By Anonymous on Sunday, June 27, 2004 - 07:35 am:

Hie to all

I would like to know concepts to be used for gradient method developement at 210 nm .

lets say my sample is having a 6 components .

components 1 and 2 elutes with very less organic around 5% at retention time of about5 minutes .

components 3 and 4 elutes with moderate organic composition at 30 to 40% .

components 5 and 6 elutes with high organic composition at 80-90%

all six compounds are having a uv spectra at 210 nm .

i need to develope gradient method .which are the buffers can be used comfortably at 210 nm to avoid the blanks and what solvent i should use to get minimum base line ramp and smooth elution .


is there any rule that if i take 0.02 m kH2PO4
as a buffer and methanol as solvent B

IF I RUN gradient from 95% buffer to 85% methanol over a 60 minutes linear . i prepare the sample in 50:50 water methanol mix .

SHOULD I GET BLANK PEAKS Or base line flutuation in above gradient with lowpressue and hight pressue systems at 210 nm .

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By juddc on Tuesday, June 29, 2004 - 01:45 pm:

In my experience, you can use a gradient at that wavelength, however there are limitations which may or may not be compatible with your analytes. You need to run using the most UV transparent buffer and organic phase possible. Phosphates are a good choice, assuming they buffer well at the appropriate pH for your analytes. Instead of methanol, I'd use acetonitrile which is more transparent than methanol, but could give you more difficulty with precipitation with a phosphate buffered mobile phase. Keep the salt concnetration as low as possible to get the job done, be careful, and with a little luck you should be OK. Good luck!

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By Ann on Wednesday, June 30, 2004 - 08:33 am:

If you use acetonitrile, make sure it is Far UV grade. I am sucessfully using a gradient assay at 208nm with a MeOH/MeCN/NaH2Po4 phase. However, I was initially troubled with mixing noise (a regular undulating baseline, which you just don't see at a higher wavelength) and I eventually had to abandon trying to mix the phases (I now just switch from 100% A to 100% B then back to 100% A to equilibrate the system). All the best :o)

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By Anonymous on Saturday, July 3, 2004 - 08:42 pm:

Still i am facinglost of probleam in terms of baseline my seperation is quite good but baseline is lots of disturbed and so many peaks arecoming
even after using gradient merck acetonitrile and hplc grade merck water .chemicall alsoi am using sigma aldrich pureset grade material.
what is wrong gradient is incorrect or what ?
i run 2 gm NH4H2PO4 -----1000 ML ph 5.6 with 1N NaOH .

A : BUFFER
B: BUFFER : ACETONITRILE (20:80

GRADIENT PROGRAMME IS AS FOLLOWS
TIME A B
0 90 10
60 35 65
70 30 70

AGAIN EQ

column is L1 250 x 4.6 5 um
at flow rate of 1.0 ml/min

wavelengthis 210 nm

basically peaks and humps starts after

50 minutes of gradient time .

is this due to 50:50(ACN:BUFFER)

HOW TO SOLVE THIS PLEASE GUIDE ME




70 70

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By tom jupille on Sunday, July 4, 2004 - 12:00 pm:

First thing to do is to run two "dummy" gradients (i.e., no injection, just run the gradient) and superimpose them.

If you see different humps and peaks (i.e., if the patterns do not coincide), then you are looking at random noise and should check for things like bubbles, flow/proportioning problems, etc.

If you see the same humps and peaks on both chromatograms, then you have established that they are part of the gradient process and not injected with the sample and/or blank. The next step is to run two dummy gradients with different equilibration times before each:
- dummy gradient (to clean off the column)
- equilibrate for 15 minutes
- dummy gradient
- equilibrate for 45 minutes
- dummy gradient

Now look at the heights of the garbage peaks in the last two gradient runs. If the heights are proportional to the equilibration time, then you have confirmed that you are seeing contamination in the "A" solvent. Contamination can come from *anywhere*:
- the pH electrode (are you putting the electrode in the mobile phase or are you reading the pH on an aliquot which is then discarded?)
- residual detergent on glassware?

If you are using a high-pressure-mixing system, you can sometimes clean things up by using a C18 guard cartridge as a precolumn between the "A" pump and the mixer.

Good luck, and let us know what you find (so we can all learn!).

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By Anonymous on Sunday, July 4, 2004 - 09:16 pm:

Thanks for sugession i will try out today sir

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By Anonymous on Monday, July 5, 2004 - 10:33 pm:

I STUDIED AS YOU SUGGESTED

DUMMY GRADIENT OF NO INJECTION VOLUME RESULTED IN SAME GRADIENT BOTH ARE OVERLAPPING EXACTELY AND THERE IS HUMPS AND SMALL PEAKS AT THE 50% OF ACETONITRILE CONCENTRATION LEVEL.

WHEN I RUN THE GRADIENT BLANK WITH DIFFERENT EQUILIBRATION TIME I FOUND THAT ITS MATCHING SO
WHAT SHOULD BE THE CAUSE OF THIS ?