Coupled Columns

[syx]

We want to develop determination method for glycolic acid and hydroquinone in ointment dosage form. The problem occurred due to low retention of glycolic acid though TBA.HSO4 has been used in the method. I want to use longer chain of the ion pair reagent, ie. dodecyltrimethylammonium HSO4 but the price will be high. I need to search other cheaper option. Is it common to use coupled columns which are having different properties? For example, anion exchange and C-18 reverse phase columns.

Best regards,
Siswanto Tanuatmojo

[Uwe Neue]

Do I understand you correctly that you are concerned about the price of this ion-pair reagent? Here is an alternative to look at: hexadecyl dimethyl ethyl ammonium, also known as cetyl dimethyl ethyl ammonium. I have not looked at this in a while but it used to be dirt cheap. 1 kg for nothing... If I remember correctly, it was the bromide salt.

[syx]

I found Dodecyltrimethylammonium from Sigma. The longer chains are Hexadecyltrimethylammonium (hydroxide, bromide, bisulfate, or phosphate) and Myristyltrimethylammonium bromide. But they have higher price.
Dr. Neue, could you give me direction where to buy the ion pair reagent as you said? What do you think if we use coupled column? Will it work?

[HW Mueller]

Syx, what about HILIC?

[syx]

I have tried it. Bad retention for both peaks...

[HW Mueller]

What have you tried?

[yangz00g]

you are talking about using two different columns in tandem if I understand right.

I used that kind of sepraration before it worked great. But you have to make the mobile phase compatiable with both columns, and pay attention to the carry over effect (in your case, I will put the ion-exchange before C-18 if the matrix is heavy). BTW, make sure the dead volume between those two columns is small enough not to disrupt peak resoluation and shape (it's very important).

[SIELC_Tech]

Syx,

You can try few alternatives:

1. Primesep D or Primesep B2 column will retain your glycolic acid based on anion exchange and RP mechanism. pKa of glycolic acid is around 2.8, so it will retain similar to fumaric acid or lactic acid.

http://www.sielc.com/compound_156.html

I also have other applications which I can send you over email. Hydroquinone will retain similar to any other RP column. You will need low organic (0-5%) to get retention for hydroquinone.

2. Coupling RP and anion-exchange column. You can use ACN-water-TFA and ELSD detection or ACN-water-phosphoric acid with UV detection

regards,

Vlad

P.S. I will see if we have glycolic acid in the lab (I know we have hydroquinone) and can run it in the lab.

[Uwe Neue]

Cetyldimethylethylammonium bromide, Acros Organics, 500g for US$ 109.40. Call 1-973-753-1940 (I have nothing to do with this company).

[Einar Ponten]

I have tried it. Bad retention for both peaks...

Compared to the use of ion-pairing agents HILIC is really an alternative. I had expected some retention for hydroquinone and good for glycolic acid.

Please tell more about the conditions you used?

[Mark Tracy]

Bromide absorbs UV. Pay a little extra for the chloride.

[syx]

I use Merck LiChrospher Silica column. The mobile phase was mixture of methanol and water (each contains H3PO4 0.1%). I have tried the composition of 40% methanol to 55% methanol (isocratic runs). Hydroquinone had retention time about 3.1 minutes and glycolic acid was retained too close to it. There was no significant change between 40% and 55% methanol.
Is LiChrospher Silica suitable for HILIC application or I should use special column?

[HW Mueller]

You gave up way too fast! In my experience (not very extensive either, though) it appears that the relationship of mobile phase composition to retention time is completely different from that in RP, needing a much larger change of composition to effect changes. Tom Jupille noted not too long ago that there is a log? type relationship. Thus, trying 20%, 10%, 5% aqueous might be an interesting experiment.

[syx]

You’re right, Mr. Muller, I gave up when I saw there was no significant different between 40 to 55% of organic solvent in mobile phase. I will try your suggestion and send the report here. Thanks!

[Einar Ponten]

In terms of solvent strength water > methanol > acetonitrile. If you have some minor retention already at 40% methanol that is a very good indicator. Typically there is almost no retention below 50% acetonitrile, so it seems that you still have some nice experiments to do.

I suggest that you try 60-80% of methanol or acetonitrile and since you are using plain silica having cation exchange properties at neutral pH an acidic pH may be required for glycolic acid, as well as "high" ion strength (25-100 mM?).

More support information is provided in "A Practical Guide to HILIC" that you can order free of charge on our website http://www.sequant.com/sn/p_notes.php?id=7

A paper that also may be useful for you is:
Retention Behavior of Small Polar Compounds on Polar Stationary Phases in Hydrophilic Interaction Chromatography
Y. Guo, S. Gaiki
J. Chromatogr. A, 1074 (2005) 71-­80