Background Of Chromatography.docx

BACKGROUND OF CHROMATOGRAPHY Chromatography literally “color writing” was first employed by Russian scientist MIKHAIL TSVET in 1900. He continued to work with chromatography in the first decade of 20th century, primarily for the separation of plant pigment such as chlorophyll, carotenes and xanthophylls. Some important terms Mobile Phase: The solvent system which carried the mixture to be separated . Stationary Phase: Immobile surface which is particulate in nature. This is the region over which the component gets separated HPLC ( High Performance Liquid Chromatography) INTRODUCTION: HPLC is the form of liquid chromatography which is use to separate the compound that are dissolved in the solution. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out the column. INSTRUMENT AND WORKING HPLC instrument consist of a reservoir of mobile phase, a pump, an injector,a separation column and a detector. The sampler brings the sample mixture into the mobile phase stream which carries it into the column. The pumps deliver the desired flow and composition of the mobile phase through the column. The detector generates a signal proportional to the amount of sample component emerging from the column, hence allowing for quantitative analysis of the sample

components. A digital microprocessor and user software control the HPLC instrument and provide data analysis. Some models of mechanical pumps in a HPLC instrument can mix multiple solvents together in ratios changing in time, generating a composition gradient in the mobile phase. Since the technique is slow and time taking so it is improved by the use of pump which pressurized the liquid in the range of 5000-10000

Various type and applications of HPLC The various types of HPLC and their characteristics are summarized in the table below.

Table 1. Various Types and Applications of HPLC TYPE

Adsorption

SAMPLE POLARITY

MOLECULAR WEIGHT RANGE 100 - 104

STATIONARY PHASE

MOBILE PHASE

Partition (reversed-phase)

non-polar to some what polar non-polar to some what polar

silica or alumina

non-polar to polar

100 - 104

non-polar liquid adsorbed or chemically bonded to the packing material

relatively polar

Partition (normalphase)

Some what polar to highly polar

100 - 104

highly polar liquid adsorbed or chemically bonded to the packing material

relatively non-polar

Ion Exchange

highly polar to ionic

100 - 104

ion-exchange resins made of insoluble, high-molecular weight solids functionalized typically with sulfonic acid (cationic exchange) or amine (anionic exchange) groups

aqueous buffers with added organic solvents to moderate solvent strength

Size-Exclusion

non-polar to ionic

103 – 106

small, porous, silica or polymeric particles

polar to non-polar

USES OF HPLC: This technique is used for Chemistry and biochemistry research analyzing complex mixtures  Purifying chemical compounds  Developing processes for synthesizing chemical compound  Isolating natural products, or predicting physical properties It is also in quality control to ensure the purity of raw material