during determination of isoniazid (INH) by HPLC, the peaks of INH and acetyl-INH are very close (differences between the retention times less than a minute), therefore the peaks are overlapped and calculating the rate of INH from such chromatogram is quite difficult and irrelevant.
mobile phase consists of (91 / 9) (0.075 M phosphate buffer / methanol)
Thank you friends for help
I tried to separate the peaks by varying the chromatographic conditions:
* I changed the proportions of buffer and methanol in the mobile phase
* I changed the wavelength
* I reduced the flow
for results, the separation is the same or separation is excellent but the result is not reproducible
Try to change the temperature to enhance the separation of two peaks as described in the attached file ... Otherwise use chemometrics and derivative techniques as described in the second file i will send you on your request...
Dear Malek,
did you try the USP Method? please try if not
http://www.uspbpep.com/usp32/pub/data/v32270/usp32nf27s0_m42980.html
Regards
Selvan
Do you validated the method?, precision, accuracy, linearity etc? Sample preparation may be the problem if your chromatographic condition are good. Prepare your sample in 50:50 mixture buffer and organic modifier.
I hope this helps!
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