Superdex Column suitable for my purpose - (Apr/18/2007 )
Hai everyone,
I will do gel filtration chromatography for my experiment. Since I am new for this stuff, I need your help. I want to know what factors should be considered to choose GFC column?
What I want to achieve is to get molecular weight distribution of humic acid solution. I will measure in terms of DOC and UV254. For those measurements, at least I have to collect minimum 5 mL for each fraction collected.
Do you have any suggestions what kind (or brand?) should I choose?
Thanks
Arie DS
-arie-
QUOTE (arie @ Apr 18 2007, 10:33 PM)
Hai everyone,
I will do gel filtration chromatography for my experiment. Since I am new for this stuff, I need your help. I want to know what factors should be considered to choose GFC column?
What I want to achieve is to get molecular weight distribution of humic acid solution. I will measure in terms of DOC and UV254. For those measurements, at least I have to collect minimum 5 mL for each fraction collected.
Do you have any suggestions what kind (or brand?) should I choose?
Thanks
Arie DS
I will do gel filtration chromatography for my experiment. Since I am new for this stuff, I need your help. I want to know what factors should be considered to choose GFC column?
What I want to achieve is to get molecular weight distribution of humic acid solution. I will measure in terms of DOC and UV254. For those measurements, at least I have to collect minimum 5 mL for each fraction collected.
Do you have any suggestions what kind (or brand?) should I choose?
Thanks
Arie DS
About choosing GFC see GEHealhcare catalog in section Chromatography media!
What is about composition of your extract I mean pH, buffer system!
The other question what is supposed Mw range of your conpounds - low or high molecular ?
First of all why do you to use GF for your extract? I think that first of all you should apply RP or NP LC depends on hydrohobicity or hydrophility of compounds in your extract, or may be better IEC to enrich your extract. Only after that you should apply GF on low resolution resins like Sepharose CL-4B, Sephadex G75 or G200, Sephacryl S-200, S-300, S 100 or S-400. Only for final polishing you should use high perfomance resins like Superose, Superdex. DON' t apply you crude extract on Superose, Superdex!
-circlepoint-
QUOTE
About choosing GFC see GEHealhcare catalog in section Chromatography media!
What is about composition of your extract I mean pH, buffer system!
The other question what is supposed Mw range of your conpounds - low or high molecular ?
First of all why do you to use GF for your extract? I think that first of all you should apply RP or NP LC depends on hydrohobicity or hydrophility of compounds in your extract, or may be better IEC to enrich your extract. Only after that you should apply GF on low resolution resins like Sepharose CL-4B, Sephadex G75 or G200, Sephacryl S-200, S-300, S 100 or S-400. Only for final polishing you should use high perfomance resins like Superose, Superdex. DON' t apply you crude extract on Superose, Superdex!
What is about composition of your extract I mean pH, buffer system!
The other question what is supposed Mw range of your conpounds - low or high molecular ?
First of all why do you to use GF for your extract? I think that first of all you should apply RP or NP LC depends on hydrohobicity or hydrophility of compounds in your extract, or may be better IEC to enrich your extract. Only after that you should apply GF on low resolution resins like Sepharose CL-4B, Sephadex G75 or G200, Sephacryl S-200, S-300, S 100 or S-400. Only for final polishing you should use high perfomance resins like Superose, Superdex. DON' t apply you crude extract on Superose, Superdex!
Thanks for the response, I do not measure such complex and small MW proteins, instead I only measure my humic acids solution MW distribution and effect of photocatalysis toward MW distribution, on pH 7, and planning to use PBS. My range would be between 100 - 100,000 kD. I suppose, this can be done on simple GFC procedures. I am only confused about picking the column..
Another thing, what does particle size refers to? I usually encounter this on the column products catalog..
Suggestions are very much appreciated
Thanks
-arie-
QUOTE (arie @ Apr 21 2007, 10:35 PM)
QUOTE
About choosing GFC see GEHealhcare catalog in section Chromatography media!
What is about composition of your extract I mean pH, buffer system!
The other question what is supposed Mw range of your conpounds - low or high molecular ?
