Sorbic Acid Determination by HPLC-UV.

[WK]

I analyse sorbic acid using RP-Amide 70% 0.1%TFA in water and 30% acetonitrile.
My standard potassium sorbate is dissolved in 0.1% TFA in water so it should pass through the column and detector as sorbic acid - right?
My sample is also dissolved in 0.1% TFA in water so it should pass through as sorbic acid - right?
If I dissolve in distilled water then I am risking analyte passing through as sorbate - is this right?
I need to keep pH of sample solution below pKa of sorbic acid - right? No problem in 0.1% TFA in water but would perhaps need to acidify a distilled water sample solution.

Many thanks for any replies

[Bill Tindall]

A reasonable sized sample will be promptly diluted by mobile phase and the form of the analytes will be entirely determined by mobile phase composition, not sample solvent composition. You may be assuming that the sample solvent accompanies the sample down the column. Presumably the sample is retained more than the sample solvent (typically,though not essential to a separation) so the greatly diluted sample solvent moves on ahead of the sample components and they elute later entirely among their mobile phase friends.

By the way this separation works just as well on many reversed phase columns. In particular I would recommend an ES Industries Aquasep. It will be more robust than an amine column. Use phosphoric acid, about 0.15%, as it is cheaper and less annoying than TFA.

[WK]

Bill,
Thanks for the reply,

I should have thought this one through properly before.
I also have to resolve sorbic from benzoic acid so an RP18 and 0.1%TFA or phosphoric acid is out of the question. Also I have used phosphoric with KH2PO4 buffer with really bad crystallization problems.
Why then do analysts go to so much trouble to dissolve in mobile phase when they can use methanol as long as its soluble in the mobile phase and doesn't cause peak shape problems?

[Consumer Products Guy]

Here we usually dissolve our consumer products in methanol or DMF, not in mobile phase, we just keep the injection volumes relatively low if the mobile phase is highly aqueous. As Bill does, we rarely adjust the pH of the dissolution solvent, the mobile phase acidity will acidfy the components of interest in the small amount of sample actually injected. I could see for RI detection, if the signal-to-noise is on the low side, that disruptions could be reduced by making up the sample in mobile phase. But in general that entails making up a mixed mobile phase, and we use the atomated pump systems to mix that for us in the pumping step, so there is a "cost" to that as well.

[Bill Tindall]

1. Do you know that benzoic acid elutes on top of sorbic with a reversed phase column? If not, try it. Almost ever organic acid can be successfully retained on a reversed phase column if the carbon load is high enough and the column can stand 100% water. For this reason I like the ES Industries Aquasep column but any RP column will retain benzoic and sorbic acids.

2. I didn't say to use a phosphate salt. Use 0.15% phosphoric acid. In most cases one wants to protonate the acids and keep them protonated. Hence, a low pH eluant such as that recommended is fine. there may be some special occasions where the extra ionic strength of the salt/acid mixture is advantageous but not in most cases.

3. For trace work it may be essential to load a large sample and under this condition the sample plug may not be sufficiently diluted by the mobile phase to get a focused injection. In this case the sample must be adjusted to more match the mobile phase. Also, the sample may be greatly different from the mobile phase. For example a basic hydrolysis of an ester may wind up in 1M base. In such case an adjustment of the sample (I add phosphoric acid) is essential before separating the acid components.

[WK]

Thanks Bill,

I knew they co-elute on C18 thats why I use RP-Amide type.Thanks for the info on H3PO4 in water

[HW Mueller]

Strange, I would have bet it takes some doing to co-elute those completely different acids on any column.
After having checked compatibilities I have never had a precipitation problem with phosphate salts or any other salts. One needs to handle potential problems all the time.

[Uwe Neue]

I need to say first that I do not have experience with this specific separation. Therefore these are just some ideas, and not gospel.

How about changing the pH to say about 4.75 with an acetic acid buffer? The pK's of sorbic and benzoic are far enough apart to seprate the beasts, even if they refuse to be separated at low pH.

As a column, I would suggest the Atlantis dC18, which works in high water and gives more retention than most other options that do the same thing.