High-Performance Liquid Chromatography (HPLC) is an advanced analytical technique used to separate, identify, and quantify components in a mixture. It is one of the most widely used methods in analytical chemistry, biochemistry, pharmaceuticals, and environmental science.
Basic Principle
HPLC works on the principle of separation based on the interactions between the sample components, the stationary phase (solid material inside the column), and the mobile phase (liquid solvent).
Different compounds move through the column at different rates depending on their:
- Polarity
- Molecular size
- Affinity to the stationary phase
This causes the components to separate and elute (come out) at different times, known as retention times.
Main Components of HPLC System
- Solvent Reservoirs (Mobile Phase):
Hold the liquid solvents used to carry the sample through the column. - Pump:
Forces the mobile phase through the column at high pressure (up to 400 atm). - Injector:
Introduces the sample mixture into the mobile phase stream. - Column (Stationary Phase):
The heart of HPLC where separation occurs. It is packed with fine particles (like silica) coated with various materials. - Detector:
Detects the separated compounds as they elute from the column (common types: UV-Vis, fluorescence, or refractive index detectors). - Data System (Computer/Recorder):
Records the detector signal to produce a chromatogram, showing peaks for each compound.
Types of HPLC
- Normal Phase HPLC:
- Stationary phase: Polar
- Mobile phase: Non-polar
- Used for separating polar compounds.
- Reverse Phase HPLC (RP-HPLC):
- Stationary phase: Non-polar (e.g., C18 column)
- Mobile phase: Polar (e.g., water, methanol)
- Most common type of HPLC used today.
- Ion Exchange HPLC:
- Separation based on ionic charge.
- Size Exclusion HPLC (Gel Filtration):
- Separation based on molecular size.
- Affinity HPLC:
- Separation based on specific binding interactions (e.g., antigen-antibody).
Applications of HPLC
- Pharmaceuticals: Drug purity testing and formulation analysis
- Food Industry: Detection of additives, preservatives, and contaminants
- Environmental Testing: Detection of pollutants and pesticides in water and soil
- Clinical Chemistry: Analysis of vitamins, hormones, and metabolites in biological samples
- Biotechnology: Protein and nucleic acid purification
Advantages
- High resolution and accuracy
- Fast and efficient separation
- Can handle complex mixtures
- Quantitative and qualitative analysis possible
Limitations
- Expensive instrumentation
- Requires trained operators
- Limited for volatile or thermally stable compounds (GC is better for those)
In short:
HPLC is a powerful, precise, and high-speed liquid chromatography technique used to separate, detect, and measure components in a mixture under high pressure for analytical and preparative purposes.