Retention Time Shift or Variation Causes || Pharma Pill

5 Causes of RT variation in HPLC

Do you know what are the main causes for retention time variations during HPLC analysis. Yes in this video I have explained the main causes for retention time variation during HPLC analysis.

1. Temperature fluctuations:Well friends to get reproducible retention time, the temperature of the HPLC column compartment must be kept constant.Not only column compartment temperature but sometimes it has been seen that fluctuations in laboratory room temperature can also cause RT variation.One important point always keep in mind that If Retention time of all the peaks is moving in same direction then you can suspect temperature fluctuation as one of the possible cause for the RT variation. Rule of thumb is with 1 degree change in temperature there will be 1-2% of variation in RT .

2. Improper degassing of mobile phase : Mobile phase degassing plays a very important role in getting reproducible RT.If mobile phase is not degassed definitely there will be airbuble generation. Which will later may obstruct continuous flow of mobile phase and ultimately it will cause retention time variation.

3.Leak in HPLC System.

4. Incorrect mobile phase composition : The most likely source of variation in RT is change in the composition of the mobile phase. So in reverse phase chromatography if you make an error of 1% in the amount of organic solvent then retention time of analyte peak will change by between 5-15%. That’s why we have to measure the mobile phase solvents accurately. The best approach to prepare a mobile phase gravimetrically rather than volumetrically.

5. Incorrect pH measurement of mobile phase : control of the pH of the mobile phase is very important. A slight change as little as 0.1 ph can result in retention time shift of 10%. So it’s very important to measure a pH accurately and to keep the pH meter well calibrated.

Hope you found this useful. Keep learning.

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