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Part 3 Chromatography
Introduction
In 1903 M.S. Tswett described a technique for the separation of plant
pigments. He called this technique “chromatography" (derived from
the Greek word which means colour writing). Today chromatography
encompasses a diverse group of methods, which permit the separation,
isolation, and identification of the components in a mixture. These
separation methods include paper chromatography (PC), thin layer
chromatography (TLC), gas chromatography (GC) and liquid
chromatography (LC).
Basically, chromatography is a physical separation technique, which
resolves the individual components of a mixture based on their
distribution between two immiscible phases:
a- Stationary phase (adsorbent). It is a solid porous media, which
consists of the rigid porous particles, usually silica based, with the
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specific surface properties (surface chemistry). It is non-moving and
may exist in a variety of forms (e.g., bed, layer, column).
B- Mobile phase (eluent): It is a liquid solvent or mixture of solvents, which
is moving through the stationary phase or chromatographic column
and carrying analytes. It may be either a gas (i.e., GC) or a liquid (i.e.
LC).
The chromatographic process occurs as the result of the repeated
sorption-desorption of the sample components as they move along the
stationary phase. When the individual component favors the stationary
phase, it is held longer and moves more slowly through the column. If
the velocities and hence the distribution coefficients of samples
components are different, the mixture could be resolved. The
distribution coefficient (Ka) is defined as:
𝐾𝑎 =Concentration of solute in stationary phase
Concentration of solute in mobile phase
The oldest form of liquid chromatography is the column
chromatography. In classical column chromatography the columns
were open tubes (e.g., burettes), which were individually packed with
coarse material. The mixture to be separated is loaded onto the top of
the column followed by more solvent. Different components in the
sample mixture pass through the column at different rates due to
differences in their partitioning behavior between the mobile phase
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and the stationary phase. Compounds are separated by collecting
aliquots of the column effluent as a function of time. Flow of the mobile
phase was achieved by gravity feeding; and components were
collected as colored fractions (Fig.1).
In the 1960's researchers began to look for ways to improve liquid
chromatography and developed high performance liquid
chromatography (HPLC). HPLC: means high performance liquid
chromatography, which refers to high speed, high-resolution
separation. Initially, pressure was selected as the principal criterion of
modern liquid chromatography and thus the name was "high pressure
liquid chromatography" or HPLC. This was, however, an unfortunate
term because it seems to indicate that the improved performance is
primarily due to the high pressure. This is not true because naturally,
pressure is needed only to permit a given flow rate of the mobile phase;
otherwise, pressure is a negative factor not contributing to the
improvement in separation.
Figure 1 Schematic of
a simple liquid
chromatographic
separation
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Comparison between classical and modern LC:
Modern LC Classical LC
1- Column:
- Stainless steel columns
- Small diameter (2-5 mm). Reusable.
- Packing with very small (3, 5 and 10
mm) particle stationary phase.
Open tube ex: a burette
-Large diameter (1-4
cm).
- Prepared each time.
-Packing with
coarse particles.
Stationary phase:
- Continuous development of new
substances to be used stationary
phase.
- Only few materials are
used as stationary phase
ex: silica and alumina.
2- Volume of sample:
- Precise sample introduction (in ul)
using micro-syringe
-Large amounts of
sample are loaded on
top of column.
4- Flow of mobile phase:
- Relatively high inlet pressures and
controlled flow of mobile phase.
-Flow of mobile phase is
achieved by specific
gravity.
5- Detection of sample:
- Special continuous flow detectors
capable of handling small flow rates
and detecting very small amounts of
samples. Then a final chromatogram is
obtained with all the information data.
-Coloured samples are
washed out and
collected in fraction,
then analyzed
separately.
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6- Resolution of sample:
- High resolution.
- Resolution is not good.
7- Analysis:
- Analysis is very rapid (in few minutes).
- Analysis is very slow.
8- Instrument:
- Automated standardized instrument
is used
- It is just a simple
burette.
II- Classification of chromatographic methods:
1- According to the states of the two phases:
Usually the mobile phase is named first
(a) Liquid-solid chromatography (LSC).
(b) Liquid-Liquid chromatography (LLC)
(c) Gas- solid chromatography (GSC).
(d) Gas- Liquid chromatography (GLC)
2-According to the shape of stationary bed:
(a) Plane or flat-bed chromatography: The stationary bed is
coated on a flat surface. The two common types of plane
chromatography are paper chromatography (PC) and thin
layer chromatography (TLC).
(b) Column chromatography: The stationary bed is contained in a
column. Examples are open-column chromatography (OCC),
gas chromatography (GC) and high pressure liquid
chromatography (HPLC).
