30-01-2013, 01:46 PM
High Performance Liquid Chromatography
[attachment=48892]
Introduction
HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution. HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector.
Compounds are separated by injecting a sample mixture onto the column. The different component in the mixture pass through the column at differentiates due to differences in their partition behavior between the mobile phase and the stationary phase. The mobile phase must be degassed to eliminate the formation of air bubbles.
FOUR TYPES OF LIQUID CHROMATOGRAPHY
Partition chromatography
Adsorption, or liquid-solid
chromatography
Ion exchange chromatography
Size exclusion, or gel, chromatography
COMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEM
Solvent
Solvent Delivery System (Pump)
Injector
Sample
Column
Detectors (Diode Array)
Waste Collector
Recorder (Data Collection)
Uses of HPLC
This technique is used for chemistry and biochemistry research analyzing complex mixtures, purifying chemical compounds, developing processes for synthesizing chemical compounds, isolating natural products, or predicting physical properties. It is also used in quality control to ensure the purity of raw materials, to control and improve process yields, to quantify assays of final products, or to evaluate product stability and monitor degradation.
In addition, it is used for analyzing air and water pollutants, for monitoring materials that may jeopardize occupational safety or health, and for monitoring pesticide levels in the environment. Federal and state regulatory agencies use HPLC to survey food and drug products, for identifying confiscated narcotics or to check for adherence to label claims.
HPLC Chromatograph injectors
The function of the injector is to place the sample into the high-pressure flow in as narrow volume as possible so that the sample enters the column as a homogeneous, low-volume plug. To minimize spreading of the injected volume during transport to the column, the shortest possible length of tubing should be used from the injector to the column.
When an injection is started, an air actuator rotates the valve: solvent goes directly to the column; and the injector needle is connected to the syringe. The air pressure lifts the needle and the vial is moved into position beneath the needle. Then, the needle is lowered to the vial.
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.
[attachment=48892]
Introduction
HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution. HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector.
Compounds are separated by injecting a sample mixture onto the column. The different component in the mixture pass through the column at differentiates due to differences in their partition behavior between the mobile phase and the stationary phase. The mobile phase must be degassed to eliminate the formation of air bubbles.
FOUR TYPES OF LIQUID CHROMATOGRAPHY
Partition chromatography
Adsorption, or liquid-solid
chromatography
Ion exchange chromatography
Size exclusion, or gel, chromatography
COMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEM
Solvent
Solvent Delivery System (Pump)
Injector
Sample
Column
Detectors (Diode Array)
Waste Collector
Recorder (Data Collection)
Uses of HPLC
This technique is used for chemistry and biochemistry research analyzing complex mixtures, purifying chemical compounds, developing processes for synthesizing chemical compounds, isolating natural products, or predicting physical properties. It is also used in quality control to ensure the purity of raw materials, to control and improve process yields, to quantify assays of final products, or to evaluate product stability and monitor degradation.
In addition, it is used for analyzing air and water pollutants, for monitoring materials that may jeopardize occupational safety or health, and for monitoring pesticide levels in the environment. Federal and state regulatory agencies use HPLC to survey food and drug products, for identifying confiscated narcotics or to check for adherence to label claims.
HPLC Chromatograph injectors
The function of the injector is to place the sample into the high-pressure flow in as narrow volume as possible so that the sample enters the column as a homogeneous, low-volume plug. To minimize spreading of the injected volume during transport to the column, the shortest possible length of tubing should be used from the injector to the column.
When an injection is started, an air actuator rotates the valve: solvent goes directly to the column; and the injector needle is connected to the syringe. The air pressure lifts the needle and the vial is moved into position beneath the needle. Then, the needle is lowered to the vial.
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.