Chromatographic Techniques

Question 1

What is Chromatography

Answer 1

Separation method to isolate, identify and/or quantify compound of interest (analyte)

Remove other compounds that could interfere with analyte

Question 2

What are the chromatographic techniques classified by

Answer 2

Type of chromatographic bed - planar or column
Type of mobile gas - gas or liquid
Type of separation mechanism - size, polarity etc.

Question 3

What are the phases of chromatography

Answer 3

Mobile Phase (MP) and Stationary Phase (SP)

Question 4

How do you work out the Partition Coefficient of an analyte (also known as Equilibrium constant K)

Answer 4

Conc of analyte (A) in Stationary Phase / Conc of analyte (A) in Mobile Phase

Question 5

What is Retention time (Tr)

Answer 5

Time from sample injection to elution from the column

Question 6

What does a higher K value or Partition Coefficient represent

Answer 6

A higher affinity in the Stationary Phase

Question 7

What does graphical column capacity and peak size mean

Answer 7

Column capacity - Maximum amount of analyte in the sample

Peak size - Greater area underneath a peak means more analyte in the sample

Question 8

What causes fronting and tailing peaks

Answer 8

fronting peak - caused by overloading column with analyte mixture

Tailing peak - caused by analyte retention (hydroxyl group)

Question 9

What are the different types of Liquid chromatography techniques

Answer 9

Ion-exchange
Size-exclusion
Affinity

Question 10

Explain Ion-exchange liquid chromatography

(What the SP consists of and how it works, How analytes are removed from the SP, what this technique separates)

Answer 10

Separates analytes based on electrical charge.

SP: resin with either positively charged (anion-exchange) or negatively charged (cation-exchange) functional groups.

Different analytes have different affinities for SP, depending on their charge.

Bound analytes removed from SP by altering pH of MP or salt concentration.

Separates: amino acids, peptides, proteins & small nucleotides.

Question 11

Explain size-exclusion liquid chromatography

(What the SP consists of, what the MP consists of, what this techniques separates)

Answer 11

Also called: Gel permeation chromatography, gel filtration chromatography & molecular sieve chromatography.

Separates analytes based on physical size.

SP chemically inert (inactive) porous beads e.g. dextran (Pore sizes vary)

MP aqueous or organic solvent-based.

Smaller molecules enter bead pores = delays passage down column; larger molecules elute first (removed or blocked)

Separates: proteins (in aqueous solution).

Question 12

Explain affinity Chromatography

Answer 12

Separates analytes based on interaction between: Antibody & antigen
Enzyme & substrate

SP is gel matrix (e.g. agarose) to which ligand is covalently bound (specific shape for desired filtrate)

Ligand binds selectively to target analyte; other (unwanted) analytes pass through column.

Target analyte washed from column by altering MP composition

Separates: nucleic acids, proteins
e.g. antibodies.

Very specific/selective
(google it rat if it don’t make sense)

Question 13

What is HPLC and what does it consists of (SP and MP)

Answer 13

High Performance (or pressure) Liquid Chromatography

Column filled with porous beads with SP compound chemically bound to them

MP containing the sample mixture pumped through the column at high pressure

Interaction through either polar / non-polar interactions as well as either any method previously described (ion-exchange, size-exclusion, affinity)

Question 14

Normal phase HPLC vs reverse-phase HPLC

Answer 14

Normal phase HPLC - polar-polar interaction
Reverse-phase HPLC - non-polar - non-polar interaction

Question 15

What are the advantages of HPLC

Answer 15

faster elution times due to higher pressure
better resolution (separation) due to small stationary phase particles

Question 16

HPLC particle size, pressure

UPLC (ultra performance Liquid chromatography) particle size, pressure

Answer 16

HPLC particle size = 2-50 micrometres
pressure = 50 - 350 bar

UPLC particle size = 1.7 micrometres
pressure = 1500 bar

Question 17

Explain Normal phase HPLC

Answer 17

Separates analytes based on polar interaction with polar SP

MP starts as non-polar, non-aqueous

Analytes dissolve in non-polar MP but sufficiently polar enough to interact with polar SP

Most polar molecules elute last

Question 18

Explain reverse phase HPLC

Answer 18

Most frequently-used phase in HPLC

Separates analytes based on non-polar hydrophobic interaction with non-polar SP

MP moderately polar; mixture of water & organic solvents

Strength of analyte bond to SP proportional to analyte hydrophobicity

More hydrophobic molecules elute last from column

MP that remains constant in composition: isocratic elution

Question 19

What equipment is used in HPLC ancillary equipment

Answer 19

Liquid handling pump: Must provide a constant flow of MP at constant (high) pressure. Also mixes solvents → elution gradient.

Detectors:
UV-Vis spectrophotometer (non-destructive).
Mass spectrometer (destructive)

Fraction collectors: Automated system of tubes collecting eluent leaving column. Can also be done manually.

Question 20

Uses for HPLC

Answer 20

Manufacturing:
Pharmaceuticals (analytical and preparative).

Legal/Forensic:
Illegal drugs/doping in urine.

Research:
Very wide range of applications in chemistry, biochemistry and pharmacology.

Medical:
Nutrients e.g. vitamins in blood serum.