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(c) 3- According to the mechanism of chromatographic
process:
According to the type of equilibration process involved between
the mobile and the stationary phases, chromatographic methods
are classified into:
(a) Adsorption chromatography:
This uses a solid stationary (like silica gel or any other silica based
packing) where the sample components are adsorbed. The mobile
phase may be a liquid (liquid-solid chromatography) or a gas (gas-solid
chromatography). The components distribute between the two phases
and the separation is based on repeated adsorption-desorption steps.
Equilibration between the
adsorbed state and the solution
accounts for the separation of
sample components, e.g. TLC,
GSC, and LSC. (Fig.2) Two
modes are defined depending
on the relative polarity of the
two phases (Fig.3). These are:
Fig. 2: Adsorption
chromatograph
1- The normal phase chromatography: in which the stationary bed
is strongly polar in nature (e.g., silica gel), and the mobile phase is
non polar (such as n-hexane or tetrahydrofuran). Polar samples
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are thus retained on the polar surface of the column packing
longer than less polar materials.
Fig.3: Graphical illustration of normal and reversed-phase
chromatography. Circles represent types of compounds present in
the sample; their relative position to direction of mobile phase flow
indicates their order of elution.
2- The reversed-phase chromatography: which is the inverse of normal
phase. The stationary bed is non polar (hydrophobic) in nature,
while the mobile phase is a polar liquid, such as mixtures of water and
methanol or acetonitrile.
Here the more non polar the material is, the longer it will be retained.
(b) Partition chromatography:
-In which the stationary phase is a liquid supported on an inert solid. The
mobile phase may be a liquid (liquid-liquid chromatography) or a gas
(gas-liquid chromatography).
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-Solute equilibrates between the stationary liquid and the liquid or
gaseous mobile phase, e.g. paper chromatography (PC), GLC and
LLC.
-PC is a type of partition chromatography in which the stationary
phase is a layer of liquid adsorbed on a sheet of paper.
This form of
chromatography is based
on a thin film formed on
the surface of a solid
support by a liquid
stationary phase. Solute
equilibrates between the
mobile phase and the
stationary liquid.
Partition Chromatography
(c) Ion exchange chromatography (IEC)
-In which an ion exchange resin is used as the stationary phase. The
stationary bed has an ionic charged surface of opposite charge to
the sample ions.
-Anions (such as -SO3─) or cations (such as -N (CH3)s+ are covalently
attached to the stationary solid phase, usually called a resin. Solute ions
of opposite charge are attached to the stationary phase by
electrostatic force.
-This technique is used almost exclusively with ionic or ionizable
samples. The stronger the charge on the sample, the stronger it will
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be attracted to the ionic surface and thus, the longer it will take to
elute. The mobile phase is an aqueous buffer, where both pH and
ionic strength are used to control elution time.
-Resins containing the active group -S03─ are called cation
exchangers, since only cations can be attracted to it. Those
containing the quaternary ammonium ions are called anion
exchangers, since only anions can be attached to it. The mechanism
of separation is based on ion exchange equilibrium as illustrated in Fig.
4 a & b.
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(d) Size exclusion chromatoqraphy (SEC)
-In which the stationary phase is sieve like structure and the solvated
molecules (the sample) are separated according to their size by their
ability to penetrate to this stationary phase (Fig.5). There is no attractive
interaction between the stationary phase and the solute.
-The column is filled with material having precisely controlled pore
sizes, and the sample is simply screened or filtered according to its
solvated molecular size.
- Larger molecules are rapidly washed through the column; smaller
molecules penetrate inside the porous of the packing particles and
elute later.
-Mainly for historical reasons, this technique is also called gel filtration
or gel permeation chromatography although, today, the stationary
phase is not restricted to a "gel".
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Fig. 5: Schematic of a size exclusion chromatography column
III- Theoretical aspects:
A- Principles of chromatography
While the mechanisms of retention for various types of chromatography
are different, they are all based on establishment of a solute equilibrium
between a stationary and a mobile phase. The more the solute
interacts with the stationary phase, the slower it is moved along the
column (component B in Fig. 6).
If a detector that response to solute concentration is placed at the end
of a column and its signal is plotted as a function of time (or volume) of
added mobile phase, a series of symmetric peaks is obtained as shown
in Fig. 6(b). Such a plot is called a chromatogram
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Fig.6: (a) Diagram showing the separation of a mixture of
components A &B by column elution chromatography. (b) The
out put of the signal detector at various stages of elution shown
in (a)
B- Parameters of chromatoqraphy:
Chromatography has a number of parameters and
equations, which qualitatively and quantitatively describe the
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shape, position, and resolution of the individual sample
components.
