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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

99 Instrumental analysis

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


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


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.


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).


(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


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).


-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


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.


(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".


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


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


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).


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).


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


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=


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).


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.


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.


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


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


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


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


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


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.


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


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

http://en.wikipedia.org/wiki/en:HPLC


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


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


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.


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.


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


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.


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).


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.


(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


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


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.


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: :……………………………… ……………………


…………………… 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 :……………………………… …………………… ……………………


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 ………


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


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.


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


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)

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