Problem with method / sample preparation or sample treatment

[Narendra]

dear all chromatographer,

I don't known the problem with method or sample preparation or sample treatment. My problem is i suppose to get two peak one is principle peak and second impurity peak with lower response than principle peak but i am getting reverse phenomenone, principle peak response lower than impurity peak. Details are as below
1 HPLC Method for Ethambutol Hydrochloride API and official method in BP
2 Chromatographic condition is as below
column : 100 cm x 4.6 mm 3 micron C18 end capped column
wavelength : 215 nm,flow rate : 1.0ml, injection volume 10 microlitre
column temp : 40 degree centigrade
Mobile phase gradient : A = 50%;50% (WATER : METHANOL)
B = 100% METHANOL
TIME A B
0.01 TO 18 71 29
18 TO 35 0 100
35 TO 40 71 29
3 TEST Solution preparation : 4 mg of subs in 4 ml of acetonitrile, sonicate for 5 minutes, add15 micrlitre triethylamine and add 100 microlitre methyl benzyl isocyanate and heat on water bath at 70 degree centrigrade for 20 min.

Can any one give what could be the problem.

NAV

[tom jupille]

I can think of a number of possibilities:

1. Are the peaks correctly identified? Have you run reference standards of the pure compound and of the impurity?

2. Coelution with contaminants in the derivatization reagent. What happens when you run a blank through the sample prep/derivatization?

3. Your sample has degraded so that is actually contains more of the impurity than of the parent compound. Have you run reference standards of the pure compound and of the impurity?

4. The impurity is more easily derivatized than the parent compound, and you are getting only partial derivatization of the parent.

[rindtr]

you may easily figure out the peak/compound correlation by adding pure standart to your vial, or spiking your sample and injecting both sample and spiked sample to compare the rise of peak. if i were you i would spike my sample in different concentrations and expect one of the peaks rise sharply.

[Safoolia]

Hi! I will be grateful for help with HILIC column adaptation for supernatant analysis. There is a mixture of proteins, peptides and dfrnt. methabolites in sample. I am interesting in polyketide analysis. Problem is to choose write buffer to make a column stable and robust during number of runs. Can you suggest something, please?

[praveenpaliwal]

can u tell me why us add methyl benzyl isocyanate? [/list]