Hi Charia,

As indicated by Markus Christ it might be that the pump lost prime. It would manifest itself in a much lower instrument backpressure, and a "bumpy" baseline. (prime the pump, and restart)

In the following, I tried to come up with some different scenarios and hope they prove as helpful.

- Is the flow rate the same? You might find it strange, but we had cases reported by others where the flow rate was changed by someone w/o properly documenting - but that is another story. (Please check the experimental conditions in the CDS and adapt as needed).

- Do you premix your eluents? If so, did you keep the eluent pressurized all the time? Reason for asking is: given the "lower" concentration of organic modifier a loss (e.g., due to evaporation) of MeOH could cause longer retention. (prepare fresh mobile phase and restart).

- If you use the gradient mixing of your pump to mix the eluent, please check that the mixing valve is working properly. (e.g., swap the eluent positions, adapt the instrument method accordingly and check the outcome).

- Not knowing the chemical nature of the components you are after, but judging from the eluent you use, something (hydrophobic) might have precipitated and "create" additional non-specific interactions. (Check the backpressure, and if that increased, carefully flush the column with higher organic modifier concentrations, e.g., by increasing the MeOH concentration slowly to 100%).

- One rather unlikely possibility: A liquid leak. However, it would need to be massive to create such a retention time shift.

My two cents.

Hope this helps,

Detlef.

Hi Charia,

first of all: Please check the pump (flow rate) and your tubings (are there any leaks?). Then check your eluent and your column. Do you use a column oven? If yes, is the temperature stable?

Regards

Markus

Hi,

Which detector are you working on?

Hello Markus Christ ,

Thank you for your answer. The pump pressure is stable, i don't have leaks and the column temperature is set from oven at 25 oC. Yesterday i set flow rate 0.1 ml/min overnight with ACN:H20 70:30 mobile phase, but my peaks is are moved again a lot to the right.

Hi Charia,

check your flow rate using a stop watch and graduated cylinder. When the flow rate is okay and you have no leaks then check you column. Maybe the column was not equilibrated. Therefore you maybe have now a shift in the retention times.

Regards

Markus

Hello Ankita,

My detector is UV-vis.

Hi Charia,

As indicated by Markus Christ it might be that the pump lost prime. It would manifest itself in a much lower instrument backpressure, and a "bumpy" baseline. (prime the pump, and restart)

In the following, I tried to come up with some different scenarios and hope they prove as helpful.

- Is the flow rate the same? You might find it strange, but we had cases reported by others where the flow rate was changed by someone w/o properly documenting - but that is another story. (Please check the experimental conditions in the CDS and adapt as needed).

- Do you premix your eluents? If so, did you keep the eluent pressurized all the time? Reason for asking is: given the "lower" concentration of organic modifier a loss (e.g., due to evaporation) of MeOH could cause longer retention. (prepare fresh mobile phase and restart).

- If you use the gradient mixing of your pump to mix the eluent, please check that the mixing valve is working properly. (e.g., swap the eluent positions, adapt the instrument method accordingly and check the outcome).

- Not knowing the chemical nature of the components you are after, but judging from the eluent you use, something (hydrophobic) might have precipitated and "create" additional non-specific interactions. (Check the backpressure, and if that increased, carefully flush the column with higher organic modifier concentrations, e.g., by increasing the MeOH concentration slowly to 100%).

- One rather unlikely possibility: A liquid leak. However, it would need to be massive to create such a retention time shift.

My two cents.

Hope this helps,

Detlef.

Dear Charia Hadjipakkou

In addition to the already-mentioned remarks, I see that you are using isocratic conditions without any additives like buffer solutions. Could you share with us the nature of the substances you are dealing with? When the retention times change, do you also see a loss in resolution? How good is the column? I would advice to monitor the baseline and the instrument back-pressure during the morning purging and the equilibrium of the column.

Hope this helps,

Greetings,

JPM

it seems unusual if there is no above stated problems,

make sure it is your target peak and mobile phase programming shall remain constant on each time. However, need to examine step by step all above parameters.

may be you have more than one analyte

It could be your organic component (Methanol and Acetonitrile) evaporated so the remaining water ensured the peak was more retained by the column. The solution is to prepare the mobile phase daily and reduce the possibility of evaporation of the organic component of the mobile phase,

Please check mobile phase temperture (Room temprature) even though you may have column oven temperature fixed. Frequently this may have obsrved to you when you load sequence at night where room temparture fall drastically.

Hello all,

Thank you for your answers,

Yesterday I think I tried everything and the problem still exists. First of all my analysts are caffeine, theobromine and theophiline and the three peaks on chromatogram are moved a lot to the right but are separated. The peaks of mobile moved the right also.

