Analytical Instruments I

Various instruments and techniques used for chemical analysis in clinical biochemistry are as follows;

1. Colorimetry

This is measurement of colors for the determination of the concentration of biochemical compounds.

Colorimetry is based on the principle that when white light passes through a colored solution, some
wavelengths are absorbed more than others.

Many compounds, not colored can be made to absorb light in visible spectrum by reaction with suitable
reagents.

Colored compounds absorb light at given wavelength at visible spectrum.

Extent at which a solution absorbs light depends on the intensity of its color.

Light travels in form of wavelength

Wavelength is the distance between two wave peak in nanometer (nm)

Wavelength between 400nm & 700nm form a visible spectrum of light

Wavelength of about 700nm are visible to the eye as red color while those of shorter wavelength gives rise to
colors; orange, yellow, green, blue and finally violet (400nm)

Wavelength greater than 700nm have vibrations known as Infrared and are not visible to the eye

Wavelengths shorter than 400nm have vibrations known as Ultraviolet and are also not visible.

Light absorption
• Light falling on a colored solution is either absorbed, reflected or transmitted
• A colored solution absorbs all the colored component of white light and selectively transmits only one color

L0 Jj La
Lt Lo = Incident light

La = Absorbed light

Lt =Transmitted light

Beers Laws
• The law states that the proportion of incident light absorbed by the molecules in a solution is directly
proportion to the number of absorbing molecules in the light path.
• It follows that the concentration of substance is directly proportion to the intensity of the color of the solution
• In this law, light path is constant while the concentration varies.

Lambert’s Law
• The law states that when a monochromatic light passes through a colored solution, the amount of light
absorbed increases with increase in the thickness of the layer of solution through which light passes
• In this law, the concentration of the solution is constant while the light path varies.

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 Beer- Lambert’s law
• The law states that under suitable condition, the amount of light absorbed by a colored solution, when
illuminated with a light of suitable wavelength is directly proportion to the concentration of colored solution
and the length of the light path through the solution.
• Therefore the amount of light decreases exponentially with the increase in the concentration of the solution
and with the increase in thickness of the layer of solution through which light passes.

NB:
• The distance travelled by the light path can be kept constant by using tubes/cuvettes identical in size and
optical density.

Transmittance

This is the ratio of the intensity of the transmitted light over the intensity of the incident light

It is usually multiplied by 100 to give percentage transmission

% transmission = Lt X 100
Lo
Absorbance
o Absorbance increases linearly with concentration
A = 2 - log %T
Application of Beer-lambert’s law
o As the test and the standard solution are compared in the same or identical cuvettes, the same
Light – path or thickness of the solution is employed.

A (test) X C (std) = C (test) X A (std)

C (test) = A (test) X C (std)

A (std)
In other words, the concentration of unknown is calculated as = A (of unknown) X C (std)
A (std)

There are 2 types of instruments used to measure absorbance;

Absorptiometer
o Also referred to as colorimeter or filter absorption spectrometer.

Spectrophotometer
o Different with this is that it can measure absorbance at specific wavelength due to presence of prism
that disperses white light into continuous spectrum.

Essential parts / system in a photoelectric absorptiometer (colorimeter)

• Light source
• Monochromator
• Wavelength selector
• Solution/sample holder
• Photosensitive detector system
• Measuring device.

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 Light source

Most common source is tungsten – filament lamp or higher powered tungsten-halogen (quartz – iodine) lamp
for ultraviolet region or deuterium lamp

Monochromator/ Wavelength selector

They are used to slit the light from the light source

This can be diffraction gratting or prism

Filters are for simple instruments such as a colorimeter and they are used for selecting a band of wavelength.

Filters have a range of 400 – 680nm

Filters have limited transmission band & are usually complementally to the color of the solution to be
measured.

Simple filters are either colored glass or suitably dye gelatin sandwiched in a glass.

