UN

NESCO-NIGGERIA TECCHNICAL &

VOCATIOONAL EDUCCATION
REVIITALISATIION PROJE
ECT-PHASE
E II

NATIIONAL
L DIPL
LOMA IN
S
SCIENCE LA
ABORAATORYY TECH
HNOLO
OGY

INSTR
RUME
ENTAL ANAL
LYTICA
AL CH
HEMIST
TRY AND
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UALITYY CON
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COU
URSE CODE
C : STC 2112

EAR 2- SE MES
YE STER 2

HEORY
TH Y

Version 1:
1 Decemb
ber 2008

1
TABLE OF CONTENTS

WEEK 1 PROPERTIES OF LIGHT………………………………………………..6

1.1 :Definition of light:.

1.2 Source of Light

1.3 Separation of Light

1.4 The spectrum

1.5 Electromagnetic spectrum

WEEK 2 BASIC PRINCIPLES OF LIGHT ABSORPTION AND EMISSION..10

2.1 Absorption and emission

2.2 laws of light absorption

2.2.1 Lambert’s law

2.2.2 Beer’s law

2.3 Limitation

2.4 Principles of UV Spectrophotometer

WEEK 3 INFRARED SPECTROSCOPY………………………………………….14

3.1 Infrared radiation

3.2 Flow analysis

3.2.1 Flow injection analysis

3.3 Principles and applications of immunoassay

WEEK 4 PRINCIPLES OF ATOMIC SPECTROSCOPY ………………………17

4.1 Principle of atomic emission spectrometry

4.2 A schematic diagram of a flame photometer

2
WEEK 5 ATOMIC ABSORPTION SPECTROPHOTOMETRY………………..19

5.1 Principles of Atomic absorption spectroscopy

5.2 Application of hollow cathode lamp

5.3 Application of A.A.S.

WEEK 6 PRINCIPLES OF ION SELECTIVE ELECTRODES………………22

6.1 Nernst equation:

WEEK 7 GLASS MEMBRANE ELECTRODE…………………………………24

7.1 Definition

7.2 Solid state membrane

7.3 Examples of solid state electrodes

7.4 Liquid – membrane electrodes

7.5 Calibration of I S E
WEEK 8 PRINCIPLES OF MASS SPECTROSCOPY…………………………27
8.1 Mass Spectroscopy

8.2 Components of Mass Spectroscopy

8.3 The schematic diagram of mass spectrometer.

WEEK 9 APPLICATION OF MASS SPECTROMETRY……………………...29

9.1 Identification of fragments

9.2 Fragmentation pattern of molecular ion

9.3 Chart of basic mass spectra

WEEK 10 NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY……...32

10.1 Proton Nuclear magnetic resonance spectroscopy

10.2 Diagram of NMR

3
WEEK 11 INTERPRETATION OF NMR SPECTRA………………………….34

11.1 Splitting in proton NMR spectroscopy

11.2 Chemical shifts for protons

11.3 H NMR chart

WEEK 12 GAS CHROMATOGRAPHY (GC)…………………………………. 36

12.1 Definition

12.2 Gas liquid chromatography

12.3 Gas Solid Chromatography

12.4 Diagram of GC

12.5 GC Column

12.6 The Stationary Phase

12.7 The Detectors for GC

12.8 Application G.C.

WEEK13 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)..40

13.1 Definition

13.2 Basic components of HPLC

13.3 The schematic diagram of HPLC

13.4 choice of column packing or material

13.5 The Mobile Phase

13.6 Column

13.7 Detectors

13.8Application

WEEK 14 THE PRINCIPLES OF QUALITY CONTROL……………………44

4
14.1 Definition

14.2 Quality Control in Laboratory

14.3 ISO Guide

WEEK 15 GOOD LABORATORY PRACTICES (GLP)………………………46

15.1 Definition:

15.2 GLP contains two unique elements:

15.3 Laboratory accreditation

5
WEEK 1 PROPERTIES OF LIGHT

1.1 Definition of light:

Light is a form of energy that is visible to the human eye which is radiated by

moving charged particles.

Literary, light is any device serving as source of illumination .Technically, light

or visible light is electromagnetic radiation of a wavelength that is visible to eye about

(400-700nm) light is composed of elementary particles called photons.

1.2 Source of Light

The sources of light are:

lamp,

lantern

electric lighting

Sun

illumination from moon light

mechanical devices uses illumination as a signal or warning.

