HPLC is a separation technique where a liquid sample is injected into a column packed with adsorbent particles and separated by interactions with a mobile liquid phase. There are three main types of HPLC based on the stationary phase: adsorption chromatography using polar or non-polar stationary phases, ion-exchange chromatography using charged surfaces, and size exclusion chromatography using precisely sized pores. HPLC instruments include pumps to move the mobile phase through the column, injectors to introduce the sample, columns for separation, and detectors to analyze the separated components.

1. Applied Chemistry
High Pressure/Performance Liquid Chromatography
HPLC

2. HPLC Theory:
• HPLC is an acronym, related to the most widely used separation technique
that involves:
• The injection of a small volume of liquid sample into a metal tube packed
with tiny particles (3 to 5 μm) called the stationary phase.
• Individual components of the injected sample are forced through the
stationary phase with the mobile phase (a liquid) delivered by a pump.
• These components are separated from one another by the column packing
that involves various chemical and/or physical interactions between the
molecules and the packing particles.

3. TYPES OF HPLC:
Based on the chemical nature of the stationary phase, and on the
retention mechanism, HPLC can be divided into three types, which
cover almost 90% of all chromatographic applications

4. 1) Adsorption chromatography:
The stationary phase is an adsorbent, and the retention mechanism based
on repeated adsorption and desorption steps.
• Normal-phase chromatography: in which the stationary phase (or
adsorbent) is more polar than the mobile phase (or eluent). For example,
the adsorbent can be polar silica-gel and the solvent can be n-hexane or
diethyl-ether.
• Reverse-phase chromatography: in which the stationary phase is nonpolar
or weakly polar and the solvent is more polar (just the opposite as in
normal-phase chromatography). Usually, the reversed stationary phases
are chemically modified silica-gel, which covalently bind alkyl chains.
Mobile phases are usually polar mixture of an organic solvent (acetonitrile,
methanol, 2-propanol, etc.) and water.

5. 2) Ion-exchange chromatography :
The stationary phase has a charged surface with
opposite charges on it compared to the sample ions.
This technique is used almost only for ionic or ionizable
samples. The mobile phase is an aqueous buffer with
controlled pH and ionic strength.

6. 3) Size exclusion chromatography:
The stationary phase is made of a material with
precisely controlled pore size. The large molecules
rapidly pass through the column, while the smaller
ones penetrate into the pores and washed out later by
the mobile phase.

7. HPLC Instrument:

8. HPLC Instruments:
HPLC involves the following instrumentation:
• Flasks For HPLC
• PUMPS
• INJECTORS
• COLUMNS
• DETECTORS

9. LC Columns:

10. LC Injectors:

11. HPLC sample Chromatogram:

12. Applications:
• Pharmaceutical Applications:
• To control, drug stability
• Tablet dissolution study of pharmaceutical dosages form
• Pharmaceutical quality control
• Environmental Applications:
• Detection of phenolic compounds in drinking water
• Bio-monitoring of pollutants
• Forensic Applications:
• Quantification of drugs in biological samples
• Identification of steroids in blood and urine etc..
• Forensic analysis of textile dyes
• Determination of cocaine and other drugs of abuse in blood and urine
• Food Applications:
• Measurement of quality of soft drinks and water
• Sugar analysis in fruit juices
• Preservatives analysis
• Clinical Tests:
• Urine analysis, Anti-biotic analysis, Analysis of bilirubin,