Home >
Community >
How to remove phosphate buffer from C18 column and reduce pressure?
Upvote
23
Downvote
+ Hplc-uv
+ Phosphates
+ Buffer
+ Urine
+ Methanol
Posted by
Marianne Komek
How to remove phosphate buffer from C18 column and reduce pressure?
Dear Shehwaz,
When buffer salts are used in mobile phase/s, be careful that the buffer salts are not allowed to precipitate out of solution and cause the pump parts to stick. Hence when the system is not used for injections, ensure that the buffer solution is always circulated through the pumps at a low flow rate, such as 0.2 ml/minute. If necessary, re-circulate the mobile phase/s. Never let the pump lie unused for more than around ten minutes, if there are buffer salts within the mobile phase/s.
When you have finished using the buffer, flush the system with 15%/85% v/v methanol /water for two hours. When buffers with a very high salt content are used, it is useful to back-flush the pump pistons, please see the pump user manual.
Using 100% water will cause the buffer salts to precipitate out of solution.
It is possible that phosphate buffer has precipitated out and blocked parts.
If there is a blockage, from the precipitation of phosphate buffer, then this would explain a pressure build up. Cleaning blockages is tricky. Disconnect the pump from the injection valve, and try, even for a few moments at a time, to pump warm 15%/85 % methanol/water (HPLC grade) through the pump, into waste. Try to clean the check valves with acetone (HPLC grade). If/when the pump starts to pump, clean the injection ports and flow cell by pumping warm 15%/85% methanol/water (HPLC grade), through the injection ports and flow cell, without any column being in place.
When buffer salts are used in mobile phase/s, be careful that the buffer salts are not allowed to precipitate out of solution and cause the pump parts to stick. Hence when the system is not used for injections, ensure that the buffer solution is always circulated through the pumps at a low flow rate, such as 0.2 ml/minute. If necessary, re-circulate the mobile phase/s. Never let the pump lie unused for more than around ten minutes, if there are buffer salts within the mobile phase/s.
When you have finished using the buffer, flush the system with 15%/85% v/v methanol /water for two hours. When buffers with a very high salt content are used, it is useful to back-flush the pump pistons, please see the pump user manual.
Using 100% water will cause the buffer salts to precipitate out of solution.
It is possible that phosphate buffer has precipitated out and blocked parts.
If there is a blockage, from the precipitation of phosphate buffer, then this would explain a pressure build up. Cleaning blockages is tricky. Disconnect the pump from the injection valve, and try, even for a few moments at a time, to pump warm 15%/85 % methanol/water (HPLC grade) through the pump, into waste. Try to clean the check valves with acetone (HPLC grade). If/when the pump starts to pump, clean the injection ports and flow cell by pumping warm 15%/85% methanol/water (HPLC grade), through the injection ports and flow cell, without any column being in place.
Hello Shehwaz. Passing 100% water through a column does not precipitate phosphate. Most likely you experienced phase collapse. Reversed phase columns require organic modifiers to keep C18 solvated. Phase collapse will change your column performance drastically. Simply passing through some aqueous methanol will restore your column. If you requite to use fully aqueous solutions I advise you make a small methanol gradient at the end of each chromatogram to keep the C18 solvated. And never store your column in water because high risk of mould growth if there is trace residual phosphate
Hello Shehwaz. Passing 100% water through a column does not precipitate phosphate. Most likely you experienced phase collapse. Reversed phase columns require organic modifiers to keep C18 solvated. Phase collapse will change your column performance drastically. Simply passing through some aqueous methanol will restore your column. If you requite to use fully aqueous solutions I advise you make a small methanol gradient at the end of each chromatogram to keep the C18 solvated. And never store your column in water because high risk of mould growth if there is trace residual phosphate
Hello Mr Anwar, may I know the result of the washing issue? I need to wash my LiChrospher rp18 column that is suspected to have phosphate precipitates inside Shehwaz AnwarAde Kujore
Hello Mr Anwar, may I know the result of the washing issue? I need to wash my LiChrospher rp18 column that is suspected to have phosphate precipitates inside Shehwaz AnwarAde Kujore
Thanks a lot Ade Kujore and Abdallah Hussien Fathy, I did backflush of column according to manual, and passed 5% methanol : 95 % water for two hours at low flow rate (0.1 ml/minute). Now it looks working and pressure is reduced from 9.9 to 6.0. Let us monitor for full night.
Thanks a lot Ade Kujore and Abdallah Hussien Fathy, I did backflush of column according to manual, and passed 5% methanol : 95 % water for two hours at low flow rate (0.1 ml/minute). Now it looks working and pressure is reduced from 9.9 to 6.0. Let us monitor for full night.
Dear Shehwaz,
When buffer salts are used in mobile phase/s, be careful that the buffer salts are not allowed to precipitate out of solution and cause the pump parts to stick. Hence when the system is not used for injections, ensure that the buffer solution is always circulated through the pumps at a low flow rate, such as 0.2 ml/minute. If necessary, re-circulate the mobile phase/s. Never let the pump lie unused for more than around ten minutes, if there are buffer salts within the mobile phase/s.
