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:
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Carl Weatherell (weathere at emr1.emr.ca)
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)
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Pier Carlo Montecucchi (100143.765 at compuserve.com)
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.
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Ann C. Rice (acrice at gems.vcu.edu)
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.
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Further investigation of various columns available netted:
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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
S3000
Rainin Hydropore- 4.6x250 mm 5u 300A 11,100 -1M 700
5-SEC
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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
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= Shaun D. Black, PhD | Internet: shaun%jason.decnet at relay.the.net =
= Dept. of Biochemistry | University of Texas Health Center, at Tyler =
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