I AM TRYING A GRADIENT SEPERATION FOR MY ONE OF THE RELATED SUBSTANCES METHOD . I AM USING

A 0.01M KH2PO4 80% 20% ACETONITRILE

B 80%ACETONITRILE 20% BUFFER MIX (0.01MKH2PO4)

BUFFER IS HAVING A PH OF 6 WITH NaOH SOLUTION

COLUMN USED IS INTERTSIL CN-3
250 X 4.6 5 um
flow rate is 1ml /min

my gradient is as followas

A B

0 80 20

8 80 20

25 20 80

30 20 80

35 80 20

40 80 20

MY DILUENT IS WHICH I AM PREPARING THE SAMPLES IS

50: 50 MIXTURE OF ACETONITRILE AND WATER
I AM GETTING SO MUCH BLANK PEAKS WHICH IS INTERFERRING IN MY IMPURITIES PEAK AND ALSO MY COMPOUND PEAK , I TRIED WITH THE BEST QUALITY OF ACETONITRILE AVILABLE . I ALSO TRIED WITH METHANOL AND BUFFER MIXTRUE, ALSO TRIED ACETONITRILE AND BUFFER MIXTRURES STILL I AM .GETTING THE BLANK PEAKS . SO HOW TO DEAL WITH THIS ANYONE CAN GUIDE ME IN THIS REGARDS

YOU CAN REPLY TO shi_ri@indiatimes.com

Shirish,

First off, it's generally not a good idea for your sample to contain more solvent than your starting condition mobile phase. If I understand your message correctly, your initial starting conditions include 32% acetonitrile which is significantly less acetonitrile than your sample solution. Of course, using excessive solvent in your sample predominately results in degradation in chromatographic performance not in baseline artifacts, so this is probably not related to your problem.

Besides the solvent, additional sources of artifacts include the reagents used to prepare your buffer solution and your deionized water used to prepare your mobile phases. You can identify which of these is the cause of the problem by simple experimentation. For example, operating with 100% buffer for 30 minutes prior to the beginning of your gradient should result in increased artifact peak areas if the problem is coming from your water or your buffer reagents. Increasing the concentration of your buffer solution should result in larger artifact peaks if the problem is due to buffer reagents. If the buffer concentration has no effect on the artifact peaks, then the culprit is most likely your water (of course there are other possible causes of these artifacts including contamination of your injection valve and/or your autosampler or component of your autosampler such as vials and septa).

chris phol

my starting point is 80% mobile phase and 20% acetonitrile . as to elute one of my impurity at 4 minutes in this isocratic condition up to 8 minutes. i am getting blank peaks at the rt of this impurities and also in linear gradient reaching to 80%of B in 25 minutes .which gives peaks at rt of main components and so many blank peaks near the elution of impurities peak near by 22 minutes.

so what should i do , i think is it advisable to make sample preparation in 90% buffer and 10% acetonitrile so that it will me the lower than all composition throught the gradient run composition . so shuold i make sample in water.

regards
shirish

Shirish,

Relative to the sample preparation issue, assuming the sample will dissolve in water, there should be no problems using water as the sample solvent. But at the very least, it's preferable to keep the solvent level in the sample solution equal to or less than that of the initial conditions of your mobile phase.

Relative to your artifacts observed during the gradient, I'm not suggesting that you should change the gradient to starting at 100% aqueous buffer but rather I'm suggesting that you use this condition as a means to determining whether or not the contaminant is in the water or in the buffer as suggested in my posting above. Once you have identified because of the problem, you should be able to return to your standard conditions for your gradient.

Refer to, "Mobile Phase Cleanup using SPE disks", M.C. Ringo et. at., LCGC North America, Vol. 21 (2), p. 168 (Feb 2003). This is an excellent article and it has the complete procedure.

We have used Empore SDB-XC (3M) Solid Phase Extraction disks to cleanup the aqueous portion (only) of the mobile phase before making the final mobile phase composition. We were able to eliminate all the unwanted system peaks. We have incorporated this cleanup procedure into two of our recent gradient methods and eliminated interference/quantitation issues. We have also used "cleaned" water by the indicated procedure prior to making the mobile phase and were able to eliminate the same system peaks. In our case, the problem was with water not acetonitrile. The disks are expensive.

Using "cleaned" mobile phase, you may also be able to investigate if the problem is due to intrumentation issues.

You may also want to simplify the gradient progarm. Mobile phase A: 68% Buffer: 32% organic and Mobile Phase B: 32% Buffer: 68% organic. Start with 100% A and end with 100% B keeping the same gradient time profile. It accomplishes the same goal with less "mixing" in the beginning and at the end of the program.

Also, prepare the sample in Mobile Phase A as indicated above. If this is possible, it will also help you reduce the system peaks in a gradient run.

Good luck
Regards
Siva

anybody can avial this article to me ?
so that i can proceed.

regards
shirish
shi_ri@indiatimes.com

Shirish,

you can download the article from the web, at the LC GC homepage:

LC GC

good luck

Bart

Shirish: do you have a system that mixes the gradient on the high-pressure side or on the low-pressure side?

i have tried same gradient on two system

1) agilent 1000series low pressure gradient

2) waters alliance system 2690 .

i had got the same gradient on both the system giving blank peaks .

both above are low pressure gradient system.

i think i got your idea , you are telling me to do it on high pressure gradient system so that mixing will be proper and gradient will improve ,

but presently in my lab only one high pressure system that is to shimadzu and its not working properly in terms of injection volume we have lots of problem with this so we are using it for washing purpose only.