First of all why do you to use GF for your extract? I think that first of all you should apply RP or NP LC depends on hydrohobicity or hydrophility of compounds in your extract, or may be better IEC to enrich your extract. Only after that you should apply GF on low resolution resins like Sepharose CL-4B, Sephadex G75 or G200, Sephacryl S-200, S-300, S 100 or S-400. Only for final polishing you should use high perfomance resins like Superose, Superdex. DON' t apply you crude extract on Superose, Superdex!
What is about composition of your extract I mean pH, buffer system!
The other question what is supposed Mw range of your conpounds - low or high molecular ?
First of all why do you to use GF for your extract? I think that first of all you should apply RP or NP LC depends on hydrohobicity or hydrophility of compounds in your extract, or may be better IEC to enrich your extract. Only after that you should apply GF on low resolution resins like Sepharose CL-4B, Sephadex G75 or G200, Sephacryl S-200, S-300, S 100 or S-400. Only for final polishing you should use high perfomance resins like Superose, Superdex. DON' t apply you crude extract on Superose, Superdex!
Thanks for the response, I do not measure such complex and small MW proteins, instead I only measure my humic acids solution MW distribution and effect of photocatalysis toward MW distribution, on pH 7, and planning to use PBS. My range would be between 100 - 100,000 kD. I suppose, this can be done on simple GFC procedures. I am only confused about picking the column..
Another thing, what does particle size refers to? I usually encounter this on the column products catalog..
Suggestions are very much appreciated
Thanks
Dear Arie!
As I work with proteins it is not easy question for me. I only can suggest the following media from GE Healhcare:
Sephacryl S-500 HR Fractionation range dextrans 40-20000kDA
Sephacryl S 1000 SF -- Fractionation range dextrans 500 kDA-100000 kDA
Concerning superdex mentioned above. It is not of your choice because Mw range is 1-100kDa (for Superdex 200)
In your case it is difficult for me to suggest what will be better because of Mw range shift will occur which deals with particular hydrodynamic radius of your polymers
Try to see literature concerning this problem.
May be this source will be usefull:
http://www.springerlink.com/content/w5p556682u1k61h3/
About particle size:
From chromatography theory: Resolution is in direct proportionality to second root of N ( theoretical plate number). Decreasing in particle size will lead to increase N. So decreasing of particle size leads to increasing resolution.
-circlepoint-
Hai,
Thanks for the reply. I have managed everything to achieve my goal. I have learned a lot in gehealthcare website, especially there is a handbook about GFC. Overall, what I want to do is to determine MW distribution of humic acid solution. I have purchase an empty column C 26/70. which of course I will have to pack it by myself with Sephadex G-75 (which I have had it already).
Thats all, thanks for your help.
Arie DS
-arie-
QUOTE (arie @ Apr 28 2007, 07:00 AM)
Hai,
Thanks for the reply. I have managed everything to achieve my goal. I have learned a lot in gehealthcare website, especially there is a handbook about GFC. Overall, what I want to do is to determine MW distribution of humic acid solution. I have purchase an empty column C 26/70. which of course I will have to pack it by myself with Sephadex G-75 (which I have had it already).
Thats all, thanks for your help.
Arie DS
Thanks for the reply. I have managed everything to achieve my goal. I have learned a lot in gehealthcare website, especially there is a handbook about GFC. Overall, what I want to do is to determine MW distribution of humic acid solution. I have purchase an empty column C 26/70. which of course I will have to pack it by myself with Sephadex G-75 (which I have had it already).
Thats all, thanks for your help.
Arie DS
Hi!
It sounds strange about your choice Sephadex G75 ( because range of Mw - 5 - 80KDA ) Is it suitable for you ( you mentioned range 100-1000KDA) Take a look here: http://www4.gelifesciences.com/aptrix/upp0...amp;zone=Labsep
Before you exp it will be better to calibrate your column with High Mw proteins in GE kit.
-circlepoint-
Hai, thanks for the reply.
I notice there was a mistake on my part, it was not 100-100,000 kDa, instead of 100-100,000 Da. Second is, it is the objective of my research that must be adjusted as such to accommodate available funding, due to funding limitation I can only buy parts that I still do not have. As a consequence, my target range is changed to around 1 kD to 50 kD.
Arie DS
-arie-