Agilent Community
Agilent Community
  • User
  • Site
  • Search the Community
  • User
Consumables
  • Technical Areas
Consumables
Wiki Carbohydrate Analyses in LC
  • Announcements
  • Forum
  • Files
  • Wiki
  • More
  • Cancel
  • Consumables
  • A Beginner’s Guide to Hydrophobic Interaction Chromatography
  • A simple tip to protect your columns
  • A Tip for Preparing Robust and Consistent Mobile Phases
  • Achieve accurate and repeatable gas flow meter measurements
  • AdvanceBio Columns Blog Series
  • Agilent 123 Meter Sodium probe maintenance
  • Agilent Collection of Columns, Supplies, and Standards Resources
  • Analysis of microplastics in the environment
  • Analyzing ADCs by HIC
  • Avoiding downtime in the lab: top tips for GC/MS success
  • Best Practices for Aqueous Mobile Phases
  • Best Practices for Making Good Connections
  • Bio LC Column User Guides
  • Bursting Tubing and Columns (GC and HPLC)
  • Calculate the Maximum Allowable Working Pressure for Tubing
  • Calculating Column Volume
  • Cannabis Potency Testing: a Reliable, Cost-Effective Method
  • Carbohydrate Analyses in LC
  • Checking Your Intuition - Sub 2 µm vs Superficially Porous
  • Choosing the right GC Injection Technique
  • Choosing the right pore size for size exclusion chromatography
  • Columns, Supplies, and Standards Knowledgebase
  • Consumables Applications and Workflows
  • Consumables Recommended Supplies Lists for Agilent.com
  • Custom Product Request
  • Extreme Makeover – Derivatizations in Chromatography – Part 1 GC
  • Extreme Makeover – Derivatizations in Chromatography – Part 2 LC
  • Fake It Until You Make It: When BioInert Isn’t an Option
  • Flipping Amino Acid Analysis on Its Head
  • Glycans at a glance:  Analyzing therapeutic glycoproteins
  • Handle and Care of Syringes
  • Help! My Peaks Look Strange - Fronting and Tailing in GC
  • Help! My Peaks Look Strange - Saddle Points - LC/GC Troubleshooting
  • How do I select a Split/Splitless liner?
  • Hydrophobic Interaction Chromatography of Proteins and mAbs
  • Importance of Silica Particle Strength for Sub-2 µm SEC Columns
  • KB: Ferrules recommended for GC self-tightening column nut
  • LC and LC/MS Columns - USP Designations
  • LC Column User Guides
  • LC Method Translation - the Dwell Volume
  • Minimize spectroscopy workflow disruptions
  • Minimizing Metals for Best HILIC Results
  • More than just a drink: Analyzing the elemental composition of beer
  • Multi-Attribute Methods – Peptide Mapping Part IV
  • Must See Webinars
  • Nomenclature of CFC's/Freons/Halons/Coolants
  • Oligonucleotide Analysis - Unexpected Details Matter
  • Optimizing Bonding Chemistry for Sub-2 µm SEC Particles
  • Pass the Salt, Please – Mobile Phase Preparation for HIC
  • Pesticides and their stability during GC analyses
  • Pre-Columns - the forgotten art of using retention gaps
  • Problematic polar analytes? Hello HILIC…
  • Protecting your laboratory productivity
  • Recommended Reading
  • Sample Prep Pointers - Peptide Mapping Part I
  • Save your results with sample filtration
  • Simplified cone inspection with the new Agilent LED measuring magnifier
  • Software - Supported Method Development - The Scanview Application
  • Software tool for the ADM Flow Meter (G6691A)
  • Stay Safe: A Win-Win for Solvent Storage
  • Streamline your sample processing
  • The importance of chemical composition for vial performance
  • Tips & Tricks for Amino Acid Analysis – Part I
  • Tips & Tricks for Amino Acid Analysis – Part II
  • Tips & Tricks for Amino Acid Analysis – Part III
  • Tips & Tricks for Amino Acid Analysis – Part IV
  • Tips for Smooth Sailing with HIC
  • Troubleshooting HPLC autosamplers
  • Troubleshooting HPLC degassers
  • Troubleshooting Sequence Coverage – Peptide Mapping Part III
  • UltiMetal Plus Flexible Metal Ferrule
  • UV, MS, TFA, and Formic Acid – What to use? Peptide Mapping Part II
  • What are the typical % Gain or EHT values for hollow cathode lamps?
  • You Need Lamps or Chemical Standards for Atomic Absorption Single-Element Analyses?
Still Need Help?

