High-Performance Liquid Chromatography (HPLC)

High-performance liquid chromatography is an improvised variety of column chromatography. Instead of the mobile phase being permitted to drip down through a column under the force of gravity, it is forced through a column under high pressures of up to 400 atmospheres, making it a much faster technique.

For the column packing material, it permits the usage of much smaller particle size, and that, in turn, helps in providing a larger surface area such that the solute can interact with the stationary phase. This promotes enhanced separation of the constituents of the mixture. Another major development over column chromatography is the use of highly sensitive detectors.

Principle

Components of interest present in the mobile phase are separated based on their differing physicochemical interactions with the stationary and mobile phases. A fine adsorbent solid is chosen as the stationary phase wherein the solid is so chosen which will clamp on the outer surface of the liquid particles.

In column chromatography, generally, the type of column used is identical to a Pasteur pipette (In small-scale column chromatography, Pasteur pipettes are normally used as columns). The separating column is made by tightly filling the adsorbent solid generally silica into the glass tube.

For high-performance liquid chromatography, it is known that the resolving power of a chromatographic column increases with column length and number of theoretical plates per unit length, although there are limitations to the length of a column because of the problems of peak broadening. As the number of theoretical plates is associated with the surface area of the stationary phase, it follows that the smaller the particle size of the stationary phase, the better the resolution and greater the resistance to eluent flow.

Instrumentation

The schematic representation in the figure below shows HPLC instrumentation.

It consists of a;
• Solvent reservoir
• Injector
• Pump
• Column
• Detector
• An integrator or acquisition and display system.
• The pump is considered the heart of this chromatography system.

The sample is injected through a port in the high-pressure liquid carrier stream between the pump and the column. The separation takes place in the column which varies from 3- 30 cm in length and 3 mm in diameter. Typical flow rates fall between 1-2 ml/min with pressure up to several thousand psi. The column effluent passes through a non-destructive detector where a property such as ultraviolet absorbance, refractive index, and molecular fluorescence is monitored.

The signal is then amplified and recorded as a detector response v/s retention time. The graph thus obtained is called a chromatogram. The effluent may be discarded, recycled, or saved for any further research or studies in a fraction collector which is synchronized with the detector.

The components of high-performance liquid chromatography include:

• Solvent Reservoir:

Mobile phase contents are stored in a glass reservoir. In HPLC, usually, a mixture of polar and non-polar liquid constituents is used as the mobile phase, or solvent, where the concentration of constituents varies based on the composition of the sample.

• Pump:

A pump sucks the mobile phase from the solvent reservoir and pushes it through the column and detector. Operating pressures of up to 42000 kPa (about 6000 psi) can be generated based on several factors that include- the dimension of the column, the particle size of the stationary phase, the composition of the mobile phase, and its flow rate, and so on.
Microprocessor-controlled pumping systems have the capability of precisely delivering a mobile phase of either constant (isocratic elution) or varying (gradient elution) composition, according to a defined program.

• Sample Injector:

The sample solution is usually introduced into the flowing mobile phase at or near the head of the column. An injection valve that can operate at high pressure is used for injection purposes. Automated injection systems are also in use these days. In the HPLC system, the injection of sample is facilitated by the injector, where the liquid sample is within the range of 0.1-100 mL of volume with increased reproducibility and pressures up to 4000 psi.

• Column:

Columns are typically made up of stainless steel. Their length ranges between 50 and 300 mm and the internal diameter between 2 and 5 mm. They are packed with stationary phase particles of size 3–10 µm. Columns are packed with solids like silica or alumina; these columns are called homogeneous columns.

If the stationary phase in the column is a liquid, the column is deemed as a bonded column. In the bonded column, a liquid stationary phase is bonded to a solid support which is generally made up of alumina or silica.

• Detector:

Since the quantity of material applied to the column is very small, the sensitivity of the detector system must be high. Commonly used detectors are UV detectors, fluorescence detectors, mass-spectrometer, and electrochemical detectors. LC-MS is also used to develop highly precise and reproducible assays. Modern-day mass spectrometers are extremely sensitive and LC-MS assays are now viable replacements for many immunoassays. LC-MS assays will possibly be most beneficial in the clinical biochemistry laboratory when used for multiplexed and screening type assays.

Applications

• Biochemical Screening for Genetic Disorders:

There are several amino acid markers and acylcarnitines which are measured in dried blood spots after extraction and derivatization in microtiter plates; screening strategies have been developed for galactosemia, sickle cell anemia, and lysosomal disorders.

• Therapeutic Drug Monitoring and Toxicology:

Assays have been developed for immune suppressants such as cyclosporine, tacrolimus, sirolimus, everolimus, and mycophenolic acid.

• Forensic Applications of HPLC includes the analysis of drugs, analysis of explosives, ink analysis in case of Questioned Documents, Dyes in illicit drugs, fibers, lipstick smears, foods, and comparison of soil samples, etc.

• HPLC can analyze very small samples and quantify trace amounts of impurities in the drug samples.

• Various Drug Enforcement Administration (DEA) agencies uses HPLC to detect trace impurities in drug samples. The profiles of these impurities in the drug samples can be matched to a specific method of manufacturing.

• Since each drug cartel has a slightly different manufacturing system and location, this information can be used to determine which cartel produced a specific sample of drugs.