HPLC- Definition, Principle, Parts, Types, Uses, Diagram

High-performance liquid chromatography (HPLC) is a technique in analytical chemistry used to separate, identify or quantify each component in a mixture. It is based on the principle of column chromatography, where a pressurized liquid (called the mobile phase) containing the sample mixture is passed through a column filled with a solid material (called the stationary phase). The different components of the sample have different affinities for the stationary phase and thus move at different speeds through the column. This results in the separation of the components as they elute from the column and are detected by a suitable detector.

HPLC is an advanced form of liquid chromatography that was developed in the 1960s to overcome the limitations of low-pressure glass columns. HPLC uses high-pressure metal columns that can withstand higher flow rates and provide better separation efficiency, versatility and speed. HPLC can also use a variety of stationary phases and mobile phases to suit different types of samples and analytical objectives.

HPLC has a wide range of applications in various fields such as pharmaceuticals, biotechnology, environmental analysis, food science, forensics and clinical chemistry. HPLC can analyze samples that are complex, thermally unstable, volatile or non-volatile, polar or non-polar, ionizable or non-ionizable. HPLC can also be coupled with other techniques such as mass spectrometry (MS) or nuclear magnetic resonance (NMR) to provide more information about the structure and identity of the analytes.

HPLC is composed of several components such as a pump, an injector, a column, a detector and a data processor.

The pump delivers a constant and controlled flow of the mobile phase through the system. The injector introduces the sample into the mobile phase stream using a syringe or an autosampler. The column contains the stationary phase that separates the sample components based on their interactions with it. The detector measures the changes in the properties of the mobile phase as it passes through the column and generates an electronic signal. The data processor records and analyzes the signal and displays it as a chromatogram, which shows the peaks corresponding to each component in the sample.

In this article, we will discuss the principle, instrumentation, types, applications, advantages and limitations of HPLC in detail.

The principle of HPLC is based on the separation of different components of a sample by their relative affinity to a stationary phase and a mobile phase. The stationary phase is a solid or liquid material that is packed inside a column. The mobile phase is a liquid or gas that flows through the column and carries the sample. The sample is injected into the mobile phase at the beginning of the column and then travels along with it. As the sample moves through the column, it interacts with the stationary phase and different components are retained for different amounts of time depending on their polarity, charge, size, shape, and other properties. The components that have a stronger affinity to the stationary phase will elute later than those that have a weaker affinity. The elution order and time of each component can be used to identify and quantify them.

The separation efficiency of HPLC depends on several factors, such as:

• The type and quality of the stationary phase
• The composition and flow rate of the mobile phase
• The temperature and pressure of the column
• The injection volume and concentration of the sample
• The detection method and sensitivity

By optimizing these factors, HPLC can achieve high resolution, accuracy, and precision in separating complex mixtures. HPLC can also be coupled with various detectors, such as UV-Vis, fluorescence, mass spectrometry, or electrochemical, to provide more information about the structure and properties of the separated components. HPLC can also be modified by using different types of stationary phases and mobile phases to suit different applications and analytes. Some common types of HPLC are normal phase, reverse phase, ion exchange, size exclusion, and affinity chromatography. Each type has its own advantages and limitations depending on the nature of the sample and the desired separation goal.

HPLC is a technique that requires a sophisticated instrument to perform the separation, identification and quantification of the components in a mixture.

The main components of an HPLC instrument are:

• The pump: The pump delivers the pressurized mobile phase (a liquid solvent or a mixture of solvents) through the system at a constant and controlled flow rate. The pump can be either a single-solvent pump or a gradient pump that can vary the composition of the mobile phase during the run. The pump should be able to generate high pressures (up to 400 atm) and provide a stable and reproducible flow rate.
• The injector: The injector introduces the sample into the mobile phase stream. The injector can be either manual or automatic. A manual injector uses a syringe to inject a fixed volume of sample into a sample loop that is connected to a switching valve. When the valve is switched, the sample loop is aligned with the mobile phase flow and the sample is carried into the column. An automatic injector uses a robotic arm to pick up a sample vial from a tray, inject a fixed volume of sample into the system, and return the vial to the tray. Automatic injectors can increase the throughput and reduce human errors.
• The column: The column is where the separation takes place. The column consists of a metal tube packed with small porous particles (2-50 micrometers in diameter) that act as the stationary phase. The stationary phase can have different chemical properties (such as polarity, charge, or affinity) that affect the interactions with the components in the sample. The column can be either analytical or preparative. An analytical column is used for separating and identifying small amounts of sample (micrograms to milligrams). A preparative column is used for isolating and purifying large amounts of sample (grams to kilograms).
• The detector: The detector measures the amount of each component that elutes from the column. The detector generates an electronic signal that is proportional to the concentration of the component in the mobile phase. The signal is recorded by a data acquisition system and displayed as a chromatogram (a plot of signal versus time). There are different types of detectors that can be used for HPLC, such as ultraviolet-visible (UV-Vis), fluorescence, refractive index, mass spectrometry, or electrochemical detectors. The choice of detector depends on the properties of the components in the sample, such as absorbance, fluorescence, refractivity, mass-to-charge ratio, or redox potential.
• The recorder: The recorder collects and processes the data from the detector. The recorder can be either a paper-chart recorder or a computer-based data processor. The recorder can perform various functions, such as peak integration, baseline correction, calibration, quantification, identification, or data storage.