Post your question in our User Forum or Contact Support.

Carbohydrate Analyses in LC

Created by Dr. Know Dr. Know 11 months ago | Last modified by Dr. Know Dr. Know 11 months ago

INTRODUCTION

Columns for carbohydrate separations use different chromatographical partitioning techniques, which separate molecules based on a number of different chemical characteristics. The most common mechanism is Ion-Exclusion Partition Chromatography (IEPC) or Ion-Moderated Partition Chromatography (IMPC).

The resin is a polystyrene-divinylbenzene co-polymer with bonded propyl benzenesulfonic acid groups, which can be loaded with a cation (sodium, calcium, lead). The negatively charged sulfonic acid groups and the positively charge metal ions form a kind of semi permeable membrane, which repels ionic species, but is permeable for neutral ones. The eluent in the resin is the second (stationary) phase and neutrals part between the eluent and the "resin-eluent". This partitioning is stimulated for carbohydrates by ligand exchange effects (see below).

Possible interactions within the "resin eluent" are:

  • HYDROGEN BONDING
  • NORMAL AND/OR REVERSED PHASE INTERACTIONS
  • SIZE EXCLUSION EFFECTS

    Capability to separate mono-, di- and oligo-saccharides:


  • LIGAND EXCHANGE



COLUMN CHOICE BASED ON APPLICATION

The Hi-Plex product range includes media that meets the USP classification for L17, L19, L34 and L58 and column dimensions as required for the specified methods.

The table below lists the main application areas with the appropriate column recommendations.

Application Areas Recommended Column
USP methods specifying L17 media Hi-Plex H
USP methods specifying L19 media Hi-Plex Ca
Hi-Plex Ca (Duo)
USP methods specifying L34 media Hi-Plex Pb
USP methods specifying L58 media Hi-Plex Na
Hi-Plex Na (Octo)
Mono-, di-, tri-saccharides Hi-Plex Ca
Hi-Plex Pb
Hi-Plex H
Hi-Plex Na (Octo)
Anomer separations Hi-Plex Ca
Organic acids Hi-Plex H
Alcohols Hi-Plex Ca
Hi-Plex K
Hi-Plex H
Hi-Plex Pb
Adulteration of food and beverages Hi-Plex Ca
Hi-Plex Pb
Food additives Hi-Plex Ca
Hi-Plex Pb
Dairy products Hi-Plex Ca
Hi-Plex H
Sweetened dairy products Hi-Plex Pb
Confectionery Hi-Plex Ca
Hi-Plex Pb
Fruit Juice Hi-Plex Ca
Wine Hi-Plex H
Wood pulp hydrolysates (cellulose/hemi-cellulose) Hi-Plex Pb
Fermentation monitoring Hi-Plex H
Oligosaccharides Hi-Plex Na
Samples with high salt content, (molasses) Hi-Plex Na (Octo)
Oligosaccharides <Dp5 with monosaccharides Hi-Plex Ca (Duo)
Corn syrups Hi-Plex Na

RETENTION TIMES OF COMMON CARBOHYDRATES

Where there are several options the retention times of the actual sample components should be compared for each column to identify the columns with the best separation. An aminopropyl column (polar interactions) has been added for comparison reasons.

Compound

Retention (mins)

