INTRODUCTION TO CHROMATOGRAPHY

PHRM 312: PHARMACEUTICAL ANALYSIS- III

SIMPLE METHODS OF SEPARATION

Evaporation

Precipitation
Crystallization Filtration Membrane separation Distillation

Sublimation
Extraction

SIMPLE METHODS OF SEPARATION (CONT)

Evaporation: Evaporation simply entails vaporizing the solvent by using heat or by utilizing air currents in a manner that the material concentrates to a solid state. Precipitation: Changing the concentration of a solute in a solution so that it exceeds solubility in a given solvent can bring about precipitation of the solute. - Solvent Precipitation - Precipitation via Chemical Reaction - Precipitation by Adjustment of pH

SIMPLE METHODS OF SEPARATION (CONT)

Crystallization: This involves concentrating a solution containing the component of interest by heating it and then allowing it to stand (i.e., cooling it) until the crystals are obtained from the solution. Filtration: Separation of particles that are visible to the naked eye with a filter paper is called filtration; however, filtration of submicron particle size is also possible. - Simple filter papers are made from cellulose and exhibit particle retention levels down to 2.5 m (e.g., Whatman Grade 5 filter paper)

SIMPLE METHODS OF SEPARATION (CONT)

- Membrane filters made from various materials can offer various pore sizes that allow separations down to 0.02 m level. Membrane separation

SIMPLE METHODS OF SEPARATION (CONT)

Dialysis A dialyzer is an apparatus in which one or more solutes are transported from one fluid to another through a membrane under a concentration driving force. Electrodialysis In electrodialysis, electrically charged membranes are used to separate components of an ionic solution under the driving force of an electric current. This process has been used for desalination of water, recovery of salt from seawater, deashing of sugar solutions, and deacidification of citrus juices.

SIMPLE METHODS OF SEPARATION (CONT)

Distillation Distillation is a method of separating mixtures based on differences in their volatilities in a boiling liquid mixture. - Origin of distillation can be related to evaporation. - In distillation, the components of interest are volatile, whereas in evaporation volatile components are separated from nonvolatile ones. - Generally liquids. Extraction - relatively simple type of separation process - a solute is distributed between two immiscible solvents.

WHAT IS CHROMATOGRAPHY?

Greek word:

Chroma (colour)

Graphein (to write).

Chromatography provides a way to identify unknown compounds and separate mixtures

HISTORY

The Russian botanist Mikhail Tsvet coined the term chromatography in 1906 to describe his experiments in separating different colored constituents of leaves by passing an extract of the leaves through a column

DEFINITION
Chromatography

is a physical method of

separation in which components to be separated are distributed between two phases

one of which does not move [(stationary

phase)S.P]

the other that moves through S.P in a definite direction (mobile phase).

CHROMATOGRAPHY
Separates components in mixture: Based on - polarity - boiling point - ionic strength - size

CHROMATOGRAPHY
Mobile

phase: phase in which sample is dissolved. It may be gas, liquid, or supercritical fluid Stationary phase: phase in which mobile phase is forced through it
Mobile

and stationary phases are chosen in a manner so that analyte will distribute itself between the two phases

TERMINOLOGY

The analyte is the substance to be separated during chromatography. The sample is the matter analyzed in chromatography. It may consist of a single component or it may be a mixture of components. When the sample is treated in the course of an analysis, the phase or the phases containing the analytes of interest is/are referred to as the sample whereas everything out of interest separated from the sample before or in the course of the analysis is referred to as waste.

TERMINOLOGY

The eluate is the mobile phase leaving the column. The eluent is the solvent that carries the analyte.

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

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.

TERMINOLOGY

A chromatograph is equipment that enables a sophisticated separation, e.g. gas chromatographic or liquid chromatographic separation. The detector refers to the instrument used for qualitative and quantitative detection of analytes after separation.

A chromatogram is the visual output of the chromatograph.

CHROMATOGRAM
Detector signal (conc. of Sample) vs. retention time or volume of mobile phase

Detector Signal

time or volume

Chromatograms
If a detector that responds solute concentration is placed at the end of the column and its signal is plotted as function of time (or of volume of the added mobile phase), a series of peaks is obtained.

Concentration of sample Vs Time (Volume of mobile phase)

Such a plot is called a chromatogram, useful for both qualitative and quantitative analysis. - positions of peaks on the time axis may serve to identify the components of the sample - areas under the peaks provide a quantitative measure of the amount of each component.

TYPES OF CHROMATOGRAPHY
According

to purpose

1. Analytical chromatography: It is used to determine the existence and possibly also the concentration of analyte(s) in a sample. 2. Preparative chromatography: It is used to purify sufficient quantities of a substance for further use, rather than analysis.

TYPES OF CHROMATOGRAPHY
In

general: 1. Column chromatography 2. Paper chromatography 3. Thin-layer chromatography (TLC) 4. Gas chromatography (GC) 5. High pressure / High performance liquid chromatography (HPLC) 6. Ion exchange chromatography 7. Gel filtration chromatography 8. Affinity chromatography 9. Super critical fluid chromatography

TYPES OF CHROMATOGRAPHY

Thin layer Paper

HPLC

Gas

Column

TYPES OF CHROMATOGRAPHY
On

the basis of stationary and mobile phase.

