retention time drift

[Jasmine]

Hi All,

I have a problem with my chromatographic runs. Equilibration was performed long enough to establish stable conditions. All the system is run at room temp. During the first 10-20 injections, the retention time is more or less near initial injection. However, as the analysis goes down further along the remaining samples from the same batch, the retention time starts to drift in one trend. Drug A which started out as approximately 5 mins end up coming out of the coming at approx. 7 mins on the 60th injection and the internal standard also changed from 14.5 mins to 18 mins on the 60th injection. What could be the problem. Help please!

Thanks!

[ntruong]

Hi Jasmine,
It seems to me that you might have problem with your active inlet valve (check valve). Have you experienced the same drift with different instrument? By the way, could you be a little more specific on what conditions you use?

[HW Mueller]

Another example of component (organic in RP?) evaporation of the mobile phase? Check the forum there are lots of examples of the rt shift problem.

[Jasmine]

Hi Ntruong and HW Mueller,

I am using a hypersil BDS18 column. The mobile phase is ACN/Water with phosphate buffer and TEA pH4. I always equilibrate the mobile phase at least 1 hour before starting to run my samples. My sample extracts are in 40% TCA solution.

What's puzzling was, I was able to achieve good reproducible retention time on my first day. Howevever, on the second day of analysis on different batch of samples and the succeeding days, the problem of retention time drift started to come in. The initial retention time for both my analyte and IS always start at early time. Howver, as the number of injection increases, the retention times for both components started to gradually lengthened and the delay reached up to 2 to 3 minutes on the 60th or so injections. I observed no leak in the system.

Please advice

[lcguy1]

As Dr. Mueller stated above, you may be having an evaporation problem. What type of degassing are you using? Are you using He sparge or vacuum? Even with vacuum degassing, just the fact of the MP sitting could change the composition of the MP. Also, the concentration, or changing of, the TFA may also be affecting the RT of your analytes. If you remake your MP do the RTs come back to "normal"?

[Uwe Neue]

I suspect that the problem is that you do not have a good buffer capacity. Phophate does not buffer at all at pH 4.

Either change to a phosphate buffer around pH 2.5 or 7, or use an ammonium acetate or ammonium formate buffer at pH 4.

[syx]

I have got problem related to this topic.
We tried to determine meloxicam in tablets.



pKa: 4.08 in water, 4.24±0.01 in water/ethanol (1:1), 4.63±0.03 in water/ethanol (1:4)

Chromatographic condition as follow:
Column: 150 mm x 4.6 mm, contains packing L1
Mobile phase: a mixture of phosphate buffer solution, methanol, and isopropanol (63:35:5)
Phosphate buffer solution: dissolve 3.30 g dibasic ammonium phosphate in 950 mL of water, adjust with 20% phosphoric acid solution to a pH of 7.0, dilute with water to 1000 mL, and mix.
Flow rate: 1.0 mL/minute
Temperature: 40°C
Detection: 365 nm
Assay preparation: transfer an accurately weighed portion of the tablet powder, equivalent to about 7.5 mg of meloxicam, to a 100 mL volumetric flask, add 10.0 mL of 0.1N NaOH solution and 15 mL methanol, sonicate for about 30 minutes. Dilute with solvent mixture of water and methanol (30:70) to volume, and mix. Pass a portion of this solution through a filter having a 0.45 um and use the filtrate as the assay preparation.

We found that the retention was very unstable. We have used 2 HPLC and got same problem.
Have anyone any idea about what the source of problem is?