Chromatography and Mass Spectrometry

A technique for separating complex mixtures based on their solubility and interaction between two phases: a stationary phase and a mobile phase.

Stationary Phase: - Immobile - Solid or liquid Through which the mobile phase flows

Mobile Phase: - Mobile - Gas or liquid Carries the sample

Some molecules are attracted to or have an affinity for the mobile phase, and some have an affinity for the stationary phase = separation.

Plane Chromatography Takes place on a flat surface or plane Mobile phase is solvent (water, alcohol, etc.)

• Paper chromatography: Stationary phase is paper • Thin Layer Chromatography (TLC): Stationary phase is a thin layer of sorbent material (silica gel, alumina gel, polyacrylamide gel, or starch gel attached to a glass plate).

• Sample spotted on stationary layer • Plate or paper placed in upright container with mobile phase (solvent) at the bottom • Migration of mobile phase through stationary phase via capillary action • Separation occurs due to the difference in time solute spends in mobile versus stationary phase • Solid phase interacts differently with sample compounds during migration • TLC (thin layer chromatography) needs no instrumentation unless quantitating with a densitometer

Drug screens, amino acids and L/S ratio

Retention Factor: Defined as the distance travelled by the solute divided by the total distance travelled by the solvent Rf = Distance traveled by solute (sample)/ Distance traveled by solvent Controls will have known retention factors

• Samples are loaded into a column with a stationary phase. • A mobile phase is added throughout the process. • Movement through column at different rates based on: Gravitational force Applied pressure Sample affinity to the two phases • Collect and detect eluate emerging from column • Eluate can be quantified using spectrophotometer

Eluate - liquid obtained from a column during separation (e.g. in column chromatography)

Each eluent fraction will contain individual components of the previously mixed sample

Stationary phase in column or tube Support particles such as aluminum silicate gel or resin particles packed into column or coated onto inner surface Mobile phase can be gas or liquid

Aka liquid-solid chromatography Mobile phase and sample compete for sites on solid stationary phase (usually silica gel) Solute will bind to adsorptive sites with attractive forces Example: Acidic support adsorbs basic substances

Aka liquid-liquid chromatography Separation is based on solubility of solute in an organic solvent and aqueous solvent Stationary phase is made of inert particles coated with liquid adsorbent Nonpolar molecules are extracted in the organic solvent Polar molecules remain in the aqueous solvent

• Separates solutes based on their solubility in two phases as well as different electrical charges • Sample is diluted with a buffer that gives the solute an electrical charge • Interact reversibly with stationary and mobile phases with the opposite charge • Varying pH of the mobile phase allows elution at different times and elution of specifically charged solute particles

• Aka size exclusion chromatography Separated in column by size and shape • Sample containing different sized molecules moves down the column • Larger molecules elute faster from the column while smaller molecules are retained in the gel longer (momentarily trapped)

High-Performance Liquid Chromatography (HPLC)

High pressure is used to push the mobile carrier through the short packed column. The column is the stationary phase is a packed with tiny particles, usually silica. The mobile liquid phase are the solvents. Separated molecules are then read by a detector and a chromatogram is created.

Column can employ adsorption, ion-exchange, etc.

UV-VIS or Mass spectrophotometer

Visible record showing the result of separation of components in a mixture by chromatography • Produces a characteristic graph for each component separated by chromatography • A chromatogram plots detector output versus time. • Can provide qualitative results in which the column retention time correlates to the identity of the compound.

• Separation based on polarity of compounds (polar or nonpolar) “Like attracts like” in polarity-based chromatography • The mobile phase and stationary phase will have different polarities • Compounds in the sample with similar polarity to the stationary phase will be delayed (attracted to packing material in column) • Compounds in the sample with similar polarity to the mobile phase will move through the column quickly

Normal Phase HPLC Stationary phase is polar (example: silica); mobile phase is nonpolar Reversed-Phase HPLC Stationary phase is nonpolar (example: silica C18); mobile phase is polar