Other components that may be present in an HPLC instrument are:

• The degasser: The degasser removes dissolved gases (such as oxygen) from the mobile phase before it enters the pump. Dissolved gases can cause bubbles in the system that can affect the pressure, flow rate, and detector signal. The degasser uses a special polymer membrane tubing that allows gas molecules to diffuse out while retaining liquid molecules.
• The column heater: The column heater controls the temperature of the column during the run. The temperature can affect the separation efficiency, resolution, and retention time of the components in the sample. Some separations require elevated temperatures (50-80°C) to improve resolution or reduce viscosity.

There are different types of HPLC based on the nature of the stationary phase and the mobile phase. The two most common variants are normal-phase and reversed-phase HPLC. Other types include ion exchange, size exclusion, and affinity chromatography.

Normal-phase HPLC

Normal-phase HPLC uses a polar stationary phase, such as silica gel or alumina, and a non-polar or weakly polar mobile phase, such as hexane or ethyl acetate. The separation is based on the different polarity of the analytes. The more polar analytes are retained longer on the stationary phase, while the less polar analytes elute faster. Normal-phase HPLC is suitable for separating water-sensitive compounds, geometric isomers, cis-trans isomers, and chiral compounds.

Reversed-phase HPLC

Reversed-phase HPLC uses a non-polar or weakly polar stationary phase, such as C18 or C8 bonded silica, and a polar mobile phase, such as water mixed with methanol or acetonitrile. The separation is based on the different hydrophobicity of the analytes. The more hydrophobic analytes are retained longer on the stationary phase, while the more hydrophilic analytes elute faster. Reversed-phase HPLC is the most widely used type of HPLC and can be used for separating polar, non-polar, ionizable, and ionic compounds.

Ion exchange HPLC

Ion exchange HPLC uses a stationary phase that contains ionic groups, such as sulfonic acid or quaternary ammonium, and a mobile phase that contains a buffer solution. The separation is based on the different charge and affinity of the analytes for the ionic groups on the stationary phase. The analytes with higher charge and stronger affinity are retained longer on the stationary phase, while the analytes with lower charge and weaker affinity elute faster. Ion exchange HPLC is used for separating anions and cations.

Size exclusion HPLC

Size exclusion HPLC uses a stationary phase that consists of porous particles with a defined pore size distribution. The mobile phase is usually an inert solvent that does not interact with the analytes or the stationary phase. The separation is based on the different size and shape of the analytes. The analytes that are larger than the pore size are excluded from entering the pores and elute first. The analytes that are smaller than the pore size can enter the pores and elute later. Size exclusion HPLC is used for separating molecules according to their molecular weight.

Affinity HPLC

Affinity HPLC uses a stationary phase that contains specific ligands that can bind to certain analytes with high specificity and affinity. The mobile phase is usually a buffer solution that can modulate the binding strength between the ligands and the analytes. The separation is based on the different binding interactions of the analytes with the ligands. The analytes that have stronger binding interactions are retained longer on the stationary phase, while the analytes that have weaker binding interactions elute faster. Affinity HPLC is used for separating biomolecules such as proteins, enzymes, antibodies, or nucleic acids.

HPLC is a versatile and powerful analytical technique that can be used to separate, identify and quantify a wide range of compounds in various fields and industries. Some of the common applications of HPLC are:

• Analysis of drugs: HPLC can be used to determine the purity, potency, stability and bioavailability of pharmaceutical products, such as tablets, capsules, injections, creams and ointments. It can also be used to detect and quantify impurities, degradation products, metabolites and contaminants in drug samples. HPLC is widely used in drug discovery and development, as well as in quality control and regulatory compliance.