1.3 Separation of Light

When there is a need to separate light of different wave length with high resolution then a

diffraction grating is most often the tool of choice. This super prism aspect of the

diffraction grating leads to application for measuring atomic spectra in both laboratory

instrument and telescopes. The diffraction grating is immensely useful tool for separation

of the spectral lines associated with atomic transitions. It acts as super prism, separating

the different colors of light much more than the dispersion effect in prism..

6
Another example is the tracks of a compact disc act as a diffraction grating, producing a

separation of the colours of white light. The nominal track separation on CD is 1.6

micrometer corresponding to about 625 tracks per millimeter. This is in the range of

ordinary laboratory diffraction gratings.

1.4 The spectrum

Spectrum is a rain bow like series of colours ,in the order of violet ,blue ,green ,yellow,

orange and red .This is produced by splitting a composite light into component colours.

In Newton’s times, physists understood that a prism could be used for the diffusion of

light rays in part ,prevailing belief was that, white light was a single colour like others,

but Newton maintained that it was a combination of all other colours. To prove this, he

directed a beam of white light through a prism. Newton gave the array of colours in

visible light the term spectrum .

The distribution of colours across the spectrum is given above.

7
1.5 Electromagnetic spectrum

Electromagnetic spectrum is the distribution of electromagnetic radiation

according to energy or equivalently, by virtue of the relations to frequency or

wavelength. Electromagnetic spectrum is just a name that scientist given to a bunch of

type radiation when they want to talk about them as a group. Radiation is energy that

travels and spreads out as it goes – visible light that comes from a lamp in your home,

radio waves that comes from radio station, other examples are microwaves, infrared and

ultraviolet light, x-rays and gamma rays. However, only extremely hot objects, or

particles moving at very high velocities can create high energy radiation like x-rays and

gamma rays.

Diagram of electromagnetic spectrum of light

8
The following table gives approximate wavelength, frequencies and energies for selected

regions of the electromagnetic spectrum.

Spectrum of Electromagnetic Radiation

Region Wavelength Wavelength Frequency Energy

(Angstroms) (centimeters) (Hz) (eV)

Radio > 109 > 10 < 3 x 109 < 10-5

Microwave 109 - 106 10 - 0.01 3 x 109 - 3 x 1012 10-5 - 0.01

Infrared 106 - 7000 0.01 - 7 x 10-5 3 x 1012 - 4.3 x 1014 0.01 – 2

Visible 7000 - 4000 7 x 10-5 – 4 x 10-5 4.3 x 1014 - 7.5 x 1014 2–3

Ultraviolet 4000 – 10 4 x 10-5 - 10-7 7.5 x 1014 - 3 x 1017 3 - 103

X-Rays 10 – 0.1 10-7 - 10-9 3 x 1017 - 3 x 1019 103 - 105

Gamma Rays < 0.1 < 10-9 > 3 x 1019 > 105

9
WEEK 2 BASIC PRINCIPLES OF LIGHT ABSORPTION AND

EMISSION

2.1 Absorption and emission

When a photon, or packet of light energy, is absorbed by an atom, the atom

gains the energy of the photon, and one of the atom’s electrons may jump to a

higher energy level. The atom is then said to be excited. When an electron of an

excited atom falls to a lower energy level, the atom may emit the electron’s excess

energy in the form of a photon. The energy levels, or orbitals, of the atoms shown

here have been greatly simplified to illustrate these absorption and emission

processes.

2.2 laws of of light absorption

2.2.1 Lambert’s law – states that the same proportions of incident light is absorbed per

unit thickness irrespective of its intensity.

Therefore the light decrease potentially through the cell and this is expressed

mathematically as:

I = Ioe-lke

Where,

Io = incident light, I = emergent light, l = path length, k = constant.

I0
Log = kcl
10 I

10
 I0
Log is called either absorbance (A) or optical density (O.D).
10 I

A is directly proportional to path length.

2.2.2Beer’s law – The law states that the absorption of light is directly proportional to the

number of absorbing molecules (Transmittance decreases with the number of absorbing

molecules)

I0
Log = kcl
10 I

Therefore

A = ECl where E is molar extinction coefficient.

Red

Orange Purple

Blue
Yellow Green

11
The diagram above shows the relationship between colour of a solution and the colour of

the light absorbed .The amount of light absorbed by a solution governed by the factors of

path length through which the light travels and the concentration of the solution. These

properties are formalized in two laws namely: Beer’s law and Lambert’s law.

2.3Limitation

Lambert’s law holds for all cases, but Beer’s law is usually only obeyed for dilute

solutions.

