As promised, here is the first 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 sample preparation and general system questions. The next post will feature the consumables (columns, electrodes, and eluent) and application/method development questions.
Can carbohydrates be cut from glycoproteins using TFA and HCl?
The enzymes, Endo H, Endo F, PNGase-F and PNGase-A, can be used to break specific bonds between carbohydrates.
If TFA or HCl can be used to cut carbohydrates from glycoproteins, why not use a mass spectrometer (MS) as a detector?
The HPAE technique can be used with MS detection but requires having a carbohydrate desalter inset to convert sodium hydroxide into water and sodium acetate into volatile acetic acid which is compatible with electrospray MS. Low molecular weight carbohydrates are best detected as lithium adducts and require lithium chloride addition after the desalter.
Have you attempted to quantify your carbohydrates in a large amount of water sample?
Yes, we have applications of carbohydrates in surface water. Read Technical Note 20 on the analysis of carbohydrates by HPAE-PAD.
Which method of total hydrolysis would you use for analysis of glucuronic acids in HPLC?
This needs to be determined experimentally. Refer to Comparison of Acid Hydrolytic Conditions for Asn-linked Oligosaccharides. Fan, J.Q.; Namiki, Y.; Matsuoka, K.; Lee, Y. C. Anal. Biochem. 1994, 219, 375–378, for a discussion on hydrolysis optimization.
What are advantages of using Reagent-Free Ion Chromatography with Eluent Generation (RFIC-EG) technology for eluent preparation versus manually prepared eluents?
Our RFIC-EG systems combine electrolytic eluent generation, continuously regenerated trap columns, and self-regenerating suppression. This means you no longer need to prepare eluents and regenerants, or take trap columns off-line to regenerate them. Eluent concentration is precisely controlled by the current applied, improving method reproducibility and eliminating errors associated with manual eluent preparation. The clear advantages are consistent results, gradient capabilities, lowest detection limits with hydroxide eluents with added water.
Is it true that Thermo Scientific chromatography instruments are better for quantitation work but not for qualitative work of carbohydrates?
No, we use our instruments for both quantitation and qualitative analysis. The only issue is that for qualitative work you will need to use a reference compound, which sometimes can be difficult to buy.
Can you recommend a system that uses anion exchange high-pressure liquid chromatography (HPLC) and a mass spectrometer for carbohydrates analysis?
Do add your questions below and we will be happy to post answers for everyone’s benefit.