Liquid Chromatography

Name
Shadish Kumar
Mohd Nazreen Taha
Keshni Devi

NPM
260110132002
260110132015
260110132026

Chromatography
A technique for the separation of a mixture
by passing it in solution or suspension
through a medium in which the
components move at different rates.
Consist of mobile phase and stationary
phase

Liquid chromatography
Liquid chromatography is a technique
used to separate a sample into its
individual parts. This separation occurs
based on the interactions of the sample
with the mobile and stationary phases.

Types of Liquid Chromatography

1. Liquid/Solid Chromatography (adsorption chromatography)
A.

Normal Phase LSC

B.

Reverse Phase LSC

2. Liquid/Liquid Chromatography (partition chromatography)

A.

Normal Phase LLC

B.

Reverse Phase LLC

3. Ion Exchange Chromatography

4. Gel Permeation Chromatography (exclusion chromatography)

Mobile Phase and Stationary Phase

Mobile Phase: Liquid
Stationary Phase
Separation Mechanism
- Solid
Adsorption
- Liquid Layer
Partition
- Ion exchange resin
Ion exchange
- Microporous beads
Size Exclusion
- Chemically modified resin Affinity

Mobile Phase
Polar Solvents
Water > Methanol > Acetonitrile > Ethanol >
Oxydipropionitrile
Non-polar Solvents
N-Decane > N-Hexane > N-Pentane >
Cyclohexane

Instrumentasi HPLC
Terdiri daripada wadah fase gerak pompa,
alat memasukan sampel, (tempat injeksi),
kolom, detektor, wadah penampung
buangan fase gerak dan komputer atau
interogator.

HPLC system

Uses of HPLC

This technique is used for chemistry and biochemistry

research analyzing complex mixtures, purifying chemical
compounds, developing processes for synthesizing chemical
compounds, isolating natural products, or predicting physical
properties. It is also used in quality control to ensure the purity
of raw materials, to control and improve process yields, to
quantify assays of final products, or to evaluate product
stability and monitor degradation.

In addition, it is used for analyzing air and water pollutants, for

monitoring materials that may jeopardize occupational safety
or health, and for monitoring pesticide levels in the
environment. Federal and state regulatory agencies use HPLC
to survey food and drug products, for identifying confiscated
narcotics or to check for adherence to label claims.

Detectors

1.

Ultraviolet Detector
200-400nm
254 nm

2.

Reflective Index Detector

HPLC UV detectors are used with high performance liquid

chromatography to detect and identify analytes in a given sample.
These detector use light to analyze samples. The analyte may be
identified by measuring the sample's absorption of light at different
wavelengths. There are two types of HPLC UV detectors viz: Single
and variable wavelength detectors. Single wavelength detectors
measure the samples absorption of a single wavelength, while
variable wavelength detectors measure absorption of multiple
wavelengths and are therefore more sensitive. WHILE
HPLC Refractive Index Detectors are also used with high-pressure
liquid chromatography when detecting substances with limited or no
UV absorption. These chemical components included alcohols,
sugars, fatty acids, polymers and carbohydrates. A limitation of
these detectors may be their lack in sensitivity and they are
temperature dependent. Refractive Index Detectors can be simple
and effective way to detect analytes that do not absorb UV light.

HPLC Parameters
The most important column parameters for
evaluation are
1. retention time
2. peak symmetry
3. theoretical plates.
Retention time to prove the reproducability of
measurements. Peak symmetry to estimate how
good the column bed is packed. A peak
symmetry value of 1.0 is optimal. Theoretical
plate numbers to determine the effectiveness of
the HPLC column.