When mobile phase or eluent emerges from the stationary bed, it is
called eluate
The process of passing liquid or gas through a stationary bed is
called elution.
1- Chromatogram is obtained by plotting detector response (or
signal) as a function of time or volume of mobile phase
added.
The greater the amount of solute passing through the
detector, the greater the detector signal. Thus peak height
(PH or hp) and peak area are used as an indication of solute
concentration. (Fig. 7)
2- Peak height is measured from the base line of the
chromatogram to the peak maximum.
3- Peak area = peak height times the peak width at half height.
As shown in Fig. 7 the separation is effected as the various
components elute from the bed at different times.
4- Retention time (tR)
The retention time (tR) is the time required for a component solute
to elute from the column. The retention time is measured from the
start of the chromatogram (i.e. from injection of a sample) to the
appearance of a solute peak, or to maximum height of the solute
peak. (Fig.7).
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5- Retention volume (VR)
The retention volume (VR) is the volume of effluent gas or liquid
required to flow through the column to elute the sample
component. It is also measured from the start of the
chromatogram to the peak corresponding to the given
component.
Similarly the void volume (v0) is the amount of mobile phase
required to elute unretained component from the column.
The relation between retention time and volume is expressed as
follows:
VR = tR F
Where F is the flow rate of the mobile phase and it is expressed in
ml / min.
Usually a very small peak appears in the chromatogram when the
mobile phase elutes from the column. This peak is called air peak
in GC and solvent peak in HPLC. Its time is called dead time t0 and
its volume is termed dead volume V0.
6- Dead time (to)
The dead time (to) or void time is the time required for an
unretained component to pass through a column (Fig. 7).
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Fig. 7: Peak height, peak area, retention time and dead time
7- Void volume (v0) is the amount of mobile phase required
to elute unretained component from the column. When the
retention time, tR, is corrected for dead time, to, the adjusted
retention time tR' is obtained.
8- Adjusted retention time (tR'): When the retention time, tR, is
corrected for dead time, to, the adjusted retention time, tR', is
obtained and is calculated as follow:
tR' = tR – t0
The corresponding required volume is the adjusted retention
volume (VR').
VR' = VR – V0
VR’ = tR’ . F
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9- Partition ratio or the capacity factor (K')
Another important term is the partition ratio or the capacity
factor (K'), which is used to describe the migration rates of
solutes on columns.
K' = tR '/to = (tR-to)/to
= VR /Vo = (VR-Vo)/V0
When the capacity factor for a solute is much less than unity,
elution occurs too rapidly that accurate determination of the
retention times is difficult.
When the capacity factor is larger than perhaps 20 to 30, elution
times become inordinately long. Ideally, separations are
performed under conditions in which the capacity factors for the
solutes in a mixture are in the range between 1 and 5. In liquid
chromatography, capacity factors can often be
manipulated to give better separations by varying the
composition of the mobile phase and the stationary phase. The
capacity factors in gas chromatography can be varied by
changing the temperature and the column packing. In PC and
TLC, retention data are expressed using the "retardation factor";
Rf value
10- Retardation factor (Rf) value
It is defined by the following equation:
Rf=Distance migrated by the solute
Distance migrated by the solvent=
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Distance from the starting line to the spot center
Distance from the starting line to the solvent front
The Rf value for each spot should be calculated and it is
characteristic for any given compound. Hence, known Rf
values can be compared to those of unknown substances to
aid in their identifications.
Rf value is usually equal to less than unity. The more sample is
retarded by the bed; the smaller will be its Rf value (Fig. 8).
11- Separation and resolution
Since in chromatography we are analyzing multi-component
samples, separation of the individual components is of prime
importance. Therefore we must investigate the separation of two
adjacent peaks. This can be expressed in two ways: the first
describes their position relative to each other (selectivity factor
a), while the other indicates the degree of resolution (resolution
Rs).
115 Instrumental analysis
a- Selectivity factor or separation factor ()
The separation factor or selectivity factor () describes the relative
position of two adjacent peaks.
01
02
'
1
,
2
'
1
'
2
01
02
'
1
,
2
'
1
'
2
'
1
,
2
'
1
,
2
'
1
'
2
K
K
K
K
K
K
VV
VV
t
t
tt
tt
t
t
V
V
t
t
R
R
R
R
R
R
R
R
R
R
R
R
Where K'2 is the partition ratio for the more strongly retained species 2 and
K'1 is the partition ratio for the less strongly held or more rapidly eluted
species 1. According to this definition, is always greater than unity.
