Look up the size ranges for the various Sephadex G-series of size exclusion colu
ID: 215751 • Letter: L
Question
Look up the size ranges for the various Sephadex G-series of size exclusion column matrices. The molecular mass of wheat germ acid phosphatase is about 55 kDa. Besides Sephadex G-75, which Sephadex matrix would be the (second) best to separate wheat germ acid phosphatase from both larger proteins as well as from smaller molecules/polyatomic ions: G-50 or G-100? Is the order in which components of a mixture will be separated different than with ion exchange chromatography? If so, why? Why is the size range of the proteins that can be separated by the Waters Protein Pak 300 SW gel filtration column different for globular vs. random-coil proteins?Explanation / Answer
Sephadex G-100 is the second best as it has a fractionation range from 4kDa - 150kDa. Whereas G-50 has fractionation range only from 1.5kDA - 30kDa. Since wheat germ acid phosphatase has a size of 55kDa, G-100 column would be better than G-50 column as the upper limit of G-50 is 30kDa which is less than the molecular weight of wheat germ acid phosphatase.
Yes the order of separation will be different than ion exchange chromatography because the molecules in gel filtration chromatography are separated based on their size, with larger molecules eluting first. In ion exchange chromatography the separation is based on the charge. Initially, molecules that do not bind or bind weakly to the stationary phase are first to wash away.
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