Chromatography Forum

Dear all,
I am trying to quantificate a contrast medium in human serum using an hplc-method.
To get rid of the serum proteins I added 400 µl 5% perchloric acid to 100 µl serum. After centrifugation 25 µl supernatant could be injected into a c18 column.
The problem is: the pH value of the sample is under 1 and this would cause resolve the functional groups of the column.
Do you have any idea, how can I adjust the pH value of the sample? Or is there other alternativ method to resolve this problem?

thanks very much

best regards

Are you actually having column life problems, or are you just worried? Unless you're using a very small column and very low flow, that 25 microliter injection should get diluted out fairly quickly (before it can damage the column). My guess is that the column will die from residual protein contamination before any deterioration from the acid becomes an issue.

If it is of concern, there are other ways to precipitate proteins (acetonitrile, zinc, etc).

I have observed the deterioration of the column.
The flowrat is 1.5 ml/min. I have also thought, that the 25 µl sample could be enoughly dilluted by the eluent (4% ACN in H2O, pH 2.5).
It takes considerably more time to use ACN or other organic solvent for the deproteination, because the samples must to be dried and then resolved in an appropriate sample buffer before the injection.

96% buffer in mobile phase is also enhance deterioration (i assume your C18 column is not designed for high hydrophilic eluents)..plus protein contamination may also be responsible..

96% buffer in mobile phase is also enhance deterioration (i assume your C18 column is not designed for high hydrophilic eluents)..plus protein contamination may also be responsible..

thanks for your reply
the column I use is the chromolith c18 column from merck, which can bear eluent pH from 2 to 7.5.
I don't really think that there is a protein contamination.

Karvezide

pH range is not the point in your case. With high aqueous mobile phases, hydrophobic C18 chains may permanently mat down to escape from water..

I have observed the deterioration of the column.
The flowrat is 1.5 ml/min. I have also thought, that the 25 µl sample could be enoughly dilluted by the eluent (4% ACN in H2O, pH 2.5).
It takes considerably more time to use ACN or other organic solvent for the deproteination, because the samples must to be dried and then resolved in an appropriate sample buffer before the injection.

Like Tom Jupille, I am also interessted in details to the observed column life time?

If your mobile phase is buffered at pH 2.5, 25 microliters of 4% perchloric acid should not have much of an effect on the column. I would still suspect that the problem is more likely due to residual protein than to the PCA.

One way to tell would be to *try* another type of deproteinization (either ACN or something like zinc sulfate) and compare column lifetime. If you also have the problem with the other techniques, then you have exonerated low pH (and if you do not, you have some idea how the alternatives perform).

By the way, what, exactly, are the symptoms that you are seeing?

or inject 25µl of perchloric acid for x times too see if this detoriates your column

If your mobile phase is buffered at pH 2.5, 25 microliters of 4% perchloric acid should not have much of an effect on the column. I would still suspect that the problem is more likely due to residual protein than to the PCA.

One way to tell would be to *try* another type of deproteinization (either ACN or something like zinc sulfate) and compare column lifetime. If you also have the problem with the other techniques, then you have exonerated low pH (and if you do not, you have some idea how the alternatives perform).

By the way, what, exactly, are the symptoms that you are seeing?

thanks a lot for your tips!
the Peaks of the substance are normmally two adjacent peaks. Their resolution is 1.
After some injections, say 100 injections, The resolution reduced by and by. At last, after about 4 months these two peaks could not be separated by the column (show as a peak).

or inject 25µl of perchloric acid for x times too see if this detoriates your column

it could be quite expensive...

If your mobile phase is buffered at pH 2.5, 25 microliters of 4% perchloric acid should not have much of an effect on the column. I would still suspect that the problem is more likely due to residual protein than to the PCA.

Full Ack and EOD. I think you have revealed the best approach to solve this problem. But unfortunately it was ignored multiple times.