Summary: HPLC gel-filtration of proteins

Wed Nov 17 01:09:19 EST 1993

Dear Colleagues,

     My original post was:
>I have occasion to do a series of molecular weight determinations of
>proteins in the presence and absence of detergents.  Years ago I used
>ToyoSoda PW and SW silica-based columns; the PW bound all of my proteins,
>and the SW worked fairly well.  My question is: what is your experience
>with HPLC gel filtration columns for proteins presently?  What/which are
>best?  Protein binding?  Silica vs plastic polymers?  Effects of detergent?

     Replies received:

Carl Weatherell (weathere at

I am presently examining secondary retention effects on ToyaSodaPW and
SW columns using factorial experimental design...  What I have found thus 
far W.R.T. the 2 stationary phases are, briefly:
     -efficiency of the PW phase is much larger than that of the SW phase
     -ion exclusion effects on the SW phase are larger than that of the PW
     -the PW phase is extremely hydrophobic... complete retention of all 
      denatured proteins is observed at 0.4M NaCl in 3.5mM SDS, pH=4 or 7
     -both phases adsorb SDS
     -a good eluent to achieve optimum resolution with no protein binding 
      to the stationary phase consists of 3.5mM SDS, pH=4 (50mM PO4 buffer
      with 0.1M NaCl)
Pier Carlo Montecucchi (100143.765 at

I have used TSK 3000 SW Spherogel column (600 x 7.5 mm I.D.) from Beckman.
Eluent: 0.01% acetic acid    Flow Rate: 1 ml/min    Detection: 220nm
Sometimes the data obtained by GP-HPLC should be treated with caution as a 
consequence (i) of aspecific interactions between the solutes and the 
stationary phase and (ii) of the effect of the mobile phase (pH value and 
salt concentration) on the tertiary structure of the molecules.  Using this 
system, I was able to determine the MW of a small molecule (MW 1000) and to 
separate this low molecular weight immunoregulin from salts.
Ann C. Rice (acrice at

I am using the Phenomenex SEC 2000 HPLC gel filtration column presently and 
think it works well.  You can go down to pH 2.5 and it will take 6M 
guanidine and 0.05% SDS.  They make 3 sizes depending on the mwt range of 
the proteins you want to characterize.

     Further investigation of various columns available netted:

Manufacturer  Column       Dimensions  Phase Dia Pores  Avg N/m  Range   $
------------- ------------ ----------- --------- ------ -------- -----  ---
ToyoHaas      TSK3000SWXL  7.8x300 mm      5u     250A   25,800  -500K  750
   "          TSK3000SW    7.5x300 mm     10u     250A    8,500  -500K  700

Beckman       Ultrasphero- 7.5x300 mm      5u     245A   24,400  -700K  620
               gel 3000

Phenomenex    BioSep-SEC-  7.8x300 mm      5u     290A   21,200  -700K  595

Rainin        Hydropore-   4.6x250 mm      5u     300A   11,100   -1M   700
All the columns were prepared from silica; the Phenomenex and Rainin are 
"hydrophilic bonded silica", and the Beckman claims a "proprietary" silica 
type.  The TSK3000SWXL had the highest plate count and also resolved large 
proteins the best of all the columns surveyed.  Plates/meter were obtained 
by averaging the plates measured for 3-4 proteins in test chromatograms; 
proteins were typically thyroglobulin, ovalbumin, cytochrome c in each case.
Recoveries were compared between the TSK3000SWXL and BioSep-SEC-S3000 
columns; the TSK showed somewhat better recoveries, especially for large 
molecular weight proteins.

Hope this is helpful.  Best regards,  Shaun
  = Shaun D. Black, PhD     | Internet:  shaun%jason.decnet at = 
  = Dept. of Biochemistry   | University of Texas Health Center, at Tyler = 

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