Introduction to HPLC
High-Performance Liquid Chromatography (HPLC) is one of the most powerful and widely used techniques in modern laboratories. It helps to separate, identify, and quantify different components of a mixture with high accuracy and efficiency.
Unlike traditional column chromatography, where the solvent moves under gravity, HPLC uses high pressure (up to 400 atmospheres) to push the solvent through tightly packed columns. This makes it faster, more precise, and suitable for complex mixtures.
HPLC is essential in fields like biochemistry, pharmaceutical industries, food analysis, forensic science, and environmental monitoring because of its ability to analyze even trace amounts of compounds.
Principle of HPLC
HPLC works on the same principle as column chromatography: separation based on interactions between the stationary phase and mobile phase.
- Stationary Phase – Solid particles (silica or polymer gels) packed in a stainless-steel column.
- Mobile Phase – Solvent or mixture of solvents forced through the column under high pressure.
- Separation – Different compounds in the mixture move at different speeds through the column depending on how strongly they interact with the stationary phase.
- Detection – As components exit the column, they are detected, and their signals are displayed as peaks on a chromatogram.
The chromatogram helps in identifying compounds and measuring their quantity.
Components of HPLC
A typical HPLC system consists of the following main parts:
1. Solvent Reservoir
- Stores the mobile phase (solvent).
- Solvents must be pure, degassed, and filtered.
2. Pump
- Generates high pressure to move the solvent through the column.
- Most common pumps: reciprocating pumps (piston-driven).
- Ensures a constant and reproducible flow rate.
3. Injector
- Introduces the sample into the system.
- Can be manual (syringe) or automatic (autosampler).
- Uses sample loops for precise volume injection.
4. Column
- The heart of HPLC where separation occurs.
- Usually stainless steel packed with silica or polymer particles.
- Different columns are used for different HPLC types (normal phase, reverse phase, etc.).
5. Detector
- Identifies and measures compounds as they exit the column.
- Common detectors:
- UV/Vis detector – most widely used.
- Fluorescence detector – for sensitive biomolecules.
- Refractive index detector – for compounds without UV absorbance.
6. Data Recorder / Computer System
- Converts detector signals into a chromatogram.
- Modern software allows peak analysis, baseline correction, concentration calculation, and molecular weight determination.
7. Degasser
- Removes dissolved gases like oxygen from the solvent.
- Prevents noise and unstable baseline in chromatograms.
8. Column Heater (Oven)
- Maintains constant column temperature.
- Some separations (e.g., sugars, organic acids) require elevated temperatures for better results.
Types of HPLC
- Normal Phase HPLC
- Stationary phase: Polar (silica).
- Mobile phase: Non-polar (hexane, chloroform).
- Used for: Water-sensitive compounds, geometric isomers, and chiral molecules.
- Reverse Phase HPLC (RP-HPLC)
- Stationary phase: Non-polar (C18).
- Mobile phase: Polar (water + methanol/acetonitrile).
- Most common type of HPLC.
- Used for: Polar, non-polar, ionizable, and ionic compounds.
- Ion Exchange HPLC
- Stationary phase: Charged groups.
- Mobile phase: Buffer solution.
- Used for: Separation of anions, cations, amino acids, proteins.
- Size Exclusion HPLC (Gel Filtration)
- Stationary phase: Porous material.
- Separation based on molecular size.
- Large molecules elute first, smaller ones later.
- Used for: Proteins, polymers, nucleic acids.
Applications of HPLC
In Chemistry
- Separation and identification of chemical compounds.
- Analysis of synthetic polymers.
- Quality control of industrial chemicals.
In Biology & Biochemistry
- Separation and purification of enzymes, nucleic acids, proteins.
- Determination of metabolites in biological samples.
- Protein sequencing and analysis.
In Medicine & Pharmacy
- Drug analysis and formulation quality control.
- Measuring drug concentrations in blood.
- Identifying metabolic disorders.
In Environmental Science
- Detecting pollutants in water, soil, and air.
- Monitoring pesticide residues in food.
In Food Industry
- Testing for food additives, vitamins, and preservatives.
- Ensuring product safety and compliance.
In Forensics
- Identifying toxins, poisons, and drugs in biological samples.
Advantages of HPLC
- High speed and efficiency.
- Very accurate and reproducible results.
- Can handle complex mixtures.
- Applicable to a wide range of samples.
- Suitable for both qualitative and quantitative analysis.
Limitations of HPLC
- Expensive instruments and solvents.
- Requires skilled operators.
- Some compounds may not be detected (irreversible adsorption).
- Less effective for highly volatile substances (better analyzed by gas chromatography).
Frequently Asked Questions (FAQ)
Q1. What is the full form of HPLC?
High-Performance Liquid Chromatography.
Q2. What is the main principle of HPLC?
Separation of compounds based on their interactions with stationary and mobile phases under high pressure.
Q3. Which type of HPLC is most commonly used?
Reverse Phase HPLC (RP-HPLC).
Q4. What is a chromatogram in HPLC?
It is the graphical output showing peaks that represent separated compounds.
Q5. What is HPLC mainly used for in biology?
Purification and identification of biomolecules like proteins, nucleic acids, and metabolites.
References
- https://www.shodex.com/en/kouza/a.html
- https://www.alphacrom.com/en/hplc-basics
- https://laboratoryinfo.com/hplc/
- https://sciencing.com/disadvantages-advantages-hplc-5911530.html
- https://www.slideshare.net/krakeshguptha/hplc-26970638
- https://www.chemguide.co.uk/analysis/chromatography/hplc.html
- https://www.azom.com/article.aspx?ArticleID=8468
- https://www.ru.ac.za/media/rhodesuniversity/content/nanotechnology/documents/chromatography%20Augustus.pdf