Lecture 2a

by

Sherif S. Ebada, Ph.D.

Instrum. Pharm. Analys.

Contact information:

Assoc. Prof. Dr. Sherif S. Ebada

Pharmaceutical Chemistry Dept.

Office hours:

Mondays and Tuesdays: 12:00–14:00 pm

E-mail: ss_ebada@mutah.edu.jo

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Course outline

Introduction.

Adsorption (CC & TLC), Flash CC,

Chromatotron etc.

Partition (Paper).

Gel Chromatography.

Ion exchange Chromatography.

Affinity Chromatography.

Electrophoresis.

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Chromatography

Lecture 2a

Adsorption

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Development Process

Elution development

This technique is widely used in the various chromatographic methods

e.g. GSC, GLC, LLC, LSC and HPLC

Mechanism

"In elution techniques a small sample mixture is introduced onto the

column and eluted with a mobile phase having a lesser affinity for the

stationary phase than the sample components".

Therefore; components move along at a rate determined by their

relative affinity for the stationary phase but at slower rate than the

eluent.

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Silica gel (Normal Phase) C18 (Reversed Phase)

Polar Stationary phase Non-polar

Si-OH Si-(C18)

Non-polar mobile phase Polar mobile phase

e.g. Chloroform, methylene chloride e.g. water, methanol,

methanol, hexane, ethyl acetate, acetonitrile.

petroleum ether.

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Adsorption Chromatography

Isocratic elution chromatography

In simple elution chromatography, the column is eluted with the same

mobile phase solvent all the time (i.e. isocratic).

It is most suitable when the components have similar affinities for the

stationary phase.

Stepwise elution

It is carried out by changing the eluent after a predetermined period of

time.

The eluents are chosen to have increasing eluting power that is, increasing

affinity of the mobile phase for the components remaining on the column

and therefore decreasing their affinity for the stationary phase and thus,

enabling them to move through the system faster.

The value of K for each components decreases at each step. Where K is

the distribution ratio.

Csp =Concentration of the component in the stationary phase.

Cmp =Concentration of the components in the mobile phase.

Csp

Cmp=K

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It means a gradual change in the composition of the eluting solvent to increase the

eluting power of the mobile phase.

Therefore, narrowing the zones and reducing tailing.

It may be a concentration changing ratio of solvent used, pH, polarity or ionic strength

gradient.

Advantages of Gradient elution analysis technique

It produces discrete bands or zones.

It reduces tailing.

Components of a mixture having widely different properties & polarities

can be separated in a single operation.

*Tailing: A phenomenon in which a component is eluted in a several

fractions (CC) or unresolved spots (TLC), it's considered as a serious

disadvantage in chromatography.

Gradient elution analysis (Modified elution development)

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Displacement development

Mechanism:

Elution is carried out by using a solvent which has greater affinity for the

stationary phase than the sample components.

The sample mixture is first introduced onto the top of the column and adheres

to stationary phase.

Elution occurs when a displacing solvent is passed through the column

progressively displacing the components from the stationary phase and the

components separated due to:

-Their varying distribution ratios (K),

-Partition coefficient or adsorption properties;

-that is their relative attraction for the stationary phase with respect to the

mobile phase.

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Displacement development

1. Components with the least affinity for the stationary phase will displace first.

2. Generally, displacement development does not produce bands completely separated by eluent.

3. Bands containing pure components are obtained but between these band other zones containing a mixture of the adjacent bands.

4. Therefore, if preparative work is being carried out to obtain pure samples only the central parts of the bands are collected.

Frontal analysis

Frontal analysis technique can be defined as,

“A preparative method primarily used for the separation of one eluted

component from others with greater affinity for the stationary phase.”

The sample mixture is continuously added to column. Initially the component with

the least affinity for the stationary phase will pass along the column whilst a

strongly adsorbed or attracted component builds up on the stationary phase at

the beginning of the column. Therefore, the first component is eluted from the

column, initially in a pure form, then as a mixture with next components to be

eluted, this process can also occur with elution development if too much sample

is placed on the column.

