L10: Chromatography

Question 1

What is the clinical utility of chromatography?

Answer 1

Used in clinical chemistry labs:
- For specialized testing (not your bread and butter assays) - via GC-FID, GC-MS, LC-MS, HPLC-ECD instruments

To measure:

• Quantify small molecules in a patient sample (ie. TDM, Steroids)
• Quantify peptides in a patient sample (ie. IGF-1, Thyroglobulin)
• Qualitative - Identify the presence of a molecule (ie. Positive/Negative for a drug)

Question 2

What is chromatography?

Answer 2

Biological fluids are complex mixtures

Chromatography is a process in which components of a mixture are separated by differential distribution between a mobile phase and a stationary phase

Question 3

Define mobile and stationary phase.

Answer 3

Mobile Phase

• Gas or liquid that passes through a column
• GC = gas carrier; LC = liquid mobile phase

Stationary Phase

• Substance that stays fixed inside the column
• Can be solid or liquid

Eluent = fluid entering the column
Eluate = fluid exiting the column

Question 4

List some peak characteristics.

Answer 4

Peak characteristics:

- Peak width, peak height, peak area, retention time

Question 5

Define resolution.

Answer 5

Resolution (RS): defined as the distance between peak maxima compared with the average base width of the two peaks

To get great Resolution (RS) requires 1 or a combination of the following parameters:

• Efficiency
• Selectivity
• Retention Factor (k’)

See calculation on slide 9

Question 6

Define efficiency.

Answer 6

Column efficiency: the ability to elute narrow, symmetrical peaks (sharper peaks)

• Stated in terms of the number of theoretical plates (N)
• Calculating plates (N): function of retention time (tR) and peak width (W1/2)

See slide 11 for formula.

Another way to describe column efficiency (instead of N), is the height equivalent of a theoretical plate  known as plate height (H) (see slide 14)

Question 7

What are theoretical plates?

Answer 7

• Increase the # of plates, improve the separation

- Increase the # of plates, increase the # of interactions with the stationary phase (better separation)

Question 8

List factors that may impact efficiency.

Answer 8

 Column length
 Flow rate
 Initial injection volume
 Mobile-phase viscosity
 Particle size of stationary phase
 Temperature
 Uniformity of the stationary phase
 Volume of connecting tubing

Question 9

Define selectivity.

Answer 9

Selectivity: degree of separation between peaks (separation to baseline)

• Characterizes the specific chemical affinity between solute and column
• Depends on nature of the column ligands and its degree of matrix substitution, the nature of the sample matrix, the nature of the target protein, type of salt and concentration of salt used for binding etc.

Question 10

Define retention factor.

Answer 10

Retention factor (k’): a measure of the time a solute resides in the stationary phase relative to the time it resides in the mobile phase.

• Mathematically, it is the ratio of the adjusted retention volume to the void volume (the time for unretained components to elute from the column)
• k’ of 0 = no binding between a solute and the stationary phase (void volume)
• Greater partitioning into the stationary phase results in longer retention times and increased k’
• In practice, it is desirable to have a k’ between 1 and 10 for optimal separations that do not take an excessive length of time

Question 11

What are the five most common types of chromatography?

Answer 11

Adsorption
Partition
Ion-exchange
Molecular exclusion
Affinity

Question 12

Explain IEC.

Answer 12

Biomolecules generally have charged groups on their surfaces, which change with pH of solution
Molecules reversibly bind to an oppositely charged group of the packing material
Molecules with higher charge density bind more strongly
Bound molecule can be selectively removed by changing pH or salt concentration of the mobile phase
IEC has high selectivity and capacity

Important factors in performance of IEC:
Type of stationary phase ionic group, charge density, stationary phase matrix, type and concentration of ions in the mobile phase, and mobile phase pH

Applications: analyses of amino acids and hemoglobins

Question 13

Explain SEC.

