HPLC (High Performance Liquid Chromatography)

LC (Liquid Chromatography) is the term generally used to describe the separation of the components of a solution following differential migration of the solutes in a liquid flowing through a column packed with solid particles.

(Riley C. M.) From “High Performance Liquid Chromatography” fundamental principles and practice. The various components that are used in a modern HPLC separation are shown in the following diagram.

The major parts are

1. Solvent Reservoir or Eluent Reservoir
2. Pump or solvent delivery system
3. Sample Injector
4. Column
5. Detector
6. Data analysis
7. Waste Bottle

A pump is used to force the eluent through the column and detector. A pump provides constant flow regardless of the back-pressure of the column, which may be anything up to 3000 lb / in².

Injector can be simply a syringe that injects the sample into the eluent stream through a rubber septum.

Column is stainless steel or heavy-walled glass, built to withstand high pressure. The inside column diameter is typically 1-3 mm., and the length is up to 1 meter. The column is packed with small particles of porous silica, alumina, or organic resin (LSC) or with a porous support coated with a liquid (LLC).

Detector : the purpose of the detector in HPLC system is to identify the presence ofcertain compounds of interest in the eluent from the HPLC column.

Types of detector

1. UV-visible absorbance detector : for aromatic compounds. Nonionic surfactant
2. RI (Refractive Index)
3. Fluorescence
4. Electrochemical
5. Conductivity: for inorganic ions eg. Cl^-, So₄^2- , Na⁺ ,anionic and cationic surfactant

Procedure in HPLC system

A sample is injected by syringe and passes through an injector by following solvent or mobile phase in a small diameter (0.009 in) tube and then pass through column. A packing in column is called chromatographic bed. When a sample passes through this bed, the components in a sample can be separated because each component has an individual ability to pass through a packing in column. Therefore each component in a sample separates into an individual band and passes through a bed of column. Each component moves out from column that is called elution. Then they go through a detector which detects amount of each component in form of a signal and sends the result or output to a data system or computer. One signal is called peak and many peaks are called chromatogram. Each peak can tell what it is by comparing with retention time that is a time for each peak or band passing through column. It must have a calibration curve which is made from knowing concentration and types of substance. We can know a retention time of a substance from a calibration. Therefore, when we have an unknown sample, we injects it in HPLC. The result from a computer shows an area and a retention time of each peak or each component in an unknown sample. Using a calibration curve compares this result, we can know what it is and its concentration.

Solvent Preparation

Solvent should be HPLC grade solvent which has a purity more than AR grade.

1. Filter solvent or mobile phase with microfilter diameter 0.45 m m 47mm. for separation an impurity which damage column and system.
2. Get rid of air bubble from solvent by sonicate (ultrasonic bath)

Sample preparation

To produce the sample in a form suitable for introduction into the measuring instrument. In HPLC , where the mobile phase is liquid, the sample should ideally be presented dissolved in the mobile phase. It also filter with microfilter dia 0.45 m m. 13mm.