When you have finished using the buffer, flush the system with 15%/85% v/v methanol /water for two hours. When buffers with a very high salt content are used, it is useful to back-flush the pump pistons, please see the pump user manual.
Using 100% water will cause the buffer salts to precipitate out of solution.
It is possible that phosphate buffer has precipitated out and blocked parts.
If there is a blockage, from the precipitation of phosphate buffer, then this would explain a pressure build up. Cleaning blockages is tricky. Disconnect the pump from the injection valve, and try, even for a few moments at a time, to pump warm 15%/85 % methanol/water (HPLC grade) through the pump, into waste. Try to clean the check valves with acetone (HPLC grade).
If/when the pump starts to pump, clean the injection ports and flow cell by pumping warm 15%/85% methanol/water (HPLC grade), through the injection ports and flow cell, without any column being in place.
Kind regards,
Ade Kujore
Marketing
Cecil Instruments Limited
Milton Technical Centre, Cambridge CB24 6AZ
United Kingdom
email:- ade.kujore@cecilinstruments.com
telephone:- +44 (0) 1223 420821
fax.:- +44 (0) 1223 420475
web site:- www.cecilinstruments.com
Facebook www.facebook.com/cecilinstruments
Registered Office: Unex House, Bourges Boulevard, Peterborough PE1 1NG, United Kingdom
Registered Number 909536
Dear Shehwaz,
When buffer salts are used in mobile phase/s, be careful that the buffer salts are not allowed to precipitate out of solution and cause the pump parts to stick. Hence when the system is not used for injections, ensure that the buffer solution is always circulated through the pumps at a low flow rate, such as 0.2 ml/minute. If necessary, re-circulate the mobile phase/s. Never let the pump lie unused for more than around ten minutes, if there are buffer salts within the mobile phase/s.
When you have finished using the buffer, flush the system with 15%/85% v/v methanol /water for two hours. When buffers with a very high salt content are used, it is useful to back-flush the pump pistons, please see the pump user manual.
Using 100% water will cause the buffer salts to precipitate out of solution.
It is possible that phosphate buffer has precipitated out and blocked parts.
If there is a blockage, from the precipitation of phosphate buffer, then this would explain a pressure build up. Cleaning blockages is tricky. Disconnect the pump from the injection valve, and try, even for a few moments at a time, to pump warm 15%/85 % methanol/water (HPLC grade) through the pump, into waste. Try to clean the check valves with acetone (HPLC grade).
If/when the pump starts to pump, clean the injection ports and flow cell by pumping warm 15%/85% methanol/water (HPLC grade), through the injection ports and flow cell, without any column being in place.
Kind regards,
Ade Kujore
Marketing
Cecil Instruments Limited
Milton Technical Centre, Cambridge CB24 6AZ
United Kingdom
email:- ade.kujore@cecilinstruments.com
telephone:- +44 (0) 1223 420821
fax.:- +44 (0) 1223 420475
web site:- www.cecilinstruments.com
Facebook www.facebook.com/cecilinstruments
Registered Office: Unex House, Bourges Boulevard, Peterborough PE1 1NG, United Kingdom
Registered Number 909536
More
VOTE
Follow the washing procedure carefully (use many solvents aq...organic...aq).
Follow the washing procedure carefully (use many solvents aq...organic...aq).
More
VOTE
Hello Shehwaz. Passing 100% water through a column does not precipitate phosphate. Most likely you experienced phase collapse. Reversed phase columns require organic modifiers to keep C18 solvated. Phase collapse will change your column performance drastically. Simply passing through some aqueous methanol will restore your column. If you requite to use fully aqueous solutions I advise you make a small methanol gradient at the end of each chromatogram to keep the C18 solvated. And never store your column in water because high risk of mould growth if there is trace residual phosphate
Hello Shehwaz. Passing 100% water through a column does not precipitate phosphate. Most likely you experienced phase collapse. Reversed phase columns require organic modifiers to keep C18 solvated. Phase collapse will change your column performance drastically. Simply passing through some aqueous methanol will restore your column. If you requite to use fully aqueous solutions I advise you make a small methanol gradient at the end of each chromatogram to keep the C18 solvated. And never store your column in water because high risk of mould growth if there is trace residual phosphate
More
VOTE
Hello Mr Anwar, may I know the result of the washing issue? I need to wash my LiChrospher rp18 column that is suspected to have phosphate precipitates inside
Shehwaz Anwar Ade Kujore
Hello Mr Anwar, may I know the result of the washing issue? I need to wash my LiChrospher rp18 column that is suspected to have phosphate precipitates inside
Shehwaz Anwar Ade Kujore
More
VOTE
Thanks a lot Ade Kujore and Abdallah Hussien Fathy,
I did backflush of column according to manual, and passed 5% methanol : 95 % water for two hours at low flow rate (0.1 ml/minute). Now it looks working and pressure is reduced from 9.9 to 6.0.
Let us monitor for full night.
Thanks a lot Ade Kujore and Abdallah Hussien Fathy,
I did backflush of column according to manual, and passed 5% methanol : 95 % water for two hours at low flow rate (0.1 ml/minute). Now it looks working and pressure is reduced from 9.9 to 6.0.
Let us monitor for full night.
More
VOTE