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Factors affecting column efficiency and separation

1-Column dimensions

The column efficiency continually improves as the length: width ratio of the column

increased. These results from the proportionality between column length and number

of theoretical plates, as well as the more uniform packing obtained in narrower

columns.

2-Adsorbent: (3 aspects are taken in consideration)

a) Particle size of the stationary phase

Uniformity of the stationary phase resulted in uniform flow rate and good

separation, as the particle size decreases the sorption equilibrium on the column

increases and separation improved.

b) Uniformity of packing of the column

If the column is not packed uniformly, the resulted zones not uniform and

separation efficiency increased with the uniform packing of the column.

c) Adsorbent activity

The role of adsorbent activity and water deactivation of the adsorbent is important

in controlling retention volumes. Changes in pH may result in changes in the

zones order.

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3-Solvent: (3 aspects are taken in consideration)

a- Selection of the solvent

The flow rate is inversely proportional to viscosity, and solvents of low viscosity

are useful in high-efficiency separations. The arrangement of bands of

components of a sample mixture may be affected by the different solvents or

mixtures of solvents. The presence of impurities in the solvents often changes

the order of the zones.

b- Constancy of flow

The flow should be continuous, and it is deleterious to interrupt the experiment

and continue it latter.

c- Solvent flow rate

A uniform and low flow rate will result in a more satisfactory separation and more

uniform zones formation than the fast flow rate, when the particle size is

uniformly small. As a result, the separation of individual components of a mixture

will be more efficient. In some instances, decreased flow rate or a decreased

particle size or a combination of both, is required to correct the situation of

uneven diffuse front.

4) Concentration of solute

At higher concentration, the solute band overshoots and moves more rapidly

through the column (Bearding). The solute concentration should not be too high;

a sample to adsorbent ratio of 1:30 or 1:50 should be used.

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5) Temperature

The chromatographic operation should be done at constant temperature

because temperature change will affect the distribution coefficient of the

components. Also affects viscosity of the solvent, the higher the temperature,

the lower the viscosity and may affect the flow rate. Also adsorption is

decreased at higher temperature.

Application of CC in Pharmacy

Separation and Purification of different plant constituents into individual

components e.g. alkaloids of opium, cardiac glycosides of digitalis etc.

Separation and purification of components of reaction mixtures in

pharmaceutical synthesis.

Purification of plant extracts prior to analysis by removing the interfering

impurities.

Chromatographic analysis of pharmaceutical preparation and help in

detecting the presence of artificial coloring matters.

Column partition chromatography.

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FLASH CHROMATOGRAPHY

(A low to medium-pressure)

What is Flash Chromatography?

Simple Definition:

Flash chromatography is a rapid form of preparative column chromatography

based on an optimized pre-packed column through which the solvent is

pumped at a high flow rate.

It is a simple and economical approach to Preparative LC.

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Advantages of flash chromatography: A quick and inexpensive technique for purifying organic

compounds.

A popular method of purification and separation using normal

phases. As well as reversed (wide range of preparative

separations).

It utilizes a plastic column filled with the stationary phase.

The use of pressure, enables the sample to run through the

column and become separated.

Flash column chromatography used air pressure initially, but

today pumps are used to speed up the separation.

This technique is considered a low to medium-pressure

technique and may be scaled up for separations from a few mg to

many tens or hundreds of grams.

Applications are many and varied from drug discovery, sample

clean-up, natural product purification and many more.

There is a direct relationship for normal phase flash column

chromatography with TLC and often the techniques are used

together prior to and post flash separations.

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Applications of flash chromatography:

Applications are many and varied from:

Drug discovery

Sample clean-up

Natural product/ Nutraceuticals applications

1. α-Santalol and β-Santalol from Sandalwood extract.

2. Flavonoids from Ginkgo biloba leaves extract.

3. Ginsenosides from red Panax ginseng extract.

4. Catechins from green tea extract.

Carbohydrate applications

1. Conjugated quercetin and rutinose.

2. Aminosugar and acarbose.

3. Flavanone glycoside purification.

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Applications of flash chromatography:

4. Aminoglycoside antibiotics.

Lipids applications

1. Fatty Acid Methyl Esters (FAMEs).

2. Mono-, Di-, and Triglycerides.

3. Purification of sterols.

Pharmaceutical/Small Molecules applications

1. Bile Acid Purification during lead generation in drug

discovery.

