Chromatographic techniques

Question 1

What are the stationary phases used in column chromatography?

Answer 1

Silica gel - most common

Alumina

Question 2

Why is water never used as a solvent in column chromatography?

Answer 2

Because it is too polar

Question 3

What is the elutropic series used for?

Answer 3

To identify which is the best solvent to use for separation

Question 4

In column chromatography polar extracts come out first. True or false?

Answer 4

False - non-polar

Question 5

Why do polar solvents come out first in column chromatography?

Answer 5

Because they have the least interaction with the matrix and more interaction with the solvent

Question 6

Column chromatography is only used on a small-scale. True or false?

Answer 6

False - small and large

Question 7

More usually, in column chromatography, the stationary phase and mobile phase are mixed together. True or false?

Answer 7

True

Question 8

What is flash column chromatography?

Answer 8

When pressure is applied through using a pump or nitrogen gas, speeding up the process

Question 9

In flash column chromatography, what influence does particle size have?

Answer 9

Small particles have a higher mesh values so need a pump to flow through mobile phase

Question 10

In TLC, what is the stationary phase that is usually used?

Answer 10

Silica

Question 11

What is the driving force in TLC?

Answer 11

Mobile phase moving through the stationery phase by capillary action

Question 12

In TLC, what does separation depend on?

Answer 12

The polarity of the solute, solvent and stationary phase

Question 13

TLC plates are treated with fluorescing dye and when placed under UV, the spots appear dark. Why is this?

Answer 13

Because the sample has aromatics and double bonds which will absorb UV light, making the spots appear dark, not green like rest of plate

Question 14

What are the 5 ways in which TLC spots can be developed?

Answer 14

UV light (indirect)
Iodine - reversibly produces brown spots
Potassium permanganate - detection of sugars
Ninhydrin - pink spots for amines - used for gentamicin
Alkaline tetrazolium - blue spots with corticosteroids

Question 15

What is TLC used for?

Answer 15

To select solvents for column chromatography 
Evaluating purity
Identification of compounds
Follow progress of a reaction
Detect impurities 
BP qualitative test of pure substances

Question 16

In gel filtration chromatograph, large molecules are separated from small ones. True or false?

Answer 16

True

Question 17

Gel filtration can be used to remove salts from large molecules. True or false?

Answer 17

True

Question 18

What are the common stationery phases used in gel filtration chromatography?

Answer 18

Dextran (sephadex)
Dextran-polyacrylamide (Sepharyl)
Agarose (Sepharose)

Question 19

In gel filtration, what is meant by void volume?

Answer 19

The volume outside the gel matrix

Question 20

In gel filtration, what is meant by elution volume?

Answer 20

Volume of buffer required to elute any given substance

Question 21

Gel filtration can be used to desalt or purify a sample. True or false?

Answer 21

True

Question 22

In ion exchange chromatography, what is separation based on?

Answer 22

Charge properties

Question 23

Ion exchange chromatography is the most popular for proteins and peptides. True or false?

Answer 23

True

Question 24

In which type of chromatography is the stationary phase usually cross-linked peptides, typically cellulose or agarose resins?

Answer 24

Ion exchange chromatography

Question 25

The stationary phase in ion exchange chromatography has positive or negative functional groups. True or false?

Answer 25

True

Question 26

In ion exchange chromatography, what happens in cation exchange?

Answer 26

positively charged molecules are attracted to a negatively charged solid support

Question 27

In ion exchange chromatography, what happens in anion exchange?

Answer 27

negatively charged molecules are attracted to a positively charged solid support

Question 28

In ion exchange chromatography, by using a linear salt gradient, molecules with the weakest ionic interactions start to elute from the column first. True or false?

Answer 28

True

Question 29

In ion exchange chromatography, the pH of the mobile phase buffer must be between isoelectric point of the charged molecule and the pKa of the charged group on solid support. True or false?

Answer 29

True

Question 30

In ion exchange chromatography, what factors affect eluting?

Answer 30

The size of the charge - divalent ions greater affinity than monovalent Intensity of the charge - small monovalent ions show greater affinity than large monovalent Conc of ions

Question 31

What are the applications of ion exchange chromatography?

Answer 31

Isolation of metabolites from biological fluids Neutral compounds separated by mixture of anionic and cationic resins, removing unwanted ions and replacing them with water Charged compounds e.g. metals can be separated from unwanted constituents