HPLC

What is HPLC :-

    1. Originally referred to as High-Pressure Liquid Chromatography.

    2. Now more commonly called High Performance Liquid Chromatography.

    3. HPLC is really the automation of traditional liquid chromatography under conditions which provide for enhanced separations during shorter periods of time, utilizing very small particles, small column diameters, and very high fluid pressure.

Principle of HPLC :-

       Separation of Compounds takes place based on polarity under high pressure of mobile-phase and column through which compound eject with respect to time (RT).

Stationary of HPLC (column) :-

Polar (“Normal”  Phase) ex : Silica, alumina

Non-Polar (“Reversed Phase”) ex: ODS Silica gel, –C18, C8  

Mobile Phase of HPLC (Solvent) :-

1. Normal chromatography

   Hexane ; dichloromethane; isopropanol; methanol, Increasing strength

2.Reverse phase chromatography

   water ; methanol; acetonitrile; tetrahydrofuran (THF)

Components of HPLC :-

1.Solvent Reservoir

2.Pumps

3.Sample Injection System

4.Columns

5.Detectors

6.Data Processing

7.Waste

Solvent Reserviour of HPLC :-

       Mobile phase

       isocratic elution - single solvent separation teachnique

       gradient elution - 2 or more solvents, varied during separation

       To carry sample into the column

Pump of HPLC :-

To produce an appropriate pressure to push solvent into the sample.

A pump capable of pumping solvent up to a pressure of 4000 psi and at flows of up to 10 ml/min

Sample injection of HPLC :-

       sample valve

       Syringe/injector

      Syringe :

                        manual, Auto-injector

       A fixed-volume loop of between 1 – 200 ml (20 ml is often used as standard)

Column of HPLC :-

straight, 15 to 150 cm in length; 2 to 3 mm i.d.

packing - silica gel, alumina, Celite

Detectors of HPLC :-

        UV/Vis, Refractive index, Fluorescence, Evaporative light scattering (ELSD), MS and Diode Array Detector (DAD)

Data processing of HPLC :-

1. Using specific software that is connected to HPLC machine

2. Receive the information from HPLC machine and present it as a graph

3. The graph describes about qualitative data (Retention time) and quantitative data (area under curve)

Application of HPLC :-

1. Pharmaceuticals industry

    To control the drug stability

    Quantity  of drug determination from pharmaceutical dosage forms,   ex. Paracetamol determination in panadol tablet

    Quantity of drug determination from biological fluids, ex: blood glucose level

2. Analysis of natural contamination

    Phenol & Mercury from sea water

3. Forensic test

    Determination of steroid in blood, urine & sweat.

    Detection of psychotropic drug in plasma

Factor influence HPLC :-

1. Internal diameter of column the smaller in diameter, the higher in sensitivity

2. Pump pressure the higher in pressure, the higher in separation

3. Sample size

4. The polarity sample, solvent and column

5. Higher in temperature, the higher in separation

Advantages of HPLC :-

1. Needs a small sample with a high accuracy and precis

2. Non-destructed sample during operation compared to GC.

Disadvantages of HPLC :-

1. Need a skill to run the instruments

2. Solvents consuming