Chromatography is the science of separating particles of a sample at an exceedingly high velocity. HPLC testing is done in high-tech labs to analyse samples that come in from companies. One might wonder what HPLC is. Well to break it down, HPLC testing stands for High-Performance Liquid Chromatography testing, which is a testing tool found in any well-established laboratory. This piece of equipment separates and quantifies a selection of analytes that are non-volatile and thermally labile enough to analyse it over a gas chromatograph.
The particle size of columns used is usually around 2um, which helps with more accurate results. It allows to test particles of the mixture and allows better separation than the columns having a particle size of 5um.
The most standard lab equipment found along with the HPLC testing tool, to further analyse the separated particles from the specimen sample is:
- RI (Refractive Index) Detector.
- FLD (Fluorescence Detector).
- PDA (Photodiode Array Detector).
- MSD (Mass Spectrometer Detector).
HPLC testing is one of the most widely used techniques to determine the particle in a mixture; for instance, it is most commonly used in the food industry, pharmaceutical industry, etc.
Domains where HPLC is used:
This form of testing analyses the components of a mixture from the following industries:
- Food industry.
- Chemical industry.
- Pharmaceutical industry.
- Colour and dye industry.
- Environment analysis sector.
HPLC is used to trace the following substances from the mixtures from the above industries as follows:
- If there are any drugs or formulations present in the mix.
- Determining the estimated number of aflatoxins.
- The residue of pesticide in the mixture.
- Method development of specimens.
- Determining the number of vitamins in the mixture.
- Profiling the number and type of impurities in pharmaceutical products.
The process of Chromatography:
- This process involves putting the specimen in different columns and then placing inside the equipment. This is the mobile phase.
- Suppose the component is of different polarities and the mobile phase passes through the columns, in that case, each component will approach the speeding column at different rates, based on its varying densities.
- Post this process, if the components of this mixture, post their separation, will appear in the form of bands. Each band represents a different particle.
- If the components are indistinguishable or partially separated, then other instruments such as UV spectroscopy, spectroscopy, etc. are used to determine each band.
The component’s mobile phase often consists of acetonitrile, water and methyl alcohol. Various component particles present in the mixture interact with the absorbent at different speeds due to density difference. Contrasting elution rates are obtained through this process, where the elusion rate is defined as the rate at which the components of a mixture separate as they pass through the column.
As compared to the process in column chromatography, HPLC testing has an advantage as it is highly automated and has high sensitivity.
HPLC Types:
1. NP – HPLC (Normal phase HPLC):
Normal phase HPLC is a method that works on the principle of separation of particles due to their varying polarity. In this, the mobile phase is usually chloroform, hexane, or even di-ethyl ether, whereas the stationary phase is silica in most cases. Due to this polarity difference, the particles end up staying near the polar surfaces of the column.
2. RP – HPLC (Reverse phase HPLC):
This process is opposite to the NP HPLC process. In this, the mobile phase is of polar type, and the stationary phase is of non-polar type. Hence, in this method, due to the polarity difference, the non-polar surface ends up retaining the particles instead of the polar surface.
Apparatus Setup:
- The pump: This is the equipment that pumps the moving phase from the solvent’s reservoir through the column and detector.
- Sample Injector: The mixture sample that needs separating, is commonly injected using the sample injector. It can either be done manually or can be automated.
- The columns: This is the section of the apparatus where the actual separation of particles takes place.
- Detector: This equipment is usually found at the end of the HPLC set up. It is mainly to detect the particle found and analyse the type, and other properties using detectors like fluorescence detector, UV detector, etc.
- Data collection: The detector is coupled with a system that decodes the detector’s signals and records the data. The result is put across the computer screen for the lab in-charge to read and report accordingly.