The HPLC chromatography has different types that are based on the principle of separation, modes of chromatography, type of analysis, the scale of operation, and elution mode.
Chromatography is an analytical technique in chemistry used to isolate and analyze complex mixtures of analytes. Chromatography works by distributing a mixture of compounds between two phases i.e. the mobile phase and the stationary phase. Liquid chromatography uses a liquid or solvent as a mobile phase, whereas the stationary phase is packed into a column or a planar surface.
The isolation of all chromatography methods, including HPLC, TLC, column chromatography, and paper chromatography works under the same basic principle. The compounds are separated because of their different affinity for the stationary phase used in the chromatography system.
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The high-performance liquid chromatography (HPLC) is a highly advanced version of column chromatography. Rather by allowing a solvent to drop under gravity via a column, it is forced at high pressure using an HPLC pump, which makes it a very rapid and highly precise method.
The stationary phases (solid) that are packed in a column (most generally silica) and the mobile phase (liquid) pass through it. It also allows the use of very small size of the particles for the packing material of the HPLC column, which gives a very high surface area for the interaction between the stationary phase and the components traveling over it.
Therefore, we obtain a better separation of the components of the sample mixture. The analytes of the sample mixture are separated based on their polarity; it's dependent on the chromatography technique that will be used for the analysis.
Different types of HPLC:
HPLC has several forms which are based on modes of chromatography, the principle of separation, elution method, type of analysis, and scale of operation, let's check it.
- Based on modes of chromatography:
Normal phase and reversed-phase chromatography
- Based on the principle of separation:
The adsorption chromatography, ion-exchange chromatography, affinity chromatography, ion-pair chromatography, gel permeation or size exclusion chromatography, and chiral chromatography, etc.
- Based on elution method followed:
Isocratic elution and gradient elution
- Based on the type of analysis:
Quantitative analysis and qualitative analysis
- Based on the scale of operation:
Preparative type HPLC and analytical type HPLC
Depend on the stationary phase in the method there are four types of HPLC i.e. reversed-phase HPLC, normal phase HPLC, size-exclusion HPLC, and ion-exchange HPLC are used to separate the molecules.
Reversed-phase HPLC:
RP-HPLC is a chromatographic technique in which the mobile phase is polar, aqueous, and the stationary phase is non-polar (hydrophobic). The particles in the reversed-phase column are usually coated with carbon chains, for example, C8/C18. Among all types of HPLC methods, we use around 70% of this process due to its wide applicability and reproducibility.
Normal phase HPLC:
NP-HPLC is the classical form of chromatography; in contrast to the reversed-phase chromatography, it uses the non-polar mobile phase and polar stationary phase (hydrophilic). If this separation mechanism depends on adsorption, it is also known as adsorption chromatography based on the selected stationary phase.
More specifically, this technique is the mobile process is 100 percent organic solvents. That means no water is used for the separation of compounds.
Size-exclusion HPLC:
Size exclusion or gel-filtration chromatography requires porous stationary particles, trapping smaller size particles within, and allowing bigger size molecules to move more rapidly. The size exclusion chromatography column is filled with precisely controlled pore-size material. The retention time of analyte increases as the size of the analyte decreases.
Ion-exchange HPLC:
Ion-exchange chromatography is a type of HPLC that used to isolate ions and polar compounds based on their affinity for an ion exchanger. To purify proteins and other charged a molecule IEC is the most popular method this is one of the major advantages of ion chromatography. IE-HPLC uses attraction by an electric charge on the surface of stationary phase particles.
The retention time of the eluted molecules relies on their inherent charge and the salt ion concentration in solution. Anion-exchange and cation-exchange are two types of ion chromatography. Anion exchange particles are positively charged cation exchange particles that are negatively charged and interact with positive ions.
Instrumentation of HPLC:
The instrumentation of high-performance liquid chromatography (HPLC) consists of a solvent reservoir, degasser, pump, sample loop (injector), column, column oven, and detector. The separation of the components happens in the HPLC column, hence is considered as a key component for separation in HPLC.
