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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ( HPLC ) - Coggle Diagram
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
( HPLC )
THEORY - The sample mixture to be separated and analyzed is introduced, in a discrete small volume (typically microliters), into the stream of mobile phase percolating through the column. The components of the sample move through the column at different velocities, which are a function of specific physical interactions with the adsorbent (also called the stationary phase). The velocity of each component depends on its chemical nature, the nature of the stationary phase (column), and on the composition of the mobile phase. The time at which a specific analyte elutes (emerges from the column) is called its retention time. The retention time measured under particular conditions is an identifying characteristic of a given analyte.
DEFINATION - formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material.
INSTRUMENTATION -
1) PUMP - he development of HPLC led to the development of the pump system.
The pump is positioned in the most upper stream of the liquid chromatography system and generates a flow of eluent from the solvent reservoir into the system.
Most pumps used in current LC systems generate the flow by back-and-forth motion of a motor-driven piston (reciprocating pumps). Because of this piston motion, it produces “pulses”.
2) INJECTOR -
An injector is placed next to the pump.
The simplest method is to use a syringe, and the sample is introduced to the flow of eluent.
The most widely used injection method is based on sampling loops.
The use of the autosampler (auto-injector) system is also widely used that allows repeated injections in a set scheduled-timing.
3) COLUMN -
The separation is performed inside the column.
The recent columns are often prepared in a stainless steel housing, instead of glass columns.
The packing material generally used is silica or polymer gels compared to calcium carbonate
4) DETECTOR - Separation of analytes is performed inside the column, whereas a detector is used to observe the obtained separation.
The composition of the eluent is consistent when no analyte is present. While the presence of analyte changes the composition of the eluent. What detector does is to measure these differences.
5) DE GASSER -
When gas is present in the eluent, this is detected as noise and causes an unstable baseline.
Degasser uses special polymer membrane tubing to remove gases.
The numerous very small pores on the surface of the polymer tube allow the air to go through while preventing any liquid to go through the pore.
6) COLUMN HEATER - In order to obtain repeatable results, it is important to keep consistent temperature conditions.
ADVANTAGES -
Speed
Efficiency
Accuracy
Versatile and extremely precise when it comes to identifying and quantifying chemical components.
APPLICATIONS -
Analysis of drugs
Analysis of synthetic polymers
Analysis of pollutants in environmental analytics
Determination of drugs in biological matrices
Pre-concentration of trace components
Ligand-exchange chromatography
Ion-exchange chromatography of proteins
High-pH anion-exchange chromatography of carbohydrates and oligosaccharides
DISADVANTAGES -
Cost: Despite its advantages, HPLC can be costly, requiring large quantities of expensive organics.
Complexity
HPLC does have low sensitivity for certain compounds, and some cannot be detected as they are irreversibly adsorbed.
Volatile substances are better separated by gas chromatography