Color of solution Color of filter Approximate range

Blue yellow 570 – 600nm

Bluish – green Red 630 – 680 nm
Violet Green 520 – 570 nm
Red Bluish- green 470 – 520 nm
Yellow Blue 420 – 570 nm
Yellowish – green Violet 400 – 420 nm

Solution/sample holder

Holds colored solution and must be scrupulously clean

Holder can be test-tube or cuvette

Cuvette are rectangular cells with one pair of opposite side optically clear while other parallel sides are opaque

Photosensitive detector system

Also referred to as photoelectric cell with meter reading proportion to light intensity. Detectors are;-
1) Barrier cell layer
2) Photoemission tubes (vacuum photo tubes)
3) Photomultiplier tube

Measuring device
• Current from the detector is fed to a sensitive suitable measuring device, usually galvanometer.
• Read out is by means of a scale or digital display
• Scale may show both the absorbance and % transmittance
• Absorbance scale ranges from 0 to infinity
• Zero absorbance is equivalence to 100% transmission & infinitive absorbance is equivalent to 0 transmission.

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 Spectrophotometer

An instrument used to measure absorbance at various wavelengths

Similar to absorptiometer except that it uses diffraction grating or glass or glass prism to produce
monochromatic light.

Types of spectrophotometers are;
(i) Single beam spectrophotometer
• Operates between 325 – 1000nm wavelength
• Uses single source of light e.g. tungsten filament lamp
• Has two photocell & light travels along only one pathway
• Test solution & blank are read in the same position.

Light source entrance slit Monochromator exit slit colored sol. Photocell Galvanometer

(ii) Double beam spectrophotometer

• Operates between 185 – 1000nm wavelength
• Has two light sources and two photocell
• Instrument splits the light from monochromator into two beans
• One beam for reference & the other beam for sample reading
• This eliminates errors due to fluctuation in light output & sensitivity of detector.

Ref. solution Photocell

Light source entrance slit Monochromator exit slit . Galvanometer

Sample solution Photocell

Setting up the colorimeter / spectrophotometer

Important step is the selection of the correct wavelength or filter to use.

Next step is to produce an absorbance curve of the color to be measured by plotting an absorbance against the
wavelength using the same solution.

After selecting the correct filter or wavelength to give maximum absorption, it is still essential the following
criteria are observed.
o Beer’s law is obeyed over rays of expected value
o Degree of sensitivity

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 Chromatography

Process of separation of a mixture of solution dissolved in a common solvent

Separation technique makes use of the differential distribution of the solutes between 2 phases

The two phases included the mobile and fixed stationary phase.

The solvent is the mobile phase which carries mixture of solutes through the stationary phase.

Under ideal conditions, the resulting difference rates of migration bring about complete separation of the
solutes.

Can be classified according to the physical states of the solutes carrier phase.
 Liquid chromatography – solute phase is liquid solution
 Gas chromatography – solute phase is gaseous state

Liquid chromatography is subdivided into;

Flat method
o Stationary phase is supported on a flat surface e.g. cellulose acetate paper or thin layer mechanically
Supported with glass or plastic
Column method
o Various types of materials e.g ion-exchange resins diatomaceous earth or internally coated fine glass
capillaries may be used.

Mechanism of separation in chromatography includes ;
 Adsorption chromatography
 Partition chromatography
 Exclusion chromatography

Adsorption chromatography

Molecules are separated due to difference in their attraction to the stationary phase and the mobile phase.

Speed of migration of a component depends on its adsorptive affinity relative to other components

Example of this technique is Thin Layer Chromatography (TLC).

In ion exchange chromatography, electrostatic forces operate between charged molecules & appositively
charged particles on the ion exchange resins or modified cellulose or dextran.

Electrostatic forces can be altered by changing the pH of the mobile phase.

Partition chromatography

Utilizes difference in the relative solubility of the solute molecules between mobile & stationary phase

The two phases may be liquid – liquid or liquid-gas

It is used in gas –liquid chromatography (GLC) or High Performance Liquid Chromatography (HPLC)

Exclusion chromatography

When a mix of small & large particles are allowed to pass over porous solid particle, smaller molecules (ions)
passes through pores of solid particles

Pore size determines which molecules are able to enter particles and others excluded

Particles with large molecular size (proteins) are excluded more easily than small particles.

Rf Value (Retardation factor)

Rf value is the ratio of distance travelled by the substance (solute) to that travelled by the mobile phase in the
same period

Mobile phase travelles faster than solute components
Rf = Distance travelled by the solute
Distance travelled by the mobile phase

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 Types of chromatography
1. Paper chromatography
o In paper chromatography, stationary phase is a paper (cellulose acetate) while mobile phase is the solvent
o A mixture of substances e.g. carbohydrate is spotted at one end of the porous filter paper strip
o In ascending type of chromatography, paper is hung vertically into the solvent
o Solvent moves up through the paper by paper action
o Various fraction in the mixture moves at different rates
o Paper is removed after the separation, dried and sprayed with a chemical for color development
o Spots of the solutes development at different sites quantitated by the area and color intensity.
o Commonly employed in separation of amino acid & carbohydrates.

