This is the second set of Q&A from our on-demand webinar focusing on HPAE-PAD analysis of carbohydrates featured earlier in the blog. (This on-demand webinar is free and be viewed at your leisure.) This post features the consumables (columns, electrodes, and eluent) questions and questions on application/method development. (The previous post on this topic featured the sample preparation and general system questions.)
Consumables (columns, electrodes, and eluent)
- Could you provide details on MALDI MS with the use of a desalter?
After the column, you can install a carbohydrate membrane desalter which converts the sodium hydroxide and sodium acetate eluent into water and acetic acid. When fractions are collected on MALDI targets, all eluent components (water and acetic acid) are volatile and can be evaporated so they do not interfere with the MS analysis.
- What is the typical lifespan for a HPAE-PAD column?
This depends on the sample matrix and sample clean-up. As HPAE-PAD is a very sensitive technology, it allows the diluting of the sample and, therefore, less matrix is injected. In addition, the carbohydrate columns are highly pH stable and can be cleaned with concentrated hydroxide or acetate eluents.
- If the column gets contaminated by a very strong anion compound how can we clean it? Can you suggest a solvent or can we use HCL? Will HCL damage the columns, for example, the PA 100 or PA 10?
Each column has its own cleaning procedure based on the contaminants. We recommend referring to the column manual for cleaning instructions. (You can find the column manuals online at www.thermoscientific.com.) Also, remember to always remove the guard before cleaning the column and then replacing it once the analytical column is cleaned. You also do not want to back-flush any of our Thermo Scientific Dionex CarboPac columns.
- How long does the gold electrode on the PAD last in terms of usable and efficient response?
This depends on the sample matrix and purity of reagents used for making the eluent and the type of electrode used. Disposable gold electrodes on Teflon can be used up to 4 weeks continuously while conventional gold electrodes can be used for years but require manual polishing.
- Can you provide more details on the electrode reactions occurring in the gold and reference systems in anion exchange chromatography of carbohydrates?
We recommend a great article written on the electrode reactions by Dennis C. Johnson and William R. LaCourse, This article, Liquid chromatography with pulsed electrochemical detection at gold and platinum electrodes, was published in the ACS journal Analytical Chemistry, Vol. 62, No. 10, May 15, 1990.
- Why are both NaOH and Na-acetate used in one method? Is it not possible to achieve a separation with NaOH only?
Acetate is a stronger eluting ion than hydroxide and acetate is required to push out strongly retained oligosaccharides while mono-, di- and trisaccharides can be eluted with just hydroxide.
- Is NaOAc stronger than OH for anion exchange?
- What is the maximum hydroxide concentration you can achieve using the Thermo Scientific Dionex eluent generator?
Using the Dionex eluent generator, the maximum hydroxide concentration for 2 and 4 mm i.d. columns is 100 mM and for capillary columns with i.d. of 0.4 mm, it is 200 mM.
- Is there a limit to the acetate concentrations? For example, do you recommend not going above 100 mM NaOH for eluent concentrations?
No, some applications for oligosaccharide separations use higher NaOH concentrations. We haven’t found any sugars that have needed more than 1.0 M NaOAc to be eluted from the column.
Application/Method Development Questions
- I noticed that maltose (reducing di saccharide) doesn’t have the same retention time as trehalose (non reducing disaccharides). In my conditions, gradient on MA1, trehalose is between inositol and mannitol and mannitol is behind saccharose, a very big difference. Why?
Trehalose lacks a reducing end, which significantly shortens its retention time.
- How can we improve the separation between isobaric oligosaccharides if they are not baseline resolved?
This would depend on the structures and the conditions already tried. There is no generic answer.
- Do you have any separation work on sucromalt?
Not at this point.
- Do you know of any method or method development for the analysis of GOS in milk powders?
We have a related application for TOS, Application Note 155, Determination of Trans-Galactooligosaccharides in Foods by AOAC Method 2001.02.
Like what you are learning?
- Could you provide the reference on Tri- and Tetra-Sialylated Oligosaccharides for the alpha 2-6 and alpha 2-3 linkages separation?
Rohrer, J. S. and Townsend, R. R. “Separation of Partially Desialylated Branched Oligosaccharide Isomers Containing a(2,3) and a(2,6) Linked Neu5Ac” (1995) Glycobiology 5, 391-395.
- The Tri- and Tetra-Sialylated oligos slide shows separation of the two glycans that differ with only a single linkage at the siallic acid. Do you see this type of separation with other linkage differences between other oligos, or this sialic specific?
Yes, though not sialic-acid specific. For example, Gal linked either Beta 1,3 or 1,4 to GlcNAc.
Further Reading Suggestions:
If you have any questions not answered here, please post below, and we will answer them for everyone’s benefit.