Chromatography (Separation)

Technique
2. Peak area
Mobile
(quantitative analysis)
phase

1. Separation

3. Retention time
Sample (qualitative analysis)
mixture

1
 Definitions of chromatography

• Chromatography:
– is a physical method of separation in which the
components to be separated are distributed between
two phases; stationary phase and mobile phase
– Those components held preferentially in the stationary
phase are retained longer in the system than those
that are distributed selectively in the mobile phase.

2
 Definitions of chromatography cont…
– The stationary phase is defined as the immobile packing
material in the column.
– the mobile phase, a fluid, is streaming through the
chromatographic system.
• Chromatographic methods are commonly used for:

– Separation, raw materials

 drug substances,
– Qualitative analysis drug in drug products (finished product)
– Quantitative analysis biological fluids

3
4
 Classification of chromatography

1. Nature of mobile phase

– Liquid Chromatography (LC)

• MP is liquid

• HPLC, TLC, HPTLC, CC, PC

– Gas Chromatography (GC)

• MP is gas

• GC:- GSC & GLC

5
 Classification cont …

2. Depending on depth of separation

– Column Chromatography:
• the stationary phase is held in a narrow
tube through which the mobile phase is
forced under pressure or by gravity.
– Planar Chromatography:
• the stationary phase is supported on a
flat plate and the mobile phase moves
through the stationary phase by
capillary action or by gravity

6
 Classification cont …

3. Mechanisms of Separation:
(Nature of stationary phase)
– Adsorption Chromatography

– Partition Chromatography

– Ion Exchange Chromatography

– Molecular Exclusion Chromatography

– Affinity Chromatography

7
 Chromatographic techniques
1. Paper chromatography
– works by the partition of solutes
between water in the paper fibres
(s p) and the solvent (m p).
– The distance a solute moves is
always the some fraction of the
distance moved by the solvent.

X Y Z
8
 2. Thin-layer Chromatography

• The surface of the plate consists of a very thin layer of silica gel on a
plastic or Aluminium or glass backing.
• The mixture is ‘spotted’ at the bottom of the TLC plate and allowed
to dry.
• The plate is placed in a closed vessel containing solvent (the mobile
phase) so that the liquid level is below the spot.
• The solvent ascends the plate by capillary action, the liquid filling
the spaces between the solid particles.
• TLC has advantages over paper chromatography in that:
– its results are more reproducible, and that separations are very
efficient because of the much smaller particle size of the
stationary phase

9
 Visualization of the spots
• Non-polar compounds
– are less strongly attracted to the plate and spend more time in the moving
phase.
– move faster and will appear closer to the top of the plate.
• Polar compounds
– more strongly attracted to the plate and spend less time in the moving phase
and appear lower on the plate.

10
 Visualization Methods
• Visualization Methods
– Most of the time, the spots don’t show unless they are visualized.
– Visualization is a method that is used to render the TLC spots visible.
– A visualization method can be:
a) Ultraviolet light b) Spraying reagent
 Iodine vapors to stain spots
 ninhydrin

11
Measuring Rf values

12
 3. Classical Column Chromatography
Column chromatography (CC):
 the stationary phase is packed into a glass tube to form a
column of granules.
 Mobile phase is liquid, water or organic solvents.

 In CC, the solutes interact with both mobile phase and

stationary phase, and the mobile phase solubilizes the
solute,
 while in GC the mobile phase (carrier gas) acts only for
the transportation of solutes 13
 Column Chromatography …
• CC is a separation technique that involves:
– the placement (injection) of a small volume of liquid sample
– into a tube packed with porous particles (stationary phase)
– where individual components of the sample are transported along the packed
tube (column) by a liquid - mobile phase moved by gravity.
– The components of the sample are separated by various chemical and/or
physical interactions between their molecules and the packing particles.
– The separated components are collected at the exit of this column and
identified by an external measurement technique.
technique

14
Column chromatography separation
mechanism

15
 Column Chromatography cont…
• Principle of chromatographic separation:
– Different distribution of the analytes between mobile and stationary phase
results in different migration velocities

Fig. Chromatogram of analytes A, B and C, in the time (t)

16
 Chromatographic nomenclature

Sample 17
injection point Source: Principles and Practice of Chromatography, 2003
 4. High Performance (Pressure) Liquid
Chromatography
High performance LC- due to it’s improved performance when
compared to classical column chromatography

High pressure LC- since high pressure is used when compared to

classical column chromatography

Developed as a result of further advances in column technology

18
 What is HPLC?
• HPLC is a separation technique that involves:
– the injection of a small volume of liquid sample
– into a tube packed with tiny particles ( 3 to 5 micron µm) in diameter
called the stationary phase
– where individual components of the sample are moved down the
packed tube (column)
column with a liquid (mobile phase)
phase forced through the
column by high pressure delivered by a pump.
– These components are separated from one another by the column
packing
• that involves various physicochemical interactions between their
molecules and the packing particles.
– These separated components are detected at the exit of this column
by a detector that measures their amount.
– An output from this detector is called a “liquid chromatogram”.
chromatogram

19
What Does a Liquid Chromatogram Look Like?

20
 HPLC cont…
The development of HPLC from classical column chromatography
can be attributed to the development of smaller particle sizes
packed in a column, used as stationary phase

Smaller particle size is important since they offer more surface

area over the conventional larger particle size

Particle size 60-200 µ - for classical column chromatography

» » 3-20 µ - for HPLC

Due to the small particle size of the stationary phases, pressure

(could be as high as 6000 psi) is required to force the mobile
phase through the stationary phase.

21
 HPLC cont…

HPLC is really the automation of traditional liquid

chromatography under conditions which provide

for enhanced separations during shorter periods of time

Probably the most widely practiced form of quantitative,

analytical chromatography practiced today due to

the wide range of molecule types and sizes which can be

separated using HPLC or variants of HPLC!!

