I am trying to quantify asparagine in my fermentation broth using HPLC, C18 column with mobile phase 0.1% tfa: Acetonitrile (95:5 ).
I'm not getting a perfect peak with the standard. The standard is prepared in water.
i'll be thankful if you can suggest me sol to this. Please check the chromaogram for better understanding.
Is this chromatogram acceptable ?
Thank you!!
Is the chromatogram acceptable? No! We need more information like chromatographic conditions (column, isocratic or gradient, flow...).
Waters and Agilent have kits (Picotag and Accutag?) for the quantitation of amino acids. Why reinvent the wheel?
As Bruce pointed out above, you have not provided us with your actual method details so there is very little that we can comment on.
One thing that is obvious though, based on the chromatogram shown, is that your peak is almost off-scale (3 Volts is too high for most UV's). This is probably because the std concentration you prepared is too high or you have injected too much volume (Note the peak fronting. That is an indication of overload And/OR possibly no retention). It does have the appearance of not being retained on the column, but this is impossible to say without knowing what your exact HPLC conditions are (which you did not provide).
I agree completely with Dr. Horak. UV is, in my opinion, not a suitable detector for amino acids unless you are derivatizing. If you want to avoid derivatization you need to move to a very polar column (HILIC or what are known as "aqueous phases") and use LC-MS/MS.
I Have a question here, Sorry for disturbance.
There are 36 total number of experiments in my case. First I will take Dry weight of fungi and after that I want to do HPLC analysis to check the concentration of different compounds present in it. For D.W I am going with 3 replications of each experiment, So how many replications will be used for HPLC? If 3 then 36*3=108 sample?
kindly guide me in this concern
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