hello

yes, I can help you.

The temperature must be very very constant.

Minimize the solution to the maximum

Filter the form before working.

Flow velocity stability.

Wash and clean the separation column before working.

My phone 009647702683703

Dr. Hussein Nasser Al-Salman.

It is possible to change your sample condition (ex -pH). First, repeatly inject few times (4-5), in same sample (ex in vial position 1- inject 3-4 times). Then you can decide the problem is with HPLC or sample

RID is not only sensitive to temperature (usually 85 Celsius HPLC water with a rigid SDVB-Ca form column) but pressure too. Be sure the HPLC is thermostated, in an air conditioned room, away from a window, a sign on the door not allowing entry when the HPLC is running.

Retention time shift has nothing to do with detector. When you said retention time shift, was it getting longer or shortern in one direction or it is random i.e. it may be short or long randomely? I need more in formation. How accurate or precise then LC pump (check the flow rate repeatedly) to see if it is not the pump issue.

Dear Narong Chamkasem

Thank you for your reply.

RT is increasing with every injection for all sugars.

Do you think it is because of pump?

Regards

Dear Bruce Neagle

Thank you for your reply.

My lab Air condition is not working properly from last few days. Due you think RT shifts because of that? I talked to Service engineer, he told me, Column and detector,both are temperature controlled so if AC is not working properly it won't affect analysis.

Regards

Dear B.K.K.K. Jinadasa

Thank you for your reply.

RT is shifting for every sample even for Calibration Standards. There is continuous shift in RTs.

Regards

Dear H. N. K. Al-Salman

Thank you for your reply.

I am already following whatever you have mentioned except my Room temperature as the Air condition has some problem from last few days.

Regards

Ok, need to do some troubleshooting

1) check the flow rate precision. If you run at 1 mL/min, collect the mobile phase from the pump say 10 min in a graduated cylinder. If the pump is good your should have more or less of 10 mL (the exact value is not important) and repeat it a few more time and you should get exactly the same volume. This is to test if it is the pump problem.

2) check temperature. In the morning say 8 am, after the instrument equilateral, run 5 injections and record the RT. At 10 am, 12 pm, 1 pm, 2 pm, and 4 pm repeat the experiment to see if the retention time will get longer in one direction. Put the data on Excel (no injection/time/retention time) and send it to me.

Also be sure to make up the mobile phase daily unless you have validation that shows the mobile phase is stable. An organic like ACN will evaporate and make your RT longer each day.

Hi Ankita,

from the composition of the mobile phase I conclude that you are running HILIC and use the RI detection. If your problem would be due to any temperature impact of the detector you might see drifting baselines, or drifting response factors.

What you mentioned are drifting retention times. That suggests two culprits:

a) column temperature is not constant. Depending on your chromatographic data system you might have a function available that would postpone and sample injection until the desired column temperature is obtained. Please check with your software supplier for help with that implementation. Implementing this has pros and cons. The pros are on the side of stabile RTs and stable baseline performance. The cons are on the time needed to achieve the stable temperature conditions. Please speak with the software supplier and get advice from their hardware team to adjust the experimental data so that you have a realistic chance to avoid too much waiting time (...to achieve the starting conditions...)

b) assuming you are running HILIC: ACN (any aprotic solvent) in HILIC is a weak eluent. Water is a strong eluent. In other words, if ACN would evaporate at large, you would see changing (longer) retention times. The solution would be (and assuming that it is not already done) to head pressure the solvents with an inert gas (e.g. nitrogen; inexpensive and excellent for the job) to avoid any loss of the volatile mobile phase constituent.

There are other possibilities (though I think you already checked):

Pump flow is increasing. Some pumps seem to increase flow over time - reason unknown - , but you can easily spot if that is the case. Just plot the column back pressure over time. If it is increasing: You identified the root cause. Solution: Get the service in, and have the pump checked.

Another possibility: Column was contaminated and now is slowly regaining its original capacity. If you suspect this: Perform a column rinsing procedure according to the column manufacturers recommendations. In case you suspect that the sample matrix contributes to the "poisoning", perform a sample preparation that prevents that issue. And before you ask: I don't know what might cause the contamination, injecting honey... May be some oligo- or poly saccharides, precipitating and slowly being eluted off the column? Purest of all assumptions...

Long message, I know...

Though I hope there is something in it for you.

Cheers,

Detlef.

Dear Narong Chamkasem

Ok. I will check flow rate and temperature and get back to you.

Thank you!

Dear Bruce Neagle

Yes i agree with you.

Mobile phase for RID detection should be prepared fresh everyday.

Thank you!

Dear Detlef Jensen

Thank you for your reply.

My column is Zorbax Carbohydrate column 4.6 mm ID x 150 mm (5 μm)

As you mentioned, it could be because of fluctuation in column temperature but the column is already present in Column Oven and i hope it's working well and keeping the temperature constant.

As there is problem with lab Air Conditioning system, might be evaporation of ACN (Mobile phase) is the reason behind drifting RTs but i am not sure.

Pump flow i have not checked yet but will check and let you know.

Column Contamination : I inject samples only after filtration with 0.2 micron nylon filter. Do you think column can get contaminated even with filtered samples?

Anyways thank you so much for your time.

I really appreciate it.

Regards

Shifting of Rt especially in a RID is basically due to fluctuation of mobile phase flow/pressure. Please check whether you are getting a steady flow (collect the mobile phase after the detector in a 5 ml graduated measuring cylinder and note the time). Repeat the same 2-3 times. If variation observed try to find out blockage is in which section (Chocked reservoir filter/Pump frit/column etc.).Most probably you can rectify this by yourself.

On a second thought:

You are running HILIC, so that a loss of ACN would result in shorter run times... (i.e. the concentration of water as the stronger eluent would increase), so that on second thought I thing we can exclude the evaporation of ACN...

Ankita,

what solvent you use for your sample? What is the injection volume?

Your mobile phase is prepared in a bottle or you program this composition in a pump?

regards,

G

One easy check is to swap the pump to eliminate the issue that it may or may not be the pump.

Dear All,

Regarding RT variation, the issue has been solved, i increased the equilibration time for the column(around 2 hours), purged the RID detector and mobile phase well. I ran the sequence and throughout the sequence i checked the pump pressure. Pump pressure was stable throughout the run.

No variation in RTs observed for all sugars in all injections.

Thank you peers for your advice and suggestions.

I am glad researchgate has brought us a platform where we can discuss various issues faced by researchers all over the globe.

Thank you everyone for being so supportive!

Glad you figured out the hardest part of HILIC, which is the equilibration. It takes 19 forevers to equilibrate properly, and 19 forevers to re-equilibrate after each run. There are better, more stable methods for sugars than HILIC. Take a look at mixed mode columns. I think you will like them.