2. Thin layer chromatography (TLC)

o Very similar to paper chromatography except paper is substituted by a thin layer of;
 Very fine powdered silica gel
 Alumina polyacrylamide gel
 Starch gel
 Kiesel guhr
Bound to glass or plastic plate
o Advantage of TLC over paper chromatography includes; -
 Easier elution of separated spot from plate by cutting thin layer
 Faster separation of constituents
 Sensitive
 Quick

3. Ion exchange chromatography

o Ion – exchange resins are suitable polymers which contain ionic groups
o Resins can be cation exchangers (N+(NH3)3) or Anion Exchangers group.
o Resins are extensively used for separation of amino acids
o If a solution containing a mixture of amino acid is applied to a column of suitable ion- exchange resins,
amino acid are linked to resins
o Degree of affinity varies with amino acids.
o After adsorption, column is washed or eluted by series of buffers of varying pH, the individual amino acid
can be separated & quantified with ninhydrin

4. Gas chromatography
o Separation process by which a mixture of compound in gaseous state is passed through a gas in stationery
phase.
o Mobile phase is usually nitrogen, helium & argon gas
o Separation is achieved by a difference in the partitioning of the various molecules between the two phases.
o If the stationary phase consist of thin layer of non-volatile liquid, the technique is called; Gas-liquid
chromatography (GLC)
o If the stationary phase is solid absorbent, then it is called ; Gas – solid chromatography (GSC)

Components of a gas chromatography

1) Sample injection area
2) Pressurized carrier gas with flow regulator
3) A column; Gas, liquid or solid
4) A detector, a recorder
5) Thermo-regulator component enclosing injection area, column & detector.

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 5. High Performance Liquid Chromatography (HPLC)
o In this, stationary phase is composed of uniform ultra-fine particles
o Large pressure of about 500 – 5000 PSI (pounds per square inch) are required to deliver constant flow rate
o The elute from the column is mounted by a variety of detectors e.g. UV or redox –potential electrode
detectors.
o HPLC is very efficiently technique for detection & quantitation of drugs.

Terminologies
• Eluate – This is the analyte material that emerges from the chromatography
• Elute – to remove by dissolving, as absorbed material from an adsorbent.
• Elution – process of extracting one material from another by washing with a solvent
• Eluent / Eluant – carrier portion of the mobile phase. It moves the analyte through the chromatography
• Elution time – time between the start of separation & the time at which the solute elutes
• Elution Volume – volume of eluent required to cause elution
• Mobile phase – A fluid which carries the solute through a structure holding another material called stationary
phase.
• Stationary phase is the substance fixed in place for the chromatography procedure. Examples include the
silica layer in thin layer chromatography

Assignment
Discuss the difference between one-dimension chromatography and two-dimension chromatography

Two-dimensional chromatography is a type of chromatographic technique in which the injected sample is separated by
passing through two different separation stages. This is done by injecting the eluent from the first column onto a second
column. Typically the second column has a different separation mechanism, so that bands that are poorly resolved from the
first column may be completely separated in the second column. (For instance, a C18 reversed-phase chromatography column
may be followed by a phenyl column.) Alternately, the two columns might run at different temperatures. The second stage of
the separation must be run much faster than the first, since there is still only a single detector. The plane surface is amenable
to sequential development in two directions using two different solvents.
If you spot your compound in one corner of a square plate and run it up in one solvent system, then turn it 90o and develop it
again in the same solvent system, all of your spots should be along a diagonal.

Reversed-phase chromatography
Reversed-phase chromatography (RPC) is any liquid chromatography procedure in which the mobile phase is significantly
more polar than the stationary phase. It is so named because in normal-phase liquid chromatography, the mobile phase is
significantly less polar than the stationary phase. Hydrophobic molecules in the mobile phase tend to adsorb to the relatively
hydrophobic stationary phase. Hydrophilic molecules in the mobile phase will tend to elute first. Separating columns typically
comprise a C8 or C18 carbon-chain bonded to a silica particle substrate.

