HPLC (High-Performance Liquid Chromatography), also known as high-pressure liquid chromatography is an improved form of column liquid chromatography. HPLC is known for its high performance and high yields as compared to other traditional forms of chromatography. This is because the sample is forced under high pressure, up to 400 atmospheres, resulting in a higher yield and performance. HPLC is a separation technique that involves the injection of a liquid sample into the column (filled with solid absorbent material), where the individual components of the sample are moved down the column by forced pressure generated through the pump. It is used to separate the components of the mixture, which are later identified and quantified with the help of spectroscopy.
Working Principle
HPLC is a separation technique used to separate individual components of a sample. Its separation is based on the distribution of the analyte (sample) between a mobile phase (eluent) and a stationary phase (packing material of the column). The stationary phase is a granular solid absorbent material, and the mobile phase is a solvent or solvent mixture that is forced under high pressure (400 atmospheres) to pass through the separating column. The sample/analyte is injected into the mobile phase with the help of a syringe, and the individual components of the sample pass through the separating column at different rates because they get retained in the stationary phase. The intermolecular interactions and the packing material (stationary phase), define their time “on-column.” Hence, different molecules of the sample are eluted at varying times, and the separation of individual components of the sample is achieved.
Types of HPLC
1. Normal Phase HPLC
Normal phase HPLC separates components on the basis of their polarity. Therefore, it uses a polar (hydrophilic) stationary phase and a non-polar (hydrophobic) mobile phase. Silica and alumina are the most commonly used stationary phases for the NP-HPLC method, whereas, hexane, heptane, methylene chloride, chloroform, diethyl ether, and mixtures of these are used as mobile phases. Less polar molecules, due to their high mobility, exit the column faster and are detected first, thus, resulting in the separation of polar and non-polar components. This technique is used for water-sensitive compounds, geometric isomers, class separation, and chiral compounds.
2. Reverse Phase HPLC
Reverse HPLC is a separation technique that works on the principle of hydrophobic (non-polar) interactions to separate polar, nonpolar, ionic and ionizable compounds. It uses a non-polar stationary phase and a polar mobile phase, such as mixtures of water and methanol or acetonitrile. The separation method of RP-HPLC relies on non-polar interactions as, the more non-polar the material is, the longer it will be retained on the surface of the stationary phase.
3. Size-exclusion HPLC
Size exclusion chromatography, also known as molecular sieve chromatography, is a technique where molecules are separated by their size and molecular weight. In this type of chromatography, the column is filled with a material having precisely controlled pore sizes. Larger molecules are easily washed off the separating column and the molecules with smaller sizes consume time, as they penetrate inside the porous of the packing particles and elute later. This method is used for separating large molecules or macromolecular complexes such as proteins and industrial polymers.
4. Ion-Exchange HPLC
It is a separation method based on the protein’s net charge. The surface charge can vary vastly between different proteins and is thus, used for separation. This method is used to separate ionic or ionizable samples. In this technique, the stationary phase is an ionically charged surface consisting of an opposite charge to sample ions. The stronger the charge on the sample, the stronger it will be attracted to the ionic surface and thus, the longer it will take to elute. The mobile phase, an aqueous buffer, also helps to control the elution time.
Instrumentation
1. Pump
The pump, also known as a solvent delivery system, is responsible for generating a flow of eluent from the solvent reservoir into the system. The main role of the pump is to provide high pressure (400 atmospheres) and allow the liquid (mobile phase) to flow through the column. It is also used to maintain a constant rate of flow throughout the column. 1 – 2ml/min is the normal flow rate in HPLC. Types of pumps used in HPLC are, constant flow reciprocating pumps, syringe-type pumps (displacement type), and pneumatic pumps. Reciprocating pumps are preferred more because of their constant rate of pressure generation.
2. Injector
An injector is an area from where the sample is injected into the flow of eluent (mobile phase) with the help of a syringe, without disturbing the flow rate and pressure of the HPLC system. The conventional type of injectors involves Rheodyne injector, Septum injector, and Stopflow injector, while the latest used injector is known as an Auto-sampler injector, high-tech automated equipment with high precision, that allows repeated injections in a set scheduled timing.
3. Column
Column, also known as separating column, is a significant part of the HPLC technique as the separation of molecular compounds is performed inside the column. C18 and C8 columns are the most commonly used columns in the pharmaceutical industry. Columns nowadays are made up of stainless steel instead of glass columns, because stainless steel columns are tolerant to a large variety of solvents. Silica and polymeric resins (polystyrene divinyl benzene) are usually used as packaging materials for columns. During analysis, the temperature of the mobile phase and the stationary phase is kept constant.
4. Detector
A detector is used to sense the presence of separated compounds obtained, as they leave the column. Detectors help to monitor the components obtained and to express them electronically. The presence of an analyte alters the composition of the eluent, and it remains constant when there are no analytes present. Ideal detectors must be sensitive, have good stability, reproducibility, be cost-effective, non-destructive, highly reliable, and withstand temperatures ranging up to 400 degrees Celcius. Types of detectors involve Refractive index detectors, Fluorimetric detectors, Conductivity detectors, and Amperometric detectors.
5. Recorder
The change of eluent is detected by detectors in the form of electric signals. These signals are interpreted into a meaningful form with the help of a recorder. The recorder/computer interprets the electric signals and expresses them in the form of a graph called a chromatogram.
6. Degasser
Noise and unstable baseline can be caused due to trapping of gases, like oxygen, during the mixing of liquids. Degasser is a highly-efficient in-line system that uses special polymer membrane tubing to remove dissolved gases from a solvent. It is reliable and easy to operate.
7. Column heater
Columns are generally kept inside a column chamber/column oven to maintain constant or controlled temperatures throughout the analysis. For example, better quality sugar and organic acids are obtained at temperatures ranging from 50 to 80°C. The precise control over the temperature during analysis, improves the sensitivity, analysis time, peak separation and ensures the accuracy of sample results. Numerous small pores on the polymeric Teflon tubing allow the gaseous exchange to the environment while preventing any liquid to pass through the pores.
Applications
1. Pharmaceutical Applications
- To test the quality of drugs
- To detect impurities
- Manufacturing of pharmaceutical drugs
- To monitor the progress of a therapy of a disease
- Qualitative and quantitative analysis
- Evaluating formulations
- Monitoring changes during scale-up
- Helps in tablet dissolution study
- To control drug stability
2. Environmental Applications
- Analytical control of pollutants
- Detection of phenolic compounds in drinking water
- Analyzing air pollution
- Oil spills analyzing
- Analyzing plastic pollution
3. Food and Flavour
- Quality check for soft drinks and water
- Sugar analysis in fruit juices
- Preservative analysis
- Polycyclic compound analysis
- Used to separate various ingredients, contaminants, and additives
4. Applications in Forensics
- Identification and quantification of drugs
- Forensic analysis of textile dyes
- Determination of cocaine and other drugs in blood
- Help to analyze volatile substances
- To determine the material used in explosives
5. Applications in Clinical Tests
- To determine illicit drugs in urine
- Antibiotics analysis in blood
- Nutrient analysis
- In hepatic disorders, for analysis of bilirubin, and biliverdin