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HPLC COLUMN SELECTION

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mohd
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Topic Started by mohd
on 7/29/2009 8:27 AM   
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1 HOW WE WILL SELECT A COLUMN FOR HPLC ANALYSIS.
2 MOLECULE IS ACIDIC IN NATUREOR BASIC IN NATURE/NEUTRAL IN NATURE.
3 IN WICH CASES WE WILL USE CN /NH2/PHENYL COLUMS.


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Dr. Analytical
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Posted By Dr. Analytical
on 7/29/2009 11:15 AM   
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There are many "Column Selection Guides" on the internet.  Search for "HPLC Column Selection Guide" and look through the options.

Here are some general rules:

  • To get a separation you must have some interaction with the stationary phase.  So, choose a stationary phase that is "like" your molecules.  If your compounds have many carbons, or differ in the number of carbons, choose a stationary phase with carbon (C18, C8, phenyl).  This works well for molecules that are soluble in solvents such as acetonitrile, methanol, etc.
  • Acids and bases can be difficult to separate.  The "neutral" form is usually retained more on a reversed phase (C18) column, while the "ionic" form is not retained as much.  You control the pH so that you have the compounds in the proper form.
  • A phenyl column is used when you have aromatic compounds that you are trying to separate.  The NH2 and CN columns can be used for separating polar organic molecules such as sugars.
You would need to give us more information before we could provide any more help.


kishoreHPLC
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Posted By kishoreHPLC
on 3/9/2010 9:37 AM   
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hello sir i have a non polar compound whose molecular weight is 488.01 and its solubility in water is 0.008 g/liter. so please let me now how to select a column depending on polarity of compound. mostly we use reverse phase columns.



Dr. Analytical
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Posted By Dr. Analytical
on 3/20/2010 10:23 AM   
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If your column is "non polar" then a reversed phase (C18) column should work.  Since this has a high molecular weight, you may want to consider a C8 column, which will produce less retention.


Ebram
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Posted By Ebram
on 4/20/2010 2:56 AM   
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Dear all,
i wanna start RPC method development for a certain protein with a molecular weight of (300KDa) i have no idea about the exact polarity .

I've already started an overview gradient with C4 (214TP54) (4.6 x 250mm) but all what i got just huge peak directly after the peak front as if that there is nothing retained.

Could the pore size of the column is the problem since large proteins need wide pore to give a chance for separation ! ..
or
just its a matter of hydrophobicity and it has nothing to do with the pore size?

thanks in advance


Last edited Apr 20, 2010, 4:57 AM by Ebram

Dr. Analytical
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Posted By Dr. Analytical
on 4/20/2010 9:10 AM   
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Please give us more information about your system: column manufacturer and brand name, mobile phase gradient, including flow rate, and detector settings.

Most protein separations are performed on "wide pore" columns.  These columns usually have a pore size of 300 Angstroms.  If you use a small pore column, the protein can not migrate into the pores, and can not be retained well.  It then comes out early.  This may be what you are seeing.

Also, a C4 column is not very retentive, and is best only for very hydrophobic proteins, and your gradient conditions will be important.



Ebram
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Posted By Ebram
on 4/21/2010 2:40 AM   
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I am using Vydac C4 column with pore size 300 Angstrome mobile phase are pure water and pure ACN each with 0.1% TFA  gradient is usual overview gradient 5%-95% through 40min.
system : shimadzu LC2010
thanks in advance



Dr. Analytical
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Posted By Dr. Analytical
on 4/21/2010 10:03 AM   
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From this information, I think you have a less hydrophobic protein.  Try using a C8 or C18 column

If you do not have other columns, you could try your separation at other pH values.  For example, try using ammonium formate at about pH 6.


viKI_14
India

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Posted By viKI_14
on 11/6/2011 23:08 PM   
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Which data of a sample must be collect for column selection and for buffer solution for mobile phase preparation?  and please tell me the chemistry of distribution and elution of sample solution from column in reverse phase liquid chromatography. 


Last edited Nov 07, 2011, 1:19 AM by viKI_14

Dr. Analytical
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Posted By Dr. Analytical
on 8/17/2012 8:42 AM   
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There are too many questions to answer.  I suggest that you find a reference book on HPLC method development or attend a training class to learn more about HPLC.


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