Chromatography Validation (13)

Mobile phase definition

A moving fluid which carries the sample mixture through or over a stationary phase in order to separate its components.

Stationary phase definition

A fixed solid or liquid medium through or over which a mobile phase passes in order to separate the components of a mixture.

Adsorption definition

The adhesion of molecules/substances to the surface of a solid/liquid.

Partition definition

The separation of components based on their different rates of dissolving (distribution) between a liquid stationary phase and a mobile phase.

Desorption definition

The breaking of the attraction between a substance and the surface to which it is adsorbed.

What are the 2 main steps to chromatography?

• Components are swept over the S/P and undergo a continual process of adsorption into it, followed by desorption and dissolving into the M/P.

• Components separate based on their individual affinities for the S/P or M/P

Chromatography definition

A method used to separate and identify the components of a mixture.

Capillary action definition

The movement of a liquid within the spaces of a porous material due to the forces of adhesion, cohesion, and surface tension.

What are the mobile and stationary phases in normal-phase TLC?

M/P = a low-polarity liquid solvent

S/P = polar silica gel (coated onto a glass/plastic/aluminium plate)

3 steps to TLC

1. Sample is dissolved into a volatile solvent and spotted onto a pencil line near the bottom of the TLC plate

2. TLC plate is placed in a sealed chamber with the M/P

3. Solvent travels up the plate via capillary action, carrying the components with it

Describe the process of separation in TLC

Polar compounds form strong hydrogen bonds with the silanol (Si-OH) groups on the silica surface, adhering strongly. They don’t dissolve bc their intermolecular forces are stronger than those in the solvent. They therefore travel short distances slowly.

Non-polar compounds form weak dispersion forces with the S/P, and dissolve readily in the solvent bc their bonds are similar in strength. They therefore travel large distances quickly.

What are the requirements for TLC to be used?

The compound must be non-volatile, completely soluble in the M/P, and unreactive with silica gel.

Examples of the use of TLC

Reaction monitoring by comparing samples of reaction mixture to samples of reactants/expected products.

Identifying illicit drugs (morphine, amphetamines)

Identifying natural pigments or contaminants in plants/food (chlorophyll, pesticides)

How to tell which components are polar/non-polar from a TLC plate?

The more non-polar a component, the greater distance it will travel, and the greater retention factor it will have.

How to calculate retention factor?

Distance traveled by compound / distance traveled by solvent front

What is the mobile phase and stationary phase in gas chromatography?

M/P = inert carrier gas (He, N2 or H2)

S/P = a porous solid coated with an ester/liquid HC with a high BP, or adsorbent solid particles (silica or alumina)

3 steps to GC?

1. Sample is dissolved/diluted in a volatile solvent, then injected into the column and vapourised.

2. The sample interacts repeatedly with the S/P in the column(a heated series of loops of glass/metal)

3. The individual components exit the column and are sensed by a detector

Explain the process of separation in GC

Components with low BPs vapourise easily and elute first with the gas phase. Those with higher BPs condense easily and spend lots of time adhered to the S/P. Non-polar components interact strongly with the S/P via London dispersion forces and stay in the column longer.

What are the 2 rules for separation depending on the polarity of the S/P in GC?

Polar S/P = non-polar compounds with low BPs elute first

Non-polar S/P = polar compounds with low BPs elute first.

What are the requirements for GC to be used?

The compound must be volatile, thermally stable, contain no water or insoluble solids, and be compatible with the S/P and carrier gas.

Applications/Examples of GC

Detecting environmental + organic pollutants - VOCs (benzene), pesticides, atmospheric pollutants (methane, sulfur dioxide)

Forensics - detecting accelerants (kerosene), drugs (methamphetamines, cocaine), steroids and stimulants.

Foods + flavours - flavour compounds (vanillin), alcohol content (ethanol, methanol)

Describe the features of a chromatogram

x-axis = retention time

y-axis = concentration/amount of compound

# of peaks = # of compounds detected

Area under each peak is proportional to the concentration of the compound in the sample

Retention time definition

The time from the sample being injected into the system until a component reaches the detector.

What are the mobile and stationary phases in HPLC?

M/P = a polar liquid solvent

S/P = a non-polar, hydrophobic silica matrix (with HC (C18) chains chemically bonded to silica beads)

3 steps to HPLC

1. M/P pumped through system at a high pressure

2. Sample solution is injected into the stream and travels through the column, separating into distinct bands

3. As each band exits the column, a detector records a signal.

Describe how separation happens in HPLC

Polar compounds readily form D-D and H bonds with the water-solvent solution, which are stronger than the dispersion forces with the S/P, so they stay dissolved and elute first.

Non-polar compounds adhere to the non-polar HC chains via widespread London dispersion forces, so they elute last.

Requirements for HPLC to be used

The compound must not contain any particulates, be compatible with the solvent, chemically stable, and in the liquid phase, at 25 degrees.

Applications/examples of HPLC

Pharmaceuticals - quality control, purity testing (penicillin, steroids, ibuprofen)

Food and beverages - nutrition labelling, detecting contaminants (caffeine, sugars, artificial sweeteners)

Forensics - toxicology, drug monitoring (cortisol, testosterone, amphetamines)

What can be inferred in regards to the polarity of a component from a HPLC chromatogram?

Short retention time = high polarity + strong bonding with mobile phase

Long retention time = non-polar, strong dispersion forces with stationary phase.

Suitability of TLC

Non-volatile and polar compounds, rapid, complex/unpurified samples, multi-sample analysis.

Limitations of TLC

Qualitative data, low resolution, volatility constraints, limited sensitivity.

Suitability of GC

Volatile + thermally stable compounds, gases + lower BP liquids, high resolution, rapid.

Limitations of GC

Thermal instability, can’t use non-volatile and high M compounds, expensive, technician needed to operate.

Suitability of HPLC

Non-volatile, large molecules, polar and non-polar compounds, high precision.

Limitations of HPLC

Expensive, technician needed to operate, wastes solvent.

What are the differences between adsorption and partition?

Adsorption only occurs on the surface of the S/P, while partition occurs throughout most of it.

Adsorption is driven by electrostatic forces and intermolecular forces, while partition is driven by solubility.

In what types of chromatography do adsorption and partition occur?

Adsorption - TLC and normal-phase HPLC

Partition - GC and reverse-phase HPLC.