7/27/2019 BIOCHEM1-Grp16 Chromatography Experiment

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DO NOT FOLLOW THE NUMBERING. MATCH THE QUESTIONS WITH WE HAVE IN OUR MANUAL. THESE ARE SOME OF

THE ANSWERS FROM LAST YEARS MANUAL.

1. Which amino acids were attached to the column initially? Explain.a. Since molecules with the same charge as the mobile phase are eluted first (in a single band)

therefore the glutamic acid is eluted first then cysteine and finally arginine. Since the buffer is acidic

both the cystein and arginine are attached to the column. When the neutral buffer was run, the

arginine is first attached and cysteine is eluted. When the basic buffer was run, arginine was eluted

with it.

2. In the paper chromartography experiment, why was it necessary to use standard amino acids to identify theunknown amino acids?

a. The Rf values with the standard amino acid is the point of comparison or essential information thatis needed to be able to identify a certain amino acid. An unknown amino acid can be identifies just

by comparing Rf values with the given standard.

3. Differentiate between ascending and descending paper chromatography.a. Ascending paper chromatography: bottom edge of the paper is immersed in the solventb. Descending paper chromatography: the paper edge is immersed in a solvent and hung down and

usually the process is stopped before the solvent reaches the opposite end and it evaporates.

4. Enumerate the factors affecting the distance travelled by an amino acid in a chromatogram.a. Polarity of the solventb. Point of the samplec. Stability of the chromatography chamberd. Nature of the amino acide. Molecular weight of the amino acidf. Quality of the stationary phase (filter paper)g. Application of chromatogram

5. Summarize the principle involved in the separation of amino acids by ion exchange column chromatographya.

Explain briefly the 2 phases present in any chromatographic procedure in the experiment, whichmaterials represent the two phases?

i. Mobile phase: the moving substance, maybe a liquid or gas, solvent and buffer solutionii. Stationary phase: the resin/ column wherein the phase and unknown solution are selectively

absorbed (paper chromatography and Dowex Mesh, water

6. Summarize the principle involved in the identification of unknown amino acid by paper chromatography.a. The sample components partition between the migrating mobile phase and the tightly bound water

The solvent is evaporated. The water from the solvent is absorbed and tightly held on the pape

fibers as they partition. The ratio of distance travelled by a particular component to the distance

travelled by the solvent is called the Rf value.