By Anonymous on Tuesday, August 20, 2002 - 05:28 am:
i am searching for hplc method of analysis of serratipeptidase from the tablet formulation.
can anybody guide me from where to start developing its method of analysis or any paper is already published .
By Armando on Saturday, August 24, 2002 - 11:17 am:
You can start with Reversed-phase HPLC using an analytical butyl column. It is very resolutive method and commonly used for analysis of proteins.
Run a gradient from TFA 0.1%/water(A) to TFA 0.1%/acetonitrile(B), starting from 5%B to 100%B in 95 min and at flow rate 1 ml/min. After the first running you can optimize the gradient slope.
For more information check at: http://www.vydac.com/vydacpubs/analytical.html
By Armando on Saturday, August 24, 2002 - 11:17 am:
You can start with Reversed-phase HPLC using an analytical butyl column. It is very resolutive method and commonly used for analysis of proteins.
Run a gradient from TFA 0.1%/water(A) to TFA 0.1%/acetonitrile(B), starting from 5%B to 100%B in 95 min and at flow rate 1 ml/min. After the first running you can optimize the gradient slope.
For more information check at: http://www.vydac.com/vydacpubs/analytical.html
By Anonymous on Thursday, August 29, 2002 - 05:37 am:
i started doing like you told and also dowlloaded the brouchure on method deve by vyadac .
my serratiopeptidase is soluble in water , and it is eluting too early i got the major peak at retention time of 2 minutes .
so i tried with phosphate buffer as mentioned by vayadc ph 2 PH 4.4 AND ph 6.5
i got the good peak shape at PH 2.0 AND GOOD Resolutions from other peak
but still i am not confirm that the peak which i got is the major peak of interest what to do?as i am totally new to the peptide chemistry.
By Benjamin on Thursday, August 29, 2002 - 11:39 am:
Dear Anonymus;
The best way to solve your problem would be to modify the good separation you already have changing the buffer to one compatible with LC-MS. I suggest you try NH4OAC at pH 4.4 or 6.5. The MW determined this way will confirm the identity of your signal.
One other approach you may take is to simply go high in ACN content under the conditions you are using, perhaps 50%ACN (phosphate buffer may precipitate at higher ACN levels). If you see only one main signal under this conditions, then quite probably you are eluting all the compounds from the column.
Good Luck
Benjamin