Question

h) Consider the separation of a mixture of glutamate dehydrogenase (molecular weight 290,000), lactate dehydrogenase (molecular weight 140,000), serum albumin (MW 67,000), ovalbumin (MW 43,000), and cytochrome c (MW 12,400) on a gel filtration column packed with Bio-Gel P-150 (fractionation range 15,000 - 150,000). •

Which protein would elute first? Explain. •

Which protein would elute last? Explain. •

List the order of elution of all five proteins, from first to last.

Answer 1

Gel filtration chromatography separates the mixture based on the molecular size of the components, the smaller molecules moves faster through the gel while the larger molecules moves on a comparatively slower rate and this helps in the separation of mixture into individual components based on molecular size.

• Cytochrome C would elute first as it has the lowest molecular weight
• Glutamate dehydrogenase would elute last as it has the highest molecular weight
• Cytochrome C - Ovalbumin - Serum albumin - Lactate dehydrogenase - Glutamate dehydrogenase