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Chromatography

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ChromatographyChromatography (from Greek

chroma "color" and graphein "to write“ = to write colors) is the collective term for a set of laboratory techniques for the separation of mixtures

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COLUMN CHROMATOGRAPHY“CG”

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Principle of operation The components to be separated are distributed between

two phases: ◦ a stationary phase bed (Adsorptive materials)◦ a mobile phase which is filtered through the stationary bed

A mixture of various components enters a chromatography process, and the different components are flushed through the system at different rates.

These differential rates of migration as the mixture moves over adsorptive materials provide separation.

Repeated sorption/desorption acts that take place during the movement of the sample over the stationary bed determine the rates.

The smaller the affinity a molecule has for the stationary phase, the shorter the time spent in a column.

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Principle of operationThe process of a chromatographic

separation takes place within a chromatography column.

This column, made of glass or metal, is either a packed bed or open tubular column. ◦A packed bed column contains particles

which make up the stationary phase. ◦Open tubular columns are lined with a

thin film stationary phase. The center of the column is hollow.

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Principle of operation The stationary phase is usually a

viscous liquid or particles coated on the surface of solid particles which are packed into the column,

The mobile phase is typically a solvent moving through the column which carries the mixture to be separated.

This can either be a liquid or a gas, depending on the type of process.

In any case, the partitioning of solutes between the stationary and mobile phases lead to the desired separations.

Eluent

Eluate

Stationaryphase

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Schematic diagram of CG

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Basic operations in a CG

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1. Separation in the Column Stationary phaseAs the sample flows through the column, its

different components will adsorb to the stationary phase to varying degrees.

Those with strong attraction to the support move more slowly than those with weak attraction. (This is how the components are separated.)

The stationary phase or adsorbent in column chromatography is a solid. ◦ The most common stationary phase for column

chromatography is silica gel, followed by alumina.

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2. Elution (االستخراج ) from the ColumnMobile phaseAfter the sample is flushed or displaced from

the stationary phase, the different components will elute from the column at different times.

The components with the least affinity for the stationary phase (the most weakly adsorbed) will elute first, while those with the greatest affinity for the stationary phase (the most strongly adsorbed) will elute last.

The mobile phase or eluent is either a pure solvent or a mixture of different solvents

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3 .DetectionThe different components are

collected as they emerge from the column.

A detector analyzes the emerging stream by measuring a property which is related to concentration and characteristic of chemical composition.

For example, the refractive index or ultra-violet absorbence is measured

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Example (a) A continuous flow of solvent carries a solution of

solutes A and B down a column. (b) As the solvent carries the two solutes down the

column, we begin to see some separation of the solution.

(c) At some later point in time, it can be seen that solute B is moving at a much faster rate than A.

In (d), solute B emerges first, while solute A finally emerges in (e).

Thus, solute A has a greater affinity for the stationary phase than solute B.

By varying the pH of the solvent or temperature of the column, the output of the column can be significantly altered, such as the timing of when individual species emerge.

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CG steps

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CG steps

Detailed steps

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Manual column CG

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The output of a CGThe ChromatogramLet's consider the output from

the detector: the chromatogram.

Since the sample is separated in the column, different peaks on the chromatogram correspond to different components in the sample mixture.

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The ChromatogramX-axis: retention time ( االحتجاز (وقتY-axis: concentration

t

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What Information Can Be Attained?

Level of complexity of the sample is indicated by the number of peaks which appear.

Qualitative information about the sample composition is obtained by comparing peak positions with those of standards.

Quantitative assessment of the relative concentrations of components is obtained from peak area comparisons.

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Definitions A chromatograph is equipment that enables a

sophisticated separation e.g. gas chromatographic or liquid chromatographic separation.

Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction.

The eluate is the mobile phase leaving the column.

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Definitions The eluent is the solvent that will carry

the analyte.

An eluotropic series is a list of solvents ranked according to their eluting power.

An immobilized phase is a stationary phase which is immobilized on the support particles, or on the inner wall of the column tubing.

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Definitions The mobile phase is the phase which moves

in a definite direction. It may be a liquid, a gas (GC), or a supercritical fluid (SFC).

The mobile phase consists of the sample being separated/analyzed and the solvent that moves the sample through the column.

The mobile phase moves through the chromatography column (i.e., through the stationary phase) where the sample interacts with the stationary phase and is separated.

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Definitions Preparative chromatography is

used to purify sufficient quantities of a substance for further use, rather than analysis.

The retention time is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions

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Definitions The sample is the matter

analyzed in chromatography. It may consist of a single component or it may be a mixture of components.

The solute refers to the sample components in partition chromatography.

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Definitions The solvent refers to any

substance capable of solubilizing other substance, and especially the liquid mobile phase in LC.

The stationary phase is the substance which is fixed in place for the chromatography procedure. Examples include the silica layer in thin layer chromatography

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CG