1- I have problem in shifting of retention time of a peak :
During analysis of rebamipide, the peak of rebamipide shift forward. for example(Std at 20.0 and sample at 20.5), and as the sample set increases the peak retention time increase more while the peak of internal standard separated for both the same.
*My mobile phase consists of 830ml of(0.058 gm Na2HPO4.12 H2O+2 gm KH2PO4 /in 1000 ml)
*Internal standard (1 gm Acetanilide in 100ml with DMF)
*Column (4.6mm x 150 ODS)
*Sample ( weigh about 0.26 gm of the powder(wt) [Equivalent to 0.15 gm Rebamipide] then dissolve it in suitable amount of (N,N DMF ) in 50 ml measuring flask then add 10ml internal standard and complete with (N,N DMF )to 50.0 ml then shake for 10 min. then take 2ml of this solution and add 40 ml( N,N DMF) in 100 ml measuring flask then complete with D.W. to 100ml.
*Standard ( As sample )
This is a common problem. Please review the linked free article to properly troubleshoot and solve the problem.
"HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it" http://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html
http://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html
Hi,
the problem is your mobile homogeneity, you can use magnetic stirrer to make your mobile solution more homogeneous. (small magnetic bar in the bottle with relatively slow rotation speed),This method helps well.
thanks,
I use magnetic stirrer to mix salts alone then filter it through 0.22 micron filter then took 830 ml from the filtrate and add 170 ml of acetonitrile then mix again using magnetic stirrer then I use it ,this what I do but the same happen every time.
Ok,
You can also keep stirring while the HPLC on use and not only in mobile preparation.
This is a common problem. Please review the linked free article to properly troubleshoot and solve the problem.
"HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it" http://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html
http://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html
This is common when one or more of the following occurs:
i inadequate degassing - which can easily be resoled with a degasser
ii temperature fluctuations - with an oven you can have constant temperature
iii pressure fluctuations - wide range pressure change can also affect the Rt , this can be influenced by a leak , which will in turn affect your Flow Rate and have great impart on the Rt.
I confess that I don't understand for sure your situation description.
what is the sample dissolved in?
" " " int. Std " " ?
Do you degas solutions?
Could you create some tiny table, containing exact conditions vs. R.T. both of sample and int. Std?
According to my understanding the reason could be a chemical modification of rebamipide linked to one or more chemical compounds in the sample.
Another explantion could be the duplication of the reference peak linked to the aging of the C18 phase: direct chemical interaction of phase or indirectly by an interaction of a disruptive chemical samples retained in the phase of the column.
The solvent does not seem to cause this progressive interaction of the column when you look at the chemistry of rebamipide.
There are also occasions in which some materials in the sample matrix interact with the stationary phase and change the chromatography. Try a different sample workup before chromatography to test the hypothesis.
Plenty of possible reasons have been proposed, but w/o more details, the reason will never be know. Sometimes it is as simple as different injection volumes or not properly washing your column after each run (*which can cause shifts in Rt). Please refer to the articles I linked.
As you are brand new to liquid chromatography, we would suggest you read some of the wonderful introductory texts on the topic and get as much practice in as possible. Additionally, this is a very hard technique to learn by yourself. Try and find someone local with ten or more years of practical experience analyzing many types of samples to assist you. You will learn much quicker this way.
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