Module 12

Question 1

What are the different phases of chromatography?

Answer 1

Mobile- carries sample

Stationary

Question 2

How are components separated in ion exchange?

Answer 2

Separated by magnitude and charge of ionic species.

Question 3

What is the stationary phase in ion exchange?

Answer 3

Ion exchanger, porous solid with surface covered in ionic sites.

Cation exchange- carries -, selects for anions

Anion exchange- carriers +, selects for cations

Question 4

What is ion exchange used for?

Answer 4

Purifying biological materials

Removing interfering substances from a solution

Separating mixes of charged molecules

Question 5

What is steric exclusion and how does it work?

Answer 5

Separation based in size and shape.

Stationary phase is porous and traps molecules, larger ones are excluded, move more quickly.

Question 6

What is steric exclusion also known as?

Answer 6

Gel filtration or permeation

Question 7

What are the uses of steric exclusion?

Answer 7

Separation of antibodies, enzymes, proteins, triglycerides.

Question 8

What is adsorption and how does it work?

Answer 8

Separation based on competition between sample components and the mobile phase for adsorptive sites on the stationary phase.

More soluble components remain in the mobile phase and move faster.

Question 9

What is adsorption used for?

Answer 9

Analysis of drugs, vitamins, fats, pesticides.

Question 10

What is partition and how does it work?

Answer 10

Separation based in polarity.

Normal- mobile phase is nonpolar, polar substances bind the stationary phase and nonpolar travel faster.

Reverse- mobile phase is polar, nonpolar substances bind the stationary phase and polar travel faster.

Question 11

Which type of partition is more common and why?

Answer 11

Reverse- safer and the sample tends to be polar based

Question 12

What is partition used for?

Answer 12

Porphobilinogen screen

Question 13

What is affinity and how does it work?

Answer 13

Separation based on specific interaction between one kind of solute molecule and a secondary molecule immobilized on the stationary phase (ligand).

Analyte of interest binds with the ligand, very specific.

Question 14

What can a ligand be?

Answer 14

Antibody, protein, specific resin group.

Question 15

What is affinity separation used for?

Answer 15

Separates glycosylated Hb from other Hbs.

Binds to boronate group of the stationary phase.

Question 16

What is plane chromatography?

Answer 16

Separation occurs on a flat plane.

Simple screening methods.

Question 17

What are the types of plane chromatography?

Answer 17

Paper

Thin layer chromatography (TLC)

Question 18

How does paper chromatography work?

Answer 18

Sheet of paper = stationary phase

Small volume of liquid = mobile phase

Mobile phase migrates up by capillary action, picks up the more soluble components.

Separation is visualized.

Question 19

How can separation be visualized?

Answer 19

Stains, iodine vapour or UV light.

Question 20

What is Rf?

Answer 20

Distance a component migrates compared with the distance the solvent front moves.

Component/solvent front

Compared with Rf of standards

Question 21

How does TLC work?

Answer 21

Thin layer of sorbent is coated on a glass or plastic plate (stationary phase).

Placed in jar with liquid mobile phase.

Separation based on preferential solubility.

Question 22

What is column chromatography?

Answer 22

Stationary phase is packed in a tube.

Mobile phase is passed through the tube.

Question 23

What can packing material be for column chromatography?

Answer 23

Aluminum silica gel or resin beads.

Question 24

What are the components of column chromatography?

Answer 24

Mobile phase reservoir

Injector port with septum

Column

Detector

Display output- chromatogram

Question 25

What extra component do liquid column chromatography systems require?

Answer 25

Pump in the reservoir

Question 26

How does high pressure liquid chromatography (HPLC) work?

Answer 26

Automated Mobile phase (liquid) is driven through the column. Eluate is collected and passes through a detector.

Question 27

What is HPLC used for?

Answer 27

Heat labile components or nonvolatile substances. Glycosylated Hb and some drugs.

Question 28

What is gas chromatography and how does it work?

Answer 28

Mobile phase is an inert gas. Stationary phase is a liquid distributed over the surface of a porous inert solid. Sample must be volatile. Temperature must be higher than the component BP to vapourize them. Analytes exit column based on different affinities. Can be coupled with a mass spectrometer for a more definitive ID.

Question 29

What is gas chromatography used for?

Answer 29

Alcohol analysis

Question 30

What types of detectors are there for chromatography?

Answer 30

Flame ionization detector (FID) Thermal conductivity detectors (TCD) e- capture detectors (ECD) UV spectrophotometers Linear diode arrays

Question 31

How do FIDs work?

Answer 31

Carrier has enters and forms a combustible mix which is ignited. Compounds enter and burn incompletely. Ions are collected. Current flow is proportional to the amount of component burned.

Question 32

How do TCDs work?

Answer 32

Carrier has flows over the electrically heated filament, dissipates heat. When sample mixes the amount of heat changes causing a change in conductivity proportional to component concentration.

Question 33

What compounds does ECD work for?

Answer 33

Halogenated compounds

Question 34

What is a chromatogram?

Answer 34

Record of all separate components detected, recognized by separate peaks. Area under peak determine quantity of components.

Question 35

What is retention time?

Answer 35

Time it takes for each peak to reach the detector, used to ID components by comparing to standards.

Question 36

What is resolution?

Answer 36

Relative separation of two separate peaks. Higher- better ID and quantification Affected by temp and column characteristics.

Question 37

What is an internal standard and why is it used?

Answer 37

A structurally similar reference compound added in constant concentration to all samples. Account for system variations.

Question 38

What internal standard is often used in gas chromatography?

Answer 38

N-propanol

Question 39

What issues could be encountered with chromatography?

Answer 39

Drift/baseline wander Noisy baseline No peaks Short retention time Ghost peaks Irregular peaks Unsymmetrical peaks Long tails

Question 40

What is chromatography used for?

Answer 40

Separation, detection and quantitation of a mix of analytes in a sample. Based on different physical interactions between the analytes and stationary phase.