22
 HPLC cont…
 HPLC has tremendous advantages in terms of speed, precision, accuracy

and ease of operation. These is due to the following special features:

 Micro particulate column packages (3 to 10µm)
-high surface area for separation

 High pressure pumping system ( 4000 to 6000 psi)

-to achieve appropriate flow rate

 Use of very small sample size ( <20µg)

- due to sensitive detectors and data handling system

 Not limited by sample volatility or thermal stability

- used for insufficient volatile and thermally unstable samples

23
 HPLC cont…
 Greater variety of stationary phase possible

- provide greater possibility of separation

 Separation enhanced at low temperature separation

- low T makes intermolecular interactions more effective ( at

room T)
 Number of unique detectors available (UVD,RID, FD etc)

- many of these detectors have little application in GC

 Relatively ease of isolate recovery

- separated fractions can easily be collected

24
 HPLC cont…
Advantage of HPLC over GC
 Only about 20% of known cpds can be handled by GC with
out prior chemical modification
 May be due to insufficiently volatile and thermally unstable

Analysis typically carried out at room T0 ; there is no volatility

or thermal decomposition problems

Wider choice of stationary phase

Wider choice of mobile phase

Retention of solutes in LC depend on their interaction with both MP
and SP phase → more flexible in optimizing separations

25
 HPLC cont…
Theoretical aspects
[Link] broadening
The tendency of a zone to spread as solutes pass through a chromatographic

column
 Formed as a result of slower rate of mass transfer and various diffusion factors

 It reflects loss of column efficiency

 Column efficiency evaluate in the form of column plate heights

26
 HPLC cont…
Theoretical plates
A measure of column efficiency;
 the more plates, the better the column efficiency
 Height Equivalent to a Theoretical Plate (HETP or H) is used to quantify the
efficiency of a chromatographic column
- It is the function of N (number of plate) and L (length of column)
- H = L/N, and N= 16(tR/wB)2,

where tR=distance from the point of injection, WB=peak width from the base

 The smaller H value, the more efficient the column

27
Calculation for theoretical plate

28
HPLC cont…

29
 HPLC cont…
Eddy diffusion (A term)
 Analyte molecules can flow multiple path ways of differing path length

 It can be due to compactness and particle size of packaging

loose packaging & voids due to large particle size causes band

broadening due to channeling

 Smaller particle packaging offers smaller differences in path length &

thus reduces peak broadening

Thus smaller SP particles contribute to smaller A values and A value

contribute less to H value

- allowing higher resolution

30
 HPLC cont…
Longitudinal diffusion (B term)
 It is due to diffusion coefficient of analyte molecules in the mobile
phase
 Faster mobile phase flow rate reduces resident time and reduces the
effect of longitudinal diffusion

- contribute better separation efficiency by minimizing band

broadening
Thus higher flow rates reduce contribution of B to H

31
 HPLC cont…
Analyte mass transfer coefficient (C term)
Depends on the time needed for the analyte molecules to
equilibrate b/n the MP & SP.
Smaller SP particles have shorter equilibration time
At short equilibration; the analyte molecule may not have enough
time to bond to the SP & it will then flow down the column with the
mobile phase
• Those bound may require time to detach from the stationary
phase causing band broadening by being left behind
• higher mobile phase flow rate contribute to spreading out of
the analyte

32
 Summary

Van Deemter plots

 A plot of plate height Vs average linear velocity of mobile phase

# Such plots are of considerable use in determining the optimum mobile phase flow rate

33
 HPLC cont…
2. Resolution
– Resolution, Rs, is generally defined as the distance between the
centers of two eluting peaks as measured by retention time or
volume divided by the average width of the respective peaks

34
Calculation for resolution

35
 HPLC cont…
3. Capacity factor
– The capacity factor, k´, is related to the retention time
– is a reflection of the proportion of time a particular solute resides in
the stationary phase as opposed to the mobile phase.
– Long retention times result in large values of k´.

– where TR and VR are the retention time and retention volume,

respectively, of the solute, and To and Vo the retention time and
retention volume, respectively, of an unretarded solute.
36
HPLC cont…

37
 Instrumentation

Basic Components of an HPLC System

Mobile Phase Reservoir

Pumping Systems

Sample Injection Systems

Columns

Detectors

Recorders and Integrators

38
 HPLC system
 Solvent Reservoir

 Degasser

 Solvent Delivery System

(Pump)

 Injector

 Column &oven

 Detectors
 Recorder (Data Collection)
39
 Instrumentation cont…
Mobile Phase Reservoir
Made of a material resistant to chemical attack by the mobile phase
and must be smooth so as to avoid growth of microorganisms on its
walls.
Glass/stainless steel

Designed to allow mobile phase degassing insitu (removal of

dissolved gases)

degassers
vacuum pumping system

heating/stirring of solvents

Sparging (helium purging)

40
 Instrumentation cont…
Purpose of degassing is

To eliminate dissolved gases particularly oxygen

Which may react with the mp or sp in the column

To reduce the possibility of bubble forming;

interfere with the separation process, steady baseline, and the shape of
the peak

Simple and effective degassing method

Bubbling helium in the solvents before actual chromatography is
commenced

A helium blanket on the surface of the solvent

Prevents oxygen from redissolving in the solvent after degassing

41
 Instrumentation cont…

Basic types of M.P delivery systems

Constant solvent composition (Isocratic elution)

Solvent composition varied with time (Gradient elution)

A. Isocratic separation
The same mobile phase combination, same polarity or
elution strength is used throughout the process of
separation

Either a single pure solvent or mixture of solvents can be

used
42
 Instrumentation cont…

B. Gradient separation
The mobile phase composition varied with time

Also known as solvent programming.

Similar to temperature programming in GC

Gradients can be continuous or stepwise.

Incorporated to achieve a better or/and faster

separation
 Decreased separation time when compared to that of
isocratic separation
43
 Gradient vs. Isocratic Conditions
• Isocratic
– mobile phase solvent composition remains
constant with time
– Best for simple separations
– Often used in quality control applications that
support and are in close proximity to a
manufacturing process
• Gradient
– mobile phase solvent (“B”) composition
increases with time
– Best for the analysis of complex samples
– Often used in method development for
unknown mixtures
– Linear gradients are most popular (for
example, the “gradient” shown at right)

44
Why Are Mobile Phase Gradients
Used in HPLC?

45
 Instrumentation cont…
Pumping systems
In HPLC, long narrow columns packed with very fine particles are used & making it resistant
to flow of mobile phase

To over come this problem high pressure is required

The two major functions of the pump in HPLC are:

To pass the mobile-phase through the column at a high pressure

To provide a constant flow rate

During the chromatographic experiment, a pump can deliver a constant mobile phase
composition (isocratic) or an increasing mobile phase composition (gradient).

the pump is Avery delicate and sensitive part of HPLC unit. [Link] buffer solution
should be removed carefully after use either by pumping water or an appropriate solvent for
several minutes.

