PCH 411/401

PHYSICAL
PHARMACEUTICAL
CHEMISTRY & ANALYSIS III

[Link]
 COURSE OUTLINE
Principles of Chromatography

• Paper Chromatography (PC)

• Column Chromatography (CC)
• Thin Layer Chromatography(TLC)
• High Performance Thin Layer Chromatography(HPTLC)
• Gas Chromatography (GC)
• High Performance Liquid Chromatography(HPLC)
• Ultra Performance Liquid Chromatography(UPLC)

Applications
 GOAL

TO UNDERSTAND HOW CHROMATOGRAPHY WORKS AS

AN ANALYTICAL TOOL AND THEIR APPLICATIONS

Historical development, Technological Progress, Sensitivity, affordability, resolution, simplicity

GENERAL PRINCIPLES

Introduction / Background

Terminologies

Instrumentation / Components

Applications
 GENERAL BACKGROUND
➢CHROMATOGRAPHY IS ONE OF THE MOST CONVENIENT AND
FREQUENTLY USED ANALYTICAL TECHNIQUE IN PHARMACEUTICAL
ANALYSIS.

➢ Mobile phase passes through the stationary phase for separation to occur

➢ Three major procedures are associated with chromatography

▪ Gas chromatography procedures

▪ Liquid chromatographic procedures

▪ Supercritical-fluid chromatographic procedures

 GENERAL BACKGROUND
➢CHROMATOGRAPHY AN ANALYTICAL TECHNIQUE IN PHARMACEUTICAL
ANALYSIS FOR SEPARATION, PURIFICATION AND IDENTIFICATION OF
COMPOUNDS.

➢MODES OF SEPARATION

▪ Adsorption chromatography

▪ Partition chromatography

▪ Ion exchange chromatography

▪ Gel Chromatography
 SOME HPLC TERMINOLOGIES

• CHROMATOGRAPH: AN ANALYTICAL INSTRUMENT THAT

SEPARATES MIXTURES OF SUBSTANCES INTO IDENTIFIABLE
COMPONENTS BY MEANS OF CHROMATOGRAPHY

• MOBILE PHASE (ELUENT/ELUANT): THE LIQUID THAT PUSHES

THE ANALYTES THROUGH THE COLUMN

• ELUATE: THE COMPONENT EMERGING FROM THE COLUMN

 SOME HPLC TERMINOLOGIES

• STATIONARY PHASE / COLUMN: THE PART OF THE CHROMATOGRAPHIC SYSTEM

THROUGH WHICH THE MOBILE PHASE FLOWS WHERE DISTRIBUTION OF THE
SOLUTES BETWEEN THE PHASES OCCURS

• ISOCRATIC MOBILE PHASE: THE SAME MOBILE PHASE COMPOSITION OVER THE
ENTIRE RUN

• GRADIENT PROGRAM: COMPOSITION OF THE MOBILE PHASE IS CHANGED

DURING THE RUN

• BASELINE: THE NAME GIVEN TO THAT PART OF A CHROMATOGRAM THAT

REPRESENTS ANY TIME PERIOD DURING WHICH ONLY MOBILE PHASE IS
PASSING THROUGH THE DETECTOR
 SOME HPLC Terminologies

• ENDCAPPING: DERIVATISATION OF SILANOL GROUPS WITH A SMALLER

MOLECULE AFTER THE PHASE IS ATTACHED

• RETENTION TIME: THE TIME BETWEEN THE TIME OF INJECTION OF A

SOLUTE AND THE TIME OF ELUTION OF THE PEAK MAXIMUM

• RESOLUTION: HOW MUCH SPACE THERE IS BETWEEN PEAKS

• CHROMATOGRAM: THE OUTPUT FROM THE SEPARATION, A VISUAL

RECORDING OF THE COMPOUNDS IN THE SAMPLE
 PAPER CHROMATOGRAPHY (PC)

Introduction / Background

Instrumentation / Components

Applications
• Principle of paper chromatography is similar to that of TLC
• Cellulose fiber of the chromatographic paper acts as the supporting
matrix for the stationary phase which could be water, non polar solvent
such as liquid paraffin
• Thickness of the paper is important
• For a thick paper, flow rate is fast thus elution might increase
• With a thick paper, there Is better loading
• When substance flows very fast, there is spot spreading
• It is better than using a thin paper even though it has a disadvantage of spot spreading.
This can be reduced by controlling the flow rate.