I have checked my system ( that I can see ) and i haven't leak.

The column temperature is set from oven at 25 oC ( how can I check if the oven is working properly?).

I checked my flow rate using a stop watch and graduated cylinder and it was okay (for 2,10 min i had 2 ml)

I set flow rate 0.1 ml/min overnight with ACN:H20 70:30 mobile phase, but my peaks is are moved again a lot to the right.

The system use mixer for mix analogy my two solvents of mobile phase and yesterday i prepare a new mobile phase with my analogy in one bottle and run with this solvent but i had same results.

Also i tried to flush my column turning it upside down with 0.1 ml/min flow rate with ACN:H20 70:30 mobile phase for 2 hours but nothing positive happened.

In the evening I set flow rate 1.0 ml/min with MeOH:H20 70:30 mobile phase for 2 hours and then i set flow rate 1.0 ml/min with MeOH:H20 30:70 mobile phase (which are analysis conditions) and I ran the sample with the three standard compounds. Suddenly something unbelievable happened, the pressure was about 65 bars, while normally it should be in the 200 bars and the peaks appear after about 40 minutes.

I am desperate. :/

Tabrez Shaikh

Bruce Neagle

Nadeem Muhammad

Jean-Pierre Mufusama

Detlef Jensen

Dear Charia Hadjipakkou

a sudden pressure drop usually means

a leak (check the pump!)

air trapped in the pump (degas the pump)

or, worst case scenario, a pump defect

Did you contact your local service organization? it might be a good idea to have a service engineer in front of the instrument assisting you with the troubleshooting.

In case you want we could also discuss offline.

Crossing my fingers,

Detlef.

Dear Charia,

I think you do not have to check your column oven.

The reason you were asked about the column temperature is, that some HPLC systems operate without termostating and the fluctuation of retention times is caused by a temperature change in the room in such case ( obviously not yours).

If you face a drop of the pressure, you can have a leak in your system, which result in time shift ( and usualy drop of sensitivity).

hope it helps a bit,

Tomas

Hi Charia,

a pressure drop maybe indicate, that you have a problem with your check valves.

Remove them and clean them in an ultrasonic bath in 10% HNO3 and then in water for about 30 minutes. Sometimes such valves can stick together and then do not open properly. Small foreign particles can also deposit on the valve seat. The result is that the valve no longer closes properly. The frit on the valve may also be clogged. Cleaning with ultrasound often brings success, but in the long run a replacement of the valves is necessary. As a rule, this does not require a service technician. You can do this yourself with on-board equipment.

Regards

Markus

Dear Charia

checked again your last respose, you wrote you had no leak (sorry...)

Turning the column upside down (as you wrote) is not a thing I would recommend, but if you had to flush the column such a way, your time shift is caused likely by an "aged" column or partially clogged system (do you filter your samples? what sort of samples do you analyze?)

regards, Tomas

Here attached my suggestion. Hopefully of help.

thank you all again for your answers. Jean-Pierre Mufusama Tomas Tomsej Markus Christ Detlef Jensen

I use only filtered and degassed solution and filtered samples. i will try remove and clean valves.

I have noticed that the injection needle created a large bubble. He also created a smaller one and removed it, but today I noticed it was very big. You can see in the photo.

You have problem with you instruments check valve

I would check any leaks particularly around the column. A pressure drops during a leak, thus check and tighten connections around the pump, injector, column, detector... When you have a partial leak you will find the peak will be very late eluting even into the next chromatogram (even if it is a blank!).

Check for the leakage if any which can be checked on the basis of backpressure at a given flow rate. Clean the check in & check out valves in iso propyl alcohol using ultrasound. Purge the system well. Equilibrate or condition the column. Calibrate the system and then check for the reproducibility in results.

Hello everyone for your suggestions. They were very helpful. We tried everything and after all we called the local teqnical. At the end we had bubbles in pump and he did purge many times with high volume. Now the pressure is around 270 bar as at the begining of the experiments and we have the peaks in the corect retection time.

Thank you all.

Best regard,

Charia & Chrysi

You may have a leak in the mobile phase tubes or you have baubles in the system.

Hi, I am using the same system, same column. Experienced same problem. For me, I think, consecutive analysis of plant extract brought the change. Everything was ok but after I run a green tea extract, I saw big difference in peak shape and position. I confirmed the change by running standard mixture. Then the column was washed with 50% ACN and I see some improvement. If you have already regenerated your column, please share how you did it though in my experience, the quality of the column can not be revived.

Hello @Ruhul Amin,

Finally we had problem with pump system and we fixed doing purge many times.

Thanks

Great..!!!