If the two peaks coincide i.e. = 1, no separation occurs; ideally should
be at least 1.25.
b- Column resolution (Rs)
The separation factor expresses relative positions of the two peaks;
however it does not give any information on actual separation of the two
peaks. The resolution Rs of a column provides a quantitative measure of
its ability to separate two analytes. The resolution of each column is
expressed as the ratio of the distance between the two maxima ( t) to
the mean value of the peak width at base.
116 Instrumental analysis
Where tR2 & tR1 are the retention times for the latest and the earliest eluting
peak and W1 and W2 are the peak widths of the two components at
baseline (Fig.9).
Fig.9 Resolution of adjacent peaks.
The significance of this term is illustrated in Fig. 10, which consists of a
chromatogram for species A and B on four columns with different resolving
powers. The resolution should exceed 1.5.
117 Instrumental analysis
1. Gas chromatography
Principle:
A pressured gas flows through heated tube coated with
liquid stationery phase or packed stationery on a solid
support. The analyte loaded on the head of the column via
heated injection port, where it is evaporated. The separation
of a mixture occurs according the relative time spent in the
stationary phase
Instrumentation
1. Injection of the simples manually or using autosampler
usually size of 0.5 – 2 ul injection volume
2. The sample is evaporated and condensed at the head of
the column
3. The column either capillary or packed column, the mobile
phase is a gas to carry the sample through the column
which is Helium or nitrogen gases.
4. The oven to heat the column up to 400 oC.
5. The detector usually flame ionization detector FID
Types of columns
1. Packed columns:
Usually glass columns silanised to remove Si….OH groups
which make tailing of peaks of polar analytes, the column
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have internal diameter of 2.5 mm, and coated with different
liquid stationary phase. The column mobile phase used in
PCGC is nitrogen at flow rate of 20 ml/min.
Limitation, cannot be used above 280 oC because of the
evaporation of the stationary phase
Stationery phase for GC
2. Capillary column
These are made of fused silica coated with polyamide to
give flexible columns
The internal diameter usually 0.15 and 0.5 mm
The inner surface is coated with orange silicon polymers
which are chemically bonded to silanol groups
The mobile phase used usually Helium at low flow of 0.5 to 2
119 Instrumental analysis
ml/min
Separation of a Mixture
Factors governing the retention of compounds in capillary GC;
1. Carrier gas type and flow: Nitrogen and helium give high
flow rates
2. Column temperature: increase of column temperature,
decreases resolution between two compounds, due to
decreases of interaction with the stationary phase as the
vapour pressure of the analyte increases
120 Instrumental analysis
3. Column length: increase the column length increases the
resolution
4. Film thickness phase loading: the greater the volume of the
stationary phase the more solutes will be retained
5. The column internal diameter: the smaller the diameter the
more efficient of the column for a given thickness due to
the good mass transfer.
Detectors for GC
1. Flame ionization detector FID
2. Electron capture detector ECD
3. Nitrogen phosphate collectors
4. Thermal conductivity detectors TCD
1. Flame ionization
detector FID
Compounds burned at
the detector produced
ions
Detects carbon –
hydrogen compounds
till 10 ng
Wide application range up to 10-6
2. Electron capture detector ECD
Highly halogenated compounds can be detected at 50 fg – 1
pg
121 Instrumental analysis
Wide application for drugs determination in biological fluids.
Have wide application in environmental analysis such as
chlorofluorocarbons in the air
3. Nitrogen phosphate collectors
Used for compounds containing nitrogen and phosphors such as
drugs and metabolities in body tissues and fluids
High selective
4. Thermal conductivity detectors TCD
Responding to cooling effect of the analyte passing over
filament
Insensitive, used for determination of water vapour such as in
peptides
Application of GC
1. Detection of impurities in drug formulation
2. limit test for solvent residues and other volatile impurities
3. used for quantification of drug substances in formulation
specially for drugs lack of chromophore
4. characterization of some row material used for drug
synthesis
5. measurements of drugs and their metabolites in biological
fluids
122 Instrumental analysis
Limitation of GC
1. only thermostable compounds can be analysed
2. the sample may require derivatisation to be volatile
3. quantitative sample introduction is more difficult due to the
small volume of sample injected
Derivatization: GC is limited to compounds
capable of being volatilized without
undergoing decomposition i.e. compounds
possessing an appreciable vapour pressure. The
technique is extended by the preparation of
volatile derivatives of the nonvolatile compounds or of the
compounds, which undergo decomposition.
