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Help me! AIR BUBBLES trapped in HPLC column...
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Bilal Majed
Jan 11, 2016, 6:11:23 PM1/11/16
to prot...@magpie.bio.indiana.edu
Hello
After I had completed my analysis, I switched to sequence flush overnight for two hours. However I made an error in the settings and realised in the next morning the method was still running and I ran out of wash phase. So air was being pumped into the system and the column.
Please can you help me correct this mistake.
Hplc system- Agilent 1220 Infinity LC System
Column- Inertsil ODS 3V
Reverse phase system
Thank you
Bilal Majed
Sent from my iPhone
After I had completed my analysis, I switched to sequence flush overnight for two hours. However I made an error in the settings and realised in the next morning the method was still running and I ran out of wash phase. So air was being pumped into the system and the column.
Please can you help me correct this mistake.
Hplc system- Agilent 1220 Infinity LC System
Column- Inertsil ODS 3V
Reverse phase system
Thank you
Bilal Majed
Sent from my iPhone
Dr Engelbert Buxbaum
Jan 12, 2016, 2:21:14 AM1/12/16
to
In article <mailman.309.145255...@net.bio.net>, majed...@gmail.com says...
> After I had completed my analysis, I switched to sequence flush overnight for two hours.
> However I made an error in the settings and realised in the next morning the method was >
still running and I ran out of wash phase. So air was being pumped into the system and the >
column.
One thing you can try is to mount the column vertically and to pump medium from below. Use
something with relatively low viscosity like acetonitrile or methanol. Afterwards measure the
number of theoretical plates of the column to see how much resolution you have lost.
The second option is to repack the column, however, for HPLC that requires a packing kit.
Open the column, empty the stationary phase into an vacuum flask with, say, 20% methanol and
connect to vacuum. Once no more air bubbles come out break the vacuum and use the slurry to
repack, following instructions that came with the kit.
> After I had completed my analysis, I switched to sequence flush overnight for two hours.
> However I made an error in the settings and realised in the next morning the method was >
still running and I ran out of wash phase. So air was being pumped into the system and the >
column.
something with relatively low viscosity like acetonitrile or methanol. Afterwards measure the
number of theoretical plates of the column to see how much resolution you have lost.
The second option is to repack the column, however, for HPLC that requires a packing kit.
Open the column, empty the stationary phase into an vacuum flask with, say, 20% methanol and
connect to vacuum. Once no more air bubbles come out break the vacuum and use the slurry to
repack, following instructions that came with the kit.
Joana Arroz
Jan 13, 2016, 2:02:38 PM1/13/16
to Bilal Majed, prot...@magpie.bio.indiana.edu
hello,
it´s not good to dry your column. to take the air off you need to increase
the pressure inside the column, so close the exit for a few seconds,
respect the maximum pressure of the column.
after this, you need to check your column, to see if it working properly.
best regards,
Joana Arroz
2016-01-10 23:32 GMT+00:00 Bilal Majed <majed...@gmail.com>:
> Hello
> Proteins mailing list
> Prot...@net.bio.net
> http://www.bio.net/biomail/listinfo/proteins
>
it´s not good to dry your column. to take the air off you need to increase
the pressure inside the column, so close the exit for a few seconds,
respect the maximum pressure of the column.
after this, you need to check your column, to see if it working properly.
best regards,
Joana Arroz
2016-01-10 23:32 GMT+00:00 Bilal Majed <majed...@gmail.com>:
> Hello
> After I had completed my analysis, I switched to sequence flush overnight
> for two hours. However I made an error in the settings and realised in the
> next morning the method was still running and I ran out of wash phase. So
> air was being pumped into the system and the column.
>
> for two hours. However I made an error in the settings and realised in the
> next morning the method was still running and I ran out of wash phase. So
> air was being pumped into the system and the column.
>
> Please can you help me correct this mistake.
>
> Hplc system- Agilent 1220 Infinity LC System
> Column- Inertsil ODS 3V
> Reverse phase system
>
> Thank you
>
> Bilal Majed
>
> Sent from my iPhone
>
> _______________________________________________
>
> Hplc system- Agilent 1220 Infinity LC System
> Column- Inertsil ODS 3V
> Reverse phase system
>
> Thank you
>
> Bilal Majed
>
> Sent from my iPhone
>
> Proteins mailing list
> Prot...@net.bio.net
> http://www.bio.net/biomail/listinfo/proteins
>
Joana Arroz
Jan 13, 2016, 2:03:01 PM1/13/16
to Bilal Majed, prot...@magpie.bio.indiana.edu
repeat several times
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