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Chromatography columns
#1
Hello,

Can anyone suggest which out of the four companies Merckmillipore, Agilent, Thermo scientific and Waters is better for chromatographic columns giving a little data supporting their answer?

According to my findings, Agilent columns are the most widely used.

Also, which columns would be more preferred for proteomics application? C18,C8,biphenyl ? and their molecular weight ranges if possible!

Thankyou!
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#2
Hi Kanny,

I don't have any data per se, but during my own research at IIT Delhi, I also used Agilent system. It has been a convention there to use Agilent columns.

My work was on bio-pesticide production, reverse phase was thus very much good to go for.

C18 is known to have highest hydrophobicity and a very broad spectrum of interaction (with a variety of compounds). C8 would just help in lower retention times (and faster elution). As C18 works with most of the organic compounds and is both cheap and very stable, I would suggest C18 for any kind of 'organic work'.

At the end of the day, Choice of stationary phases is very much dependent upon the analyte at hand. And choice of molecular wight range/pore size for your column would thus depend upon the type(s) of molecule(s) you are going to work upon and the pressure you are going to expose the column to.

The manual for columns usually helps sufficiently to address most of your needs..
Sunil Nagpal
MS(Research) Scholar, IIT Delhi (Alumnus)
Advisor for the Biotech Students portal (BiotechStudents.com)
Computational Researcher in BioSciences at a leading MNC


Suggested Reads:
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Biotechnology Competitive Exams in India
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#3
Thankyou Very much Sunil. Smile

Actually, I did read quiet a few manuals and details of the columns too..just that they haven't stated the mol wt. ranges.
Have tried contacting agilent and thermo scientific; Awaiting their replies.

Thanks once again.
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#4
Pleasure is all mine Kanny!

I will do some research to look for information on Mol wt ranges for various columns.

If you get to know the same, do share it..that would be a useful one.
Sunil Nagpal
MS(Research) Scholar, IIT Delhi (Alumnus)
Advisor for the Biotech Students portal (BiotechStudents.com)
Computational Researcher in BioSciences at a leading MNC


Suggested Reads:
Top Biotech Companies | Top places to work
Indian Biotech Companies and Job Openings
Aiming a PhD in Top Grad School? | These are the Important Points to Consider
Careers in Biotechnology | A list of various Options
Biotechnology Competitive Exams in India
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#5
Sure!

Infact : http://www.seaviewsci.com/vydac/vydacpub...k1429.pdf))
Got some data from here, though not convincing enough:

C18 : 2000`3000Da
C4 : > 3000Da (some even mention upto 20000Da)
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#6
Hi Kanny,

In general, molecular weight range in case of RP should be associated with 'pore size' of the column (larger peptides would need a wider column, while smaller peptides may be dealt with a narrow one as well). Since it's not a 'molecular sieve' chromatography, the stationary phase (C18) should be expected to have a universal range.

At the same time, it would be an interesting piece of information, as to what's the 'handling limit' of C18 stationary phase in general.
Sunil Nagpal
MS(Research) Scholar, IIT Delhi (Alumnus)
Advisor for the Biotech Students portal (BiotechStudents.com)
Computational Researcher in BioSciences at a leading MNC


Suggested Reads:
Top Biotech Companies | Top places to work
Indian Biotech Companies and Job Openings
Aiming a PhD in Top Grad School? | These are the Important Points to Consider
Careers in Biotechnology | A list of various Options
Biotechnology Competitive Exams in India
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#7
Just found an interesting piece of layout for deciding the pore-size of C18 chromatography column (rom glsciences):


   
Sunil Nagpal
MS(Research) Scholar, IIT Delhi (Alumnus)
Advisor for the Biotech Students portal (BiotechStudents.com)
Computational Researcher in BioSciences at a leading MNC


Suggested Reads:
Top Biotech Companies | Top places to work
Indian Biotech Companies and Job Openings
Aiming a PhD in Top Grad School? | These are the Important Points to Consider
Careers in Biotechnology | A list of various Options
Biotechnology Competitive Exams in India
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#8
Nice!
Thankyou.

I have come up with another question though Tongue

Each column might have its own best suited solvents and their gradient concentrations, So was looking for C18, Biphenyl and C4 solvents for proteomics applications!
If anyone could help on this.

Thankyou! Smile
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#9
(06-30-2015, 04:46 PM)Kanny Wrote: Nice!
Thankyou.

I have come up with another question though Tongue

Each column might have its own best suited solvents and their gradient concentrations, So was looking for C18, Biphenyl and C4 solvents for proteomics applications!
If anyone could help on this.

Thankyou! Smile

Hi Kanny,
Quote:The answer is two way:
1) Columns are indeed suited to a particular set of solvents
2) One must take care of the chemical nature of the analyte to avoid its reaction/deterioration/dissolution in/with solvent(s).
  • C18 is a highly non-polar stationary phase, so using a polar solvent is the most recommended one (often [Water: HPLC grade water with 0.1% acid] with different proportions of [metahnol/acetonitrile/propanol: again HPLC grade] are sufficient).
  • Acetonitrile and Acetic acid (low conc) can be used with C4. People recommend/suggest THF too (but that can be nasty for your column. You may use higher proportions of THF/Acetic acid in C18s or C8s too for the cases where protein/peptide is resisting elution.
  • A mix of Methanol (~70%) and water (~30%) is often considered ideal for Phenyl packings.


Cleaning Practice for Reverse Phase Columns (like C18, C8, C4, C1, C30, CN or Phenyl packings)

Regeneration of RP packings RP- packings like C18, C8, C4, C1, C30, CN or Phenyl  stationary phases should be cleaned through following procedure (source: nestgrp): 

• Flush the column with 20 column volumes Water
• Flush the column with 20 column volumes Acetonitrile
• Flush the column with 5 column volumes Isopropanol
• Flush the column with 20 column volumes Heptane
• Flush the column with 5 column volumes Isopropanol
• Flush the column with 20 column volumes Acetonitrile
Sunil Nagpal
MS(Research) Scholar, IIT Delhi (Alumnus)
Advisor for the Biotech Students portal (BiotechStudents.com)
Computational Researcher in BioSciences at a leading MNC


Suggested Reads:
Top Biotech Companies | Top places to work
Indian Biotech Companies and Job Openings
Aiming a PhD in Top Grad School? | These are the Important Points to Consider
Careers in Biotechnology | A list of various Options
Biotechnology Competitive Exams in India
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