Methods In Clin Path: Chromatography

Question 1

Chromatography refers to the group of techniques used to

Answer 1

separate complex mixtures on the basis of different physical interactions between the individual compounds and the stationary phase of the system.

Question 2

The basic components in any chromatographic technique are 4

Answer 2

the mobile phase (gas or liquid), which carries the complex mixture (sample);

the stationary phase (solid or liquid), through which the mobile phase flows;

the column holding the stationary phase;

and the separated components (eluate).

Question 3

Adsorption chromatography aka

Answer 3

Liquid- solid chromatography

Question 4

In adsorption chromatography?

Answer 4

It is based on the competition between the sample and the mobile phase for adsorptive sites on the solid stationary phase.
The molecules that are most soluble in the mobile phase, move fastest; the least soluble, move slowest.
Thus, a mixture is typically separated into classes according to polar functional groups.

Question 5

In adsorption, the stationary phase can be?

The mobile phase can be?

Answer 5

The stationary phase can be either acidic polar (e.g., silica gel), basic polar (e.g., alumina), or nonpolar (e.g., charcoal).
The mobile phase can be a single solvent or a mixture of two or more solvents, depending on the analytes to be desorbed.

Question 6

Partition chromatography aka

Answer 6

Liquid-liquid chromatography

Question 7

In partition chromatography?

Answer 7

Separation of solute is based on relative solubility in an organic (nonpolar) solvent and an aqueous (polar) solvent
Molecules containing polar and nonpolar groups in an aqueous solution are added to an immiscible organic solvent.
After vigorous shaking, the two phases are allowed to separate.
Polar molecules remain in the aqueous solvent; nonpolar molecules are extracted in the organic solvent

Question 8

Continuing

Answer 8

This results in the partitioning of the solute molecules into two separate phases.
The ratio of the concentration of the solute in the two liquids is known as the partition coefficient:

K= solute in stationary phase/ solute in mobile phase

Partition chromatography is applicable to any substance that may be distributed between two liquid phases.
Because ionic compounds are generally soluble only in water, partition chromatography works best with non-ionic compounds

Question 9

Steric exclusion, a variation of _______ chromatography

Answer 9

liquid-solid chromatography,

Question 10

In steric exclusion

Answer 10

It is used to separate solute molecules on the basis of size and shape.
The chromatographic column is packed with porous material.
A sample containing different-sized molecules moves down the column dissolved in the mobile solvent.
Small molecules enter the pores in the packing and are momentarily trapped.
Large molecules are excluded from the small pores and so move quickly between the particles.

Question 11

In ion exchange chromatography

Answer 11

In ion-exchange chromatography, solute mixtures are separated by virtue of the magnitude and charge of ionic species.
The stationary phase is a resin, consisting of large polymers of substituted benzene, silicates, or cellulose derivatives, with charged functional groups
The mobile phase may be made of cation exchange resins or anion exchange resins.
Ion-exchange chromatography is used to remove interfering substances from a solution, to concentrate dilute ion solutions, and to separate mixtures of charged molecules, such as amino acids.
Changing pH and ionic concentration of the mobile phase allows separation of mixtures of organic and inorganic ions.

Question 12

read thin layer and high performance abeg

Question 13

Thin layer chromatography is a variant of

Answer 13

column chromatography.

Question 14

What happens in TLC

Answer 14

A thin layer of sorbent, such as alumina, silica gel, cellulose, or cross-linked dextran, is uniformly coated on a glass or plastic plate.
Each sample to be analyzed is applied as a spot near one edge of the plate.
The mobile phase (solvent) is usually placed in a closed container until the atmosphere is saturated with solvent vapor.
One edge of the plate is placed in the solvent, as shown.
The solvent migrates up the thin layer by capillary action, dissolving and carrying sample molecules.
Separation can be achieved by any of the four processes previously described, depending on the sorbent (thin layer) and solvent chosen.
After the solvent reaches a predetermined height, the plate is removed and dried.
Sample components are identified by comparison with standards on the same plate.

Question 15

In TLC, what is Retention factor

Answer 15

The distance a component migrates, compared with the distance the solvent front moves, is called the retention factor, Rf:

Distance leading edge component moves/ total distance solvent front moves

Each sample-component Rf is compared with the Rf of standards.

Question 16

Example for RF

Answer 16

Using the figure above as an example, standard A has an Rf value of 0.4, standard B has an Rf value of 0.6, and standard C has a value of 0.8.
The first unknown contains A and C, because the Rf values are the same.
This ratio is valid only for separations run under identical conditions.
Because Rf values may overlap for some components, further identifying information is obtained by spraying different stains on the dried plate and comparing colours of the standards.
TLC is most commonly used as a semiquantitative screening test.

Question 17

In High-Performance liquid chromatography

Answer 17

High-Performance liquid chromatography uses pressure for fast separations, controlled temperature, in-line detectors, and gradient elution techniques.

Question 18

The basic components are

Answer 18

The basic components are: (1) Pumps: A pump forces the mobile phase through the column at a much greater velocity than that accomplished by gravity-flow columns.
(2) Columns: The stationary phase is packed into long stainless steel columns. Fine, uniform column packing results in sharper peaks but requires pressure to force the mobile phase through.

Question 19

More components

Answer 19

(3) Sample Injectors : A small syringe can be used to introduce the sample into the path of the mobile phase that carries it into the column. loop injectors can also be used and can be programmed for automatic injection of samples.
(4) Detectors: Modern HPLC detectors monitor the eluate as it leaves the column and, ideally, produce an electronic signal proportional to the concentration of each separated component.
Spectrophotometers that detect absorbances of visible or UV light can be used as detectors
Unknowns can be compared against library spectra

Question 20

5th component

Answer 20

(5) Recorders: The recorder is used to record detector signal versus the time the mobile phase passed through the instrument, starting from the time of sample injection.
The graph is called a chromatogram. The retention time is used to identify compounds when compared with standard retention times run under identical conditions.
Peak area is proportional to concentration of the compounds that produced the peaks.