05-02-2013, 02:18 PM
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
[attachment=49693]
INTRODUCTION:
In 1941 Martin and Synge, described the discovery of liquid-liquid partition chromatography and also laid the foundatio9n of Gas liquid chromatography and High performance liquid chromatography. They also introduced the concept of the Height Equivalent to the Theoretical Plate , which has since been adopted as the measure of Chromatographic efficiency.
In Classical Column Liquid Chromatography, the mobile liquid phase flows slowly through the column by means of gravity. The method is generally characterized by low column efficiencies and long separation times. Since ab0out 1969, there has been a very marked revival of interest in the technique of liquid column chromatography because of the development of HPLC by Kirkland and Huber. They proposed high pressure systems capable of operating at pressures up to 3000psi. In HPLC, small diameter columns(1-3mm) with support particle sizes in the region of 30µm are used and the eluent is pumped through the column at a high flow rate. It has been fond that separation by HPLC may be effected about 100 times faster than by the use of conventional liquid chromatography.
by operating at high pressures these instruments for liquid chromatography over come the effect of higher liquid viscosities relative to gas viscosities and gave analysis times comparable with GLC.
INSTRUMENTATON:
1. Solvent delivery system
2. Pumps
3. Sample injection system
4. Column
5. Detectors
6. Recorders and Integrators
SOLVENT DELIVERY SYSTEM:
The solvents or mobile phases used must be passed through the column at high pressure at about 1000 to 3000 psi. this is because as the particle size of stationary phase is few µ (5-10µ), the resistance to the flow of solvent is high. Hence such high pressure is recommended.
The choice of mobile phase is very important in HPLC and the eluting power of the mobile phase is determined by its overall polarity, the polarity of the stationary phase and the nature of the sample components.
Mixing unit is used to mix the solvents in different proportions and pass through the column. There are 2 types of mixing units.
[attachment=49693]
INTRODUCTION:
In 1941 Martin and Synge, described the discovery of liquid-liquid partition chromatography and also laid the foundatio9n of Gas liquid chromatography and High performance liquid chromatography. They also introduced the concept of the Height Equivalent to the Theoretical Plate , which has since been adopted as the measure of Chromatographic efficiency.
In Classical Column Liquid Chromatography, the mobile liquid phase flows slowly through the column by means of gravity. The method is generally characterized by low column efficiencies and long separation times. Since ab0out 1969, there has been a very marked revival of interest in the technique of liquid column chromatography because of the development of HPLC by Kirkland and Huber. They proposed high pressure systems capable of operating at pressures up to 3000psi. In HPLC, small diameter columns(1-3mm) with support particle sizes in the region of 30µm are used and the eluent is pumped through the column at a high flow rate. It has been fond that separation by HPLC may be effected about 100 times faster than by the use of conventional liquid chromatography.
by operating at high pressures these instruments for liquid chromatography over come the effect of higher liquid viscosities relative to gas viscosities and gave analysis times comparable with GLC.
INSTRUMENTATON:
1. Solvent delivery system
2. Pumps
3. Sample injection system
4. Column
5. Detectors
6. Recorders and Integrators
SOLVENT DELIVERY SYSTEM:
The solvents or mobile phases used must be passed through the column at high pressure at about 1000 to 3000 psi. this is because as the particle size of stationary phase is few µ (5-10µ), the resistance to the flow of solvent is high. Hence such high pressure is recommended.
The choice of mobile phase is very important in HPLC and the eluting power of the mobile phase is determined by its overall polarity, the polarity of the stationary phase and the nature of the sample components.
Mixing unit is used to mix the solvents in different proportions and pass through the column. There are 2 types of mixing units.