46
 Instrumentation cont…
HPLC makes use of two types of pumps

(a) Constant Pressure Pump

Acts by applying a constant pressure to the mobile-phase

The flow rate through the column is determined by the flow

resistance of the column
the flow rate will change if the flow resistance changes

(b) Constant Flow Pump

affords and maintains a given flow of liquid

The pressure developed entirely depends upon the flow

resistance
the changes in flow resistance are compensated by a change of
47
pressure.
 Instrumentation cont…

types of pumps
pneumatic pumps (constant pressure pump)- Pulse free

Mechanical pumps
a screw-driven syringe type - Pulse free ( constant flow rate)

a reciprocating pump - Pulsed flow( due to back &forth motion of

piston)

48
 Instrumentation cont…

 Requirements

– ability to mix solvents and vary polarity of mobile phase during run

– generation of pressures up to 6000 - 9000 psi (400 – 600 bar)

– flow rates ranging from 0.1 to 10 mL/min( Variable flow rate device must

be available to monitor flow rate)

– flow reproducibility’s of 0.5 % or better

– resistance to corrosion by a variety of solvents

– pulse-free output( solvent flow must be non pulsing)

49
– It should be easy to dismantle and repair.
 Instrumentation cont…
Injection system
 This allows an introduction (injection) of the liquid analytes
mixture into the stream of the mobile phase.

i. Manual Syringe injection

 Earliest and simplest means but with the bad reproducibility.
 Modern HPLC uses Sampling loop technology.
The typical operating pressure of an HPLC is sufficiently high that it is
impossible to inject the sample in the same manner as in gas
chromatography.
Instead, the sample is introduced using a loop injector

50
 Instrumentation cont…
 In the load position the sampling loop is isolated from the
mobile phase and is open to the atmosphere.
 A syringe with a capacity several times that of the sampling loop is used to
place the sample in the loop.
 Any extra sample beyond that needed to fill the sample loop exits through
the waste line.

 After loading the sample, the injector is turned to the inject

position.
 In this position the mobile phase is directed through the sampling loop, and
the sample is swept onto the column.

51
 Injection system…

from Pump to Column from Pump to Column

Needle

Vial
LOAD INJECT
Measuring Pump
52
 Instrumentation cont…
ii. Automatic injection
 It is frequently used by highly sophisticated modern HPLC

 Good precision compared to manual injection

 The syringe plunger is usually operated pneumatically &
the syringe is first washed with solvent, then it is rinsed
with the sample, then reloaded with the sample and
finally the contents are discharged into the column.

After the analysis the next cycle commences with the

next syringe washing procedure.

53
 Manual Vs automatic
Manual Injector:
User manually loads sample into the injector using a syringe
 and then turns the handle to inject sample into the flowing mobile phase
… which transports the sample into the beginning (head) of the column, which
is at high pressure
Autosampler:
User loads vials filled with sample solution into the autosampler tray (100
samples)
 and the autosampler automatically
measures the appropriate sample volume,
injects the sample,
then flushes the injector to be ready for the next sample, etc., until all
sample vials are processed … 54
 Instrumentation cont…
In general the sample injection system should enable the following
 Very low dispersion and high accuracy characteristics
 Must allow delivery of very constant smaller sample size
 Reproducibility of the sample into the column must be ensured
 There should not be adsorption of samples
 Must tolerate pressure up to 10,0000 psi
 Sample volume can varies 5 to 500 micro litter
 Instrumentation cont…
Column
An HPLC typically includes two columns:

I. Guard column
A short column

Set b/n injection port and column.

The purpose of the guard column is
Traps strongly retained species or particulate matter originating in the
mobile phase, the samples or from wearing of the injection valve.
To increase the life of the analytical column.

To saturate the mobile phase with the liquid making up the stationary
phase
to prevent stripping of the SP from the packing of the analytical column
56
 Instrumentation cont…
II. Analytical Column
“heart of the chromatograph”

the column’s stationary phase separates the

sample components of interest using various
physical and chemical parameters.

made up of either stainless steel, glass, …

Most widely used are stainless steel which can

with stand high pressure

length: varies from 10 to 30 cm (usually: 25 cm)

Internal diameter: ranges from 4 to 5 mm

(usually: 4.6 mm)

57
 HPLC Columns Packing Materials
• Columns are packed with small diameter porous particles.
particles
– The most popular sizes are: 5μm, 3.5μm and 1.8μm
• Columns are packed using high-pressure to ensure that they are stable
during use
– most users purchase pre-packed columns to use in their liquid chromatographs
• These porous particles in the column usually have a chemically bonded
phase on their surface which interacts with the sample components to
separate them from one another
– for example, C18 is a popular bonded phase
• The process of retention of the sample components (often called
analytes) is determined by:
– the choice of column packing
– the selection of the mobile phase to push the analytes through the packed column.
• The correct selection of the column packing and the mobile phase are the
most important factors in successful HPLC.
58
 Instrumentation cont…

• Detectors
– The detector can see (detect) the individual molecules that come out
(elute) from the column.
– A detector serves to measure the amount of those molecules so that
the analyst can quantitatively analyze the sample components.
– The detector provides an output to a recorder or computer that
results in the liquid chromatogram.
chromatogram

59
 Instrumentation cont…
Detector Can be broadly divided into the following two classes:

I. Bulk property detectors

Measure the difference in some physical property of the solute in
the mobile phase compared to the mobile phase alone
e.g. refractive index and conductivity detectors

They are generally universal in application but tend to have poor

sensitivity

Such detectors are usually affected by even small changes in the

mobile-phase composition
Which precludes the use of techniques such as gradient elution

60
 Example of bulk property detector
• Refractive Index (RI) Detection
– The ability of a compound or solvent to deflect light provides a way to
detect it.
– The RI is a measure of molecule’s ability to deflect light in a flowing
mobile phase in a flow cell relative to a static mobile phase contained
in a reference flow cell.
– The amount of deflection is proportional to concentration.
– The RI detector is considered to be a universal detector but it is not
very sensitive.

