Chromatographic techniques

Question 1

What are the 6 different types of chromatography?

Answer 1

1. Column Chromatography
2. Thin Layer Chromatography
3. Gel Filtration (Size Exclusion)
4. Ion Exchange
5. High Pressure Liquid Chromatography
6. Gas Chromatography

Less well used: Paper & Affinity chromatography

Question 2

What are the common features of all chromatography?

Answer 2

• All have a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas).
• The mobile phase flows through the stationary phase and carries the components of the mixture with it.
• Different components travel at different rates dependent on their attraction to the mobile phase and the stationary phase

Question 3

What is column chromatography?

Answer 3

Column either loaded dry and filled with the mobile phase, which is then flushed through the column, or, more usually, loaded with a slurry of stationary and mobile phase together (care taken to avoid bubbles)

Question 4

What stationary phases are used in column chrom?

Answer 4

•	Silica Gel (most common)
o	Typically 40-63 μm particle size
o	Granular & porous 
o	high surface area (around 800 m²/g)
•	Alumina (less common)
o	Typically 50-200 μm particle size

Question 5

What are the 5 steps in column chromatography?

Answer 5

1) Load Stationary Phase material to column
2) Equilibrate Column Stationary Phase with Mobile Phase
3) Load sample in as small a volume as possible
4) Add more Mobile Phase to column
5) Collect sample fractions from column

Question 6

What substances will separate first?

Answer 6

Non-polar

Question 7

What affects solvent flow?

Answer 7

Particle size affects solvent flow. Smaller particles (higher mesh values) need a pump to flow the mobile phase

70–230 silica gel for gravity columns & 230–400 mesh for flash columns

Question 8

What is elution?

Answer 8

The separation (development) of the organic classes. With increasing polarity, the slower they move

Question 9

How are components of interest determined?

Answer 9

by methods such as colour, TLC ,Ultraviolet , fluorometry etc

• Column can then be washed with eluting solvent and reused or discarded
• Used in the laboratory and industry to separate and purify compounds on a preparative scale from mg to kg

Question 10

What is Thin Layer Chromatography? (TLC)

Answer 10

Stationary phase: usually silica, but aluminium and cellulose sometimes used
Mobile phase: Single solvent or mixture
Driving force: Mobile phase moves through stationary phase by capillary action
Affinity: polarity

Same principle as column chromatography using silica but on a smaller scale (thin layer)

Question 11

What does separation depend on in TLC?

Answer 11

Separation depends on polarity of solute and solvent and stationary phase

Question 12

Describe what happens as the mobile phase moves up the plate

Answer 12

As the mobile phase (solvent) moves up the plate, each component is carried at a different rate.
When the solvent reaches the top end of the plate, the plate is removed from the beaker and the solvent allowed to evaporate

Question 13

What allows for the components to be seen as dark spots?

Answer 13

The plates are treated with fluorescing agent so show up as dark spots under UV light - this can help to detect components which are colourless

Question 14

What are 5 different ways of developing spots?

Answer 14

• UV light (indirect): If analyte absorbs UV, gives dark spot on yellow/green background
• Iodine vapour: Reversibly produces brown spots with many organic compounds, spraying with starch makes permanent
• Potassium permanganate: used for detection of sugars and sugar-like molecules
• Ninhydrin: gives purple/pink spot with amines, used for gentamycin
• Alkaline tetrazolium: Blue spots with corticosteroids

Question 15

What are Rf values?

Answer 15

Measure the distances from the starting point to the solvent front and from the starting point to the centre of each spot, to calculate the Rf values

High Rf = less polar

Question 16

What is TLC used for?

Answer 16

Quality control and purification process etc

Also used in BP monographs as qualitative identity test on pure substances

Question 17

What is Gel filtration chromatography?

Answer 17

• Chromatographic method : size exclusion or gel filtration
• Large molecules are separated from small ones by their size
• It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers.
• It is also applied to remove salts from large molecules

Question 18

How does gel filtration chrom work?

Answer 18

• Markers of known molecular mass used to calibrate the column
• Thus, unknown molecular mass can be found from calibration curve

Question 19

What stationary phase is used for gel chromatography?

Answer 19

• The most common stationary phases are Dextran (Sephadex), dextran-polyacrylamide (Sepharyl) and agarose (Sepharose).
• These are inert
• Each is available with a variety pore sizes to suit fractionation range
• Very large molecules are eluted in the void volume (space between the beads)

The upper limit is known as the exclusion limit of the gel - the size above which proteins will elute in the void volume of the column.

Question 20

What is gel filtration used to find?

Answer 20

Used to find the molecular mass of unknown
V0 = Void volume = Volume outside the gel matrix
Ve = Elution volume = volume of buffer required to elute any given substance

Also used to desalt or purify a sample

Question 21

How is salt removed in gel filtration?

Answer 21

• Salt is small molecular weight compared to protein so will have longer retention time

• Removal of NaCl from albumin solution. Sephadex G-25 column equilibrated with distilled water. Apply human serum albumin (25 mg) dissolved in 2.5 ml 0.5M NaCl solution. A total of 23.8 mg albumin was recovered in 3.5 ml eluent
corresponding to a yield of 95.3% (between arrows).

Question 22

What is ion exchange chromatography?

Answer 22

• Separation based on charge properties.

* Most popular for proteins and peptides.

Question 23

What stationary phase is used in ion exchange chromatography?

Answer 23

• Cross linked polymers, typically cellulose or agarose resins
• Stationary phase has –ve or +ve functional groups
• Binds to oppositely charged ions in the sample or the aqueous buffer

Question 24

What are the features of cation exchange? (-)

Answer 24

• In cation exchange - positively charged molecules are attracted to a negatively charged solid support

E.g. Suphonate, carboxylate, carboxymethyl

Question 25

What are the features of anion exchange? (+)

Answer 25

• In anion exchange -negatively charged molecules attracted to a positively charged solid support. • By using a linear salt gradient the molecules with the weakest ionic interactions start to elute from the column first E.g. Quaternary ammonium, tertiary ammonium, Diethylaminoethyl

Question 26

What must the pH of the mobile phase buffer be ? (ion exchange)

Answer 26

must be between the pI (isoelectric point) of the charged molecule and the pKa of the charged group on the solid support

Question 27

What 3 factors affect elution in ion exchange?

Answer 27

1. Size of Charge Divalent ions show greater affinity than monovalent 2. The intensity of the charge Small monovalent ions (eg H+) show greater affinity than large monovalent ion (eg.K+) 3. Concentration of the ions A high concentration of a low affinity ion can displace a low concentration of a high affinity ion Careful control of these factors provides the selectivity of the technique • Usually salt concentration is progressively (gradient) increased to elute compound of interest

Question 28

What are the applications of Ion exchange resin?

Answer 28

* Neutral compounds can be separated by a mixture of anionic and cationic resins, removing unwanted ions and replacing them with water (H+ + OH-). Water purification works this way * Charged compounds e.g. metals can be selectively bound whilst unwanted constituents that are, either uncharged or with the same charge as the resin, can be washed off the resin, before the required compound is eluted * A mixture of amino acids e.g. In amino acid analysis of protein * Isolation of metabolites from biological fluids