Hi all
I’m working with RP HPLC
With injecting the sample from the same vial on a 4.6x250mm column (C18, 5um, 1ml/min) for several injections I get varying peak integrity
Is there a rational explanation for this?
Sanaz Hashemipour,
first of all, varying data should not be there because analysis by HPLC gives constant peak and RSD for retention time(RT) should be less than 1%. The variation in Retention time is mainly due to flow variation. Generally for Assay and dissolution peak quantification only two integration parameters used like one is for system suitability and second for sample integration.
kindly check your system flow and temperature of the column oven.
Dear Vijay Arjun Bagul thank you for your response,Actually RT is constant but peak area is different for one sample .!!
do you think the problem is flow rate of instrument or the column is not working efficiently?
In this case, I think, the problem is either the sample or the injection system.
In your sample, physical or chemical reactions might take place causing a constant raise or decrease of your analyte's concentration. Alternatively, your sample is not homogenised sufficiently (shake it?).
Concerning the injection system, I recommend to use a sample loop and to inject sample volumes that are sightly greater than the loop's volume. Then, you are sure that the sample volume always equals exactly the loop's volume (which shouldn't change) and no variations can originate from inconstant injection volumes.
Dear Sanaz Hashemipour,
I got your point if RT is constant then problem is not with the flow rate of instrument. In such case there might be the changes of air bubble generation from mobile phase if it is not filter through 045 micron and degassed. Even sometime after taking precaution air bubble can generate and area may vary. Also, when there are number of injections from same vial it can create the issue due to septa puncture.
my suggestion is to you load new set and keep number of vials for sample. If it is system suitability then need to inject from same vial.
Hopefully, you got some solution. If query feel free to ask on my WhatsApp number +91 9833618555.
Dear Paul Korner
Thank you for your time, I use 20 microliters loop and my loading volume is constant (0.4 ml) and no leaking from injection system
Dear Vijay Arjun Bagul,
I appreciate your point of view. So, it seems that the degasser is not working effectively.
- "HPLC Retention Time Drift, Change, Area Variability or Poor Reproducibility. Common Reasons for it"; https://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html
Sanaz Hashemipour: By "peak integrity" (? no meaning) we assume you are referring to a variation in peak integration area values. Poor reproducibility is a training and method issue which are best addressed by seeking the help of an experienced chromatographer.
There could be many reasons:
1. Integrating on a downslope because of a placebo peak peculiar to this sample.
2. A sample that has a higher fat (oil) content than used for your SS calibration.
3. ....
Dear Bruce Neagle,
Thank you for your response. I would appreciate it if you could explain more, as I have extracted a sample of oil. What steps should I take to accurately measure the area for several injections from one vial?!
Assuming, this issue is peculiar to this 1 sample, you can try a 'spike and recovery' experiment. Thus, you can add 1.00 ppm, 2.50 ppm, 5.00 ppm, and 10.0 ppm of your standard to the sample. Using only 1 type of integration (valley to valley for instance), you should get 1.00 + x ppm (where x is the natural concentration), 2.50 + x ppm, 5.00 + x ppm, 10.0 + x ppm. This assumes your recovery is 100%. This series is linear and offset by x from your normal linear curve (detector response to concentration).
I should explain the need for the above procedure! Many chemicals are amphoteric and have side groups that are both polar and non-polar. Thus a peak that is clean and sharp in your system suitability standards may appear as 'squashed' (has the same retention time) in your peculiar sample because the micro-environment is different (more fatty maybe). A 'spike and recovery' study will give you a concentration for 1 sample but not explain why this sample is different from the others.
I should have a 'better' explanation since I forgot my math! Your offset is 1.00 ppm + y (the detector response). Since most HPLC detectors are linear (y = mx + b), x (the natural concentration) can be calculated.
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