Derivatization may be used also for improvement of peak shape,
relocation of an interfering peak, improvement of sensitivity or
improvement of separation of closely related compounds. An
example of derivatization is silylation by addition of trimethylsilyl
group to carboxylic acids, amines, imines, alcohols, phenols and
thiols by treatment with hexamethyldisilazane.
123 Instrumental analysis
Amines, alcohols and thiols may be acylated using acid anhydrides in
a solvent such as pyridine (to bind the acid produced). Anhydrous
conditions are essential since derivatives are easily hydrolysed.
2- High pressure liquid chromatography HPLC
The conventional open-column liquid chromatography is inefficient
and extremely slow, requiring from 30 min to several days for an
effective separation while GC is much more rapid and efficient, its
direct applications are limited to those samples which have a low
vapour pressure and may be heated without decomposition. The
disadvantages of these techniques are rapidly overcome with HPLC.
A schematic diagram of a typical HPLC unit is shown in Fig. 17.
The system consists of main parts:
1- Mobile phase or solvent reservoir.
2- A high pressure pump.
3- A sample inlet port.
4- Column
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5- Detector
6- Recorder
Fig. 17: Schematic diagram of an HPLC unit
(1) Solvent reservoirs, (2) Solvent degasser, (3) Gradient valve,
(4) Mixing vessel for delivery of the mobile phase, (5) High-
pressure pump, (6) Switching valve in "inject position", (6')
Switching valve in "load position", (7) Sample injection loop, (8)
Pre-column (guard column), (9) Analytical column, (10) Detector
(i.e. IR, UV), (11) Data acquisition, (12) Waste or fraction
collector.
The pump, capable of maintaining high pressures draws the solvent
(mobile liquid phase) from the reservoir and pushes it through the
column. The sample is injected through a port into the high pressure
liquid carrier steam between the pump and the column. The
separation takes place on the columns, which vary, from 25-100 cm
length and 2-5 mm in internal diameter. Typical flow rates are 1-2
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ml/min with pressures up to several thousand psi. The column effluent
passes through a non-destructive detector where a property such
as UV absorbance, Rl or molecular fluorescence is monitored
amplified and recorded as a typical detector response vs retention
time chromatogram. The effluent may be either discarded or saved
for further studies in a fraction collector, which is synchronized with
the detector.
To increase the efficiency of separation, the mobile phase may be
altered by changing its polarity, pH or ionic strength. HPLC offers the
advantages of speed, resolution and sensitivity.
There are two types of HPLC procedures:
1- LLC: the column consists of an inert support usually silica gel on
which the stationary partitioning phase is adsorbed. The mobile
phase flowing through the column is in contact with the stationary
phase. Equilibrium distributions of the solute between the two phases
take place rapidly. In the normal phase mode, the stationary phase
is polar (e.g. methanol, acetonitrile or water) while the mobile phase
is less polar (e.g. iso-octane, chloroform or n-hexane). This mode is
usually used for the separation of polar components. In the reverse
phase LLC, the stationary phase is less polar and the mobile phase is
polar. It is usually used for the separation of non-polar components.
2- LSC: The packing may be silica (polar packing) or octadecylsilica,
ODS (C18-silica, non-polar packing). Adsorption mechanism is
126 Instrumental analysis
involved here. In the normal phase LSC, the packing is polar (silica)
and the mobile phase is less polar (e.g. n-hexane). In the reverse
phase LSC, the packing is non-polar (eg. ODS) and the mobile
phase is polar (e.g. acetonitrile-water or methanol-water). Again,
as under LLC, normal phase LSC is used for polar solutes while reverse
phase LSC is used for separation of non-polar compounds. There are
two types of elution in HPLC:
I- Isocratic elution: Using one and the same solvent during the whole
chromatogram.
Il-Gradient elution: To effect separation of complex mixtures, the
polarity of the mobile phase is increased after certain time increments.
This is usually done in a mixing chamber before the pressure pump.
3- Supercritical fluid chromatography (SFC)
HPLC and GC are complementary. HPLC does not require the analyte
to be volatile or thermally stable, but it lacks the universal-type
detectors of GC. The technique of SFC is intermediate between these
two and offers advantages of both. A great advantage is the ability
to use FID for the measurement of non-volatile analytes. Some
important definitions are given below.
A supercritical fluid: is a substance above its critical temperature
and pressure.
Critical temperature (Tc): is that above which it is impossible to
liquefy a gas, no matter how great a pressure is applied.
Critical pressure (Pc): is the minimum pressure necessary to
127 Instrumental analysis
bring about liquefaction at Tc.
Critical volume (Vc): is the volume occupied by one mole of
gas or liquid at the critical temperature and pressure.
