Related Substances Testing of Peptides

[adoyle]

Hi everybody (Hi, Doctor Nick!)

We are successfully using the following 'standard' system for assay testing of a peptide consisting of 9 amino acids.

Column: Grace Vydac (218TP54) 250 x 4.6 mm
Mobile Phase A: 0.1 % TFA in Water
Mobile Phase B: 0.1% TFA in Acetonitrile

However, related substances testing of the peptide using the above system is not suitable, as the TFA makes the baseline much too noisy (and LOQ's are not near low enough for impurity detection). The same gradient ran without TFA in the mobile phases produces a much better baseline.

Could anybody suggest an alternative mobile phase/buffer/chromatographic system?

thanks in advance

[Mark Tracy]

You may not like this, but 10 mM perchloric acid has similar chromatographic properties to 0.1% TFA, but a clean UV baseline. Phosphoric acid does not have any ion-pairing activity, so the selectivity is rather different for basic peptides, but it too gives good UV baselines.

I don't recommend putting concentrated acids into 100% acetonitrile, too much chemistry can happen. Dilute the acetonitrile to 70% which is enough to elute most everything from your column. Either that or use a ternary gradient with plain solvents in A & B, and the acid in C.

There are only a few things you can do to minimize the baseline hash with TFA. One is to use a larger gradient mixer. Another is to avoid proportioning near 0 or 100%. A third is to use slow gradients. And of course use the highest quality TFA you can get; it really makes a difference.

[Bryan Evans]

Hi Adoyle -

If I'm not mistaken - the column you are using is a 5um, 300A C18.

It might be helpful to switch to a smaller pore size and smaller particle size. This should result in better retention / efficiency for the smaller peptides, and will increase your LOQ.

Below is a peptide separation done on our Cadenza CD-C18 (3um, 120A)
column and our Intrada WP-RP (3um, 300A) column:

http://www.imtakt.com/TecInfo/TI291E.pdf

Also, an excellent LCGC article was previously posted on baseline disturbance using TFA. I've had customers in the past use the suggestions in this article to help with baseline noise:

http://www.lcgceurope.com/lcgceurope/da ... rticle.pdf

[DR]

As Mark mentioned, tno all TFAs are created (or packaged) equal.

You might try chromatography grade TFA packed in sealed ampoules (4ml, among other size are available).

[Mark Tracy]

Bryan,
Thanks for the reference.