2.4 Principles of UV Spectrophotometer

Spectrophotometer consist of two instruments, namely spectrometer for producing

light of any selected colour (wave length) and a photometer for measuring the sensitivity

of light. The instruments are arranged so that liquid in a curvette can be placed between

the spectrometer beam and the photometer. The amount of light passing through the tube

is measured by the photometer. The photometer delivers voltage signed to a display

device, normally galvanometer.

The UV-Visible spectrophotometer uses two light sources, a deuterium (D2) lamp for

ultraviolet light and a tungsten (W) lamp for visible light. After bouncing off a mirror

(mirror 1), the light beam passes through a slit and hits a diffraction grating. The grating

can be rotated allowing for a specific wavelength to be selected. At any specific

orientation of the grating, only monochromatic (single wavelength) successfully passes

through a slit. A filter is used to remove unwanted higher orders of diffraction. One of the

beams is allowed to pass through a reference curvette (which contains the solvent only),

the other passes through the sample curvette. The intensities of the light beams are then

measured at the end.

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NOTE:

In spectrophotometry, the monochromator is provided by a prism or gratings. The

bandwith of light passed by filter is quite broad, so it may be difficult to distinguish

between two compounds of closely related absorption with colorimeter. Hence,

spectrophotometer is needed. Some compounds absorb strongly in ultraviolet region of

the spectrum, hence, their concentration can be determined by using a more expensive

type of spectrophotometer which operates down to 190nm. This instrument is called

uv/visible spectrophotometer. Example, concentration of uric acid can be estimated at

293nm, while that of protein at 280nm and nucleic acid at 260nm.

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WEEK 3 INFRARED SPECTROSCOPY

3.1 Infrared radiation

Infrared Radiation, emission of energy as electromagnetic waves in the

portion of the spectrum just beyond the limit of the red portion of visible radiation .

The wavelengths of infrared radiation are shorter than those of radio waves and

longer than those of light waves. They range between approximately 10-6 and 10-3 .

Infrared radiation may be detected as heat. This type of spectroscopy depends on

the vibration of atoms with respect to one another .The most important are the

mutual vibrations of two atoms bonded to one another .The frequency of the

vibration corresponds to the frequency of absorbed radiation. This depends on the

masses of the atoms and the bond strength .The frequency is characteristics of the

two bonded atoms. The IR spectroscopy of an organic compound reveals whether it

contains a specific functional group,such as carbonyl, hydroxyl etc.

3.2 Flow analysis

Flow analysis is an automated method in which samples are analysed as they are pumped

in a sequence through a series of tubing containing a continuously flowing reagent

stream.

Continuous flow analysis is an autoanalysis that can perform all the basic steps involved

in a simple quantitative photometric procedure. These include pipetting of the sample and

reagents, mixing, heating at specific temperatures, detection and presentation of results.

In addition to these some other procedures such as digestion of sample, phase separation,

removal of interfering substances may be included when required.

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In flow analysis, colorimetric method of detection is frequently employed, but other

methods such as fluorimetry, refractive index, e t c. are also used.

This type of analysis takes place as the samples and reagents are pumped through a

system of tubing instead of being retained separately. The stream passes through the

tubing to the different units of the analyser each of which performs a specific analytical

function. In this case, the liquids involved are mixed carefully in a controlled proportions,

which are determined by using delivery tubes of varying internal diameter with a constant

speed pump. The samples are aspirated into the reagent stream with a small volume of

water separating each one.

Quantitation in continuous flow analysis is based on the direct comparison of the peak

heights on a recorder trace of the samples and standard.

3.2.1 Flow injection analysis

In this technique, microliter volumes of liquid samples are technically injected at

intervals into a continuously flowing carrier stream which is not air segmented. Various

reagent streams are introduced as required, while controlled mixing of reagents and

samples occurs.

3.3 Principles and applications of immunoassay

Immunoassay is an analytical technology introduced by Rosalind yalow &

Soloman Berson in 1960 with their use of anti-insulin antibodies to measure the

concentration of the hormone in plasma. The basic principle of immunoassay is that the

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process relies on the competition between labeled and unlabelled antigens for a fixed and

limited number of antibody – combining sites.

The principles of immunoassay have been exploited by commercial manufacturers to

provide Kits which are packages of reagents,designed to analyse samples for a wide

variety of analytes. The commercial exploitation of immunoassay technology and the

current development of suitable automated instrumentation has had a profound effect on

the analysis of biological samples.