61
 Instrumentation cont…
II. Solute property detectors
Respond to a particular physical or chemical property of the
solute
e.g. Spectrophotometric, fluorescence and MS

Being ideally independent of the mobile phase or Signal

discrimination is usually sufficient
permit operation with solvent changes, e.g. gradient elution

They generally provide high sensitivity

Since they are more selective

62
 Solute property detectors …
• Ultraviolet (UV) Absorption
– An ultraviolet light beam is directed through a flow cell and a sensor
measures the light passing through the cell.
– If a compound elutes from the column that absorbs this light energy,
it will change the amount of light energy falling on the sensor.
– The resulting change in this electrical signal is amplified and directed
to a recorder or data system.
– A UV spectrum is sometimes also obtained which may aid in the
identification of a compound or series of compounds.

63
 Solute property detectors …
• Mass Spectroscopy (MS)
– An MS detector senses a compound eluting from the HPLC column
first by ionizing it then by measuring it’s mass and/or fragmenting
the molecule into smaller pieces that are unique to the compound.
– The MS detector can sometimes identify the compound directly since
its mass spectrum is like a fingerprint and is quite unique to that
compound.

64
 Solute property detectors …

• Fluorescence Detection
– Compared to UV-Vis detectors; fluorescence detectors offer a higher
sensitivity and selectivity
• allows to quantify and identify compounds and impurities in complex
matrices at extremely low concentration levels (trace level analysis).

– Fluorescence detectors sense only those substances that

fluoresce(sensitive to compounds that are inherently fluorescent or that can be
converted to fluorescent derivatives either by chemical transformation of the
compound or by coupling with fluorescent reagent s at specific functional groups.)

65
 Instrumentation cont…

Computer
Frequently called the data system,

Controls all the modules of the HPLC instrument

It takes the signal from the detector and uses it to determine the
time of elution (retention time) of the sample components
(qualitative analysis)
analysis and the amount of sample (quantitative
analysis).
analysis

Record the responses obtained from detector

Chromatogram

66
 HPLC Instrumentation…
Approximation
HPLC Chromatograms of peak area by
triangulation
Absorbance 

Peak A Peak B

height

0 1 2 3 4 5 6 7
Time (minutes) base

Rt = 3.0 min. Rt = 5.2 min.

faster moving slower moving base x height
Area =
less retained more retained 2

67
 HPLC type
Types of HPLC techniques
Based on principle of separation
1. Adsorption HPLC
2. Partition HPLC
3. Ion exchange HPLC
4. Size exclusion or Gel permeation HPLC
5. Affinity HPLC
reading assignment
6. Chiral HPLC

68
 1. Adsorption HPLC

Adsorption HPLC
 Liquid-solid chromatography: adsorption of analyte on a solid surface

 Stationary phase: polar solid, finely divided silica - SiO2(more common

higher sample capacity) or alumina - Al2O3

 Analyte polarity ↑→ tR↑

 Mobile phase: the variable

 Application: relatively non polar, water-insoluble organic compounds

 Decreased application due to normal phase LC

69
 2. Partition HPLC
 Partition chromatography, now normally referred as to Bonded-phase
chromatography, has two types:

a. Normal-phase HPLC:
 Classic form of liquid chromatography using:
 polar stationary phases (silica or chemically modified surface)

 non-polar mobile phases ((e.g. hexane, iso-octane, methylene chloride,

ethyl acetate).

 The analyte is retained by the interaction of its polar functional groups with
the polar groups on the surface of the packing
 Analytes elute from the column starting with the least polar compound
followed by other compounds in order of their increasing polarity
– Good for separation of non-polar solutes. 70
 Chromatography Stationary Phases
Silica Gel Derivatized Silica Gel

O O O O O O
| | | | | |
OSiOSiOSiOH OSiOSiOSiOR Where R = C18H37
| | |
| | | hydrocarbon chain
O O O
| | |
O O O (octadecylsilyl
OSiOSiOSiOH | | | deriv.
| | | OSiOSiOSiOR silica or “C18”)
O O O | | |
O O O

bulk (SiO2)x surface bulk (SiO2)x surface

relatively polar surface

relatively nonpolar surface
“normal phase”
“reversed phase”

71
72
 b. Reversed-phase HPLC:
• The column packing is non-polar (e.g. C18, C8, C3, phenyl,
etc.) and the mobile phase is water (buffer) + water-miscible
organic solvent (e.g. methanol, acetonitrile)
– RPC is, by far, the most popular mode …
• over 90% of chromatographers use this mode

– The technique can be used for non-polar, polar, ionizable

and ionic molecules …
• making RPC very versatile

73
 Partition HPLC…
– The analytes are attracted to the surface by their non-polar
functional groups
– The most polar analyte elutes from the RP column first
followed by other analytes in order of decreasing polarity
• compound polarity ↓ or solvent polarity ↑ → tR ↑
– For samples containing a wide range of compounds,
gradient elution is often used …
• One begins with a predominantly water-based mobile phase and
then adds organic solvent as a function of time.
• The organic solvent increases the solvent strength and
elutes compounds that are very strongly retained on the
RPC packing

74
 Types of HPLC…

• The mobile phase in reversed-phase HPLC generally

comprises water and a less polar organic solvent modifier.
– E.g. Methanol or acetonitrile

75
A B

D
C

76
 3. Ion Exchange HPLC
 Stationary phase is ion exchange resin

• In ion exchange, the column packing contains ionic groups (e.g.

sulfonic, tetraalkylammonium) and the mobile phase is an aqueous
buffer (e.g. phosphate, formate, etc.).
– Ion exchange is used by about 20% of the liquid chromatographers

– The technique is well suited for:

• the separation of inorganic and organic anions and cations in aqueous
solution.
• Ionic dyes, amino acids, and proteins can be separated by ion exchange
because such compounds are salt in brine water,

77
 Ion Exchange HPLC…
 Stationary phase
i. Cation exchange resins
– sulfonic acid group ( ), carboxylic acid group( )
– For cation analysis
ii. Anion exchange resins
– quaternary amine group ( ), primary amine group ( )
– For anion analysis
 Mobile phase
– Dilute mineral acid for cation analysis and hydrogen carbonate buffer for
anion analysis
 Detector :
– electric conductivity detector

78
 Ion Exchange HPLC…
a polymer containing fixed charged groups and replaceable
counter ions of the opposite charge .

+
+
+
+

+
+

79
 Ion Exchange HPLC…
i. Cation exchange resins
– sulfonic acid group, carboxylic acid group
ii. Anion exchange resins
– quaternary amine group, primary amine group