2. Anti-malarial drug purification.

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Thin Layer Chromatography TLC

1 2 3

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Thin Layer Chromatography (TLC)

• TLC is a simple, quick, and inexpensive procedure

• Gives the chemist a quick answer as to how many components are in a

mixture.

• TLC is also used to support the identity of a compound in a mixture when

the Rf of a compound is compared with the Rf of a known compound

(preferably both run on the same TLC plate).

• A TLC plate is a sheet of glass, metal, or plastic which is coated with a thin

layer of a solid adsorbent (usually silica, alumina or cellulose).

• A small amount of the mixture to be analyzed is spotted near the bottom of

this plate.

• The TLC plate is then placed in a shallow pool of a solvent in a developing

chamber so that only the very bottom of the plate is in the liquid.

• This liquid, or the eluent, is the mobile phase, and it slowly rises up the TLC

plate by capillary action.

• In principle, the components will differ in solubility in the mobile phase and

in the strength of their adsorption to the adsorbent and some components

will be carried farther up the plate than others.

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When the solvent has reached the top of the plate, the plate is removed from the

developing chamber, dried, and the separated components of the mixture are

visualized.

If the compounds are colored, visualization is straightforward. Usually the

compounds are not colored, so a UV lamp is used to visualize the plates.

(The plate itself contains a phosphor additive which fluoresces green at 254 nm

wavelength except where an organic compound is on the plate).

Visualization

There are various techniques to visualize the compounds:

•Destructive

10% H2SO4 in ethanol/heat: charred spots, general purpose.

Ceric stain/heat: dark blue spots, polar compounds.

KMnO4/heat: Yellow spots on purple background, olefins and readily oxidized groups.

•Semi-destructive

Iodine:

Iodine absorbs onto the spots, not permanent.

For unsaturated and aromatic compounds.

•Non-destructive

UV light

Short wavelength (254 nm): background green, spots dark

Long wavelength (365 nm): compounds glow.

For compounds only absorbing in UV

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Analysis

Rf (Retardation factor) is defined as the distance the center of the spot

moved divided by the distance the solvent front moved (both measured

from the

origin).

The Rf for a compound is a constant from one experiment using

constant chromatography conditions:

Solvent system

Adsorbent

Thickness of the adsorbent

Amount of material spotted

Temperature

Since these previous factors are difficult to keep constant from

experiment to experiment, relative Rf values (Rst) (the values are reported

relative to a standard), are generally considered.

Analysis

The separation between two analytes on a chromatogram can be

expressed as the resolution (Rs).

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Troubleshooting TLC

Examples of common problems encountered in TLC:

The compound runs as a streak rather than a spot

The sample may be overloaded so, run the TLC again after diluting your sample.

The sample runs as a smear or an upward crescent.

Compounds which possess strongly acidic or basic groups show up on a TLC

plate with this behavior.

Overcome by adding few drops of acid or alkali to the eluting solvent to obtain

clearer plates.

The sample runs as a downward crescent.

The adsorbent was disturbed during the spotting, causing the crescent shape.

The plate solvent front runs crookedly.

Either the adsorbent has flaked off the sides of the plate or the sides of the plate

are touching the sides of the container as the plate develops.

Crookedly run plates make it harder to measure Rf values accurately.

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Troubleshooting TLC

Random spots are seen on the plate.

Make sure that you do not accidentally drop any organic compound on the plate.

No spots are seen on the plate.

If the solution of the compound is too dilute.

Some compounds do not show up under UV light; try another method of

visualizing the plate.

If the solvent level in the developing jar is above the origin (spotting line) of

the - TLC plate, the solvent will dissolve the compounds.

You see a blur of blue spots on the plate as it develops.

If an ink pen instead of a pencil is used to mark the origin.

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Advantages of TLC:

Minimum sample preparation. Sensitivity is less than μg

sample amounts.

Simultaneous analysis of multiple standards and

samples can carried out under identical conditions in a time

comparable to HPLC.

All components are locatable, of HPLC, where highly

polar materials may be overlooked as the peaks are very

broad.

