This document discusses high performance liquid chromatography (HPLC) and its applications in forensic science. It begins with introducing HPLC and its principles, which involve separating components in a mixture using a stationary and mobile phase under high pressure. It then describes HPLC instrumentation, methodology, types based on separation mode (e.g. normal phase, reverse phase), columns, and forensic applications such as identifying drugs, dyes, and metabolites in samples. Specific examples given are using HPLC to estimate the age of a human bloodstain and matching a lipstick smear from a crime scene to a specific brand.

1. AND THEIR APPLICATION IN FORENSIC SCIENCE
SAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE ,
TECHNOLOGY & SCIENCES
NAME : Pallavi kumari
STANDARD: Msc.FORENSIC SCIENCE

2. CONTENT
 INTRODUCTION
 PRINCIPLE
 INSTRUMENTATION
 FLOW SCHEME FOR HPLC
 METHODOLOGIES IN HPLC
 TYPES OF HPLC
 COLUMNS OF HPLC
 FORENSIC APPLICATION OF HPLC

3. INTRODUCTION
 HPLC stands for “High performance liquid
chromatography”or “High pressure liquid chromatography
 HPLC is an extention of conventional liquid chromatography.
 Developed in 1970s.
 The sample is forced through a column that is packed with a
stationary phase composed of irregularly or specially shaped
particles, a porous membrane by a liquid (liquid phase) at high
pressure.

4. CHROMATOGRAPHY
• Chromatography is a technique which separates components
in a mixture due to the differing time taken for each
component to travel through a stationary phase when carried
through it by a mobile phase.

5. PRINCIPLES OF HPLC
 The main principle of separation is adsorption.
 Separation of components with high performance parameters
under high pressure takes place between two phase- a stationary
phase and mobile phase.
 Stationary phase: The substance on which adsorption of the
analyte (substance to be separated) takes place.
 Mobile phase : Solvent which carries the analyte(a liquid or gas).
analyte adsorbed on the surface of
stationary phase

6. INSTRUMENTATION
 Pump
 In-line filters
 Injector
 Column
-Guard column
-HPLC/Main column
 Detector
 Recorder or data system

7. A Flow Scheme for HPLC
PUMP TO
PRODUCE SIGNAL TO PROCESSER
HIGH PRESSURES
SOLVENT
RESERVOIR
SAMPLE
INJECTION
HPLC TUBE
DETECTOR
PROCESSING
AND DISPLAY
WASTE

8. METHODOLOGIES IN HPLC

9. TYPES OF HPLC
 BASED ON MODE OF SEPARATION
1.Normal phase chromatography: stationary phase is polar
(hydrophilic)and mobile phase is non-polar(hydrophobic).
2.Reverse phase chromatography: stationary phase is non-
polar(hydrophobic) and mobile phase is polar(hydrophilic).
This chromatography is more commonly used in drugs are usually
hydrophilic.
sample
sample

10. TYPES OF HPLC
3.Size exclusion chromatography: Size exclusion
chromatography (SEC), also called as gel permeation
chromatography or gel filtration chromatography
mainly separates particles on the basis of size.
4.Ion exchange chromatography: In Ion exchange
chromatography, retention is based on the attraction
between solute ions and charged sites bound to the
stationary phase. Ions of the same charge are excluded.

11. COLUMNS OF HPLC

12. FORENSIC APPLICATION OF HPLC
 HPLC is used to detect trace impurities in drugs which
can be matched to a specific method of
manufacturing.
 Samples or evidences, such as cosmetics,biological
specimen,microbiology,drugs,industrial chemical,
forensic sample ,food or any type of mixture of
chemicals can be analysed in HPLC.
 Identification of anabolic steroids in serum, urine,
sweat, and hair.
 Forensic analysis of textile dyes.
 Determination of cocaine and metabolites in blood.

13. For instance:
a. Estimation of age
human bloodstain
b.Tying lipsticks smear from
Crime scene to specific brand