HILIC/ELSD [August 25, 2004]
Posted: Sun Aug 29, 2004 8:41 pm
By GoranKarlsson on Wednesday, August 25, 2004 - 08:11 am:
Hi,
I am using a TSK Amide-80 HILIC HPLC column together with an ELSD detector (Sedex 75). Mobile phase is 75% acetonitrile in water. This column gives rather much noise by ELSD. I have tried to wash the (new) column with 100 % water to clean it, but after about 20 column volumes of water there is still rather much noisy response (the response should be lower with 100% wather compared to 75% acetonitrile, I have understood).
Could someone please tell me how to handle this problem. Is it sutable eg to wash the column with an "regeneration solution", 0.3 M sodium acetate/0.2 M sodium phosphate, pH 5-6,
which is recommended for Polyhydroxyethyl A Hilic columns?
In the instruction for the column from Tosohaas it is said that a temperature up to 80 C is OK. I think this is strange - does the amide group really stand this high temperature? pH up to 7.5 is also recommended - this makes sense because deamidation is stimulated by alkaline pH.
I am grateful for all information/suggestions concerning this problem.
Regards,
Goran Karlsson
Octapharma
Stockholm
Sweden
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By Kostas Petritis on Wednesday, August 25, 2004 - 03:36 pm:
Hi Goran,
I do not have experience with the Amide-80 column but I have used the PolyLC polyhydroxylethyl A with ELSD. I always start by using the regeneration solution you mentioned in the brand HILIC new columns as these columns tend to leak when they are new or if you store them for long time (even if they are stored in MeOH and in the refrigerator). After regeneration I wash with several volumes of pure water and I start with a blank gradient before analysing the compounds of interest.
Even after regeneration the background noise will be a little bit higher than C18 columns and you might experience some system peaks during gradient elution (depending on your gradient).
Kostas
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By Einar Pontén - SeQuant AB on Thursday, August 26, 2004 - 02:05 am:
Phase bleed from polar embedded and some other polar phases are well known. Lengthy and tedious washing procedures may reduce the interference problems.
In fact, ELSD has been used for the comparision of phase bleed between columns;
"Determination of HPLC Column Phase Bleed Using Evaporative Light Scattering Detection"
Scott C. Anderson, Raaidah Saari-Nordhaus, Ian Chappell
Poster presented at Pittcon 2005, available from Alltech Associates Inc.
By the same method, these authors have also shown that the ZIC®-HILIC column is just as stable as several other non-polar reversed phase columns (C8-C18).
(More information on the ZIC®-HILIC column is available at http://www.sequant.com)
Hi,
I am using a TSK Amide-80 HILIC HPLC column together with an ELSD detector (Sedex 75). Mobile phase is 75% acetonitrile in water. This column gives rather much noise by ELSD. I have tried to wash the (new) column with 100 % water to clean it, but after about 20 column volumes of water there is still rather much noisy response (the response should be lower with 100% wather compared to 75% acetonitrile, I have understood).
Could someone please tell me how to handle this problem. Is it sutable eg to wash the column with an "regeneration solution", 0.3 M sodium acetate/0.2 M sodium phosphate, pH 5-6,
which is recommended for Polyhydroxyethyl A Hilic columns?
In the instruction for the column from Tosohaas it is said that a temperature up to 80 C is OK. I think this is strange - does the amide group really stand this high temperature? pH up to 7.5 is also recommended - this makes sense because deamidation is stimulated by alkaline pH.
I am grateful for all information/suggestions concerning this problem.
Regards,
Goran Karlsson
Octapharma
Stockholm
Sweden
-------------------------------------------------------------------------------------------------------
By Kostas Petritis on Wednesday, August 25, 2004 - 03:36 pm:
Hi Goran,
I do not have experience with the Amide-80 column but I have used the PolyLC polyhydroxylethyl A with ELSD. I always start by using the regeneration solution you mentioned in the brand HILIC new columns as these columns tend to leak when they are new or if you store them for long time (even if they are stored in MeOH and in the refrigerator). After regeneration I wash with several volumes of pure water and I start with a blank gradient before analysing the compounds of interest.
Even after regeneration the background noise will be a little bit higher than C18 columns and you might experience some system peaks during gradient elution (depending on your gradient).
Kostas
-------------------------------------------------------------------------------------------------------
By Einar Pontén - SeQuant AB on Thursday, August 26, 2004 - 02:05 am:
Phase bleed from polar embedded and some other polar phases are well known. Lengthy and tedious washing procedures may reduce the interference problems.
In fact, ELSD has been used for the comparision of phase bleed between columns;
"Determination of HPLC Column Phase Bleed Using Evaporative Light Scattering Detection"
Scott C. Anderson, Raaidah Saari-Nordhaus, Ian Chappell
Poster presented at Pittcon 2005, available from Alltech Associates Inc.
By the same method, these authors have also shown that the ZIC®-HILIC column is just as stable as several other non-polar reversed phase columns (C8-C18).
(More information on the ZIC®-HILIC column is available at http://www.sequant.com)