• This is done by cutting the paper at the edge so that the amount of solvent entering is
controlled. Although the surface is not linear, before the mobile phase gets to the spots it
becomes uniform
• Another way is to attach a thin paper to the edge of the chromatographic
paper so that the amount of solvent entering is controlled by the thin paper
thereby reducing the flow rate
• Paper can be made to behave as an adsorption system by use of solvents such formamide,
methyl or dimethyl formamide.

• Paper can also be impregnated with non polar solvents such as paraffin, alpha bromo
naphthalene or ion exchange.

• In paper chromatography, the stationary phase is a very uniform absorbent paper. The
mobile phase is a suitable liquid solvent or mixture of solvents.

• The paper is suspended in a container with a shallow layer of a suitable solvent or mixture of
solvents in it. It is important that the solvent level is below the line with the spots on it.
TWO TECHNIQUES ARE EMPLOYED
• ASCENDING TECHNIQUE

• Paper is folded and clipped

• Introduced into the TLC chamber after equilibration.

• It is allowed to stand so the base of the paper is in contact with the solvent.

• Since the spots are above the solvent, they can be separated as solvent moves up by capillary
action
• For 2 dimensional chromatography, first run should be on the machine direction. Second run will
be against it
• DESCENDING TECHNIQUE

COURTESY: PHARMA INFORMATION ZONE

• IN DESCENDING TECHNIQUE, SOLVENT MOVES DOWN UNDER GRAVITY.

• PAPER IS NOT ALLOWED TO TOUCH THE SOLVENT AT THE BASE OF THE TANK
• Ascending technique is preferred because of the simplicity of set up

• Flow of solvent is faster in descending technique

• Detection is similar to that for TLC but spraying with H2SO4 is not recommended as this
causes the paper to disintegrate

• Identification is made on the basis of distance moved by component during development

relative to the distance moved by the solvent front i.e.
Rf = Distance moved by solute
Distance moved by solvent front
 VISUALIZATION

• UV LIGHT, WHICH IS NON DESTRUCTIVE

• IODINE WHICH IS SEMI DESTRUCTIVE

• CERIC STAIN WHICH IS DESTRUCTIVE- LEAVES BLACK SPOTS

BEHIND FOR POLAR COMPOUNDS
COLUMN CHROMATOGRAPHY
 COLUMN CHROMATOGRAPHY
Column chromatography is a method used to purify individual
chemical compounds from mixtures of compounds.

It is often used for preparative applications from micrograms up to

kilograms.

The main advantage of column chromatography is the relatively low

cost and disposability of the stationary phase used in the process.
The latter prevents cross-contamination and stationary phase
degradation due to recycling.
• Two methods are used to prepare a column:
✓the dry method
✓ the wet method.
• For the dry method, the column is first filled with dry stationary phase powder,
followed by the addition of mobile phase, which is flushed through the column until it
is completely wet, and from this point is never allowed to run dry.

• For the wet method, a slurry is prepared of the eluent with the stationary phase
powder and then carefully poured into the column. Care must be taken to avoid air
bubbles.

• A solution of the organic material is pipetted on top of the stationary phase.

• This layer is usually topped with a small layer of sand or with cotton or
glass wool to protect the shape of the organic layer from the velocity of
newly added eluent.

• Eluent is slowly passed through the column to advance the organic

material.

• Often a spherical eluent reservoir or an eluent-filled and

stoppered separating funnel is put on top of the column.
• The sample is introduced at the top of the column as a narrow band.
Ideally, the solute’s initial concentration profile is rectangular.

• As the sample moves down the column the solutes begin to separate,
and the individual solute bands begin to broaden and develop a
Gaussian profile.