Answer 13

Also referred to as Gel Permeation Chromatography (GPC) for non-aqueous elution systems or Gel Filtration Chromatography (GFC) for aqueous systems
A method in which the separation is achieved based on molecular size. Large biomolecules that cannot penetrate the pores of the packing material elute first. Smaller molecules partially or completely enter the stationary phase

A simple method for separating proteins, peptides and oligonucleotides

Low resolution technique; separation of components requires substantial differences in MW

Question 14

Explain AFC.

Answer 14

Based on the specific adsorption of a molecule to a ligand or macromolecule

Advantage: almost all biomolecules can be purified on the basis of specific interaction between their chemical or biological structure and a suitable affinity ligand

AFC has ligands that are bonded to the packing material. Adsorbed biomolecule can be eluted from the column by competitive displacement or by a change in the conformation of the molecule through a change in pH or ionic strength

Provides high purification yields

Single-step gradient elution

Question 15

Explain RPC.

Answer 15

Employs a polar (aqueous) mobile phase

Hydrophobic molecules in the polar mobile phase tend to adsorb to the hydrophobic stationary phase, and hydrophilic molecules in the mobile phase will pass through the column and are eluted first

Used to separate small molecules, nucleotides, and peptides; not suited for proteins (would need to add mobile phase additives to increase hydrophobicity of proteins)

Question 16

Explain HILIC.

Answer 16

HILIC is used for separation of polar and hydrophilic compounds

Stationary phase is polar (hydrophilic), with reversed-type eluents (eg. ACN with small amount of water)

The aqueous content of the mobile phase creates a water rich layer on the surface of the stationary phase. This allows partitioning of solutes between the more organic mobile phase and the aqueous layer

HILIC is suited for MS analysis of water soluble polar compounds, because the high organic content in the mobile phase increases MS detection sensitivity

Question 17

Explain GC.

Answer 17

Gas-Solid Chromatography (GSC)
Adsorption: supports made of silica or alumina
Size exclusion: porous materials, mixtures of silica and alumina or porous polymers
Separation dependent on surface area of support, size of pores, and/or functional groups

Gas-Liquid Chromatography (GLC)
Partition: Liquid coating on the solid support (hollow down the middle for gas flow)
Eg. liquid supports such as Polysiloxanes with low volatility
Molecules of interest will interact with liquid layer (slow elution)

Question 18

Describe planar chromatography.

Answer 18

• Paper chromatography and thin-layer chromatography (TLC) are classified as planar
  techniques (…not because they are boring!)
• TLC uses a thin layer of sorbent, such as silica gel, microparticulate cellulose, or alumina, spread uniformly on a glass plate, plastic sheet, or aluminum foil
• Sample is added as a small spot or band on the plate, which is placed in a closed container with the lower edge within, and the sample band just above, the mobile phase
• after the mobile phase travels the desired distance by capillary action, the plate is removed from the tank and dried
• separated components are located and identified by procedures such as UV, spraying with colour-generating reagents, or autoradiography

Question 19

Describe the main components of an LC-MS and a GC-MS.

Answer 19

See slides 33-39.

Question 20

Describe the importance of chromatography gradients.

Answer 20

see slide 41.

Question 21

Describe qualitative analysis.

Answer 21

Analyte Identification (qualitative)
The retention time that an unknown solute elutes from a column, or the distance traveled on a plate, is often compared and matched with that of a reference compound
But, appearance does not prove identify (could be a similar structure that elutes at the same time)
HPLC-ECD and GC-FID assays can be affected by false positives (ie. patient on certain meds)
Mass spec detector allows for specificity

Question 22

Describe quantitative analysis.

Answer 22

Analyte quantification
External calibration: Standards with known quantities are processed in a manner identical to unknown samples containing the analyte (i.e. patient sample)
Calibration curve of (1) peak area or (2) spot density versus calibrator concentration used to calculate concentration of the unknown analyte in patient sample
Internal calibration: An internal standard of known concentration is added to each calibrator and patient sample
The internal standard can correct for systematic losses through the extraction or chromatography process (allows for less variability/imprecision)

Question 23

Describe trouble shooting.

Answer 23

Refer to slides.