High performance liquid chromatography simplified for better understanding

2. Introduction
• HPLC is an abbreviation for High Performance Liquid Chromatography.
"Chromatography" is a technique for separation, "chromatogram" is the result
of chromatography, and "chromatograph" is the instrument used to conduct
chromatography.
• The purpose of high performance liquid chromatography (HPLC) analysis of
any drugs is to confirm the identity of a drug and provide quantitative results
and also to monitor the progress of the therapy of a disease.
• Its earlier name was High Pressure Liquid Chromatography because it involved
use of liquid mobile phase requiring higher pressures than gases used in Gas
Chromatography.
• HPLC columns are mostly four main types- Normal phase, Reverse Phase, and
Ion Exchange columns. In the pharmaceutical industry mostly reverse
columns are used for analysis.
• Used in analysis of sugar, vitamins and amino acids.

3. • Standard silica-based phases are stable within a pH range from 2-8.
Columns with special modifications or endcapping can be operated in an
enlarged pH range.
• Standard columns for reversed phase, normal phase, and ion exchange
chromatography typically range from 3.9 to 4.6 mm internal diameter and
15, 25, and 30 cm in length. Standard columns for gel permeation
chromatography (GPC) are 7.8 mm by 30 cm.
• HPLC consists of a variety of components, including a solvent delivery
pump, a degassing unit, a sample injector, a column oven, a detector, and a
data processor.
• HPLC can be used to analyze any compound with solubility in a liquid that
can be used as the mobile phase.
• Separation can be achieved by utilizing principles of partition , ion
exchange , size exclusion and affinity chromatography.

5. Components of HPLC system
• The main components of HPLC system are :
- Pump
- Injector
- Column
- Detector
- Recorder

6. 1. Pump
• Deliver mobile phase, flow rate- 1ml/ min
• Two types- constant pressure and constant volume
• Constant pressure pump
- Reciprocative or syringe type
- Pumping connecting lines are made of stainless steel
- Withstand high pressure and corrosion resistant (organic solvents-
bases, acids and halides)
- Always rinsed thoroughly with water

7. Reciprocative pump Syringe type pump

8. 2. Injector
• Place sample into the flowing mobile phase
• loop type injection valves used
• Syringe used to inject sample b/w two ports multiporated valve
• Mobile phase directed through loop carrying sample on to head of
column
• 10to 100 microlitre sample
• Auto samplers may be used
• Help reduce labor cost
• Sample stability is key factor in HPLC

9. 3. Column
• constructed of stainless steel tubing , terminals to
attach to detectors and injectors
• Column made from glass, fused silica, titanium and
PEEK(poly ether ether ketone)
• Smaller guard columns used to protect analytical
column, b/w injector and analytical column
• Packing material identical to hplc column
• Larger packing materials used for ease of repacking
10-25cm
4.6mm
Pore size- 5 to
10micro m

10. 4. HPLC column
• Packing material- chromatographic bed and may or may not be involved in
actual separation process
• Column packing material act as both support and stationary phase
• 3 types of column packing- silica based, pellicular packing and polymeric
column packing
• Requirements for HPLC column
- Availability in well defined particle size
- Narrow particle size distribution
- Sufficient mechanical strength- withstand pressure during packing and use
- Good chemical stability

11. 5. Detector
• Translates concentration changes into electrical signals
• Choice of detector depends on solute type and concentration and linear
sensitivity , detector sensitivity and compatibility with solvent and elution
mode used.
• Most commonly used detectors include
- UV Visible Detectors
- Fluorescence detectors
- Refractive index detector
- Amperometric detectors
- Mass spectrometers (LCMS)

12. a. UV Visible Detectors
• Measures absorption of radiation in wavelength
range 190 to 800 nm based on Beer’s law
• Variants also measured by detectors with fixed
wavelengths , variable wavelength and
photodiode array
• Sensitivity directly proportional to wavelength
• Does not respond to non-uv absorbing
compounds
• Selective detector

13. b. Fluorescence detector
• Greater sensitivity and
selectivity than UV Visible
detector
• Sensitivity depends on
fluorescence property of
components in the elute
• Similar in design to
fluorometers and Spectro
fluorometers

14. c. Refractive Index Detector
• Measure change in refractive index of
mobile phase due to solutes
• Used for non UV absorbing analytes
such as carbohydrates and lipids
• RI detectors are sensitive to change in
temperature and flow rates , hence
can’t be used for gradient elution

15. d. Amperometric Detector
• Measure change in
current as the analyte is
oxidized or reduced by
application of volatage
across electrodes of flow
cell
• Pulsed amperometric
detection (PAD), is used in
the HPLC analysis of
carbohydrates and related
substances. At a high pH
carbohydrates can be
oxidized and analyzed
with high sensitivity.

16. e. Conductivity detector
• Respond to presence of ions
eluting from column
• Used in ion chromatography

17. f. Mass Spectrometer (LCMS)
• Mass spectrometry (MS) ionizes atoms or molecules to facilitate their
separation and detection in accordance with their molecular masses and
charges (mass to charge ratio). MS is used in various applications, e.g.,
biochemicals and atomic physics
• The combined technique between MS and HPLC is commonly known as LC-
MS. Combining the two analytical methods reduces experimental error and
improves accuracy. The application of LC-MS is very useful in situations that
involve a huge number of compounds, such as environmental effluents.
• LC-MS involves separating mixtures in accordance with their physical and
chemical properties, then identifying the components within each peak and
detecting based on their mass spectrum. The flow rates used in LC-MS
should be less than those used for HPLC. This is to ensure complete
ionization and to maintain the detection sensitivity of the MS, which starts to
decrease beyond 200 µL/min. Therefore, the column in LC-MS is much
smaller to accommodate the smaller solvent flow rates and sample volumes.

19. 6. Recorder / Data System
• Results of chromatographic
separation are displayed on a
strip chart recorder
• From the chart- retention, peak
height or area under curve are
obtained
• These data help in quantitative
identification sample
• Computer controlled systems
and electronic integrators may
be used for additional data
handling capacity

20. APPLICATIONS OF HPLC
• The HPLC has developed into a universally applicable method so that it finds its use
in almost all areas of chemistry, biochemistry, and pharmacy.
• Analysis of drugs
• Analysis of synthetic polymers
• Analysis of pollutants in environmental analytics
• Determination of drugs in biological matrices
• Isolation of valuable products
• Product purity and quality control of industrial products and fine chemicals
• Separation and purification of biopolymers such as enzymes or nucleic acids
• Water purification
• Pre-concentration of trace components
• Ligand-exchange chromatography
• Ion-exchange chromatography of proteins
• High-pH anion-exchange chromatography of carbohydrates and oligosaccharides

21. ADVANTAGES
• Speed
• Efficiency
• Accuracy
• Versatile and extremely precise when it comes to identifying and
quantifying chemical components.
LIMITATIONS
• Cost: Despite its advantages, HPLC can be costly, requiring large quantities
of expensive organics.
• Complexity
• HPLC does have low sensitivity for certain compounds, and some cannot
be detected as they are irreversibly adsorbed.
• Volatile substances are better separated by gas chromatography