Ca Ca (Duo) K Pb H Na (Octo) Aminopropyl
300 mm x 7.7 mm 300 mm x 7.7 mm 300 mm x 7.7 mm 300 mm x 7.7 mm 300 mm x 7.7 mm 300 mm x 7.7 mm 250 mm x 4.6 mm
Water with 0.005N Sulfuric Acid ACN/H2O 3:1
(0.6 mL/min, 85 ºC) (0.4 mL/min, 85 ºC) (0.6 mL/min, 85 ºC) (0.6 mL/min, 70 ºC) (0.6 mL/min, 70 ºC) (0.6 mL/min, 70 ºC) (1.0 mL/min, ambient)
Adonitol (Ribitol) 15.2 14 10.3 20.9 11.5 11 -
Arabinose 13.7 13.7 12.7 16.7 11.4 12.4 7.5
Arabitol 17.5 15.6 10.5 30.5 11.7 11.3 7.2
Dulcitol 19.7 16.6 10.1 45.9 11 11 9
Erythritol 15.5 14.7 10.9 20.7 12.7 11.7 5.9
Fructose 13.5 13.3 11.6 19.3 10.6 11.6 8.3
Fucose 13.8 13.7 12.5 16.7 12.2 12.3 -
Galactose 12.3 12.4 11.5 15.3 10.7 11.3 10.3
Glucose 11.1 11.4 10.9 13.3 9.95 10.6 9.8
Glycerol 16.1 15.5 11.8 19.6 14.1 12.7 not recommended
Lactose 9.68 9.77 8.6 12.1 8.5 8.58 19.5
Maltitol 12.7 11.4 8.19 27.1 8.78 8.66 15.5
Maltose 9.34 9.59 8.54 11.7 8.4 8.5 17.4
Maltotriose 8.46 8.76 7.55 11.1 7.7 7.61 31
Mannitol 17.4 15.1 9.96 30.7 11 10.6 9.2
Mannose 12.6 12.7 11.9 20 10.5 11.4 9.1
Melezitose 8.29 8.56 7.22 9.86 *8.33 7.27 -
Raffinose 8.46 8.66 7.31 10.4 *8.2 7.38 29.7
Rhamnose 12.7 12.7 11.2 19.8 11.6 11 -
Sorbitol 21.5 17.2 10.3 - 11.1 11.2 9
Stachyose 7.82 8.16 6.77 9.84 7.4 6.83 67.3
Sucrose 9.25 9.51 8.24 11 *9.8 8.37 14
Xylitol 20.3 17.5 10.9 42.7 11.9 11.9 7.3
Xylose 12.1 12.5 11.8 14.4 10.6 11.5 -
* denotes partial hydrolysis

PART NUMBERS

Product Crosslink Content Particle Size Counter Ion Column Dimensions Part Number
Columns
Hi-Plex Ca 8% 8 µm Ca2+ 300 mm × 7.7 mm PL1170-6810
Hi-Plex Ca 8% 8 µm Ca2+ 250 mm × 4.6 mm PL1570-6810
Hi-Plex Ca (Duo) 8% 8 µm Ca2+ 300 mm × 6.5 mm PL1F70-6850
Hi-Plex Ca USP L19 8% 8 µm Ca2+ 250 mm × 4.0 mm PL1570-5810
Hi-Plex Pb 8% 8 µm Pb2+ 300 mm × 7.7 mm PL1170-6820
Hi-Plex Pb USP L34 8% 8 µm Pb2+ 100 mm × 7.7 mm PL1170-2820
Hi-Plex K 8% 8 µm K+ 300 mm × 7.7 mm PL1170-6860
Hi-Plex H 8% 8 µm H+ 300 mm × 7.7 mm PL1170-6830
Hi-Plex H 8% 8 µm H+ 300 mm × 6.5 mm PL1F70-6830
Hi-Plex H 8% 8 µm H+ 220 mm × 4.6 mm PL12506
Hi-Plex H USP L17 8% 8 µm H+ 100 mm × 7.7 mm PL1170-2823
Hi-Plex Na 4% 10 µm Na+ 250 mm × 4.6 mm PL1571-6140
Hi-Plex Na 4% 10 µm Na+ 300 mm × 7.7 mm PL1171-6140
Hi-Plex Na (Octo) 8% 8 µm Na+ 300 mm × 7.7 mm PL1170-6840
Guard Columns
Hi-Plex Ca 8% 8 µm Ca2+ 50 mm × 7.7 mm PL1170-1810
Hi-Plex Ca (Duo) 8% 8 µm Ca2+ 50 mm × 7.7 mm PL1170-1850
Hi-Plex Pb 8% 8 µm Pb2+ 50 mm × 7.7 mm PL1170-1820
Hi-Plex K 8% 8 µm K+ 50 mm × 7.7 mm PL1170-1860
Hi-Plex H 8% 8 µm H+ 50 mm × 7.7 mm PL1170-1830
Hi-Plex Na 4% 10 µm Na+ 50 mm × 7.7 mm PL1171-1140
Hi-Plex Na (Octo) 8% 8 µm Na+ 50 mm × 7.7 mm PL1170-1840
Guard Cartridges
Hi-Plex Ca 8% 8 µm Ca2+ 5 mm × 3 mm (2) PL1670-0810
Hi-Plex Ca (Duo) 8% 8 µm Ca2+ 5 mm × 3 mm (2) PL1670-0850
Hi-Plex Pb 8% 8 µm Pb2+ 5 mm × 3 mm (2) PL1670-0820
Hi-Plex K 8% 8 µm K+ 5 mm × 3 mm (2) PL1670-0860
Hi-Plex H 8% 8 µm H+ 5 mm × 3 mm (2) PL1670-0830
Hi-Plex Na 4% 10 µm Na+ 5 mm × 3 mm (2) PL1671-0140
Hi-Plex Na (Octo) 8% 8 µm Na+ 5 mm × 3 mm (2) PL1670-0840

