New column bad results

[dragosb]

Tailing can be related to plumbing, hardware problems, or else column related. Try reproducing the QA conditions with neutral solutes and see if tailing persists.

Phosphate buffer has been shown to lead to shorten the life of ODS

What would You suggest to use instead of phosphate?
I will check again all the connections and see if there is a void volume problem.

Thanks again

[danko]

The phosphate per se is not your problem. If it was.... you would've seen the vice versa effect - i.e. "new column good results"

The plumbing doesn't seam to be the problem either - if you just install the new column instad of the old one on the same system etc.

Best Regards

[dragosb]

I sent a message to waters, they will replace de column for know to verify if there is a column problem or something else.

As for my method, could there be the problem?

I start with a 10% ACN then after 5 minutes I go to 7.5% and then at 15 min I do a wash at 40% for 5 minutes and then I come back to 10%.

Please let me know if this is OK for the column.

Thanks again all of you

[danko]

The column will deal with the described gradient all right.
But I must say I’ve never seen such kind (sort of reversed gradient) of elution on a RP column. It resembles Normal Phase (HILIC if you like) combined with Reversed Phase gradient And that is wrong!

Best Regards

[dragosb]

You are saying that the reversed gradient is not good. Please explain as I must say I do not really know why.

Should I better start at a 4-5% and then go for a plateau at 7.5 and then for the wash at 40 or more?

Thanks

[Camisotro]

You are saying that the reversed gradient is not good. Please explain as I must say I do not really know why.

Should I better start at a 4-5% and then go for a plateau at 7.5 and then for the wash at 40 or more?

Thanks

How did you arrive at that gradient... was it in the literature or did you come up with it from scratch? What are you particularly trying to accomplish with that drop?

The idea is, at each solvent composition, each analyte will have a certain rate of migration. For each analyte there are solvent compositions that basically don't move it along the column at all, some that will push it along the column at the same rate as the mobile phase, and a narrower range in the middle where its rate changes rapidly.

Normally in RPLC, all analytes move faster at higher organic composition. So you'd expect that once the column has started at 10% ACN, it will elute everything that elutes at any composition lower than 10%. When you drop down to 7.5%, you are essentially slowing down the elution of anything that has not already come off the column.

[dragosb]

Hi,

Actually I have some peaks that come out at the begining that I do not need so I started at 10% ACn to make them come out faster, but the sepparation for the ISTD and My peak of aminoacid is better at 7.5%.
If I run a isocratic 7.5% ACN it takes too much to have the peaks so that is why I tried to shorten the time for each sample.

Please let me know if there is anything else I can try.

Thanks a lot

[danko]

Depending on the retention times of the peaks of your interest (we don’t know them) you can estimate the acetonitrile concentration at which they elute.
Quick and dirty suggestion: Try isocratic elution (8 % acetonitrile) from 0 to 20 min, then 40 % acetonitrile for 5 min and then again 8% acetonitril for 5 – 6 min (equilibration step).
The peaks you aren’t interested in will probably elute a couple of minute later, but as long as they don’t disturb the peaks of interest you should be happy. The elution will be much more stable and reliable this way.

Best Regards

[dragosb]

Hi, danko,

I tried with the 8% but the separation is not that good and the peaks come out at 22 min for the ISTD and 23 for the Tranexamic acid.

I have a better sepparation at 7.5 but then the retention time will be even longer.
Could a gradient help to improve my situation?

Thanks

[danko]

A shallow gradient starting from let's say 6% and ending at 12% ACN over perhaps 20 min. could be worth trying.
Then a couple of min. at 40 - 50% ACN and finaly equilibration stem at 6% ACN

Best regards

[dragosb]

Thanks again.

Concerning the pH of the mobile pahse, I use a pH of 6.75.

The pKa for the acid is 4.3, 10.6 and for the ISTD is 2.36 and 9.76.

Do you think a different pH would be better?

Thanks again

[danko]

Definitely! You want/need the pH to be a couple of units below the pKa.
In your case I'd recomend pH 2.5. Phosphate would be a good choice.

Be prepared to use significantly more ACN in order to elute the components. But you'll acheive better efficiency and parhaps better separation.

Best Regards

[HPLCaddict]

Definitely! You want/need the pH to be a couple of units below the pKa.
In your case I'd recomend pH 2.5. Phosphate would be a good choice.

Erm, one of the pKa's of the internal standard is 2.36, right? So, pH 2.5 would be VERY close...
May I suggest a pH of ~3.5? Should be reasonably away from those two lower pKa's (4.3 and 2.36) and as it's right between them it will leave the acid mainly protonated and the ISTD unprotonated - that should give a selectivity boost.

[danko]

Oops, I missed the 2:36 part.
HPLCaddict's suggestion seems reasonable.

Best Regards

[dragosb]

Thanks again.

Using a phosphate buffer, how do I get down to a pH of 3.5?

I am using for know a NaH2PO4/Na2HPO4 buffer.