Chromatography Forum

Hey there, let me start off by saying that i am very new to HPLCs and how they operate. Let me start with my problem. Up until recently our HPLC which is used with our RI detector has been testing samples fine. However, we have a had a problem with calibration as of late. We calibrate our unit once per month, however this time we ended up with funny results. Our "amounts" are not reading anywhere close to what they were the previous month. Our standards are prepared by a 3rd party and the standards are used kept cold and used for several months until more is needed. I will say that the samples that are being run now have the same retention time as the previous months samples, however the area and peak heights are different. Unfortunately this is happening to all samples we run on this machine. I will consider changing out syringe and injection port/needle. I just want to see if anyone has had this problem before and what they might have done to fix it. I know this is vague but will answer any more questions that might be helpful. Thanks

I would suggest to find a separate check standard, one that is known to be stable and works with your same column and mobile phase if possible.

We calibrate our unit once per month, however this time we ended up with funny results.

Please explain exactly what this "calibration" entails.

We did pharmaceuticals but even for non-regulated assays we would run and quantitate v. standards every day/every sequence.

Was your standard a different batch or lot? Did you compare to a standard run previously but now injected at same time as current standard?

Our calibration uses 5 levels of known ion standards, and then typically we run one "check" standard to see if our HPLC is running optimally. So for example, our level 1 typically reads around a 11.6 in ethanol, our check standard should read close to the same amount, but instead we are getting 5, 9's, this fluctuates periodically when ran. The standards we are using are of the same lot/batch we have been using in the prior months. This problem has only just occurred. Currently just changed out syringe and needle/needle seat to see if this fixes anything. Thanks for the quick reply!

Did you purge the reference cell ,with fresh mobile phase , before starting ?

So this is what has been done so far, the system has been cleaned with acetonitrile and then flushed with DI water. The problem still occurred, so then a new column was installed, however the problem still occurs. New injection needle along with syringe has been installed with no luck. The system was purged after every new installation with fresh mobile phase. I think i will dive into the RI detector today, maybe the lamp life is near its end.

Any large changes in room temperature lately? I have heard RI detectors are very sensitive to temperature variations.

Hows retention time and back pressure? Could be a leak or blockage in the autosampler, not pulling correct amount. What model RID and autosampler?

Most RI detectors are temp controlled. Flush the ever living hell out of the reference cell, like 2 days at 2ml/min. Do you have another RI detector you can swap on to confirm it is the detector? Otherwise you need to a known good sample that runs on same conditions(MP/Columm/temp/Flow rate) confirm tat is working correctly, that will implicate the samples/standards.