 The strength & exchange capacities of ion exchange resins

depend on:
– The acidic or basic strength of the fixed charged group.

o Resins of sulphonic acid & quaternary ammonium groups are

strong ion exchange resins with a high exchange capacity.

o Resins containing the weakly acidic carboxylic acid (-COOH) or

weakly basic derivatives of tertiary amine (NHR2+Cl-) generally have
a lower exchange capacity
80
 4. Size exclusion HPLC
 Cross-linked polystyrene-divinyl benzene resins or silica microspheres (5–
15 µm id) are used to fractionate materials of high molecular weight.
 Usually only a single aqueous or organic solvent is used as the mobile
phase.
 In SEC, there is no interaction between the sample compounds and the
column packing material.
 Instead, molecules diffuse into pores of a porous medium.
 Depending on their size relative to the pore size, molecules are
separated.
 Molecules larger than the pore opening do not diffuse into the particles,
while molecules smaller than the pore opening enter the particle and are
separated.
 Large molecules elute first. Smaller molecules elute later

81
 Size exclusion HPLC …

• The SEC technique is used by 10-15% of chromatographers, mainly

for polymer characterization and for proteins.

• There are two modes:

– non-aqueous SEC [ Gel Permeation Chromatography (GPC)]
• an organic solvent is used to transport the sample through the column

– aqueous SEC [ Gel Filtration Chromatography (GFC)].

• aqueous solution is used as a mobile phase

82
Size exclusion HPLC

Time
83
 Mobile phase (MP)
Must be:
Compatible with the instrument (pumps, seals, fittings,
detector, etc)

Compatible with the stationary phase

Readily available (often use liters/day)

Of adequate purity

Not too compressible (causes pump/flow problems)

Free of gases (which cause compressibility problems)

84
 Mobile Phase in HPLC…

 The elution order of solutes in HPLC is governed by polarity.

 In a normal-phase separation the least polar solute spends proportionally
less time in the polar stationary phase and is the first solute to elute from
the column.
 Retention times are controlled by selecting the mobile phase, with a less
polar mobile phase leading to longer retention times.
 In a reverse-phase separation the order of elution is reversed, with the
most polar solute being the first to elute.
 Increasing the polarity of the mobile phase leads to longer retention times,
whereas shorter retention times require a mobile phase of lower polarity.

85
 What is HPLC used for?
1. Qualitative Analysis
The identification (ID) of individual compounds in the sample;
 the most common parameter for compound ID is its retention time
 the time it takes for that specific compound to elute from the column after injection

 depending on the detector used, the chemical structure, molecular

weight or some other molecular parameter.
 Under identical conditions, the tR of standard and sample are the same
 but for new chemical entity, a single retention-time measurement is
not a very satisfactory parameter.
 because of its dependence on such variables as flow rate, pressure,
and composition of the stationary phase etc.
86
Qualitative Analysis

87
 What is HPLC used for? ….
2. Quantitative analysis
The quantitative determination of a component in HPLC is based upon:
measurement of the recorded peak area or peak height

88
 Quantitative analysis …

The following different methods can be used

1. Internal standard method

The internal standard should meet the following requirements
 it should give a completely resolved peak from the analyte and any
excipient
It should be eluted close to the components to be measured;
 its peak height or peak area should be similar in magnitude to those
of the components to be measured; and
it should be chemically similar to but not present in the original
sample.
89
 Internal std cont….
 The procedure comprises the addition of a constant amount of
internal standard to a fixed volume of several synthetic mixtures;
Which contain varying known amounts of the component to be determined
 a calibration curve is constructed by plotting
 The ratio of component PA to internal std PA against component conc.
 From the observed ratio of PA , the solute concentration is read off using the
calibration curve equation.
Eg. Determination of miconazole cream by using econazole as internal std
Y = 0.048x – 0.006; r = 0.998

90
Internal standard calibration

91
 Quantification cont….
2. Calibration with external standard
 Steps: -
 prepare stock solution ( Reference std)
 Prepare appropriate dilution from the stock to produce a calibration series
of solution
 Inject the calibration solution in to HPLC starting with the lowest conc.
 Plot calibration curve for the area of the peak against the conc. of solution
(r >0.99)
 Prepare formulation for extraction, filtration & dilute the sample
 Inject the sample solution in to HPLC system => peak area
 Determine the concentration of the sample from calibration curve
equation.

92
Data obtained from the analysis of paracitamol std solution by
HPLC
Conc. of Paracitamol std solution Peak area Peak area
(mg/100ml) Ref. std sample
0.5044 17994
1.009 36109 45205
1.513 54121
2.018 71988
2.522 89984
Solution:
45205=35656x + 80
X = 45205-80/35656
X = 1.266mg/100ml
Dilution Step:
25 ml in to 100 ml (4x)
10 ml in to 100 ml (10x)
Total = 40x
Conc. In undiluted sample
= 1.266mg/100ml x 40
93
= 50.64mg/100ml
Single point calibration : the HPLC chromatogram of an extract
from linctus containing pseudoephedrine (30mg), dextromethorphan
(10mg), and methyl paraben. ODS column 25 cm x 4.6mm, MP –
ACN:0.05M phosphate buffer ph=6.8 (35:65), FR = 1 ml/min, UV : 260 nm

Data obtained:
Volume of elixir extracted = 5 ml
Final volume of extract = 100 ml
Conc. of PSD = (318915/325178) x
31.23
5ml of elixir = 30.63 mg/100 ml
Conc. of DXT= (469293/ 479918) x
10.51 94
3. Standard addition

95
 Application of HPLC
• Separation and analysis of non-volatile or thermally-unstable
compounds
• NOTE: If a compound is volatile gas chromatography is a better separation
technique.
– Tablet dissolution of pharmaceutical dosages
– Shelf life determinations of pharmaceutical products
– Identification of counterfeit drug products
– Method of choice for checking the purity of new drug candidates
– Pharmaceutical quality control etc.

96
 Application of HPLC …
• Preparation of Pure Compound(s)
– By collecting the chromatographic peaks at the exit of the detector,
– and concentrating the compound (analyte) by removing/evaporating
the solvent,
– a pure substance can be prepared for later use (e.g. organic synthesis,
clinical studies, toxicology studies, etc.).
– This methodology is called preparative chromatography.