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WEEK 4 PRINCIPLES OF ATOMIC SPECTROSCOPY

4.1 Principle of atomic emission spectrometry

The principle is that atoms of element in an excited state emit radiation at a

specific wave length as they return to the ground state. The amount of light that is

emitted is proportional to the concentration of the material in solution to be tested. In this

method heat radiation is applied instead of light as in the case of atomic absorption

technique. A flame photometer, is designed to cause atomic excitation of the analyte from

the sample to be tested and subsequently to measure the emitted radiation. In this case, a

monochromating system is essential to distinguish between the emission of the test

element and other radiation from the flame .The flame combines both the source of

radiation and the atomized sample and it must be very table to obtain a steady result. The

flame temperature must be high enough to excite the atom under investigation. The hotter

the flame, the greater the proportion of the atoms to be excited.

4.2 A schematic diagram of a flame photometer is represented as below:

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In quantitative analysis, a number of standard solutions containing the metallic element to

be tested are prepared and aspirated into the flame. The meter deflections or readings

which depend on the intensity of light emitted by the metal atom that reach the detector,

are plotted against the concentrations. This gives a straight line graph called a calibration

curve. Then the meter readings for the solution of unknown concentration can now be

converted into the desired concentration using the graph.

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WEEK 5 ATOMIC ABSORPTION SPECTROPHOTOMETRY:

5.1 Principles of Atomic absorption spectroscopy

In atomic absorption analysis, the absorption of light by free atomic species is measured.

This is done by the use of an instrument called Atomic absorption spectrophotometer

(AAS). In this process, a flame system is generally employed to dissociate elements form

their chemical bonds. The atoms absorb light at characteristic wave length when present

in their ground state. A mixture of air and acetylene produce a flame which is of a

sufficient high temperature to ensure the presence of free atoms of most elements. The

use of nitrous oxide in place of air result in a higher temperature and this is necessary for

the estimation of certain elements.

The sample solution is passed in to the flame via a nebulizer, which forms a fine spray or

mist. The source of a light is a hollow cathode lamp containing the element under

examination. The lamp produces radiation at the appropriate wavelength for absorption

by the free atoms of the sample. After passing through the flame the light impinges on a

monochromator which removes unwanted emission but transmits light of the test

wavelength which passes to a photo detector. This device produces a signal which allows

for the measurement of light intensity and hence, of absorption caused by the sample

elements which are determined by atomic absorption analysis. The schematic diagram is :

19
5.2 Application of hollow cathode lamp

In the A. A. S., the source of radiation is a hollow cathode lamp which consists of two

electrodes seated in a glass envelop filled with an inert gas, usually argon or neon. The

cathode lamp is either made of the element whose spectrum is required or coated with the

element to be tested. The application of a potential between 300 and 400 volts is usually

required to cause excitation of the atoms and discharge of the appropriate radiation.

A hollow cathode electrical energy excites the atoms of the element in the tube to a

higher electronic states and some return to the ground state emitting radiation, the

wavelength of which are characteristics of the element in the solution. Obviously, each

element to be determined in a sample requires its own hollow cathode lamp. The diagram

of a hollow cathode lamp is given below.

5.3 Application of A.A.S.

Atomic absorption can be used to determine the concentration of metallic elements such

as copper, Arsenic, lead, cadmium etc, in varieties of samples. The instrument is very

20
sensitive and specific and is not affected by changes in the flame. Atomic absorption is

widely used in food analysis , environmental impact analysis ,etc.

21
WEEK 6 PRINCIPLES OF ION SELECTIVE ELECTRODES:

6.1 Nernst equation:

The potential developed by a single electrode in a solution is caused by the tendency of

the solution to donate or accept electrons and can be calculated using the Nernst equation:

23026 RT
E = E0 – x log a
nF

where: E is the electrode potential at the specified concentration,

E0 is the standard electrode potential

R is the gas constant

T is the absolute temperature

n is the number of electrons involved

F is the Faraday constant

a is the activity of the ion.

NOTE:

For measurements made at 250C the equation is simplified as:

0.059
E = E0 – log a
n

The Nernst equation expresses the potential developed by an electrode in terms of the

activity and number of ions involved in the reaction. There is no chemical reaction

without an electron donor or acceptor. Each of these electrode system is known as a half-

cell and the potential developed by a half cell cannot be measured in absolute terms but

22
can only be compared with that of another half cell. The chemical reaction occurring at

each half cell is known as a half reaction.An active electrode is composed of an element

(m) that is in an equilibrium with its ions in the surrounding solution.

