CHROMA P1

CHROMATOGRAPHY

Science of the study of separation of molecules based on differences in their structure and/or composition

CHROMATOGRAPHY

is a collective term for a family of laboratory techniques for the separation of mixtures

“mobile phase” through a “stationary phase” which separates the analyte to be measured from the other molecules in the mixture

explain chromatography principle

MOBILE PHASE

gas or liquid that passes through the column.

ETHANOL

CHLOROFORM

examples of mobile phase

STATIONARY PHASE

A solid or liquid that does not move

STATIONARY PHASE

chromatographic support

PREPARATIVE CHROMATOGRAPHY

seeks to separate the components of a mixture for further use and thus is a form of purification

ANALYTICAL CHROMATOGRAPHY

seeks to measure the relative proportions of analytes in mixtures

ANALYTICAL CHROMATOGRAPHY

small scale chromatography

PREPARATIVE CHROMATOGRAPHY

large scale chromatography

RATE THEORY

realistic theory of chromatography

RATE THEORY

describes the process of peak dispersion

RATE THEORY

provides an equation to calculate the variance per unit length of the column.

PLATE THEORY

describes the separation in a chromatographic technique in the form of hypothetical plates.

CAPILLARY ACTION to move the solvent through the stationary phase

rathe theory and plate theory exhbit the…

RETENTION SPEED

Measure of speed at which a substance moves in a chromatographic system.

RETENTION TIME

In continuous development systems (HPLC & GC), where the compounds are eluted with an eluent, it is expressed as…

RETENTION FACTOR

In interrupted development systems (TLC & PC), it is expressed as…

ADSORPTION

Retention and separation depends on the ability of the atoms on the surface to remove analytes from the mobile phase and ____ them temporarily by means of electrostatic forces

ELECTROSTATIC FORCES

Retention and separation depends on the ability of the atoms on the surface to remove analytes from the mobile phase and adsorb them temporarily by means of…

PARTITION COEFFICIENTS

Retention and separation occur due to the relative solubility of the analytes in the two fluids as determined by their…

ION-EXCHANGE

Retention and separation is mainly due to the electrostatic bonds with the functional groups

MOLECULAR EXCLUSION

aka size exclusion

MOLECULAR EXCLUSION

aka gel permeation

MOLECULAR EXCLUSION

aka gel filtration

MOLECULAR EXCLUSION

Retention and separation depends on the differential migration of solute molecules based on molecular size

ELECTROSTATIC BONDS WITH FUNCTIONAL GROUPS

o Retention and separation is mainly due to the…

COLUMN

PLANAR (PAPER AND THIN LAYER)

chromatography based on bed shapee

GAS

LIQUID (HPLC)

chromatography based on physical state of mobile phase

-bomb detection in airports

-identify/quantify drugs and alcohol

-forensics to compare fibers found on a victim

gas chroma application (B DA F)

test water samples for pollution

application of LC

separation of amino acids and anions

RNA fingerprinting

separation and testing of histamines and antibiotic

PC application (AA R HA)

detection of pesticides and insecticides in food

analyze dye composition of fibers in forensics

TLC application (PI DC)

COLUMN CHROMATOGRAPHY

A separation technique in which the stationary bed is within a tube

COLUMN CHROMATOGRAPHY

It is a method used to purify individual chemical compounds from mixtures of compounds

suction flask

cylindrical glass or quartz

chromatographic column: the simplest type is consists of

CHROMATOGRAPHIC COLUMN

The size of the column is determined by the quantity and adsorbability of the substance being separated.

quantity

adsorbability

The size of the chromatographic column is determined by...

silica gel

alumina

calcium carbonate

purified sileceous earth

cellulose powder

diatomaceous earth (celite)

cross-linked dextrans (SephadexLH20)

stationary phase examples (CCC SADP)

SephadexLH20

other term for cross-linked dextrans

celite

diatomaceous earth other term

MOBILE PHASE

either a pure solvent or a mixture of different solvents

ALCOHOLS AND AMIDES

PURIFIED WATER

mobile phase examples

dry powder or slurry suspended in mobile phase

The adsorbent is introduced into the column either as a

benzene

hydrophobic mobile phase

air bubbles

important to avoid in chromatography

COLORED SAMPLES

the adsorbent column is forced intact from the tube and the fractions are easily divided by a knife.

