Dear all,
I develop a method for my product, everything was perfect but at last there is peak in BLANK at the component RT. sometime it happen only like a baseline drife. But it comes, where 75 min baseline is going accurate except that.
My effort for that is following:
1. Changed in column for carry over,Also tried new column.
2. changed in reagent for MP preparation(A-OPA:ACN 80:20, B-OPA:ACN 20:80 (Buffer pH 5.00)
3.Changed in System and make also.
4. Changed in solvent make,needle wash,pH meter,water and much more
Please suggest what can i do more?
Thanks
Take the column offline and wash the system in sequence (10x column volumes) with 100% Water, Methanol, Acetonitrile IPA, Hexane and back. Make multiple 100 uL injections of those solvents at the same time.
You have a contaminant somewhere in your system. Good luck!
Sir, My method is gradient.
Time in minute Mobile phase-A (%v/v) Mobile phase-B (%v/v)
0 100 0
35.0 60 40
55.0 0 100
65.0 0 100
68.0 100 0
75.0 100 0
Peak is having area about 20000 at about RT 28.0.
I have checked system blank and changed system for contamination(6-7 system and make also).
I have also used the solution said by Mr.Bruce, thrice. I though that it is system carry over or column carryover. but i used new column and a system on that first time i am injecting the compound.
Bu i surprised that it comes each and every time at every where.
Please suggest.
Seems like carryover; try using pure methanol as your needle wash solvent!
Hi, in addition of those noce comments above, I would like to ask you whether you used DAD or MS for your detector? I think you can try to measure the UV spectrum or MS of the peak of your blank. By this you can predict the possible compound of the peak. Have you evaluate the stability of your compounds and all others in blank preparation? Best regards
Assuming you have performed the above suggestions, you may have a 'poor' method where a peak/contaminate is late eluting and being dumped into the next chromatogram. Therefore, increase your organic strength or decrease the amount of aqueous (water).
As Bruce said, I think you have a late eluting peak. It means that if you inject your samples for a very long period of time (eg 2 hours), you will see the late eluting peak and its retention time. You will be able to calculate your retention time for each injection to avoid this peak around the RT you are looking for. To flush this peak, you can also use a gradient. If you find the source of this peak (carryover, late eluting peak, contamination, etc) you will be able to find how to get rid of this peak.
If you have carryover, check your injector. Maybe you have to change the rotor seal of the injector or change the autosampler rinsing solution. Carryover can also be created by your column. Wash your column, change you column or clean it with a Mobile Phase gradient. Check your tubing too. Some compounds stick on stainless tubing.
Change your blank matrix if your blank samples are treated biological matrix such as human plasma or human blood.
Does OPA is Orthophtaldehyde??? I hope for you that can can use another Mobile phase composition...
You might want to rethink your gradient composition and the way you bring it back to starting conditions. It looks, according to the chart, that you sweep through compositions abruptly, and this can cause a number of different kinds of problems, such as incipient phase separation, failure of the column to equilibrate, and transient . One way to check for this is increase the flow rate and see if the problem disappates or changes location; another is modify the return to original conditions. Simple experiments like run gradient without action of the injector. This will eliminate or isolate one possible source of the problem.
some time i am facing this problem gradient type of analysis by hplc ,first u try 90 % water 1 %acid acetonitrile 9 % is mobile phase
yes sir this is Ortho-phosphoric acid.
about late eluting peak, i thought about that initially, but when i tried this thing on such a system on that i started this first time and also i am not injecting my main component. Injecting only blank, with new column, carried out by system blank with gradient only.
same problem persist.
Now trying to do with change in flow rate.
if continues than i have to rethink on my gradient.
Thank you all for your suggestion .
Regards
Not a whole lot to add to others suggestions. I would think it is either system carryover (often from injector) or impurity in mobile phase additive. I would suggest, if you haven't, not making an injection and run your gradient. If you still see it you know it isn't from the injector. Also, try different mobile phase additives or higher purity of same additives. Just some thoughts in addition to the good ones already provided.
I would have to say that it is probably contamination in the injector. If you are describing an issue with a mass spec then you could also look to the inlet of the mass spec.
Have you tried preparing the blank with solvents and reagents from different batch?
If it is a component of the solvent or the reagent little or nothing can be done
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