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 Atomic Absorption Spectroscope

Based on the principle of flame test used in quantitative analysis

When an alkali metal salt or Calcium, Strotium or Barium salt are heated in a Bunsen burner flame, a
characteristic color is observed.. such as;
 Na+ - Yellow
 Li+ - Crimson
 Ca2+ - Brick red
 Sr - Crimson
 Ba – Green

In the flame, the concentration are reduced to gaseous metal atoms

The high temperature of the flame excites a valence electrons to high energy orbital

The atoms then emit energy in the form of visible light as the electrons fall back into the lower energy orbital
(ground state)
Excited state

Absorbed energy Emitted energy

Ground state

Ground state atoms absorb light of the same characteristic wavelength as it emits when returning from excited
state to ground states.

Intensity of absorbed light is proportional to the concentration of the element in the flame

In quantitative analysis, absorbance or emission of atomic vapor is measured.

Absorbance = - log (transmitted radiation)

(Incident radiation)

Transmission = - log (Intensity of radiation reaching the detector in absence of the sample)
(Intensity of radiation reaching the detector in presence of the sample)

Schematic diagram of Atomic Absorption Spectroscopy

Light source lens atomized lens monochromator detector amplifier read out
sample
Nebulizer

Sucks up the liquid sample

Creates fine aerosols (fine sprays) for introduction into flame

Mixes aerosols, fuel & oxidant thoroughly creating heterogeneous mixture

The smaller the size of the droplet produced, the higher the element sensitivity
Advantages of AAS
• Inexpensive in equipment and day to day running
• Higher sample throughput
• Easy to use
• High precision

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 Disadvantage of AAS
• Only solution can be analyzed
• Relatively large sample quantity required (1 -2 Liters)
• Less sensitive (compared to graphite furnace)
• Possess problems with refractory elements
Duboscq Visual Colorimeter

The principle of visual comparison is made use of in Duboscq comparator or colorimeter which is an
instrument that allows variation of the depth of liquid through which the light must pass to reach the eye

The depth is adjusted until the visual intensity through sample and standards are equal.

Concentration is then calculated by the above relation

The duboscq colorimeter is equipped with an optical system which permits the ready comparison of the beams
passing through an eye piece with a split field.

Disadvantages of Visual Colorimeter Methods

Requires availability of standards or series of standards

Eye unable to match the color in presence of a second colored substance

Eye not sensitive enough to small difference in absorbance

Concentration difference smaller than about 5% relative are difficult to be detected.
Light Path of a Visual Colorimeter

View

eye piece

cell

light

light

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 Electrophoresis

It is the movement of charged particles such as molecules or ions under the influence of electric current.

Electric field is applied to a solution by placing two opposite charged electrodes in a solution

Depending on nature of charges, the molecules moves through the solution towards the electrodes of opposite
charge

The positive charged particles (cations) move towards negatively charged electrodes (Cathode) and vice versa.

At fixed pH (isoelectric pH), the rate of migration of particles with similar charge will depend on;
 Magnitude of charges carried on them
 Molecular weight or charge size ratio

Because of these variations in migration, electrophoresis can be used to separate complex mixture of plasma
proteins into number of fractions or zones

This is termed as Zone electrophoresis.

Turbidimetry & Nephelometry

When light strikes a particle in a liquid, light can either be ; absorbed, transmitted, reflected or scattered

In TURBIDIMETRY, turbidity causes a reduction in the intensity of incident light as it passes through a
solution of particles

Turbidimetry is the measurement of this loss of intensity because of scattering, absorption or reflectance of
incident light

Turbidity is measured at 180o from incident beam.

Most colorimeters can measure turbidity with good precision and accuracy.

In NEPHELOMETRY, this is the measurement of light scattered or reflected towards a detector which is not
in the direct path.

The instrument used for nephelometry is called nephelometer

Most nephelometers measures scattered light at 90o to the incident light.