M = M+ + e-

The activity of an ion reflects the proportion of the molecules that are actually ionized at

the time. The standard electrode potential of an element is defined as its electrical

potential when it is in contact with a molar solution of its ions. For redox system, the

standard redox potential is that developed by a solution containing molar concentration of

both ionic forms ion-selective electrode (ISE).

ISE is a glass electrode that is selectively permeable to hydrogen ions which is used for

measuring the pH of solutions. Varying the glass membrane can change the permeability

of the glass and several action sensitive electrodes have developed in this way. The ISE

may be specific and selective for ions such as Na+, K+, Ca+ and NH4+. but there is

significant interference from other ions, particularly hydrogen ions.

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WEEK 7 GLASS MEMBRANE ELECTRODE

7.1 Definition

Glass membrane electrode is the type of electrode that is commonly used for measuring

pH of a given solution.

7.2 Solid state membrane

This electrode consists of silver- silver chloride electrode in a reference solution of

hydrochloric acid (usually 0.1moldm-3 ) contain in a glass membrane .The membrane is

made of a special glass , usually a hydrated aluminosilicate containing calcium or Sodium

ions. It is selectively permeable to hydrogen ions and the potential that develops across

the membrane depends on the hydrogen ion concentration of the test solution compared

with the reference acid solution inside the electrode. This potential can be measured

against a reference calomel electrode using a high sensitive voltmeter .In pH measurement

, calibration of the instrument is essential and may be achieved with either buffers whose

pH has been previously measured . But for measurement over a range of pH values, it is

necessary to standardize the instrument on at least two standard buffer solutions which

cover the required range. The simplest solid state membranes are designed to measure

test ions , which are also the mobile ions of the crystals and are usually single substance

crystals. Alternatively, the test substance may involved in one or two reactions on the

surface of the electrode which alter the activity of the mobile ion in the membrane.

24
 7.3Examples of solid state electrodes
Test ion Membrane material major interfering ions

Fluoride LaF I- Br- Cl-

Chloride AgCl/Ag2S S- I-
Bromide AgBr/Ag2S S- I-
Iodide AgI/Ag2S S-
Sulphide AgI/Ag2S Hg+ Ag+
Cupric Ag2S/CuS Hg+ Ag+
Lead Ag2S/PbS Hg+ Ag+

7.4 Liquid – membrane electrodes

On the other hand, liquid – membrane electrodes consist of an ion –selective material

dissolved ina solvent that is not miscible with water . The liquid is held in a porous, inert

membrane which allows contact between the test solution on one side and the reference

electrolyte on the other.The ions from the reference solution will partition them selves

between the two immiscible solvent, giving the electrode a particular potential. The

presence of the test ion in the sample affect the activity of the reference ions in the

membrane resulting in a change in the potential difference across the membrane.

Example of liquid-membrane electrode are:

Test ion Membrane material

Potassium Valinomycin in diphenyl ether
Ammonium Macrotetrolides in tris phosphate
Calcium Calcium dialkylphosphate

7.5 Calibration of I S E
Because, potentiometric measurement reflect the activity of an ion rather than its

concentration ,it is therefore necessary to calibrate the system with a standard solution of

the value of the activity coefficient for the ion is known. That is,

25
Log = log

Where, a is the activity of the ion ,c is the concentration of the ion,and is the activity

coefficient. But ,the equation is more useful in the form of:

Log C = log a – log .

26
WEEK 8 PRINCIPLES OF MASS SPECTROSCOPY

8.1 Mass Spectroscopy

This technique is based on the fact that charged particles in motion is detected by a

magnetic field . The amount of deflection on the momentum of the particle .The mass

spectrometer uses differences in mass to charge ratio to diffentiate ionized molecules or

fragment of molecules from one another. Each molecule has distinctive fragmentation

pattern that provides structural information .The MS is useful in determining the structure

of unknown molecule.

