CHROMATOGRAPHY

DEFINITION:

the separation of individual compounds with in a complex mixture by partitioning between a stationary phase , and a mobile phase.

CHROMATOGRAPHY TECHNIQUES

GAS CHROMATOGRAPHY
GC is used used as an analytical tool .

It is used to separate volatile components of

mixture . In this the stationary phase is a high boiling liquid and the mobile phase is an inert gas.

TYPES OF GAS CHROMATOGRAPHY

1. Gas Solid Chromatography

2. Gas Liquid chromatography

INSTRUMENTAL COMPONENTS

CARRIER GAS
The most important requirements of carrier gas: It should be inert. It should be available at low cost. It should allow the detector to respond in an adequate manner.

SAMPLE INJECTION PORT

COLUMNS: TWO TYPES COLUMNS

DETECTORS
Types Of Detectors FID TCD NPD ECD

FACTORS EFFECTS OF GC
Volatility of compound

Polarity of compound
Flow rate of the gas

APPLICATIONS
Analysis of purity of organic compound

Analysis of pesticide
Biochemical and clinical field In cosmetics and perfumes field In plastic industry

GAS CHROMATOGRAPHY
It is an analytical tool

It is used to separate volatile components of a

mixture. In this stationary phase is a high-boiling liquid &the mobile phase is an inert gas

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

INTRODUCTION:HPLC
HPLC is a form of liquid chromatography used

to separate compounds that are dissolved in solution. HPLC instrument consists of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector. Compounds are separated by injecting a sample mixture on the column. The different component in the mixture pass through the column at differentiates due to differences in their partition behavior between the mobile phase and the stationary phase. The mobile phase must be degassed to eliminate the formation of air bubbles.

FLOW CHAT

INSTRUMENTATION

MOBILE PHASE
The mobile phase in HLPC refers to the solvent

being continuously applied to the column or stationary phase.

STATIONARY PHASE
The stationary phase in HPLC refers to the solid support contained within the column over which the mobile phase continuously flow

INJECTORS FOR HPLC

Samples are injected into HPLC via injection port

Injection port consist of a injection valve and

sample loop The sample dissolved in mobile phase before injection The sample drawn into syringe and injected into loop via injector valve Injection of sample range 10ul to over 500ul In modern HPLC sample injection is typically automatic

HPLC COLUMN

TYPES OF DETECTORS
Absorbance

(UV with filters, UV with monochromators) IR Absorbance Fluorescence Refractive-index Evaporative Light Scattering Detector (ELSD) Electrochemical Mass Spectrometric Photo-diode Array

THE COLUMN AND THE SOLVENT

They are two varients in use in HPLC depending

on the relative polarity of the solvent and the stationary phase 1. NORMAI PHASE HPLC 2.REVERSED PHASE HPLC

NORMAL PHASE
In this column type, the retention is governed by

the interaction of the polar parts of the stationary phase and solute. For retention to occur in normal phase, the packing must be more polar than the mobile phase with respect to the sample.

Reverse phase
In this column the packing material is relatively

nonpolar and the sample. Retention is the result of the interaction of the nonpolar stationary phase. Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons (such as C18, C8, etc) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water.

ISOCRATIC AND GRADIENT ELUTIONS

A Separation in which the mobile phase

composition remain constant throughout is termed isocratic The mobile phase composition does not have to remain constant is termed Gradient elution

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 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

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