Light scattering instruments are useful in immunoassay of serum proteins, Ig, coagulation factors and drugs

Schematic diagram of light scattering Instrument

Light Source

Excitation optics

Excitation filter

Specimen

Light scattering optic

Turbidimeter Detector filter

o
30 forward Detector 90o Nephelometer
Scatter nephelometer

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 Osmometry

Measurement of Osmolarity of a solution

Osmolarity depends on the number of particles in a solution

The size, charge or mass of a molecule does not affect the measurement

One (1) Osmole of a substance is equals to the gram molecular weight divided by number of particles or ions
in which the substance dissociates into solution

For example, NaCl dissociates into 2 ions (Na+ and Cl-)

1 Osmole of NaCl = 58.5/2 = 29.25g = 0.5 mole

One Osmole can be measured as a mass of the solute which when dissolved in 1kg of pure water produces
osmotic pressure of 22.4 atm at normal temperature and pressure.

Can also be defined as mass of solute which when dissolved in 1kg water depresses the freezing point by
1.86oC.

Osmometers are the most often used to measure osmolarity of urine. This differs from Specific Gravity

S.G is the mass concentration of solute

Osmolarity is the molecular concentration of solute.
ξm = νmφ
Calculation of osmolality
A 0.90% w/w solution of sodium chloride (mol. wt. # 58.5) has an osmotic coefficient of 0.928.
Calculate the osmolality of the solution.
Answer
Osmolality is given by ξ_m = νmφ so,

ξ_m = 2 ×9.0× 0.928 = 286 mosmol kg_−1

58.5
Fluorometry

Fluorometry is the measurement of fluorescence

When a compound (fluorescence compound) absorbs light energy from one wavelength, an electron is raised
to higher energy level (excited)

Part of the absorbed energy is used up

When the electron fall back to its original state, excess energy is emitted as light and the substance is said to
fluoresce.

The emitted light has a lower energy level (higher wavelength) than the absorbed light.

Some fluorescent substance absorbs light energy from the invisible Ultraviolet rays (340nm wavelength) and
emit light in the visible spectrum (400nm -700nm)

Fluorescein Isothiocyanate absorbs UV light & fluoresces green light.

Basic components of fluorometers are;
(i) Excited source
(ii) Excitation monochromator
(iii) Sample cell (cuvette)
(iv) Emission monochromator
(v) detectors

This is extremely sensitive technique & widely used for immunochemical measurement for analysis of drugs,
peptides, hormones and other compounds present in very low concentrations.

Flame emission Spectroscopy

This is also referred to as flame photometer or flame spectrometry

When some elements are heated directly in a flame, their atoms become excited and emit energy at wavelength
characteristic of that element.

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Intensity of the flame color is directly proportional to the concentration in the sample

Commonly used for elements such as;

Symbol Name Color Image

As Arsenic Blue

B Boron Bright green

Ba Barium Pale/Apple green

Ca Calcium Brick red

Cs Caesium Blue-Violet
Cu(I) Copper(I) Bluish-green

Cu(II) Copper(II) (non-halide) Green

Cu(II) Copper(II) (halide) Blue-green

Fe Iron Gold
In Indium Blue

K Potassium Lilac

Li Lithium Red

Mn (II) Manganese (II) Yellowish green

Mo Molybdenum Yellowish green

Na Sodium Intense yellow

P Phosphorus Pale bluish green

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 Pb Lead Blue/white

Ra Radium Crimson red

Rb Rubidium Red-violet

Sb Antimony Pale green

Se Selenium Azure blue

Sr Strontium Crimson

Te Tellurium Pale green

Tl Thallium Pure green
Zn Zinc Colorless (sometimes reported as bluish-green)

Flame intensity can be measured in a flame photometer using appropriate filter

There are two methods of flame photometry
• Flame emission photometry
• Flame absorption photometry
Principle

Using compressed air, dilute serum/plasma is sprayed as fine droplets into a non-luminous gas flame
which becomes colored by the characteristic emission of sodium/potassium metallic ions in the sample.

Using light filter or prism system, light of wavelength corresponding to the metal being estimated is
selected.

The amount of light emitted is directly proportional to the concentration of metallic ions present in the
sample.

Atoms
in sample burning

photocell galvanometer
Atomizer Entrance Filter Exit slit
(Sample+air+fuel) slit

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 Basic principle of flame photometer

Nebulizer;
• Mechanism of mixing sample with air and spraying it to the burner at a constant rate as a fine spray.

Mixing chamber with baffles

• Atomized sample and fuel are mixed in mixing chamber
• Baffles deflect only large droplets to waste while small droplets are allowed to get through the flame

Burner;
• Means of reducing metal to atomic state and exciting the atoms to emit light.