• Analysis of synthetic polymers: HPLC can be used to characterize the molecular weight distribution, composition, structure and functionality of synthetic polymers, such as plastics, rubbers, fibers and coatings. It can also be used to monitor the polymerization process and the effects of additives, solvents and environmental factors on the properties of polymers.

• Analysis of pollutants in environmental analytics: HPLC can be used to detect and quantify trace levels of organic and inorganic pollutants in environmental samples, such as water, soil, air and biota. It can also be used to study the fate and transport of pollutants in the environment and their effects on ecosystems and human health.

• Determination of drugs in biological matrices: HPLC can be used to measure the concentration of drugs and their metabolites in biological fluids and tissues, such as blood, urine, saliva, hair and nails. It can also be used to study the pharmacokinetics, pharmacodynamics, bioequivalence and drug interactions of drugs in vivo.

• Isolation of valuable products: HPLC can be used to isolate and purify valuable products from complex mixtures, such as natural products, peptides, proteins, nucleic acids and antibodies. It can also be used to separate enantiomers, isomers and stereoisomers of chiral compounds.

• Product purity and quality control of industrial products and fine chemicals: HPLC can be used to ensure the purity and quality of industrial products and fine chemicals, such as pesticides, herbicides, dyes, pigments, flavors, fragrances and cosmetics. It can also be used to monitor the production process and the stability of the products.

• Separation and purification of biopolymers such as enzymes or nucleic acids: HPLC can be used to separate and purify biopolymers based on their size, charge, hydrophobicity or affinity. It can also be used to study the structure, function and interactions of biopolymers.

• Water purification: HPLC can be used to remove organic contaminants from water by using reverse phase or ion exchange columns. It can also be used to analyze the quality of drinking water or wastewater.

• Pre-concentration of trace components: HPLC can be used to pre-concentrate trace components from large volumes of samples by using solid phase extraction or liquid-liquid extraction techniques. This can improve the sensitivity and selectivity of the subsequent analysis.

• Ligand-exchange chromatography: HPLC can be used to separate metal ions based on their complexation with a ligand that is immobilized on the stationary phase. It can also be used to study the thermodynamics and kinetics of metal-ligand interactions.

• Ion-exchange chromatography of proteins: HPLC can be used to separate proteins based on their net charge at a given pH by using an ion exchange column that has either positively or negatively charged groups on the stationary phase. It can also be used to study the charge distribution and pI values of proteins.

• High-pH anion-exchange chromatography of carbohydrates and oligosaccharides: HPLC can be used to separate carbohydrates and oligosaccharides based on their charge at high pH by using an anion exchange column that has quaternary ammonium groups on the stationary phase. It can also be used to study the structure and linkage of carbohydrates and oligosaccharides.

  Advantages and Limitations of High-Performance Liquid Chromatography (HPLC)

High-performance liquid chromatography (HPLC) is a widely used technique for the separation, identification and quantification of various components in a mixture. HPLC has many advantages over other chromatographic methods, but it also has some limitations that need to be considered. Here are some of the main advantages and limitations of HPLC:

Advantages

• Speed: HPLC can perform fast and efficient separations using high-pressure pumps and small particle sizes in the stationary phase. A typical HPLC run can be completed within an hour or less, with high resolution and sensitivity.
• Accuracy: HPLC can provide reliable and precise results for a wide range of analytes and matrices. HPLC can achieve up to 0.1% relative standard deviation (RSD) and less than 0.01% quantitation for trace impurities. HPLC can also detect any contamination or interference in the system and correct for it.
• Versatility: HPLC can be applied to both analytical and preparative purposes, and can separate various types of compounds, such as polar, non-polar, ionizable, ionic, water-sensitive, geometric isomers, chiral compounds, etc . HPLC can also be coupled with different types of detectors, such as UV-Vis, fluorescence, mass spectrometry, etc., to provide more information about the analytes.

Limitations

• Cost: Despite its advantages, HPLC can be costly, requiring large quantities of expensive organics, a power supply and regular maintenance . The columns, pumps, detectors and other components of the HPLC system can also be expensive to purchase and replace.
• Complexity: HPLC can be complicated to troubleshoot problems or develop new methods. The choice of the mobile phase, stationary phase, column temperature, flow rate, injection volume, detection wavelength, etc., can affect the separation and detection of the analytes. The lack of a universal detector for HPLC also means that different detectors may have different sensitivities and selectivities for different analytes.
• Sensitivity: HPLC can have low sensitivity for certain compounds that are not chromophoric or fluorescent, or that are irreversibly adsorbed on the stationary phase . Volatile substances are also better separated by gas chromatography than by HPLC.

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