97
 Application of HPLC…
• Trace analysis
– A trace compound is a compound that is of interest to the analyst but
it’s concentration is very low, usually less than 1% by weight, often parts
per million (ppm) or lower;

• the determination of trace compounds is very important in

pharmaceutical, biological, toxicology, and environmental studies since
even a trace substance can be harmful or poisonous;
• in a chromatogram trace substances can be difficult to separate or
detect;
• high resolution separations and very sensitive detectors are required.

98
Application of HPLC…

99
 4. Gas chromatography

Introduction

GC is a technique used for separation and analysis of

volatile substances.
• Gases, liquids, dissolved solids
• Organic and inorganic materials

Separation occurs by interaction of analyte differentially with

stationary phase and temperature

The stationary phase can be either solid (GSC)] or liquid (GLC)

Mobile phase does not interact with analyte

05/30/24 100
 Introduction cont…
• Advantages of GC
– Fast analysis
• Typically minutes

• Can be automated

– Small samples (μl or μg needed)

– High resolution - Record: N ~ 1.3 x 106

– Non-destructive (relatively)

– Allows on-line coupling, e.g. to MS

– Sensitive detectors (easy ppm, often ppb)

05/30/24 101
 Introduction cont…

• Disadvantages of GC
– Limited to volatile samples

• T of column limited to ~ 380 °C

– Not suitable for thermally labile samples

– Requires spectroscopy (usually MS) to confirm the peak identity

05/30/24 102
 Instrumentation

05/30/24 103
 Carrier gas
The mobile phase gas is called the carrier gas and is the used to transport
the sample through the column at a selected steady rate
Commonly used gases include N, He, Ar, and H
Properties of carrier gas:
 It should be Inert and available at low cost
 Does not chemically interact with sample component, column (SP) and
detector
 Compatable with the detector
 No noise or less risk of explosions or fire hazard
 Purity and availability
 Impurities will degrade column and cause noise in detector
 Pressure : inlet 10 to 50 psi
05/30/24 104
 Carrier gas
Selection of the best carrier gas very important, because it affects both the column
separation and detector performance.
performance

He (or H2) is preferred for TCD because of its high thermal conductivity but He is
expensive

N2, though readily available, is not too useful with thermal conductivity detector & not
sensitive.

If FID is used as the detector hydrogen is used as fuel along with air while He or N2
can be used as a carrier gas

N2 - ECD

Hydrogen has the disadvantage of fire and explosive hazard and also react with
unsaturated sample components

05/30/24 105
 Carrier gas
• Impurities in the carrier gas such as air, water vapour and trace gaseous
hydrocarbons can cause sample reaction, column character and affect the
detector performance.
– The carrier gas system should contains a molecular sieve to remove water
and other impurities.

• Carrier gases are available in pressurized tanks.

– Pressure regulators and flow meters are required to control the flow rate of the gas.

• The gases are supplied from the high pressure gas cylinder , being stored
at pressure up to 300psi
• Carrier gas should be better then 99.99% and 99.999% is often used
05/30/24 106
 Flow regulators and meters :
• Flow Rate of Carrier Gas
– Flow regulators are used to deliver the gas with uniform pressure
or flow rate
– Flow rates of carrier gas:
• Linear flow rate (cm/s): u = L/tr
• Volumetric flow rate (mL/min): u= (π r2)
– L is length of column, tr is retention time, r is the internal radius of
column
– Flow rate depends on type of column
• Packed column: 25-100 mL/min
• Capillary column: 1-25 mL/min
– Flow rate will decrease as column T increases
• Viscosity of carrier gas increases with T
107
 Sample injection port
Injection port (Injector)

Located ahead of the column, is designed to allow the introduction of a sample

rapidly and in a reproducible manner in to the column

These are composed of a glass tube where vaporization of the sample takes
place.

The sample is introduced into the injector through a self-sealing silicone

rubber septum.

The carrier gas flows through the injector carrying vaporized solutes.

Provide proper mixing of sample vapor and carrier gas

Prevent non-volatile material from reaching the column

05/30/24 108
 Instrumentation cont…

The temperature of the injector should be

adjusted so that flash vaporization of all
solutes occurs.

If the temperature of the injector is not high

enough, band broadening will take place.
at least 50 degrees above highest
boiling component

05/30/24 109
 Instrumentation cont…

Open tubular columns have very low sample capacity

 to avoid overloading of the stationary phase of Capillary columns
specialized injection system, split/splitless injection, is used.

For best peak shape and max resolution,

resolution the smallest possible sample size
should be used.

05/30/24 110
 Instrumentation cont…
The injector can be used in one of two
modes; split or splitless.
split injection
– Mechanism by which a portion of the
injected solution is discarded.
– to prevent overloading the column with
sample and yet utilize routine syringes.
– Only a small portion (1/1000 - 1/20) of
sample goes through the column
– Used for concentrated samples (>0.1%)
splitless injection
– Most of the sample goes through the column
(85-100%)
– Used for dilute samples (<0.1%)
– Trace analysis

The injector contains a heated chamber

containing a glass liner into which the
sample is injected through the septum.

The septum purge outlet prevents septum

bleed components from entering the
column.

05/30/24 111
 Column

Column Configurations
The column is the “heart” of the
chromatograph.
There are two general types of column,
Packed column (2-5m)
Capillary (also known as open tubular)
column (few meter to 100m)

They are constructed of stainless steel,

glass, fused silica, or Teflon.

05/30/24 112
 Types of column
Packed column
• Packed columns are fabricated from glass, metal (stainless steel, copper,
aluminum), or Teflon tubes that typically have
– Lengths => 2 to 5 m
– Inside diameters =>2 to 4 mm.
• These tubes are densely packed with a uniform, finely divided packing
material,
material or solid support that is coated with a thin layer of the stationary
liquid phase.
phase
– They offer more resistance to the flow of carrier gas
• In order to fit in a thermostating oven, the tubes are formed as coils having
diameters of roughly 15 cm.
• Have high sample loading capacity
– Sample size ranges from 0.1 up to 20 μL.
• Limited resolution (N < 8,000), slow and inefficient

05/30/24 113
 Types of column….
Capillary columns (open tubular column)
 Most common and efficient

 Offer least resistance to the flow of carrier gas

 Hence they are more efficient than packed columns

 Need much less sample, typically around 10-3 µL

 Stationary phase is a thin, uniform liquid film coated on the wall of the
tubing

Capillary columns can be one of two types;

 Wall-coated open tubular (WCOT) or

 Support-coated open tubular (SCOT).

