Retention Time Shift or Variation Causes || Pharma Pill
ฝัง
- เผยแพร่เมื่อ 1 ก.ค. 2024
- 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.
Best
Pharma Pill….✍🏻
Good information
Good
Sir want more troubleshooting videos like that........Thank you for this information.
Thanking you☺️
Such a informative video...thanku...post every day so that we can learn more😊
Thanks Soumya Mishra …everyday little difficult… 🙂
Useful video but not fresh all are old only
Good information
Keep continue 🙏
Thank you, I will
I have two doubts.
1. We have to add buffer to organic solvent while making mobile phase or organic to buffer
2.RT some times shifting about 1 min in one sequence what is the reason.
Mahesh Pds thanks for the nice questions.
Answer of first question is: While preparing mobile phase the order of addition of solvents should be as given in individual specification. If specification says to mix organic phase and aqueous phase, it has to be ensured that organic phase is to be added to the aqueous phase and not vice versa.
1.temperature fluctuation
2. improper degassing of MP.. air bubbles.. obstruct MP flow
3. leakage in hplc system.. if peak rt has increased due to leakage then flow rate will decrease
4. incorrect mp composition
in reverse phase.. error of 1% in org solvent then rt shift of analyte can be 5-15%.. prepare mp gravimetrically
5. incorrect ph measurement of mp
Instead of these any other possibilities for rt fluxation
These are the major causes of RT fluctuation. Sometimes insufficient column saturation can also cause RT fluctuation.
Nice explanation
Can u explain about DMF and types of DMF.
Saikrishna Lakkimsetty Thanks
Honestly speaking I don’t know much about DMF but I will learn about it make video in future..
I have a doubt can you guide me.
RRF once arrived for a impurity 1.5.
After that another person doing same the RRF is not same 1.5 . It is higher or lower got. How much percentage is allowable for RRF variation acceptance criteria anything in any guidelines
No variation accepted in RRF values… whatever given in your standard Method same you have to apply during processing.
Can you Say the guidelines number for this RRF variation not accepted
How much is the acceptable RT variation limit
10%