In analysis of a weak acid with pKa of 4.6, using mobile phase of phosphate buffer pH 7.7 and methanol (63:37, v/v) and hypersil C18 column, the retention time of this analyte is not stable (it is decreasing with time), although different types and composition of buffer and different pH were tested, but the problem was the instability of the retention time (decrease in retention time).
Do you think that the problem is the column? Is it possible that the instability would be due to the sample? An acid of pKa around 4.6 should not attack hypersil C18 column, but it is possible that you have an equilibrium between the dissociated and non-dissociated form, and both forms could have different retention time. On the other hand, even if the HPLC is isocratic, you should regenerate the column with movile phase for a period before injecting a new sample. Good luck!
Mobile phase compostion should be constatnt. If you make an error of 1% organic solvent, the retaintion time can change by between 5-10%. Mobile phase should be prepare by volumertrically and degas properly. Instument should purge, clean cloumn with hieghly organic than equliberate till straghit basline. If sill problum is coming than check the pump and mixing device can be verify through flow rate. Temperature is also important factor which may cause flactuations in retaintion time.
Please try saturating the column before injecting the sample. It may help.
Improper column saturation might be one of the reasons for ur observed complications...
Try with some different column. And compound pka away plus/minus 2 pH of buffer. Saturate your column with initial condition for 1hr. Take A and B buffer in different line purge well. If you HPLC pump or some air is trap in line then there are chances. And even if your compound is not stable in buffer in this case go for Water/Acn or Water/Methanol without buffer.
1- decrease in temp. or increase in temp. will change the retention time.
2- change in mobile phase pH can cause large changes in retention time.
3- the mobile phase should be prepared once
4- look for air bubbles in the system
You should use DryLab HPLC modeling to understand the influence of a number of variables and allow yourself to make changes in conditions if necessary without a new validation.
Take a look at www.molnar-institute.com
Many factors can cause the instability:
1. pH of the mobile phase.
2. Could be due air bubble in the system. Purge the system before the analysis.
3. Ensure proper mixing of mobile phase if you use isocratic elution or using single pump
4. Column -
Are you using a column heater. If you do not the room warms up during the day and the retention times come out early because the mobile phase is stronger at warmer temps. I have seen this with only a couple of degrees C change. The way around this is to use a column heater and keep the column about 5C hotter than the highest temperature that the room gets up to.
Another advantage is the higher temp means lower viscosity and lower back pressure. Also your analytes diffuse faster at higher temps so your peaks will be a bit sharper. You may have to weaken your mobile phase a bit since the peaks elute faster at warmer temps.
Some people like to go really hot. We had a Waters UPLC and ran some methods at 60C. Very fast separations. (your compound have to be thermally stable, though)
There are many things that could cause this and the other posters have addressed several already. If you are finding retention shifts from injecting the sample and not a standard, it could be the matrix is not fully eluting and is impacting the phase chemistry of the column. You may need to run it longer or use a gradient with adequate equilibration time. However, if you are seeing this with a pure standard of your compound, then this points to some other issue. I would investigate the system as a whole and ensure that you are not dealing with a mixing issue, stuck check valve, gas in the system (degas mobile phases), etc. If you can inject six replicate injections of the standard and get good retention time and area reproducibility and only find this with samples, it likely indicates the matrix is the problem and you will need to either clean up the sample or use a gradient or longer hold time to elute the matrix. I would also check into the stability of the analyte and be sure it is not undergoing changes in structure. Technically, your pH is far enough away from the pKa however, you might have issues that are due to the column chemistry. Often, in labs where columns are used for a variety of samples and analytes, they can become contaminated from irreversible binding. I would see if the column (providing it is not a new one) is contaminated by running a regeneration according to the guide with the column. In addition, if there is a guard cartridge i would remove that before regeneration to ensure you are not contaminating the column during this cycle. If you rule out these items, then temperature of the column or slight changes in the mobile phase ratio or pH are the likely culprits.
The organic concentration of the mobile phases might be off which could lead to rt shift. The pH of the mobile phase could have changed from batch to batch. I have had the pH be off and that caused me a rt shift.
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