Liquid ChromatographyName NPM

•Shadish Kumar 260110132002•Mohd Nazreen Taha 260110132015•Keshni Devi 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.

1.1. Ultraviolet DetectorUltraviolet Detector

200-400nm 200-400nm 254 nm254 nm

2.2. Reflective Index DetectorReflective Index Detector

Universal DetectorUniversal Detector

DetectorsDetectors

• 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 time2.peak symmetry3. 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.