COLORLESS SAMPLES

visualization is done by first spraying or painting the extrude column with a color forming agent

RADIOACTIVE SAMPLES

their position in the column is determined using a Geiger-muller counter.

FLASH COLUMN CHROMATOGRAPHY

A modified version of column chromatography

William Clark Still in 1978

who introduced FC CHROMATOGRAPHY

FLASH COLUMN CHROMATOGRAPHY

It is very similar to the traditional column chromatography, except for that the solvent is driven through the column by applying positive pressure

PLANAR CHROMATOGRAPHY

A separation technique in which the stationary phase is present as or on a plane

paper

glass plate

plane can be…

paper

serving as such or impregnated by a substance as the stationary bed

glass plate

example of a layer of solid particles spread on support

PAPER CHROMATOGRAPHY

An analytical technique for separating and identifying mixtures that or can be colored, especially pigments

Archer John Porter Martin

Richard Laurence Millington Synge

invented PC

STATIONARY PHASE IN PC

It is consists of a sheet of paper with controlled texture and thickness usually made up of cellulose [filter paper]

Mixture of alcohol and water with added ammonia or acetic acid

Chloroform

Benzene

Cyclohexane

mobile phase in PC

VOLATILE SOLVENT

PC samples are applied as a solution in…

capillary pipetes of microliter syringe

Samples are applied to a strip of chromatography paper at origins using..

ascending chromatogram,

the spots are applied at 3-5 cm from the lower edge

descending chromatogram

the spots are applied at 7-9 cm from the upper edge.

radial chromatogram

the spots are applied on a circle with a radius of 1-3 cm

distance traveled solute over distance traveled solvent

Rf value formula

elution

cutting the spot and soaking the paper in an appropriate solvent

TLC

A widely employed technique similar to paper chromatography

TLC

It involves spotting of sample or a mixture of samples at one end of an adsorbent-coated glass plate followed by passage of a solvent through the adsorbent for the purpose of separating the components of a mixture

 Rapid

 Sensitive

 Excellent resolution

 Simplicity

 Cheap

advantages of TLC

TLC STATIONARY PHASE

consists of a thin layer of adsorbent material immobilized onto flat, inert carrier sheet

TLC Plates

made by mixing the adsorbent, such as silica gel with a small amount of inert binder like calcium sulfate [gypsum] and wa

silica gel + calcium sulfate (gypsum) and water

TLC plates are made by mixing

gypsum

calcium sulfate other term

TLC plate

The mixture is spread as thick slurry on an unreactive carrier sheet, usually glass, thick aluminum foil or plastic, and the resultant plate is then dried and activated by heating in an oven for thirty minutes at 110C.

chloroform

ethanol

benzene

common mobile phase TLC

TLC MOBILE PHASE

For mixtures of unknown composition, benzene or chloroform with 10% ethanol is the best solvent for exploratory runs

Rf values

Once the chromatogram has been developed, the spots must be made visible in order to determine the…

CHARRING

Involves the spraying of concentrated sulfuric acid and heating the plate

IODINE VAPOR

n this method, the chromatogram is placed in a closed container holding a few iodine crystals.

reversible

iodine vapor

reversible or irreversible

BROWN SPOTS

result of iodine vapor

charring

use of iodine vapor

examination under uv radiation

enum special detection methods

EXAMINATION UNDER UV RADIATION

Useful for compounds that fluoresce

EXAMINATION UNDER UV RADIATION

Two UV light sources are useful and commercially available

Amino acids

identified by ninhydrin

lipids

identified by rhodamine b

steroids

terpenoids

identified by antimony chloride

Universal visualizing agent for most organic substances

identified by sulfuric acid + heating

Hydrocarbons

identifed by potassium permanganate in H2SO4

Carbohydrates

identified by Anisaldehyde in sulfuric acid

Olefins

bromine vapor identifies

Analgesics

 Antipyretics

 Anti-inflammatory drugs

 Uricosuric drugs

 Caffeine and caffeine-containing drugs

drugs analyzed by tlc