Question

Briefly explain how chromatography technique works. What type of chromatography is being used in this lab activity? ___________________________________________________ __________________________________ What are the mobile phase and stationary phase substances/Compounds, which are used in this experiment? Briefly explain the purpose of each phase. ____________________________ ______________________________________ If the following mix of molecules were purified using size exclusion chromatography, what would be the order in which the molecules pass through the opening in the bottom of the column? Mixture containing: Hemoglobin, 65,000 Daltons; Ribonuclease A, 13, 700 Daltons; Myosin, 180,000 Daltons. First molecule to appear: ____________________________ Second molecule to appear: __________________________________ Third molecule to appear: ________________________

Answer 1

Ans 1.

The Chromatography is the technique which is used for the separation of the mixture.

Chromatography Technique works as, basically on the 2 Phases mainly the mobile phase and the stationary phase. The mixture is passed through the column , the sample is passed alone with the mobile phase , if the sample has the affinity with the mobile phase then the it will elute first , and if the sample has the affinity towards the stationary phase then that will take more time to elute.

Ans 2

Mobile phase commonly used are water , organic solvent , polar solvents - methanol, acetonitrile, tetrahydrofuran ; non polar solvents such as hexane, heptane, iso-octane ; high polar solvents such as acetone , alcohols, DMSO.

Stationary Phase - Paper for the Paper chromatography, Glass plate is coated with the silica for the thin layer chromatography, Packed Column for the HPLC with is coated inside with the high boiling polymer.

Purpose - Mobile phase is that it carries the components of the mixture through the medium. and stationary phase remains constant.

Ans 3

First molecule to appear - Hemoglobin 65,000 Daltons

second molecule to appear - Ribonulease A 13,700 Daltons

Third Molecule to appear - Myosin 180,000 Daltons

Reason- The Hemoglobin was excluded from the column beads , it gets eluted 1st , then the later 2 gets eluted a bit later.