FREQUENTLY ASKED QUESTIONS

Q. How do I choose between the 4% and 8% cross linked media?

A. The difference is in the pore size of the material, the lower the crosslink density the bigger the pore size is, so the 4% material is used for oligosaccharide analysis and the 8% for oligosaccharide <dp 3.


Q. How do I choose the most appropriate counter ion for the analysis?

A. The resin must be selected based on the solute of interest – refer to the table of elution times to identify the most effective column.


Q. Can I change the counter ion on the Hi-Plex column?

A. Because the Hi-Plex materials are microporous the swell will change with the counter ion – therefore if the counter ion is exchanged on a pre-packed column swelling or shrinking will occur and the column will either over pressure or void – these materials are termed fixed-ion exchangers.


Q. Can I use eluents other than water with the Hi-Plex columns?

A. It is important to ensure that the counter ions are not stripped from the Hi-Plex columns so eluent containing cations that are different to those on the media should never be used – however, with the Hi-Plex Na it is possible to run at high pH with sodium hydroxide as the eluent and the Hi-Plex H can be used with acid eluents.


Q. To protect my analytical column, should I use a guard cartridge or a guard column?

A. This depends on the sample matrix – the cartridges have a small volume of 35 µL. The interstitial volume (remember: interstitial volume = volume of empty column tubing - volume of packing) is ca. 14 µL, so the capacity is low and they will quickly foul and should not be used if the sample contains high concentration of salts or any concentration of a salt with a metal ion that is stronger than that bound to the phase (see counter-ion selectivity's).


Q. Why do I have to run my column at lower flow rate when first installed on the HPLC?

A. As the Hi-Plex media is microporous, the columns are packed at lower pressures than silica based HPLC columns so when installed it is important not to exceed the maximum operating pressure. Water is viscous and the pressure is high at room temperature but as the temperature increases so viscosity reduces and the flow rate can be increased.


Q. How should I remove the Hi-Plex column from the HPLC instrument?

A. As the column will have been operated at high temperature – 60 °C to 95 °C it must be allowed to gradually cool to room temperature – switch off the oven and let the thermal mass of steel cool. While the column is being decommissioned, the eluent flow should be reduced to 0.2 to 0.1 mL/min


Q. How do I store the column?

A. Once the column is at room temperature, the end caps should be replaced and the column stored at 4 °C or in a cool lab. The columns should be stored in water without sodium azide.


Q. The column has lost performance – can it be regenerated?

A. There are two main reasons why a column will fail, the maximum operating pressure has been exceeded and it has voided or the counter ions have been stripped. If the column has voided then it cannot be regenerated, but if the counter ions have been stripped the regeneration procedure detailed in the Column User Guide should be followed.


Q. My sample has no UV chromophore, what detector should I use?

A. As the Hi-Plex separations are isocratic it is possible to use a refractive index detector, such as the Agilent 356-LC Refractive Index Detector. If more sensitivity is required then an ELSD can be used, Agilent 385-LC ELSD or Agilent 380-LC ELSD.

DOCUMENTATION

  • Agilent Hi-Plex Ligand-Exchange HPLC Columns
  • Agilent Hi-Plex Columns Applications Compendium
  • Share
  • History
  • More
  • Cancel
Anonymous
Related

Agilent Community Feedback

Agilent Community Feedback

×
We are glad this was helpful! We are sorry this was not helpful. If you still need assistance please create a community post or contact support. To help us improve, please provide any additional feedback. For full details of how we will treat your information, please view our privacy policy.
Submit Cancel
Submit Cancel
Recommended
Privacy Statement
Terms of Use
Contact Us
Site Help