High Performance Liquid Chromatography 3k4i41

High Performance Liquid Chromatography

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 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)

Detector Column Pump

Eluent (mobile phase)

Column oven (thermostatic column chamber)

Sample injection unit (injector)

Drain Data processor

Degasser

Advantages of High Performance Liquid Chromatography • High separation capacity, enabling the batch analysis of multiple components • Superior quantitative capability and reproducibility • Moderate analytical conditions • Unlike GC, the sample does not need to be vaporized.

• Generally high sensitivity • Low sample consumption • Easy preparative separation and purification of samples

Fields in Which High Performance Liquid Chromatography Is Used • Biogenic substances • Sugars, lipids, nucleic acids, amino acids, proteins, peptides, steroids, amines, etc.

• Medical products • Drugs, antibiotics, etc.

• Food products • Vitamins, food additives, sugars, organic acids, amino acids, etc.

• Environmental samples • Inorganic ions • Hazardous organic substances, etc.

• Organic industrial products • Synthetic polymers, additives, surfactants, etc.

Solvent Delivery Pump • Performance Requirements • Capacity to withstand high load pressures. • Pulsations that accompany pressure fluctuations are small. • Flow rate does not fluctuate. • Solvent replacement is easy. • The flow rate setting range is wide and the flow rate is accurate.

HPLC system

Picture of an HPLC column

Solvent Delivery Pump: Schematic Diagram of Plunger Pump

Motor and cam

Pump head

Check valves

Plunger Plunger seal

10 -100µL

Solvent Delivery Pump: Single Plunger Type

Check valves

Plunger head

Solvent Delivery Pump: Dual Plunger Type Check valves

Plunger heads

Type

Type

Manual Injector

From pump

To column

LOAD position From pump

INJECT position

To column

Autosampler (Pressure Injection Method)

From pump

To column

From pump

To column

Sample Loop

LOAD

INJECT

Picture of HPLC instrument