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I am a student of M.Pharm, working on HPLC, since last three days I m having problem in the HPLC, the problem is pressure wont getting low, so I would like to know how to reduce the pressure, actually I have run plasma sample before in the column, so is there any standard method to clean the column after using plasma sample? If then please suggest me.
Birendra chaurasiya
Kathmandu University,Nepal
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The following message was posted to: PharmPK
It could be number of different things.
1. Take one module at a time and identify the source of the problem. It could be LC pumps, auto sampler, tubing, guard column, column.
2. What type of column are you using? Are you using guard column or frits (filter)to remove residual proteins or particulate matters from the sample.
3. Lastly column manufacturer should have information regarding instructions for cleaning the column. This information can be available on line at the column manufacturer website.
I hope this is helpful.
Regards,
Anila
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Dear Birendra Chaurasiya,
As you said there is problem with your HPLC, is because of using old column which may increases its back presure of column, it may not be come down.
And as far cleaning procedure is concerned you should wash your column with mobile phase except buffer solution.
ex: if your mobile phase is 80:20 :: Acetonitril: 5mM ammonium formate, your washing solution must be 80:20:: Acetonitril: water.
Hope you get the answer for you.
do not use the column, if column exceeds the injection of 3000- 3500 times.
expecting more answers from experts.
regards,
MUTHU SWAMY.C,
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Dear Birendra,
Usually, column pressure increases because the flow in the column is being restricted by small particles, such as may be present in your plasma samples.
Often this can be partially corrected by simply reversing the column in the instrument and flowing mobile phase through it (in the reverse direction from usual) for a while. However, some of the particles will remain, some particles will have changed the flow paths in the column, and flowing mobile phase through the column in the reverse direction will also change the flow paths in the column, so the column will not be restored completely.
Often a guard column is used to protect a new main column from particles in either the sample or the mobile phase, although the guard column may slightly reduce the overall resolution and plate count obtained.
Sometimes methods can be changed to incorporate filtering or centrifuging the sample, in order to reduce the possibility of introducing particles into the column in the future. However, these approaches are also likely to reduce the recovery of the target analytes. This can be evaluated using spiked samples (blank plasma spiked to a calculated level), even though spiking is may not be completely realistic.
Regards,
Frank Bales, Ph.D.
Email: frankbales.at.msn.com
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You are already past the point where pre-filters will help. The point is trying to rescue the column you have rather than purchasing another.
To rescue the column is not without risk of ruining the column.
1. disconnect the column from the detector.
2 Now connect the column to your system in the reverse direction
3 Run several cycles of your gradient at elevated temperature 45-40oC.
4 Has this brought the pressure down to your pre use level? 5 If yes, continue to run in the reverse direction, keep watch for an change in peak shape or retention time.
6 If no, try 10 injection of 50:50 Water DMSO-100 uL, You may need to drop the flow rate to tolerate the increase in pressure with the DMSO. Did this decrease the pressure? If yes go to step 5. If no, step 7.
Step 7. With the column in a vertical oreintation, Carefullly remove the original inlet endfitting on your column. Be sure not to bend any of the fittings or the tube. Using air pressure. remove the end frit from the fitting and clean. Check the end of the colunm packing for discoloration. Use a syringe needle or other small tool to remove some of the column packing and replace it with a methanol bead slurry of similar material from a used column or micro glass beads. Replace the frit and the end fitting. Reinstall column and check performance.
Step 7 is fairly heroic, you want to order a replacement column/
To limit over pressurization or the column you may want to insure that additional proteins are not precipitating in the samle vials. Perhaps increase the organic % you use to precipitate, increase the number of gs, decrease the temperature of precip or sample storage, Be sure to transfer the supernatant from ppt to a fresh vial.
Adding filtration steps and guard columns will also extend the lifetime of the column, so if your column does come back or if you order a new one be sure to add those steps/items.
-- Edward F. O'Connor
78 Marbern Drive
Suffield, Ct 06078-1533
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Hi Birendra,
You should check without column whether the pressure is low or high? If high then keep four channel in 100% methanol and purge, then you keep flow 3-5ml/min to clear the line without column. I do not know what kind of column , Mob phase you are using. Wash your column with 70:30 ACN or MeOH: Water after all samples run for at least 30min if possible in back direction with 0.5-1.0ml/min.
Rammohan
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Dear Birendra
You should check without column whether the pressure is low or high? First you can wash the system with 100% Methanol followed by (Iso propyl Alcohol). If pressure is with in limit. You can wash column with 100% Milli-Q water for 30 to 60 min initially which clean all plasma components which are polar in nature then followed by 100% methanol or Acetonitrile which will take of non polar components of plasma. Further column can be stored as per the manufacturers instruction. But before starting this procedure kindly refer the instruction manual for the column.
Hope this will help you. If you want further assistant do not hesitate to ask.
With Reagrds
NIL
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Dear birendra,
First you check how many injection over on your column if it is over than limit so its show back pressure and check pressure without column if it is ok then try to put reverse your column another wise keep overnight washing with 0.5 ml flow by using methanol:water(70:30). And I have to suggest that whenever you use column after complete analysis put wash method for 2 hr its give long life to your column and if your using any type of matrix it will not create problem in your column only thing is keep proper washing and filter your buffer if use high concentration
Regards
Mahendra sahu
Torrent research centre ahmedabad
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