It is apparent from the phase diagram of CO2 (Fig. 18) that at
temperatures of 31 °C and above with pressures of 75.3 atm. or above,
CO2 exists as a supercritical fluid. Some substances used as
supercritical fluids are listed in Table 1 with their critical parameters.
Concerning the typical physical properties, supercritical fluids
are intermediate between gases and liquids.
The densities of supercritical fluids are nearer those of liquids but
viscosities are closer to those of gases.
The diffusion coefficients of substances in supercritical fluids are
much less than those in gases and much more than those in liquids.
Fig. 18: Phase diagram of carbon dioxide.
128 Instrumental analysis
Table 1: Critical parameters of some substances commonly used
as supercritical fluids.
Compound
Tc, °C
Pc, atm.
Carbon dioxide
31.05
72.9
Nitrous oxide
36.4
71.5
Ammonia
132.4
111.3
2-Propanol
235.1
47.6
Methanol
239.4
79.9
Acetonitrile
274.8
47.0
Water
374.1
217.6
An advantage of supercritical fluids as mobile phases in
chromatography compared with liquid chromatography is that
solutes generally have much higher diffusion coefficient in them
than in liquids. This leads to enhanced speed of separation and
possibly greater resolution with complex mixtures, especially for
large molecules. SFC possesses also advantages over GC in that
solutes do not have to be volatile or thermally stable.
Mobile phases used for SFC are frequently cooled to maintain them
in a liquid state for easier pumping to a column, which is heated in
an oven above the critical temperature (100-200°C). The most
common supercritical fluid used in chromatography is CO2, because
of its compatibility with FID, and because of its low Tc and non-toxic
nature.
129 Instrumental analysis
4. Electrophoresis (Electrochromatography)
Electrophoresis involves the differential migration of charged species
in an electrolyte solution under the influence of an applied potential
gradient. A supporting medium is used to provide an inert porous
structure for the electrolyte solution. Filter paper and polymerized
cellulose acetate are used as sheets while agar, starch and
polyacrylamide are used as gels. Both are used in the form of thin flat
beds or in columns. This technique is widely used for charged colloidal
particles or macromolecular ions such as those of proteins, nucleic
acids, enzymes and polysaccharides. The rate of migration
(electrophoretic mobility) of each species is a function of its charge,
shape and size. Other factors actually affect the migrate rate of
charged particles e.g. the applied voltage, electrolyte concentration,
ionic strength, pH, temperature, viscosity of the electrolyte solution
and other physicochemical properties of the migration medium but
these are kept constant. Apparatus and Methodology:
In zone electrophoresis, sheets of filter paper moistened with the
electrolyte solution (usually a buffer) and stretched horizontally
between two electrodes vessels to which a potential difference is
applied are usually employed. The sample is placed in the centre of
the strips or the strip is sandwiched between two glass plates in order
to avoid warming and evaporation of the electrolyte. Application of a
direct current potential across the solution at 100-300 V for a period of
130 Instrumental analysis
time. Cations migrate towards the cathode (negative electrode) and
anions to the anode (positive electrode). Carbon and platinum
electrodes are usually used. The components of the mixture are
separated into individual bands or spots. Detecting techniques such
as spraying with a chromogenic reagent or staining with a dyestuff
are then used to visualize the developed electropherogram.
Cellulose acetate has the added advantage that it can be made
water clear by treatment with oil or with a mixture of acetic acid
and ethanol. Gel electropherograms cannot be dried before
spraying or dipping, and precipitant is incorporated into the
visualizing reagent to prevent materials dissolving during treatment.
A schematic diagram of a paper electrophoresis apparatus is
shown in Fig. 19.
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Electrophoresis differ from chromatography in that only a single phase
is involved, i.e. the electrolyte solution which essentially remains
stationary on the supporting medium.
High Performance Capillary Electrophoresis (HPCE):
This is a recent development that is proving to be a very powerful
separation technique of growing importance. It involves high voltage
electrophoresis in narrow bore fused-silica capillary tubes and on-line
detectors similar to those used in HPLC. Components of the mixture
injected into one end of the capillary migrate along it under the
influence of the electric field (potential gradient) at rates
determined by their electrophoretic mobilities. On passing through
the detector, they produce response profiles that are sharper than
chromatographic peaks. A schematic diagram of an HPCE system
is shown in Fig. 20. Potentials of between 10 and 30 KV are applied
across the capillary during electrophoresis. The on-line detector
positioned close to the cathodic end of the capillary is commonly
a UV absorbance or fluorescence monitor. Several types of
conductivity, amperometric and mass spectrometric detectors
have also been designed. Samples are injected into the capillary at
the opposite end from the detector. A very small volume of the
sample solution is introduced (usually 1-50 µl).
