HPLC

Chromatography is used to separate mixtures of substances into their components. All forms of chromatography work on the same principle. They all have a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows through the stationary phase and carries the components of the mixture with it. Different components travel at different rates.

TLC- Thin layer chromatography is done exactly as it says - using a thin, uniform layer of silica gel or alumina coated onto a piece of glass, metal or rigid plastic. The silica gel (or the alumina) is the stationary phase. The stationary phase for thin layer chromatography also often contains a substance which fluoresces in UV light - for reasons you will see later. The mobile phase is a suitable liquid solvent or mixture of solvents.

The diagram shows the plate after the solvent has moved about half way up it. The solvent is allowed to rise until it almost reaches the top of the plate. That will give the maximum separation of the dye components for this particular combination of solvent and stationary phase

What separates the compounds as a chromatogram develops?

How fast the compounds get carried up the plate depends on two things: What separates the compounds as a chromatogram develops? As the solvent begins to soak up the plate, it first dissolves the compounds in the spot that you have put on the base line. The compounds present will then tend to get carried up the chromatography plate as the solvent continues to move upwards. How fast the compounds get carried up the plate depends on two things: How soluble the compound is in the solvent. This will depend on how much attraction there is between the molecules of the compound and those of the solvent. How much the compound sticks to the stationary phase - the silica gel, for example. This will depend on how much attraction there is between the molecules of the compound and the silica gel

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY - HPLC High performance liquid chromatography is basically a highly improved form of column chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much faster.

Fast Protein Liquid Chromatograph (FPLC) 1 2 3 5 4 No air bubbles (Priming) Use degassed buffers Injector Module Column Inlet Detector Fraction Collector

HPLC is a separation technique that involves: •the injection of a small volume of liquid sample •into a tube packed with tiny particles (3 to 5 micron (μm) in diameter called the stationary phase) •where individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump.

Definitions 􀂙 Stationary phase -- common name for the column packing material in any type of chromatography. -- those samples which have stronger interactions with the stationary phase than with the mobile phase will elute from the column less quickly, and thus have a longer retention time, while the reverse is also true 􀂙 Mobile phase -- liquid media that continuously flows through the column and carries the analytes. a carrier for the sample solution -- normally use mixtures of solvents as mobile phase.

HPLC system

FOUR TYPES OF LIQUID CHROMATOGRAPHY Partition chromatography Adsorption, or liquid-solid chromatography Ion exchange chromatography Size exclusion, or gel, chromatography

WHAT AFFECTS SYSTEM Column Material Deactivation Stationary Phase Column Parameters Column Material Deactivation Stationary Phase Coating Material Instrument Parameters Temperature Flow Signal Sample Sensitivity Detector

WHAT AFFECTS SYSTEM Sample Parameters Concentration Matrix Solvent Effect Sample Effect

Several column types (can be classified as ) Normal phase Reverse phase Size exclusion Ion exchange

Normal phase In this column type, the retention is governed by the interaction of the polar parts of the stationary phase and solute. For retention to occur in normal phase, the packing must be more polar than the mobile phase with respect to the sample

Reverse phase In this column the packing material is relatively nonpolar and the solvent is polar with respect to the sample. Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase. Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water.

Size exclusion In size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size. Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores. The large molecules elute before the smaller molecules.

Ion exchange In this column type the sample components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase. Separations are made between a polar mobile liquid, usually water containing salts or small amounts of alcohols, and a stationary phase containing either acidic or basic fixed sites.

Types of Detectors Absorbance (UV with Filters, UV with Monochromators) IR Absorbance Fluorescence Refractive-Index Evaporative Light Scattering Detector (ELSD) Electrochemical Mass-Spectrometric Photo-Diode Array