BIO-360 BIOCHEMISTRY LABORATORY II

BIO-360 BIOCHEMISTRY LABORATORY II

• XII. WEEK

12th WEEK

 CHROMATOGRAPHIC TECHNIQUES

 Thin Layer Chromatography

 Paper Chromatography

(Application)

Ion Exchange Chromatography

•Molecules are separated according to their net charge.

•Solid phase: ion-exchange resin

•Mobile phase: aqueous solution

Ion Exchange Chromatography

•Example: resins with sulfonic acid groups (-SO3-) are generally used in salt form (SO3Na). The positively

charged portion in the sample interacts with the resin

electrostatically.

http://elte.prompt.hu/sites/default/files/tananyagok/IntroductionToPracticalBiochemistry/ch 06s02.html

Size Exclusion

Chromatography

•Molecules are separated according to their shapes and sizes.

•Solid phase: spherical gel parts

•Mobile phase: aqueous solution

•Smaller proteins move more slowly, while adults move quickly through the column.

• Dextran gels: Sephadex

http://archive.cnx.org/

Affinity Chromatography

•Molecules are separated according to biological selectivity.

•Solid phase: ligand or substrate bonded matrix

•Mobile phase: aqueous solution

•It is especially used for the

purification of enzymes.

http://www.bio-rad.com/en-us/applications-technologies/introduction-affinity- chromatography

THIN LAYER CHROMATOGRAPHY



Thin layer chromatography is solid-liquid

adsorption chromatography. In this method, the

solid phase is a thin layer of solid absorbent

material on glass plates of various sizes.

THIN LAYER CHROMATOGRAPHY

 In this type of chromatography, the mobile

phase travels through the solid phase, from

bottom to top. The solvent runs on the thin

layer plate immersed in the capillary effect.

https://en.wikibooks.org/wiki/Structural_Biochemistry/Chromatography/T hin_Layer

 In application, amino acid

solutions will be separated based

on thin layer and paper

chromatography techniques. Each

amino acid will be determined

according to RF values.

 Students will be organized into groups of three before the study.

 All consumables, chemicals,

laboratory equipment, especially

glassware, and machinery and

equipment will be provided as

required.

 It will be reminded that each student will be responsible for the issues given to all other groups within the application.

 Students will be asked to report

their observations and results

within the scope of the

application to the test report

immediately given to them.

 Students will be asked to remove any materials they use, and they will be expected to assist in cleaning.

 Students will be allowed to leave

after the laboratory safety and

cleanliness is ensured.