132 Instrumental analysis
Fig 20 Schematic of capillary
electrophoresis
Schematic of the double
layer on the capillary
surface
Quantitative chromatographic analysis
(a) For Open-column Chromatography: Two techniques are used
1- Fraction analysis: this is performed on the effluent, which is
collected in fractions in graduated receivers. When many small
fractions are to be collected, an automatic fraction collector is
employed. The concentration of components in the fractions is
estimated by titration or spectrophotometry.
2- Extrusion analysis: when the zones are completely resolved on the
column, the eiuent is stopped. The chromatogram column is
extruded out, cut into sections, every one containing a zone, and
the component is extracted for analysis.
133 Instrumental analysis
(b) For PC, TLC and Zone Electrophoresis: Two
techniques are used
1- Elution method: after location and identification, the spots are
cut from the chromatogram, eluted by a suitable solvent
(eluent) and the recovered component is estimated in the
eluate by UV or VIS spectrophotometry.
2- Densitometry: This is based on measuring the fluorescence or
light absorption properties of each component of the mixture
(Fig.21) directly on the paper or silica or cellulose acetate
after visualization.
Fig. 21: Block diagram of an absorption densitometer
(c) For GC, HPLC and HPCE:
The size of a single component peak is proportional to the
quantity of the detected component. Peak size is estimated
either by measuring peak height (the distance from baseline to
peak maximum) with a ruler or by measuring peak area with a
134 Instrumental analysis
planimeter, a digital electronic integrator or a microcomputer.
After peak size measurement, data calibration is carried out
using one of the following methods:
1-External standard method: a calibration plot is first constructed
based on samples that contain known concentrations or weights
of the compounds of interest. A fixed volume of each standard
is then injected and processed as in the assay procedure.
Peak size is plotted versus concentration or weight. The
concentration of the unknown is obtained from the linear
standard plot. (Fig. 22).
2-lnternal standard method (more reliable): a known weight of
an internal standard is added to each of a series of known
sample weights. This known compound is used as an internal
marker to compensate for the effects of minor variations in
135 Instrumental analysis
separation parameters on peak size.
The internal standard must be similar in structure to the sample
components and the retention times of the standard and
sample components must be close to each other but must be
resolved from each other.
A linear calibration graph is prepared by plotting the compound
to internal standard peak height or peak area ratio versus the
concentration of the compound.
The weight of unknown is determined from the graph as shown
in Fig.23.
136 Instrumental analysis
Instrumental analysis PC 407 Clinical Pharmacy - Faculty of pharmacy – Zagazig University
Periodic examination for second year student for clinical pharmacy Name of student: …………
Try to solve the following Problems:
1. Balance the following redox reactions using the half reaction method.
MnO4- + Fe2+ = Mn2+ + Fe3+
2. What is the Oxidation Number of iron in the following compounds?
K4Fe(CN)6, K3Fe(CN)6, Fe2O3, Fe,
2. If a Cu/Cu2+ (0.01N) electrode is connected to a Zn /Zn2+(0.1N) electrode,
what reaction occurs? What is E°cell? Write the schematic digram represent
the battery, label the cathode and anode and show the direction of electron
flow.(EoCu/Cu2+=+0.34 , Eo
zn/zn2+=-0.76)
3. NHE, mention the half redox reaction, Nernst equation, advantage and
disadvantage and draw Sketch for it.
Instrumental analysis PC 407 Clinical Pharmacy - Faculty of pharmacy – Zagazig University
Periodic examination for second year student for clinical pharmacy Name of student: …………
Complete the following statement:
1. The wave number is defined as:……………………………… …………………… ……………………
2. The energy of the photon (E) is: :……………………………… ……………………
137 Instrumental analysis
…………………… 3. Types electronic transitions is an organic molecule are:
:……………………………… …………………… …………………… 4. Absorption spectrum is a plot of:……………………………… ……………………
…………………… 5. Bathochromic and blue shifts are :……………………………… ……………………
……………………
And give examples of red shift and blue shift 6. Chromophore is :……………………………… …………………… …………………… 7. Beers- Lambert’s law is formulated as follow :………………………………
…………………… …………………… 8. The general requirements of the coloured product to be analyzed
colorimetry are :……………………………… …………………… ……………………
138 Instrumental analysis
Student Name: ……………………………………………….. Instrumental Analysis (PC 407) Practical Exam May 2011
Q.1 Determination of iron samples using NH4SCN A- Construction of Calibration Curve and calculating Regression equation In test tube Pipette (0.5-2.0 ml (50 µg-200 µg)) standard iron solution + 2 ml dil. HNO3 --- boil -- cool ---- add 3 ml NH4SCN reagent and complete to 10 ml with distilled water--- measure absorbance.
