Dialysis Membrane Solvent Compatibility
  Membrane Part Numbers & Prices
  
  
• Dialyzer and Equilibrium Dialyzer
  Bibliography of Equilibrium Dialysis Applications
  Equilibrium Dialysis Formulas and Protocols (Note: 5.7M file using Harvard Apparatus p/n, not Nest p/n)
  
  Dialyzer and Equilibrium Dialyzer Prices