8.2 Components of Mass Spectroscopy

1. The sample inlet system and ion source

2. Mass analyzer

3. Detector system

4. Control and signal processing electronics

27
8.3 The schematic diagram of mass spectrometer.

diagram of mass spectrometer

28
WEEK 9 APPLICATION OF MASS SPECTROMETRY

9.1 Identification of fragments

In many cases, the relative molecular mass can be found quickly and with a very high

degree of precision by mass spectrometry. The vapour of the substance, at a low pressure

(10- 5_ 10_6 mm Hg), is bombarded by a stream of electrons. This causes the loss of one

electron from a molecule of the substance, giving the molecular ion (sometimes called the

parent ion):

M + e -* M + 2e

These positive ions are accelerated in an electrostatic field and pass through a slit in the

negatively charged plate into an electrostatic analyser. This causes them to be deflected

by amounts depending on their kinetic energies, and only one component of the beam of

ions, with a well defined kinetic energy, can emerge from the slit at the other end of the

analyser. This component then enters a magnetic field in which it is again deflected, and

the strength of the field is altered until the ions imping1e on a detector; this generates an

electrical signal which is shown on an oscilloscope and/or recorded on photographic

paper.

The abundances of the different positively charged fragments vary widely. For

convenience in interpreting the spectrum, the relative abundances of

the fragments are plotted against their masses, the most abundant ion being

given an arbitrary value of 100 units (the base peak); the plot is known as

a stick diagram. The molecular ion is rarely the most abundant one; indeed,

with some compounds, the molecular ion is not detected and the formula

weight cannot then be determined .

29
The fragmentation pattern of a compound depends on its structure. The

presence of particular groupings is associated with specific fragmentation

patterns, and so the determination of the fragmentation pattern for a compound

of unknown structure can often enable the structure to be deduced.

For example, the stick diagram is shown below.

9.2 Fragmention pattern of molecular ion

30
9.3 Chart of basic mass spectra

31
WEEK10 NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY

10.1 Proton Nuclear magnetic resonance spectroscopy

The atoms of some of the elements have a nuclear structure which causes them to behave

like small magnets. In organic compounds, the commonest of these elements is hydrogen;

the hydrogen nucleus—the proton—when placed in a magnetic field, aligns itself so that

its magnetic moment (sometimes referred to as its ‘spin’) is either in the same direction

as the applied field or in the opposite direction.

The energies of these two possible states’differ. The protons whose spins are aligned with

the field have a lower energy than those aligned against the field; the energy difference,

1E, is proportional to the strength of the field, B:

zIE = 2it

where y is a constant for a particular nucleus and h is Planck’s constant. If

electromagnetic radiation is incident on the protons, then, providing

that its frequency v is such that its energy, hv, is exactly equal to the energy difference,

4E, for the proton’s two orientations, some of the radiation will be absorbed and some of

the protons in the orientation of lower energy will ‘flip’ over to the orientation of higher

energy; the total energy of the system remains constant. Thus, the condition for

absorption of radiation (the ‘resonance’ condition) is

yB

32
10.2 Diagram of NMR

33
WEEK11 INTERPRETATION OF NMR SPECTRA

11.1 Splitting in H NMR spectroscopy

The number of protons directly on adjacent carbon atoms can be determined by

evaluating the appearance and number of peaks in each signal. Spitting with non

equivalent carbon atoms can be observed in proton NMR spectroscopy. This type of

splitting is called vicinal splitting. Hence, the proton on a carbon “see” the number of

protons on directly adjacent carbon atoms .The number of peaks in a signal equals the

number non equivalent protons on adjacent carbon ie n + 1 .

11.2Chemical shifts for protons

Type of protons Chemical shift[ppm]

R-CH3 0.9

R-CH2-R 1.3

R-O-CH3 1-5

34
11.3 H NMR chart of methanol

35
WEEK 12 GAS CHROMATOGRAPHY (GC)

12.1 Definition

Gas chromatography is a technique for carrying out the separation and measurement of

mixtures of materials that can be volatilized. It is a separation method in which the

mobile phase is a gas. In gas chromatography, mixture is separated by adsorption

between two immiscible phases, one of which is a mobile (carrier gas) and One of which

is stationary (column packing).

Gas chromatography is divided into two:—

12.2 Gas liquid chromatography:— The stationary phase is a viscous liquid coated

onto a solid support material. Separating principle depends on the difference in the

partition coefficients between the liquid and gas phases of the constituents of a mixture.

Those that are highly soluble in the stationary phase will tend to dwell on the column.

12.3 Gas Solid Chromatography:— In this adsorption chromatography the column

packing is a solid material with surface active properties. The separation depends to the

extent to which the components of a mixture are adsorbed by the solid, i.e silica.

The materials that can be examined using G.C. may be gases, liquids, or solid that have

appreciable vapour pressures at temperatures up to a few hundred degrees (i.e, substances

that can be easily volatilized).

