Negative diflection/peak

[vikmurush]

Hi,
I am running an isocratic method. Mobile phase is 35% ACN and 65% aqueous pH 5.3 solun. Aqueous has 0.3%TEA and adjusted pH to 5.3. Wavelength 250nm, column 250x4.6, c8(2). LC system Agilent 1100 series.

Sample in 90% ACN 10% water, 10ul inj

Problem: randomley seeing negative deflection after active peak. This is coming some times where the imp/degradants RT. I could not find any specific reason why this is happening? I know that there is incompatability between MP and sample diluent, but by increasing aqueous in the sample we are making a known imp/degradant. I tried ACN and TEA from different manufacturers but no use. It bothers me that some times i dont see the negative peak and some times i do. I even noticed this pattern using differnet instruments.

any suggestions greatly appreciated.
[/b]

[Mark Tracy]

I have seen this when the mobile phase is slightly contaminated with a UV absorbing species and an analyte elutes that absorbs less than the contaminant. If you zero your detector on pure water, what is the absorbance of the mobile phase? How do you adjust to pH 5.3?

When you say random, do you mean that replicate injections from the same vial sometimes have the negative peak and sometimes don't? Or is it that the negative peak is randomly distributed among the samples? In the first case, you probably have a problem related to your HPLC system; in the second it is perhaps related to the sample prep.

[vikmurush]

Thank you Mark for your reply.

I think you are right about mobile phase but i am not sure. Random meaning from different runs on different days. I am pretty much sure that this is not due to instruments and columns. I have used different columns and different instruments. I have a strong feeling that it is from MP only. But i need to isolate the problem. Here is how i am making MP. Adding 6ml of TEA to 2L of HPLC grade water, adjusting pH to 5.3 with phosphoric acid.

I am using online mixing of 65% of pH 5.3 solun and 35% of ACN

Yesterday i made two MPs.
Column: the one i did not seen negative peak using this column.

MP1: same as above preparation and pH adjusted with 85% H3PO4 (pH electrode dipped when i adjusted pH for MP1)

MP2: same as above preparation and pH adjusted with 6% H3PO4 (pH electrode not dipped into MP2 when adjusted pH).

here are the results:

MP1 has negative peak after active
MP2 not seen.

In my previous runs i remember even i do not dip the electrode to MP i have seen negative peaks. Do you think that it could be possible using 85% H3PO4 giving negative inflection? I could not imagine if that is the case.

Any thoughts and ideas are appreciated.

[Bruce Hamilton]

As it's isocratic, I would suggest premixing the mobile phases, and pH adjust the aqueous buffer with 1:1 acid before the mixing.

If you are adding concentrated acid, you need to ensure the acid dilution is rapid, so the aqueous + TEA should be well agitated before, and during, the addition of concentrated acid.

If you measure how much acid is required, you can eliminate the pH electrode, and just quantitatively add the acid. In that situation, I would add slightly dilute acid ( eg 1:1 with water ), as it lowers the viscosity.

Also, check that your solvents, including water, aren't being contaminated by filters or glassware.

Please keep having fun,

Bruce Hamilton

[vikmurush]

Thank you for the reply Bruce

I did the studies with premixing MP vs online mixing and they both gave similar chromatography. But i could not imagine that the negative inflection is due to adjusting pH with strong acid. I tried to eliminate adjusting pH by adding premeasured diluted acid. It is not possible due to small diluted acid volume changes +/-0.2 pH in the MP. I believe i minimized contamination from glassware and solvents. During the method development and validation i did not seen this negative diflection in the chromatography. Now i am testing stability samples and found this problem since last 4 to 5 runs.

[Bruce Hamilton]

OK, can you post an example ( details on how to post are at the top of the forum )?. The most common reason for lower absorbance is a component that elutes and also absorbs less than the mobile phase at the detection wavelength. Amines can also behave in strange ways.

If you are doing stability testing, is it consistent?. In other words, if you inject samples 6 times, do the sample that doesn't show the peak, and the sample that does have the peak, behave consistently. You could analyse with a DAD to obtain the spectra of the samples and mobile phase. Is the size of the baseline depression related to any spectral changes in the main peaks, sample storage conditions, or even a more aggressive sample dissolution technique?.

Maybe it's a consequence of a partially degraded formulated sample - are your samples formulated with excipients, or are they pure API?
Do you have some control samples that are stored at lower temperatures?. If so, what happens if you dissolve the sample in the mobile phase, or dissolve less of the sample and inject a larger volume?.

The best way to solve the problem it to perform a systematic series of analyses to identify what creates the negative peak, using suitable samples and changing each of the possible causes.

Please keep having fun,

Bruce Hamilton

[Mark Tracy]

Your mobile phase formula is unstable. Phosphate has no buffering capacity at pH 5.3, and as you noticed, it is excessively tricky to get it adjusted. Are you certain that you can't use acetate? Then you could just weigh the components and use them straight away.

[HW Mueller]

Elsewhere it was hurry up, here it´s rush, thus the repeat of the suggestion above: Calm down, think of some simple experiments as suggested in the other chain. The problem will almost explain itself.

[vikmurush]

Thank you everybody for your comments/suggestions.

we validated the method and for now we are not able to change the method. This method orginated from EP monograph. I should have mentioned before that when ever i saw negative inflection in the chromatography i get the same from diluent. I will do couple of experiments to see i can determine what it casues the negative inflection. Once again i appreciate your time.

Thank you