Chromatography Forum

Dear members,
I am making to analyze Alkyl glycose Vitamin C. This stuff can not be retained at conventional ODS.
Considering its many hydroxy groups, I prefered the carbohydrate column which designed for saccharides. According to description, the void time is about 16.2min. But I can see the first peak at 5.3 min and repeatable. Any one has idea about this? The flow is correct.
Give me some comments if someone has experience in analysis Vitamin C.
TKS.
Michelle

What about trying a HILIC column for the separation?

Dear James,
Have you had any experience in this kind of analysis?

Hi Michelle,

Here are a few methods for Vitamin C using mixed mode Primesep columns. Depending on the buffer nature you can retain Vitamin C even on a very short column (50 mm):

http://hplcmethods.com/compound_149.php

void on 3.2x50 mm column is about 0.4 min (at 0.5 ml/min)

Can you send me a structure of your particular compound?

Hi, thank your reply.
I can post the structure. It is Vitamin C derivative with one glucoside. I think it is highly hydrophilic so no retention.
And can I get some instruction about your column? I am interested in.
Thank you.
michelle

Members,
do you know how to preparation the "Dulbecco's Ca- and Mg- Free phosphate buffered saline(CMF)"?
I read it in a paper, but no details. Thank you.

no personal experience with HILIC, but a lot of info if you google it.

Michelle,

Ascorbic acid derivative will retain on Primesep column based on anion exchange mechanisms. If your substitution has any hydrophobicity, then you can use it to add RP mechanism, although it might be weak and you need to go to low organic (0-5%).
Ascorbic acid pKa is 4.1 which is close to some organic acids (benzoic for example):

http://www.petrik.com/PUBLIC/library/mi ... ase_pk.htm

If you can send us sample we will develop a method for you (free of charge).

Dear all,
I'd like to agree with James Little, who suggested that HILIC It is a feasible alternative for your compounds.

In the below link you'll find a chromatogram showing a separation of Vitamin C from its oxidation product dehydroascorbate on a ZIC-HILIC column.
http://www.sequant.com/products/zichili ... 00-12A.pdf.

/Patrik

Michelle,
on the buffer, I vaguely remember seeing something on this, nothing special, you should find it with google or in some catalogs, maybe Pierce? etc.

Now, if I read you correctly, you are not interested in vitamin C for which a million methods have been published (still can be very difficult depending on the matrix). So where is your sugar attached? Especially if it is attached to the acidic OH (C-3) then surely its chemistry will have almost nothing in common with ascorbic. HILIC or carbohydrate methods (no experience here) would also be my suggestion.

Hi, thanks to all answers,
Muller, you are right. The sample I am interested in is a condensation result by dehydration 2-hydroxy of Vc and glucoside. Maybe it is so highly hydrophilic that I can not find retention.
Michelle

Strange bird? If I remember correctly the substitutions are mostly at the OH of C-3 and C-6. Anyway, if there is no substutution at the C-3 acidic hydroxyl my guess is that you should get very good retention in HILIC at pH toward neutral. (Is this also unstable near neutral and especially at higher pH?)

Mueller. I check the structure again. the substitution is at 2-carbon.
However, I will try the HILIC seperation.

I will bet that a Biorad 87H will retain this compound, dilute sulfuric acid eluant. This column retains aliphatic acids, alcohols, and carbohydrates so it seems a natural for this compound.

Ascorbic is a so called vinylogue of a carboxylic acid.
In our hands its analysis in tomatoes was a breeze with an Aqu C18 column, acidic mobile phase. Analysing it in human plasma/serum proved so difficult that I gave it up after some years. (There must have been some discussions on this before).
But michelle´s compound is not ascorbic acid, though it should still be acidic. Thus, playing with pH on HILIC could be the next choice, if it doesn´t work there are still the mentioned carbohydrate columns.