Permit the use of corrosive spray reagents.

Wider range of solvents could be applied than in case of

PC.

Greater speed and better resolution.

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Some Common Uses of TLC

Thin-layer chromatography (TLC) is a very commonly used

technique in:

Identifying compounds,

Determining their purity and

Following the progress of reaction.

Optimization of the solvent system for a given

separation problem.

In comparison with column chromatography, it only requires

small quantities of the compound (~ng) and is much faster

as well.

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Reversed phase thin layer chromatography

The sorption mechanism predominant on silica gel (polar stationary

phase) is adsorption and the plates with suitable choice of eluent can be

used to separate neutral, basic, and acidic hydrophilic substances.

This mode of separation is referred to as normal phase.

In contrast when a non-polar stationary phase is eluted with a more

polar eluent the order of the elution of analytes is reversed and this

referred to as reverse phase chromatography.

Advantage of RPTLC (Reversed Phase Thin Layer

Chromatography):

RPTLC has a number of advantages as compared with normal phase

TLC including:

Superior selectivity.

Better for separation of non-polar compounds.

Better recovery in preparative mode.

Simplified sample clean-up and activation is unnecessary.

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Development in TLC

l) Horizontal development: (Disc chromatogram)

A) Horizontal developing plates as in HPTLC.

B) Radial development

The sample is applied to the center of the disc.

The solvent is supplied through a hole in the plate via a wick which dips

into a solvent reservoir.

As development proceeds the components move out radially forming

circles of increasing diameter.

The relationship between linear Rf values and circular RRf is : Rf =(RRf)2

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2-Vertical developmentAscending & Descending

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C- Continuous development• In ascending chromatography the eluant is allowed to

wash off the bottom of the plate and a continuous flow of

solvent is allowed to wash off the top of the plate and a

continuous flow of solvent is obtained.

• In descending chromatography the eluent simply being

allowed to wash off the plate into the bottom of the tank.

• Descending development has the advantage of quicker

solvent flow, due to the action of capillary and gravity

forces on the eluent, though it has the disadvantage of

requiring additional equipment and additional experience to

set up.

• Continuous development can be used to good effect for

the separation / resolution of compounds using low polarity

solvents.

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D- Multiple and stepwise development

The plate removed from the tank, solvent allowed to evaporate off and

the whole sequence repeated.

If the same eluant is employed and the elution distance kept constant

from one run to another, this is termed multiple development.

Alternatively, the subsequent elution procedure may be modified: for

instance, different solvent systems can be utilized and the solvent

allowed to migrate to different extents. The sequential use of a series

of eluent of differing elutropic strength can be used for the separation

of mixtures of wide-ranging polarity. Depending upon the nature of the

mixture either an increasing or decreasing series of solvent strengths

may be used.

This latter approach is termed stepwise development. Both these

development procedures are complementary to gradient techniques

and offer the following advantages:

a) Enhanced efficiency due to the refocusing /re-concentration of the

analyte spots on each run.

b) Improved detection due to smaller separated zones.

c) Separation of wider polarity range of analytes.

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E- Two-dimensional development

A technique used a second eluent system run at right angles to the

first.

In the case of large groups of compounds of similar chemical

structures and properties, such as amino acids, the Rf values are too

close to give a good separation using one-dimensional linear

development techniques.

The use of standards enable identification of the unknown

components.

Where the pure components of a mixture are not available or are

unknown then the chromatogram obtained may serve as a fingerprint

map in identifying and characterizing the sample.

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Features of HPTLC

It is a sophisticated & automated TLC

Simultaneous processing of sample and standard

Better analytical precision and accuracy

Less need for Internal Standard

Several analysts work simultaneously

Lower analysis time and less cost per analysis

Low maintenance cost

Simple sample preparation - handle samples of divergent nature

No prior treatment for solvents like filtration and degassing

Low mobile phase consumption per sample

No interference from previous analysis - fresh stationary and mobile phases for each analysis - no contamination

Visual detection possible - open system

Non UV absorbing compounds detected by post-chromatographic derivatisation

High Performance Thin Layer Chromatography (HPTLC

) (sophisticated & automated TLC)

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