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Dear Sir,
I am getting problem with HPLC. Pump pressure has suddenly risen
upto 200 kgf. We were injecting plasma samples after extraction with
methanol. Syringe filters were being used. Last time when I was using
HPLC
there was fluctuation in power supply. Due to this I shut down the
machine. Next morning I fund that pressure has hiked up. I washed the
column for two whole days with 50% methanol. Pressure was still the
same.
Today I backwashed the column using 30% methanol. The pressure is still
105 kgf.What shall I do to bring down the pressure.
Please reply
Sincerely
Rajani
--
Rajani Shakya
Assistant Professor
Department of Pharmacy
Kathmandu University
Dhulikhel, Kavre, NEPAL.
GPO Box 6250, Kathmandu, NEPAL.
Email: rajani.-at-.ku.edu.np
Homepage:http://www.ku.edu.np/pharmacy
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The following message was posted to: PharmPK
Dear Rajani,
If the backpressure is more you can suspect two things
A) Column blockage
b) pump-line blockage
i) You didn't mention the mobile phase that you had used for the method.
If in the mobile phase more buffer content is there than organic
content, it leads to precipitation of the buffer when you wash with 50%
methanol. So it is suggested to wash the column first with pure water
followed by pure organic and then ratio of both.
ii) You can flush all the lines starting with high polar solvent water
(moderately warm), followed by acetonitrile/methanol, methanol:water
(50:50) Isopropyl alchohol, methanol:water(50:50). (remove the column)
iii) Remove the column and backflush the detector line by connecting in
the reverse side.
iv) Remove the frit present on the pump head and soniacte in
water/methanol.
Hope so I answered your query. Any doubts revert back.
With Regards,
Veeravalli Vijaya Bhaskar,
Research Associate,
Aurigene Discovery Technologies Limited,
Bioanalytical Division,
Bangalore,
Email: vijayb.-a-.aurigene.com
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The following message was posted to: PharmPK
Dear rajani,
Go through the link given below.
http://www.lcresources.com/resources/TSWiz/idx.htm
With Regards,
Veeravalli Vijaya Bhaskar,
Research Associate,
Aurigene Discovery Technologies Limited,
Bioanalytical Division,
Bangalore,
Email: vijayb.-at-.aurigene.com
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The following message was posted to: PharmPK
Dear Rajani,
Sometimes its not the column thats the problem, have
you systematically check your lines for some blockage
or kinks.
What is your back pressure in the absence of the
column?
Backtrack and check each segment of the lines to find
where the blockage occurs. If you found out that it
is really the column. Try higher organic such as
acetonitrile or even 10% DMSO to clean the HPLC
columns.
Hopefully this help
Julius
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The following message was posted to: PharmPK
Hi Rajani,
First thing you need to know.. where is the problem, Remove the column
and place connector between inlet and outlet, and check pressure,
similarly, if you may have to look for other parts to find main cause.
If it is really the column, I'll suggest to reverse the column and wash
with 90% Acetonitrile.
Good luck
Bharat
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As others have mentioned in their responses, it can any part of the
lines that is suffering blockage or kinks. You want to go sequantially
from inlet to outlet points in your flow and check. The best scenario
being a blockage of the inlet frtit that is in contact with mobile
phase. Simply sonicate or replace these. worst case being the pump or
colum blockage. Drastic measures where nitric acid flushing could be
needed, but this owuld be a last resort.
I hope this helps.
Murad Melhem
Assistant Director
Cognigen Corporation
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