05/30/24 114
 Types of column…..
Wall-coated columns (WCOT) consist of a capillary tube whose walls are
coated with liquid stationary phase.

In support-coated columns (SCOT), the inner wall of the capillary is lined

with a thin layer of support material, onto which the stationary phase has
been adsorbed

05/30/24 115
The stationary phase

116
 The Stationary Phase :

• Hundreds of SP have been used

– Only requirements are:
• Low vapor pressure
• Thermal stability
• Low viscosity (for fast mass transfer)
• High selectivity for compounds of interest

05/30/24 117
 Instrumentation cont…
Column ovens
– Column temperature is very important in GC

– The column is ordinarily housed in a thermostated oven.

– They are usually formed as coils having diameters of 10 to 30 cm.

– The optimum column temperature depends upon the boiling point of

the sample and the degree of separation required.
required
• Roughly, a temperature equal to or slightly above the average boiling point
of a sample results in a reasonable elution time (2 to 30 min).

– If a sample has a wide boiling range, then temperature programming

can be useful.

05/30/24 118
 Isothermal Vs Temperature Programming

Isothermal refers to maintaining constant temperature of the oven

during the chromatographic run

Temperature programming refers to increasing the temperature of the

oven in linear fashion during the chromatographic analysis.

05/30/24 119
 Isothermal Vs Temperature Programming …

Low temperature
Low T • Weakly retained components (low
boiling points) resolved
• Strongly retained components (high
boiling points) not eluted in desired
time
High T
High temperature
• Strongly retained components
eluted and detected
• Weakly retained components poorly
resolved

Programmed temperature
• Temperature is increased during run
• Good retention and resolution for
wide range of boiling points

05/30/24 120
 Isothermal Vs Temperature Programming …

Disadvantages result from isothermal mode of operation.

Early peaks closely spaced (i.e. resolution is relatively poor in this region
of the chromatogram),

whereas late peaks tend to be, broad and widely spaced (i.e. resolution is
excessive).

Compounds of high boiling point are often undetected

particularly in the study of mixtures of unknown composition and wide boiling
point range cpds.

=> Avoided by using temperature programming!!!

05/30/24 121
 Isothermal Vs Temperature Programming …
Programmed-temperature gas chromatography permits
 the separation of compounds of a very wide boiling range more rapidly than
by isothermal operation of the column.
 The peaks on the chromatogram are also sharper and more uniform in shape
so that, they may be used to obtain accurate quantitative analysis

There are a few disadvantages of using temperature programming

This increases the expense for instrumentation.

If the vapor pressure of the stationary phase is too high for the oven
temperature used, then the stationary phase can slowly elute from the
column, a phenomena called column bleed

Column bleed results in high background noise from the detector

05/30/24 122
 Detector
Detectors
A detector uses some property by which it can detect the difference between a
pure carrier gas and a eluted component

The requirements of an ideal detector are:

 Applicability to wide range of samples

 High sensitivity to even small concentrations

 i.e, less response to low concentration and proportional response to high

concentration
 Rapidity of response

 Large Linearity dynamic range

 Nondestructive to the sample in case of preparative work

 Inexpensive operation from RT to 400 oC etc

05/30/24 123
 Detector …
Most commonly used detectors in GC are:
Thermal conductivity detector (TCD)
Flame ionization detector (FID)
Argon ionization detector (AID)
Electron capture detectors (ECD)
MS, etc.

a) Thermal conductivity detector (TCD)

Also called Katharometer or Hot wire detector
The principle is based upon thermal conductivity difference between carrier
gas and that of component
it has two platinum wires of uniform dimensions

05/30/24 124
 TCD Detector …
Through one of them, pure carrier gas always flow through and through
the other, the effluents of the column
The two platinum wires are heated electrically and hence assume
equilibrium conditions of temperature and electrical resistance
When pure carrier gas passes through both of them, there is no difference
in temperature or resistance and hence a base line is recorded.
When a component emerges from the column, it alters the thermal
conductivity and resistance of the wire
Hence this produces a difference in resistance and so conductivity between
two wires, which is amplified and recorded as a signal

05/30/24 125
 Detector …
Advantage of katharometer
Applicability to most cpds (universal
detecctor)
Linearity is good
The sample is not destroyed and hence
used in preparative scale
Simple, easy to maintain and
inexpensive
Disadvantage of katharometer
Low sensitivity (ng/ml)
Affected by fluctuations in temperature
and flow rate
Note:
Hydrogen and helium have higher Diagram of A Thermal Conductivity Detector [TCD].
thermal conductivity and carrier gas
provide best sensitivity
Poorer sensitivity than FID, but more
universal
05/30/24 126
 Instrumentation cont…

Flame ionization detector (FID)

Most widely use H2/air flame

The burner tip made up of platinum capillary, which acts as one electrode
(cathode)

the anode is placed little above the burner tip

When pure carrier gas alone passes, there is no ionization and no current flows

When a component sample emerges from the column, number of ions are
produced because of ionization by the thermal energy of the flame

This cause a potential difference and causes a flow of current which is

amplified and recorded as signal

05/30/24 127
 Instrumentation cont…

Advantage
 changes in mobile phase flow rate do
not affect the detector's response.
is a useful for the analysis of organic
compounds;
it has high sensitivity, a large linear
response range, and low noise.
It is also robust and easy to use
Disadvantage
Destroy the sample
less sensitive for non-hydrocarbon
groups
Insensitive to noncombustible
gases(CO2, SO2, NO2 and H2O) Schematic Diagram of A Flame Ionization Detector [FID].

