Introduction to Chromatography

Chromatography

separative process in which a mixture of solutes carried in a moving phase are separated as a result of differential distribution of solute molecules between a moving phase (either liquid or gas) and a solid stationary phase

Non Covalent

the interactions in chromatography

Resolution

separation

No Resolution

no separation

Mixture of Solutes

are separated as a result of their differential distribution between a stationary and mobile phase

Applications of Chromatography

• source

• identification

• action

• formulation/ analysis

• clinical

• food and beverage

Conjugated System

must be present in a compound for UV detection

• e.g. benzene ring

Planar Techniques

TLC (thin layer chromatography) and paper chromatography

Column Techniques

gas-liquid chromatography and HPLC (high performance liquid chromatography)

Dispersive Forces

molecular interactions with a strong effect when scattered throughout the molecule, are quite weak when not scattered

Carbonyl Group

influences electron density

Strong Molecular Interactions

• hydrogen bonding

• coulombic forces

Coulombic Forces

the attraction of oppositely charged ions

• the smaller the size, the bigger the coulombic

• the bigger the charge, the bigger the coulombic

• bonding occurs due to difference in coulombic

Alkyl Chain

decreases as polarity increases

Isocratic Conditions

maintains constant mobile phase composition

Gradient Elution

mobile phase changes throughout separation

Normal Phase Chromatography

• stationary phase is polar

• mobile phase is relatively non polar

Reverse Phase Chromatography

• stationary phase is non polar

• mobile phase is polar

Polar

mobile phase in reverse phase chromatography

Polar

stationary phase in normal phase chromatography

Non Polar

mobile phase in normal phase chromatography

Non Polar

stationary phase in reverse phase chromatography

Aromatic Ring with Alkyl Chain

• weak instantaneous reactions in the alkyl chain

• pi pi interactions in the double bonds

Size Exclusion Chromatography

compounds are separated as a result of differences in molecular weight

• compounds with a smaller molecular weight are trapped in the pores of the polymer beads before moving through

Ion Exchange Chromatography

used for highly polar samples

negative ions are attached to the resin

Ion Pair Chromatography

can convert a reverse phase column to an ion exchange column by the addition of an ion pairing agent to the mobile phase

• ion pairing agent must have a degree of similarity to the stationary phase (long alkyl chain)

Normal Phase Chromatography

elution order:

least polar first, most polar last

Reverse Phase Chromatography

elution order:

most polar first, least polar last

Polar Stationary Phase

silica

Non Polar Stationary Phase

C18 silica

Normal Phase Chromatography

mobile phase:

low to medium polarity

Reverse Phase Chromatography

mobile phase:

medium to high polarity

Normal Phase Chromatography

effect of increase in solvent polarity:

reduced analysis time (decreased retention)

Reverse Phase Chromatography

effect of increase in solvent polarity:

increased analysis time (increased retention time)

Retention Factor

distance travelled by solute / distance travelled by solvent

Development Distance

the distance up to which you allow the solvent to run up to

Spot Size

the smaller, the better the resolution

Edge Effect

due to saturation, solute on edges travels faster

• don’t load sample too close to the edge to avoid

Visualisation Reagents

destructive technique, molecules cannot be recovered

Physicochemical Visualisation Methods

non destructive techniques

• ultraviolet (conjugated compounds)

• aromatic compounds

• enones

• fluorescence-polyaromatic compounds

0.25mm

thickness of TLC plate

Silica Particle Size

5-20 micrometres

• in TLC

HPTLC Advantages

• higher resolution

• rapid development time

• shorter development distance

• detection limit is increased tenfold (able to find out minimal amount of sample)

• three times the number of theoretical plates (concept similar to pixels - the more, the higher the resolution)