Chromatography Forum

I'm tring to set a method for those 3 preservative in juice, I using column PE C18 Value line, mobile phase acetonitrile 25% and KH2PO4 25mM pH 2,3 75%.

I had a really bad peak....with long tail.

Any suggestion???

mmegliol,

I would suggest that you do a search of a couple of chromatography vendor web sites for examples of analysis conditions for these compounds. You should come up with several possible mobile phases to try. Good luck!

I've done those in soft drinks with two different columns. Check the links:
http://www1.dionex.com/en-us/acclaim_li ... 29720.html
http://www1.dionex.com/en-us/acclaim_li ... 29721.html

Fruit juice has a lot of colorful stuff in it that is a nuisance for HPLC. You might want to try the OnGuard II P cleanup cartridge; it was designed for decolorizing red wine before analysis for organic acids.

With your conditions, ascorbic acid will practically be in the void. With any RP method it elutes very early; for best results use an aqueous-compatible RP column and start in 100% buffer. You will want a gradient to elute the sorbic and benzoic acids in a convenient span of time.

You can also separate benzoate and sorbate salts via RP if necessary. Prepare your buffer with a pH of about 5.5 (I've used ammonium acetate) and decrease the organic content. Roughly 10-12% MeCN works well with a fairly retentive C18 - Inertsil or equivalent. Ascorbate will elute in the void under these conditions, however, so you'll need a separate method for that should you choose to try what I describe. Just a reminder that pH can be used to alter selectivity for two of your three analytes, though most choose to keep them protonated...

It's very difficult retaining ascorbic acid on a C18 column without an
ion pairing reagent.

For benzoic and sorbic acids on C18:
http://www.silvertonesciences.com/modul ... TI033E.pdf

For ascorbic acid on C18:
http://www.imtakt.com/TecInfo/TI155E.pdf

For ascorbic acid on silica column:
http://www.imtakt.com/TecInfo/TI155E.pdf

Hope that helps!

A couple of minor things:

1. The links posted to your ascorbic acid application on a C18 and a silica are the same application. I'm interested in seeing the silica separation.

2. What's the pH for the IP separation on the C18? I assume basic or at least above the pK of the ascorbate...detection at 260 mn seems to indicate this. Please specify pH with IP separations...

3. I've had success with aqueous 0.1% H3PO4 (pH 2.2) on an Inertsil C8 250x4.6. Ascorbic acid elutes in about 4 min at 1 mL/min. Many other applications exist from a variety of manufacturers as well. YMMV...

This is probably not the answer for which you are looking, but I've done these by GC, and the benzoic acid by HPLC/UV. If I was trying do do 2 or 3 by HPLC, I think I'd try Alltech Prevail column and conductivity detector (with dilute H2SO4 eluent), and possibly route to UV detector for the benzoic acid as well.

My apologies for displaying the wrong chromatogram.

http://www.imtakt.com/TecInfo/TI154E.pdf

As for the pH of the aqueous solution, the data is as is
and I would only be guessing if i were to give a pH value.

Alltech has an excellent organic acid column that I have used for very similar separations. You have to remove all interferences from other products by SPE, but once done the separtion is excellent and you are using simple buffers to do it.

This was mentioned before: Quantitating ascorbic acid in tomatos was a breeze with C-18 (don´t remember, but it was probably an aqua type), wheras attempts to analyze ascorbic in serum/plasma was given up (actually didn´t hae time to try one more derivatization technique). This could be a case were two analyses are best: Ascorbic at quite low pH (see also juddc above) the others at higher pH and higher organic modifyer.