Solvent reservoir:
It is used to store the mobile phase or solvents. HPLC usually consists of polar and non-polar liquid mobile phases such as water, methanol, acetonitrile, and buffer or a suitable combination whose specific concentration is changed depending on the properties of the sample.
Pump:
By providing high pressure the pump of HPLC gives a constant, pulseless flow and reproducible flow of the mobile phase. A standard HPLC pump needs to be pumped against pressures of up to 6,000 PSI, hence generally reciprocating piston pumps are used in the HPLC. Reciprocating pump is used as isocratic and gradient mode, and a syringe pump, a pneumatic pump is used as an isocratic mode. The different types of pumps are available as single, binary or quaternary as needed.
Degasser:
The vacuum degasser is an in-line component that removes dissolved gases from solvents or the mobile phase. Inside the vacuum degasser, the solvent moves through tubing that is situated in a vacuum chamber. The partial vacuum inside the chamber is maintained by a continuously running vacuum pump. The solvent moves within the coil. The dissolved gases are drawn by vacuum through the tubing wall.
Injector:
A sample injector is a device used to inject the sample solution into the system. Typically the injection volume of the sample uses between 5 -100 microliters. The injectors should have high reproducible and work on the high back pressure of HPLC (up to 4000 psi). Rheodyne injectors, septum injectors, and stop flow injectors are examples of HPLC injectors, of which Rheodyne is commonly used.
Column:
The selection of HPLC column dimensions to perform the analysis will depend on chromatographic applications, properties, and the number of molecules in the sample. Generally, modern columns are made of stainless steel tubes; they packed by spherical silica gels that are coated with a hydrophobic stationary phase with a molecule size of 3 to 10 µm. Columns are around 50 to 300 mm long and the around 2 to 5 mm has a diameter.
Column oven:
Nowadays the column oven in LC is an essential component that is used to control the temperature of the column. For separations of molecules with the marginal resolution of the critical peak pair, a proper column temperature control is necessary. It is also useful when the viscous buffer solution is used in the mobile phase.
As the viscosity decreases, consequently, the backpressure of the system decreases. In general, when the column temperature increases, the process of chromatographic separation becomes more rapid and more efficient.
Detector:
A detector is a tool used to detect solutes eluted from the HPLC column. The detector converts the effluent into an electrical signal and the computerized system record it. The UV/VIS and PDA detector are most commonly used in HPLC; different types of HPLC detectors are the mass detector (LC-MS), fluorescence detector, and Infrared detector, etc.
Data acquisition and control system:
Each parameter of the system is controlled through a computerized system. HPLC parameters such as mobile phase composition, flow rate, wavelength, run time, temperature, sample sequence, and injection volume, etc. It is used to constantly monitor the system and check the back pressure and it allows integrating the obtained data.
What is the HPLC principle?
The basic principle of HPLC is based on the distribution of the compounds among the stationary phase (column packing) and a mobile phase (solvent). Compounds are separated according to their chemical structure, and affinity towards the column.
Where is HPLC used?
HPLC is more commonly used in the analytical field and pharmaceutical applications. It plays a significant role in the pharmaceutical analysis as it is used for qualitative and quantitative analysis of test samples. Some other major applications of HPLC include forensics applications, environmental applications, clinical applications, food, and beverage applications.
Why UV detector is used in HPLC?
UV detector is used in HPLC to detect and identify molecules in the sample mixture. The ultraviolet detector is based on UV absorbance and works in the range of 200 nm to 400 nm. It is the most commonly used detector for liquid chromatography, as it is nondestructive and measures the amount of light absorbed by the molecule.
Why c18 column is used in HPLC?
There are different types of columns are used in HPLC separation, but the most frequently used column is C18 (Octyldecylsilane). It consists of 18 carbons bound to the silica that is more carbon and a longer carbon chain than other columns such as C8 or C4. Due to the more carbons chain, C18 has a big surface area that provides more interaction time among the bonded phase and the solutes. Consequently, the analytes are more slowly eluted from the column and more separation occurs.
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