Insensitive to functional group

(carbonyl, alcohol, halogen and amine)

05/30/24 128
 Instrumentation cont…
Argon Ionization detector (AID)
• this type of detector depends on the exitation of argon atoms to a
metastable state, by using radioactive energy
• this is achieved by irradiating the carrier gas with either α particles or β
particles
• These high energy particles ionize the argon atoms and hence they are
exited to a metastable state
• These molecules collide with the effluent molecules and ionises them
• These ions when they reach the detector will cause an increase in current
• thus the cpds can be detected

05/30/24 129
 Instrumentation cont…
Electron capture detectors (ECD)
Consists of a small chamber with two electrodes and a source of radioactivity
placed close to the cathode
This radioactivity source (nickel-63: radioactive β-emitter-electron) ionizes the
carrier gas (N2)

When potential (2-3 volts) is applied, electrons flow to & collected at the
anode
Produce a steady background current

When solute with a high affinity to electrons - High electronegative group

(halogen, peroxide, quinones and nitro group)] in the sample capture the
electrons.
Causing a fall in current
this fall in current will be recorded
05/30/24 130
 Instrumentation cont…

 ECD is selective in its response and is

highly sensitive to ward electronegative
functional groups such as
 halogens, carbonyl cpds, and nitro groups
 ECD is insensitive to amines, alcohols and
hydrocarbons
 An important application of ECD
 Selectively to halogen-containing organic
sample for the detection and determination of Diagram of an Electron Capture Detector [ECD]

chlorinated insecticides.

05/30/24 131
 Instrumentation cont…

Recorders and integrators

Used to record the responses obtained from detectors after amplification, if

necessary

They can record the individual peaks with retention times, height and
width of peaks, peak area, percentage of area etc.

05/30/24 tr 132
 Pyrolysis and derivatization in GC

GC separation is designed for compounds that can be made volatile while

passing through the GC column

Many samples are, however, unsuitable for direct injection into a gas
chromatograph because of their high polarity, low volatility or thermal
instability

In this respect the versatility and application of gas chromatography can be

greatly extended by

Derivatization

Pyrolysis

05/30/24 133
 Derivatization

Derivatization

is the process of chemically modifying a compound to produce a new

compound which has properties that are suitable for analysis using a GC

Why Derivatization???

To permit analysis of compounds not directly amenable to analysis due to,

for example, inadequate volatility or stability

Improve chromatographic behavior or detectability

The introduction of ECD detectable groups, such as halogenated acyl groups,
allows detection of previously undetectable compounds

05/30/24 134
 Derivatization in GC cont…

The low volatility may result from

the size of the molecule and

the resultant large dispersion forces holding the molecule together.

Some molecules may have a low volatility

due to the strong intermolecular attractions between polar groups.

In such case, masking the polar groups by derivatization can yield

dramatic increases in volatility.

Some compounds, which can be volatilized, undergo partial thermal

decomposition in the GC
so they need to be made more stable.

05/30/24 135
 Derivatization in GC cont…
Main Types of Derivatization

Silylation - Most prevalent method

Introduce trimethylsilyl group to make sample volatile readily

Alkylation

Used as the first step to further derivatizations or as a method of

protection of certain active hydrogens

Acylation

commonly used to add fluorinated groups (ECD)

05/30/24 136
 Derivatization in GC

Pyrolysis

Pyrolysis is the breaking of chemical bonds through the use of thermal

energy
For compounds of high molecular mass, the formation of derivatives does not
help to solve the problem of involatility

This difficulty may be overcome by breaking the large molecules up into

smaller and more volatile fragments
Which may then be analysed by gas-liquid chromatography

Technique known as pyrolysis gas chromatography (PGC)

05/30/24 137
 Application of GC
Qualitative Analysis by GC

Retention time data should be useful for identification of mixtures

Comparing the retention time of the sample as well as the standard

 Checking the purity of a compound: compare the standard and sample

 Additional peaks are obtained…..impurities are present….compound is not pure

GC is much less useful, however, as a tool for identifying components

A single retention-time measurement is not a very satisfactory parameter upon

which to base qualitative analysis
 because of its dependence on such variables as column temperature, flow rate,
pressure, and composition of the stationary phase

05/30/24 138
 Quantitative Analysis by GC

The quantitative determination of a component in GC is based upon

measurement of the recorded peak area or peak height

In order that these quantities may be related to the amount of solute in the
sample two conditions must prevail:
 the response must be linear with respect to the concentration of the solute

 factors such as the rate of carrier gas flow, column temperature, etc., must be
kept constant or the effect of variation must be eliminated, e.g. by use of the
internal standard method
The following different methods can be used

05/30/24 139
 Quantitative Analysis by GC

• Quantitative analysis:
– Direct comparison method: -comparing the area of the peak, peak
height, width of peak.
– Calibration curves: -standards of varying concentration are used
determine peak areas .
– Internal standard method: -A known concentration of the internal
standard is added separately to the standard solution -The peak area
ratio of sample and internal standard….
 unknown concentration is easily determined .

05/30/24 140
 Quantitative Analysis by GC cont...
Area normalization

In area normalization, the composition of the mixture is obtained

 By expressing the area of each individual peak as a percentage of the total area
of all the peaks in the chromatogram;

Correction should be made for any significant variation in

 Sensitivity of the detector for the different components of the mixture

In a mixture containing component X, Y and Z, the %X can be give

where, A = Peak area, and
f = Response factor

where, W = Weight or conc. of component in the mixture

r = A reference component present in the mixture which
is assumed to have response factor of unity.

05/30/24 141
 %WS= PA S/PARS x %WRS
05/30/24 142
 Quantitative Analysis by GC cont...

Internal standard method

fluctuations of the carrier gas flow rate, the column and detector
temperatures can affect the quantitative result

The effect of such variations can be eliminated by use of the internal

standard

05/30/24 143
 Quantitative Analysis by GC cont...

The procedure comprises the addition of a constant amount of internal

standard to a fixed volume of several synthetic mixtures
 which contain varying known amounts of the component to be determined

a calibration curve is constructed by plotting

 the ratio of component peak area to internal standard peak area against component
conc.

The analysis of the unknown mixture is carried out by

 addition of the same amount of internal standard to the specified volume of the
mixture

From the observed ratio of peak areas the solute concentration is read off
using the calibration curve

05/30/24 144
 Reference std
Internal standard spiked with Int.
std
Reference standard

Sample spiked with internal std

where WC and WA = weights of C and A, respectively, and AC and AA are the areas of C
and A, respectively; R is the response ratio. Since the weight of A added to the sample is
known, the weight of C in the sample can be calculated:
( AC A [Link] ) sample
WC   [Link]
( [Link] [Link] ) [Link]

05/30/24 145
 Applications

• Elemental analysis
– Determination of C,H ,O ,S and N .

– Determination of mixture of drugs

– Isolation and identification of drugs

– Isolation and identification of mixture of

components(amino acids ,plant extracts ,volatile oils)

05/30/24 146