1. Introduction to Liquid Chromatography
What is Liquid Chromatography?
Liquid chromatography is a separation technique that involves:
- The placement (injection) of a small volume of liquid sample
- Into a tube packed with porous particles (stationary phase)
- Where individual components of the sample are transported along the packed tube (column) by a liquid moved by gravity
- The components are separated by various chemical and/or physical interactions between their molecules and the packing particles
- The separated components are collected at the column exit and identified by external measurement techniques
Note: The modern form of liquid chromatography is now referred to as "flash chromatography".
2. What is HPLC?
High Performance Liquid Chromatography
HPLC is an abbreviation for High Performance Liquid Chromatography (previously referred to as High Pressure LC).
Key difference from LC: In principle, LC and HPLC work the same way except the speed, efficiency, sensitivity and ease of operation of HPLC is vastly superior.
HPLC involves:
- Injection of a small volume of liquid sample
- Into a tube packed with tiny particles (3 to 5 μm diameter stationary phase)
- Components are moved down the column with a liquid (mobile phase) forced through by high pressure from a pump
- Separation occurs via chemical/physical interactions between molecules and packing particles
- Separated components are detected at column exit by a flow-through detector
- The detector output is called a "liquid chromatogram"
History of HPLC
- 1960s: Beginning as High Pressure Liquid Chromatography
- 1970s: Improvements in column material and instrumentation led to High Performance Liquid Chromatography
- 1980s: "Boom" in HPLC started
- Since 2006: New terms like UPLC, RRLC, UFLC, RSLC emerged
3. HPLC Components and Their Functions
Proper understanding of HPLC components is essential for effective operation and troubleshooting.
1. Pump
Forces the mobile phase through the chromatograph at a specific flow rate (mL/min).
- Normal flow rates: 1-2 mL/min
- Typical pressure range: 6000-9000 psi (400-600 bar)
- Can deliver constant (isocratic) or increasing (gradient) mobile phase composition
2. Injector
Introduces the liquid sample into the mobile phase flow stream.
- Typical sample volumes: 5-20 μL
- Must withstand high system pressures
- Autosamplers automate injection for multiple samples
3. Column
The "heart of the chromatograph" where separation occurs.
- Stationary phase separates sample components
- Small particles cause high backpressure
- Most critical component for successful HPLC
4. Detector
Detects individual molecules eluting from the column.
- Measures amount of molecules for quantitative analysis
- Provides output to recorder/computer (chromatogram)
- Various types: UV, RI, fluorescence, MS, etc.
5. Computer/Data System
Controls HPLC instrument modules and processes detector signals.
- Determines retention time (qualitative analysis)
- Determines sample amount (quantitative analysis)
- Records and analyzes chromatographic data
4. HPLC Applications
Primary Uses of HPLC
Separation and Analysis of Non-volatile or Thermally-unstable Compounds
HPLC is optimal for chemical and biological compounds that are non-volatile.
- Pharmaceuticals (aspirin, ibuprofen, acetaminophen)
- Salts (sodium chloride, potassium phosphate)
- Proteins (egg white, blood protein)
- Organic chemicals/polymers (polystyrene, polyethylene)
- Heavy hydrocarbons (asphalt, motor oil)
- Natural products (ginseng, herbal medicines, plant extracts)
- Thermally unstable compounds (TNT, enzymes)
Qualitative Analysis
Identification of individual compounds in a sample.
- Primary parameter: Retention time
- Additional identification: Chemical structure, molecular weight, other molecular parameters (depending on detector)
Quantitative Analysis
Measurement of compound amount/concentration in a sample.
Two main quantification methods:
- Peak height measurement from baseline
- Peak area determination
Calibration with known standards establishes relationship between peak height/area and sample amount.
Preparation of Pure Compounds (Preparative Chromatography)
Collecting chromatographic peaks and concentrating analytes by removing/evaporating solvent to obtain pure substances for:
- Organic synthesis
- Clinical studies
- Toxicology studies
Trace Analysis
Determination of compounds present at very low concentrations (90% of chromatographers)
6. HPLC Columns
Proper choice of column is critical for success in HPLC.
Column Types
- Analytical: i.d. 1.0-4.6 mm; lengths 15-250 mm
- Preparative: i.d. >4.6 mm; lengths 50-250 mm
- Capillary: i.d. 0.1-1.0 mm; various lengths
- Nano: i.d.