During the process of operation, the carrier gas supplied from a pressurized tank, passes

through one or more pressure regulators which control the flow rate through the

apparatus. The sample is introduced into a heated chamber either through a silicons

rubber septum with hypodermic syringe if it is liquid, or by means of a special sampling

valve if it is a gas.

36
12.4 Diagram of GC

carrier gas detector gas

sample in

The sample vapourized immediately and introduced into a moving stream of the helium

or nitrogen (carrier gas). The carrier gas then takes the vapour sample into the column

containing the stationary adsorbent, where the various components are separated. The

volatile components of the sample get separated from each other according to their

partition coefficient between the liquid and mobile gaseous phases. When each

compound leaves the column, a detector sends an electrical signal to a recorder which

plots the output of the recorder on a continuously moving roll of chart. The effluent gases

are fed into a thermal conductivity detector, a hot wire detector or an ionization detector.

The detector records the emergence of the volatile components of the sample as a series

of peaks in a chromatogram. Individual components are identified by comparing this

chromatogram with that of a standard mixture of known composition. The concentrations

of the component of the sample are obtained from the areas under respective peaks. The

effluent gas may also be fed into a mass spectrometer for analysis.

37
The factors affecting separation include:— Carrier gas, stationary phase, temperature and

sample injection.

12.5 GC Column:

The column is the most important feature of a GC. It contains the stationary phase and

has a great influence on the separation of mixture. The column is made of glass or metal.

A packed column contains solid particles of uniform size generally coated with a

stationary phase stainless steel or glass tubing is used for most columns this is to avoid

reactions of some sensitive molecule in the column.

12.6 The Stationary Phase

Separation of components occurs by partition between the mobile phase and s suitable

stationary phase. The stationary phase for GC needs to be:

- thermally stable

- unreactive

- negligible volatility

- should have a reasonable column life over the operating range.

The choice of stationary phase depends on the selectivity and degree of polarity of the

compounds.

38
12.7 The Detectors for GC

The purpose of a detector is to monitor the GC column effluent and to measure the

variations in the composition of the eluted components. In GC different types of detectors

are used. These are:

1) Flame ionization (FID)

2) Nitrogen phosphorous (NPD)

3) Flame photometric

4) Kathermeter (Thermal conductivity – TCD)

5) Electron capture (ECD)

- The flame ionization detector is almost the ideal GC detector and hence is

used for routine and general purpose analysis. This type of detector is capable

of detecting virtually all organic compounds.

- The thermal conductivity or katherometer is the simplest detector but

commonly used nowadays. It measures the variations in resistance of a solid

electrical conductors which is induced by changes in temperature.

- The electron capture detector also depends on ionization of the carrier gas. It

is extremely sensitive which detects electrophilic compounds.

12.8Application: G.C. can be. used for separating., identifying and estimating volatile

compounds of fatty acids, steroids, other liquids, vitamins, sugars and amino acids. It is

also extensively used in the analysis of drugs, pesticides, petroleum products and many

other substances.

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WEEK13 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

(HPLC)

13.1 Definition

High performance liquid chromatography is the type of column chromatography

in which the mobile phase is a liquid material. Separation of mixture is achieved by

differential distribution of the sample components between the stationary and the mobile

phase. HPLC is now a highly developed technique and a wide range of stationary phases

are available. These enable partition, gel permeation, affinity and ion exchange

chromatography to be performed in conjunction with the HPLC.For effective separation,

it is essential to have a very small and regular shaped support media, a supply of mobile

phase pumped at a pressure that is adequate to give suitable constant flow rate through

the column and a convenient efficient detector system .The solvent delivery system

included a pump delivers the appropriate solvent from the reservoir at pre selected rate.

13.2 Basic components of HPLC

Basically, the HPLC instrument consist of five sections:

1) the solvent reservoir

2) pump

3) chromatographic column and oven

4) detector unit

5) Amplifier and signal processing unit or recorder.

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13.3 The schematic diagram of HPLC

13.4 choice of column packing or material

A wide range of column materials have been developed. This enable the basic HPLC

instrument to be used for the major chromatographic techniques.Thus, in selecting a

column material for the separation of a specific substance it is necessary for consider the

physical characteristic of the molecule. The first thing to consider is the polarity of the

molecule. So, for ionic specie, the column packing should be ion exchange resins, while

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for molecule with moderate polarity adsorption column packing is the right choice. But

for the separation of large molecules, gel permeation should be considered.

13.5 The Mobile Phase

The liquid which can be used for HPLC separations may comprise of water, aqueous

buffer solutions, organic solvents such as methanol, aceto nitrile, etc.All solvents should

be of high degree purity, dust free, and should be free from any gaseous impurity.

