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Dear All,
I want to know Is it neccessary to prefer isocratic method? even
though a gradient method for the same molecule is available with less
run time.Are there any basic rules for setting up a gradient method?
What does the term Linear/Generic Gradient signifies?
Thanks in advance
T.S.Chandran
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The following message was posted to: PharmPK
Dear Chandran,
Isocratic or gradient is a personal preference of analyst. Each method
has its own baggage- Isocratic methods may shorten column life due to
retention of un-eluted peaks, may get interference with slow eluting
components.
Gradient needs longer stabilization time, but gives superior
chromatography.
Generic gradient is the one which you normally use as a start point. A
typical 3 min generic gradient may look like this:
0-0.5 min 5 % B
0.5-1.0 min Increase B to 70 %
1.0-2.0 min Increase B to 98 %
2.0-2.5 min Hold at 98 % B
2.5-2.6 min Return to 5% B
2.6-3.0 min Stabilise at 5 % B
Depending on the peaks you get, you may vary time programme and % B, so
that you need not unnecessarily run a longer gradient,but still achieve
desired separation.
Hope this helps.
Vinayak
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Dear T.S.Chandran,
There are no hard and fast rules to select isocratic method all the
time.
Depends upon the chromatography, we can choose isocratic and gradient
method.
Most of the time, gradient method works fine as it increase resolution
and gives better separation.
What ever gradient method you choose, you have to give proper
equilibration time otherwise we will observe fluctuation and peak shift.
You can read any basic chromatography book like "Practical HPLC method
development by L.R Snyder" to get more idea about gradient method.
Regards,
Sudipta Basu
Senior Research Scientist (DMPK-Bioanalytical)
Sai Advantium Pharma Ltd.,
Pune - 57.
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Dear T.S.Chandran,
The other posters to this thread have already correctly described the
advantages of the two approaches.
However, please remember that although the chromatogaphic run of a
gradient method takes less time than that of an isocratic method, a
gradient method does not normally improve throughput in the laboratory
because of the needed equilibration time at the end.
Regards,
Frank Bales, Ph.D. Email: frankbales.-a-.msn.com
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The following message was posted to: PharmPK
Isocratic could minimize carry over. Also for chiral separation,
people tend to use isocratic.
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Hi Bales,
We use gradient methods with 3.5 min Inj to Inj cycle time on a normal
LC. (I have modified agilant LC to reduce system volume.)
For a high throughput method, what should be inj to inj cycle time?
Regards,
Vinayak
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