HPLC is a new age technology and this presentation gives insight about it.It is used in agriculture,medical,pharmaceutical etc.for detection of various molecules of economic importance. Reference:-https://www.advancechemjournal.com/apdf/aac-aid1026.pdf

1. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
Submitted by:-Ashish Ajrawat
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INTRODUCTION :
• HPLC is an analytical technique used to separate, identify or quantify
each component in a mixture.
• The mixture is separated using the basic principle of column
chromatography and then identified and quantified by spectroscopy or
UV Detectors.

3. PRINCIPLE :
• It is the technique in which the components of a mixture are separated
based upon the rates at which they are carried or moved through a
stationary phase (column) by a gaseous or liquid mobile phase.
• The separation of a mixture into its components depends on different
degrees of retention of each component in the column.
• The extent to which a component is retained in the column is determined
by its partitioning between the liquid mobile phase and the stationary
phase.
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4. INSTRUMENTATION IN HPLC :
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• The Pump
• Injector
• Column
• Detector
• Recorder
• Column Heater

5. PUMP :
• The pump is positioned in the most upper stream of the liquid
chromatography system and generates a flow of eluent from the solvent
reservoir into the system.
• High-pressure generation is a “standard” requirement of pumps besides
which, it should also to be able to provide a consistent pressure at any
condition and a controllable and reproducible flow rate.
• Most pumps used in current LC systems generate the flow by back-and-
forth motion of a motor-driven piston (reciprocating pumps). Because of
this piston motion, it produces “pulses”.

6. INJECTOR :
• An injector is placed next to the pump.
• The injector can be a single injection or an automated injection system.
• An injector for an HPLC system should provide an injection of the liquid
sample within the range of 0.1-100 mL of volume with high
reproducibility and under high pressure (up to 4000 psi).
• The injector must also be able to withstand the high pressures of the
liquid system.

7. COLUMN :
• The separation is performed inside the column.
• Columns are usually made of polished stainless steel and are between
50 and 300 mm long, and have an internal diameter of between 2 and 5
mm.
• They are commonly filled with a stationary phase with a particle size of
3–10 μm. Columns with internal diameters of less than 2 mm are often
referred to as microbore columns.
• The temperature of the mobile phase and the column should be kept
constant during an analysis.

8. DETECTOR :
• The HPLC detector located at the end of the column detects the analytes
as they elute from the chromatographic column.
• The detector can see (detect) the individual molecules that come out
(elute) from the column.
• Commonly used detectors are evaporative light scattering, UV-
spectroscopy, fluorescence, mass-spectrometric and electrochemical
detectors.

9. ADVANTAGES :
• Separations are fast and efficient (high-resolution power).
• Continuous monitoring of the column effluent.
• It can be applied to the separation and analysis of very complex
mixtures.
• Accurate quantitative measurements.
• Repetitive and reproducible analysis using the same column.
• It provides a means for the determination of multiple components in a
single analysis and etc.

10. DISADVANTAGES :
• Very costive
• Low sensitivity for certain compounds
• Some cannot be detected as they are irreversibly adsorbed.

11. APPLICATIONS :
• For analysis of:-
• Fat-soluble vitamins (A, D, E, and K)
• Water-soluble vitamins (B-complex vitamins such as B1, B2, B3, B6, Folic acid, Pantothenic
acid, B12, Vitamin C)
• Residual pesticides such as 2, 4-D, and Monochrotophos.
• Antioxidants such as TBHQ, BHA, and BHT.
• Sugars: Glucose, Fructose, Maltose, and other saccharides
• Mycotoxins such as Aflatoxins B1, B2, G1, G2, M1, M2, and ochratoxin
• Amino acids
• Residual antibiotics,Steroids and flavonoids.
• Aspartame and other artificial sweeteners.

12. APPLICATIONS IN MEDICAL:
• Quantification of drugs in biological samples.
• Identification of steroids in blood, urine, etc.
• Determination of cocaine and other drugs of abuse in blood, urine, etc.
• Useful for diagnosing vitamin D deficiencies in children.
• Detection of molecules that are useful for clinical studies.
• Nutrient analysis.

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