Regression Equation: …………………………………………..
B- Determination of Unknown sample
Sample No. Absorbance Conc.
…………………. …………………… …………………..
Q.2 Chromatographic Separation of mixture of dyes
Separation is done using 90% methanol and 10% chloroform as mobile phase to separate the sample spotted over a filter paper.
Concentration
µg/ml Absorbance
50 …………….
100 ……………
150 …………
200 ………
139 Instrumental analysis
Zagazig University Faculty of pharmacy Clinical Pharmacy PC 405
Instrumental methods of analysis Time allowed 1 hours 24/8/2010 – Summer Corse
Q1: Tabulate the most suitable answer
1 6 11 16
2 7 12 17
3 8 13 18
4 9 14 19
5 10 15 20
1. The plots of absorbance against concentration is called: a) Calibration curve b) Polarogram c) Spectrum d) None of the above 2. Phenol in sodium hydroxide exhibits: a) Red shift b) Hyperchromic effect c) Hypochromic effect d) Both a and b. e) Both a and c
3. The energy of photon (E) is related to "C" , "" and ""
a)
hcE b) E =hc
c) E= h d) none of the above 4. Beer- Lambert’s law may be formulated as:
a) bC aI
Ilog o b) A=abC
c) bC εabcI
Ilog o d) all of the above
5. Monochromator is act as
a) Wave length selector b) Electron transducer. c) Source of radiation d) None of the above
140 Instrumental analysis
6. Grating is functioning by: a) Diffraction and interference b) Refraction c) Selective absorption d) All of the above.
7. Tungsten halide filaments are used in U.V. molecular absorption to:
a) Acts as wavelength selector. b) Deliver constant and uniform radiant energy. c) Act as light detector. d) All of the above.
8. Photomultiplier tube consists of:
a) Photo emissive-cathode b) Several dynodes. c) An anode. d) All of the above.
9. High concentration of analyte may cause the following deviation from Beer’s law:
a) Real deviation. b) Irregular instrumental deviation. c) Regular instrumental deviation d) Chemical deviation.
10. The method of continuous variation (Job’s method) is applied for determination of:
a) Ligand/metal ratio in a complex b) Impurity index (I.I.) c) Determination of some physical constant. d) Quantitative analysis of a single component.
11. Light detector is used to measure
a) Resistance. b) Potential difference. c) Conductance. d) Light intensity.
141 Instrumental analysis
12. Barrier-Layer cell detector consists of: a) Selenium Collector cathode. b) Iron anode. c) Silver collecting electrode. d) All of the above
13. The most suitable electrode for pH measurements is: a) silver-silver chloride electrode b) Calomel electrode c) Glass electrodes d) platinum or gold electrode
14. Conductometric titrations is not suitable for:
a) Neutralization reaction b) Complexation reaction c) Precipitation reaction d) Redox reaction
15 The unit used for measuring resistance is:
a) Quinhydrone electrode
b) wheatstone bridge.
c) Ac galvanometer
d) potentiometer
16 The main limitation for Standard hydrogen electrode is
due to: a) It is toxicity and environmental problems with
consequent cleanup and disposal difficulties b) It is difficult to be used and to keep H2 gas at one
atmosphere during all determinations. c) The dependence of its potential upon KCl concentration d) All of the above
17 Cannot be used in the presence of oxidizing or reducing
agent: a) Glass electrodes b) Quinhydrone electrode c) Ag/AgCl, saturated KCl electrode d) All of the above
142 Instrumental analysis
18. Used as indicators electrodes for redox reaction (for )4+example, titration of Ce
a) Glass electrodes b) Quinhydrone electrode c) Ag/AgCl, saturated KCl electrode d) platinum or gold electrode
19. Glass electrode is a type of:
a) Metallic electrode b) Membrane electrode c) Second order electrodes for anions d) First-order electrodes for cations
20. The main application of direct conductatometry:
a) Checking purity of distilled water or other chemicals b) Measuring the ability of sample to absorb light. c) Measuring pH of solution d) All of the above
Q2: Write short notes on the following:
1. Beer-Lambert’s law and deviation from the law.
2. Effect of PH on absorption spectra.
3. Antimony electrode, Draw the sketch diagram and mention
the half redox reaction, Nernst equation, advantage and disadvantage
4. Conductometric titration curve of HCl against NaOH (the
conductance of H+ = 350, OH- = 200, Na+=50 and Cl-=76)