13.6 Column

The column for analytical HPLC are typically 10-25cm long and 4 – 6mm internal

diameter. The columns are made of stainless steel to cope with the operating high

pressure. It is also lined with glass internally to prevent metal catalysis of solvent – solute

reactions.

13.7 Detectors

Various types of detectors are used in HPLC for the detection of eluted solute. These are

classified as:

1. Detectors which monitors a specific property of the solute, e.g. UV

absorbance and fluorescence.

2. Detecting system which monitors a bulk property of the eluant, e.g refractive

index.

3. Detectors which function by separating the solvent from the eluant, e.g flame

ionization (FID) or mass spectrometry (MS) detectors.

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NOTE: The choice of detector is governed by the properties of the solute and the

sensitivity required from the analysis.

13.8Application

HPLC is used for both qualitative and quantitative analysis of variety of substances ,such

as drugs, pesticides, herbicides, vitamins, natural products, e.t.c.

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WEEK 14 THE PRINCIPLES OF QUALITY CONTROL

14.1 Definition:-

Quality control is the analytical process by which we measure the “fitness for purpose” of

a particular product or process. It ensures that a product is manufactured within certain

defined limits of purity or that an analytical process is working within a particular range

of accuracy. In this way we make sure that the product being manufactured is good

enough to do the job we want (fit for purpose) or that the analytical process is accurate

enough to tell us what we need to know (fit for purpose). So, quality can be defined as the

body of characteristics, properties, attributes or abilities of an entity that make it fit for

purpose.

14.2 Quality Control in Laboratory

Quality control involves the examination of laboratory and its results. But quality

assessment involves inspecting quality control, the laboratory, the results it supplies and

their relationship to the solution to the analytical problem. Quality assurance activities

should lead to the implementation of corrective actions, which should initially focus on

the laboratory.

14.3 ISO Guide

ISO- International Standard Organization. The ISO has issued a variety of standards and

guides that are directly or indirectly relevant to the implementation of quality in the

analytical Laboratory. The parent standards are those in the ISO series, which describe, in

broad terms, the framework elements for the management of quality in the business

world. But at the Laboratory level, these standards have materialized in ISO guide 25

which is currently being revised.

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The European centre for standardization has also developed a series of standards for the

implementation of quality which is based on ISO 9000 and ISO guide 25 that are directly

applicable to the laboratory.

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WEEK 15 GOOD LABORATORY PRACTICES (GLP)

15.1 Definition:

Good Laboratory practice can be defined as a body of rules, operating procedure and

practices established by a given organization that are considered to be mandatory with a

view to ensuring quality and correctness in the results produced by a Laboratory.

Good Laboratory practices constitute an alternative to the standards derived from ISO

guide 25. They have been established world wide by institutions such as the organization

for Economic Cooperation and Development (OECD) and the European Union (EU). The

GLP has also been gazetted as imperatives rules for Laboratories engaged in analysis and

assessment of substances with potential social Implications, which as such require

regulation. Typical examples include pharmaceutical formulations, foods, cosmetics and

environmentally toxic products.

15.2 GLP contains two unique elements:

• Standard operating procedures (SOP)

• Quality Assurance Unit (QAU)

The Standard operating procedures are detailed description of all the activities performed

by a Laboratory. Examples SOPs must exist for sample handling; reagent usage;

reference materials; methods; instrument and apparatus supervision, etc. SOPs must be

followed literary without any alteration.

The Quality assurance unit is the unit that is responsible for instituting quality,

controlling and assessing it, and proposing actions to enhance it. This unit provides an

audit that is external to the laboratory but internal to its parent body.

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15.3 Laboratory Accreditation

Laboratory accreditation with respect to quality is defined as the formal written

acknowledgment that a Laboratory is fit and competent to perform one or more type of

analysis. This type of accreditation is provided by a public or private national

organization and relies on internationally established standards.

Laboratory accreditation can be voluntary conducted when requested by the laboratory.

The accreditation process is started if the laboratory already possess a quality system

which is materialized in a quality manual. The auditor conducts a qualitative visual and

documentary inspection and generates a report that can be appealed by the laboratory.

If the report is eventually favorable, an accreditation certificate is issued to the

laboratory. Also free access to the laboratory is given to the auditor (supervisor) in order

to conduct periodic controls over the period where accreditation is to hold. The excises, is

subject to renewal after the previous one has expired.

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