• If the strength of each solute’s interaction with the stationary phase is

sufficiently different, then the solutes separate into individual bands.
 Figure : Progress of a column chromatographic separation of a two-component
mixture. In (a) the sample is layered on top of the stationary phase. As mobile phase
passes through the column, the sample separates into two solute bands (b–d). In (e)
and (f), we collect each solute as it elutes from the column.
Figure: An alternative view of the separation in figure above showing the concentration
of each solute as a function of distance down the column.
The progress of the separation is followed either by

• collecting fractions as they elute from the column, or

• placing a suitable detector at the end of the column.

A plot of the detector’s response as a function of elution time, or as a function of the

volume of mobile phase, is known as a chromatogram, and consists of a peak for
each solute.
Figure :. Chromatogram for the separation shown in previous figures
showing the detector’s response as a function of the elution time.
• A solute’s chromatographic peak may be characterized in many ways, two of which are
shown.
• Retention time, tr, is the time between the sample’s injection and the maximum response
for the solute’s peak.
• Another important parameter is the baseline width, w, which, is determined by extending
tangent lines from the inflection points on either side of the chromatographic peak through
the baseline.
• Although usually we report tR and w using units of time, we can report them using units of
volume by multiplying each by the mobile phase’s velocity, or in linear units by measuring
distances with a ruler.
For example, a solute’s retention volume, VR, is
VR = tR x u
where u is the mobile phase’s velocity through the column
tR is retention time
Figure: Chromatogram showing a solute’s retention time, tr, and
baseline width, w, and the column’s void time, tm, for
non retained solutes.
 SELECTIVITY

• Selectivity is a relative measure of the retention of two solutes, which

we define using a selectivity factor, α

α = kB / kA = (tr,B− tm)/(tr,A − tm)

where solute A always has the smaller retention time. When two
solutes elute with identical retention time, α = 1.00; for all other
conditions α > 1.00.
 COLUMN EFFICIENCY

A sample consisting of a single component is injected. At the moment

of injection the sample occupies a narrow band of finite width. As the
sample passes through the column, the width of this band increases in
a process we call band broadening.

Column efficiency provides a quantitative measure of the extent of

band broadening.
• In their original theoretical model of chromatography, Martin and Synge
divided the chromatographic column into discrete sections—what they
called theoretical plates—in which there is an equilibrium partitioning
of the solute between the stationary phase and the mobile
phase. They described column efficiency in terms of the number of
theoretical plates, N,
N = L/H (i)

where L= the column’s length

H = the height of a theoretical plate
Factors affecting column chromatography

• The polarity of the mobile phase

• Nature of the solvent
• Quality of solvents
• Solvent selection
• Flow rate
• Packing of the column
• Column dimension
• The particle size of the adsorbent
• Pressure
• The temperature
THE DIMENSION OF THE COLUMN WOULD DEPEND ON:
• SEPARATION EFFICIENCY REQUIRED
• SIZE OF THE SAMPLE
• TYPE OF CHROMATOGRAPHY EMPLOYED

THE FLOW RATE DEPENDS ON :

• TYPE OF CHROMATOGRAPHY
• COLUMN SIZE
ADSORBENTS USED IN COLUMN CHROMATOGRAPHY

• CHARCOAL STRONGEST
• ALUMINA
• MAGNESIUM SILICATE
• SILICA GEL
• MAGNESIA
• CALCIUM CARBONATE
• TALC
• INULIN
• STARCH
• SUCROSE WEAKEST
 Factors taken into consideration when choosing an adsorbent

• Adsorptivity
• Surface area
• Particle Size
• Surface Activity
 Solvents used as mobile phase
Heptane strongest
Hexane
Isoctane
Petroleum Ether
Cyclohexane
Carbon Tetrachloride
Toluene
Benzene
Ethyl Ether
Chloroform
Dichloromethane
Terahydrofuran
Acetone
Ethyl Acetate
Acetonitrile
Pyridine
N-Propanol
Ethanol
Methanol
Acetic Acid
Water
weakest
 THANK YOU
AND
PREPARE YOUR QUESTIONS