- liquid-solid chromatography
- liquid-liquid chromatography

- gas-solid chromatography
- gas-liquid chromatography

TYPES OF CHROMATOGRAPHY
On

the basis of type of equilibrium process:

1. Adsorption chromatography
2. Partition chromatography

3. Ion Exchange chromatography

4. Molecular/size exclusion chromatography

5. Affinity chromatography
6. Gel Electrophoresis

ADSORPTION CHROMATOGRAPHY
The

stationary phase is solid on which the sample components are adsorbed. The mobile phase may be a - liquid (liquid-solid chromatography) or, - gas (gas-solid chromatography) The components distribute between the two phases through the combination of - adsorption & - desorption e.g. Column chromatography, TLC

PARTITION CHROMATOGRAPHY
The stationary phase is a liquid supported on an inert solid The mobile phase is a - liquid (liquid-liquid partition chromatography) or, - gas ( gas-liquid partition chromatography)

Paper chromatography is a type of partition chromatography in which the stationary phase is a layer of solvent adsorbed on a sheet of paper.

ION EXCHANGE CHROMATOGRAPHY

An ion exchange resin is used as the stationary phase. Separation of either cations or anions Separtion based on relative strength of ionic bond Mobile phases used is always liquid (liquid chromatography)

MOLECULAR/SIZE EXCLUSION CHROMATOGRAPHY

Separation based on size Stationary phase is polymeric substance containing numerous pores of molecular dimension.

Larger molecules that will not fit into the pores remain in the mobile phase and are not retained.
Small molecules get trapped in pores & take longer to get out

AFFINITY CHROMATOGRAPHY
A method of separating biochemical mixtures Based on a highly specific biological interaction such as that between - antigen and antibody, - enzyme and substrate, or - receptor and ligand. Stationary phase is typically a gel matrix, often of agarose.

GEL ELECTROPHORESIS
a separation technique in which the flow of the mobile phase or buffer is driven through a chromatographic column by an electric field, rather than by an applied pressure Separation based on size and charge Smaller molecules will migrate further, less tangled

APPLICATIONS OF CHROMATOGRAPHY

Forensics

Research

Pharmaceutical industry

APPLICATION OF CHROMATOGRAPHY
It

is a technique for

separating mixtures of compounds identifying unknown compounds establishing the purity or concentration of compounds monitoring product formation in the pharmaceutical and biotechnology industries.
It

is widely used by forensic teams to analyse blood and urine samples for drugs.

TYPES OF CHROMATOGRAPHY (CONT.)

Classification

on the basis of the sample introduction and migration through the chromatographic bed: 1. Frontal Analysis

2. Displacement Analysis
3. Elution Analysis

FRONTAL ANALYSIS
Sample

is introduced into the bed continuously,

rather than in small portions and sample itself

constitutes the mobile phase.

The

sample components are selectively retarded;

and fronts, rather than bands, are formed as a result of the separation.
The

less retained component emerges from the

column first, followed by the mixture of the first

component plus the next most strongly retained component.

FRONTAL ANALYSIS (CONT.)

Frontal

analysis

cannot accomplish complete recovery of

the pure sample;

however, it is useful for concentrating trace impurities and for purifying large

volumes of liquids

DISPLACEMENT ANALYSIS
Sample mixture, dissolve in a small volume of solvent
C O L U M N

Mobile phase, containing a displacing agent

The displacement agent is a substance that is retained more strongly by the stationary phase than any of the components in the sample mixture and therefore forces them off the surface of the stationary phase into the mobile phase.

DISPLACEMENT ANALYSIS (CONT.)

As each of the displaced solute pass through the column in the mobile phase, it in turn acts as a displacing agent

for less strongly retained compounds.

The final result is that, the compound that is least firmly bound is eluted first, followed in order by those more tightly bound and finally by the displacing agent.

ELUTION ANALYSIS
Elution analysis is carried out by introducing the sample in as small volume as possible onto the head of the column. The mobile phase is then allowed to flow through the system. The components with larger partition coefficients will be retarded and will elute later. Compounds can be isolated in a relatively pure state. - Isocratic elution - Gradient elution

STATIONARY PHASES
Alumina: Aluminum oxide - Surface is highly polar - Capable of adsorbing practically all polar molecules. - Judicious selection of the eluent allows to separate any pairs of solute on alumina column. - Adsorbent power of alumina is markedly affected by its water content, with freshly dried alumina having the highest adsorbent activity. - Adsorbent power of alumina is specified by its ability to retard the migration of certain dyestuffs through a column prepared with the alumina. Grades I-V

STATIONARY PHASES

Silicic acid or silica gel, SiO2 - can be activated by heating or prewashing the bed with an anhydrous solvent to remove adosorbed water.

Some others stationary phase: 1. Fullers earth 5. Potassium carbonate 2. Activated charcoal 6. Talc 3. Magnesium oxide 7. Starch 4. Calcium carbonate