Photosensitive element;
• Means of detecting and recording intensity of light emitted.
• Diode is used to convert the emitted light into electric current.

Wavelength selector
• Device for selecting the wavelength of the emissions to be measured
• Lens focuses light from flame and a narrow band filter selects wavelength.

Working system of flame photometer

Sample and the standard are diluted manually or by peristaltic pump

Diluent contains lithium

Diluted sample is drawn through a capillary tube by stream of air supplied by the compressor

Air current causes the sample to be split into a haze of tiny droplet (Nebulization)

Large droplets fall to the bottom of nebulization chamber and goes to waste.

Remaining haze of small sized droplets is mixed with propane

Mixture on passing through a baffle plate, fuel-air stream is ignited & burns, thus converting the metal present
to their atomic form

Light from the flame is measured at appropriate wavelength by photodetector.

Potentiometry

This compares the difference between two electrodes immersed in a solution under zero current conditions

By using a reference electrode known as a potential, the charge on the other indicator electrode can be
determined

Commonly used reference electrode is saturated calomel electrode & silver chloride electrode

Indicator electrode can be platinum wire, carbon rod or a thin stream of mercury

Potentiometry is used for measurement of pH

Ultraviolet spectrophotometry

Wavelength used in spectrophotometry depends on the wavelength at which there is significant absorption by
the substance to be measured

If the wavelength of the light absorbed is in UV region of the spectrum which is non-visible (200-400nm),
analysis is called UV spectrometry.

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 Coulometry

This measures the current passing through between electrodes

When a constant current is applied to generate a titrating agent, time required to titrate the sample is related to
the amount of analyte in the sample.

Clinical application is the measurement of chloride in serum and other body fluids

If a constant current is applied across silver electrode, it liberates silver ions into the solution at a constant rate.

When all the chloride ions in solution have been combined with silver ions, excess silver ions in solution activate
a relay to stop titration.

The length of time of this titration is directly proportion to the concentration of chloride in the sample.

Ion Selective Electrodes (ISE)

If a metal rod is placed in one of its salt, it acquires an electric potential

If two different metals are immersed separately into its own salts, the difference in their potential can be
measured

An ion selective membrane is required to separate the solution of unknown activity from the detecting system

If unknown solution has different concentration of the particular ion from that of the solution filling the
electrode, the ions migrate towards the lower concentration.

This brings about a change in potential across the membrane

Process goes on until the equilibrium is established across the membrane

Number of ions transported across the membrane is related to the original concentration of the ion in unknown
solution

ISE are used to measure dissolved gasses e.g. pCO2 or serum electrolytes such as Na+ & K+

Immunochemical assay

They make use of antigen-antibody reactions.

Antigenic properties of some drugs & hormones are used to raise specific antibodies in experimental animals

These antibodies are used as specific reagents for the detection & quantitation of drugs & hormones in patient’s
specimen

Serum immunophoresis is useful in detection of abnormal fraction in the serum which combines immunoassay
with electrophoresis.

Radioactivity

Atomic number of an element is the number of protons in the nucleus

Mass number is the total number of protons and neutrons

Nuclide is an atomic species (element) with a given atomic number & mass number

Isotopes are nuclides with the same atomic number but different mass number

Isotopes of lighter elements are stable

Isotopes of heavy elements are unstable

Unstable nuclides undergo spontaneous decay to produce stable nucleus by a process known as radioactive
decay.

Decay is accompanied by emission of energy inform of radiation

Half-life of radioactive substance is the time required for the isotope to decay to half its activity

Radiation from an atom or its nucleus may either be

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 1) Particulate
2) Electromagnetic

Particulate radiation (decay)

Consist of small particles of nuclear fragment such as Alpha (∞) & Beta particles

Alpha decay emit alpha particles made up of 2 neutrons and 2 protons

Beta decay nuclide losses one neutron and gains one proton.

Electromagnetic Radiation

This is divided into two groups;
o Radiation with very low energy & long wavelength (radio waves)
o Radiation with high energy and high wavelength (light waves)

Radiations with highest energy are called X-rays & gamma rays.

Gamma rays can penetrate materials which blocks alpha and beta radiation.

Detection and measurement of radioactivity

Darkening of photographic emulsion is used for x-rays

Scintillation counting system are in common use in clinical chemistry

Isotopes used in radioactivity measurement includes; 125I & 3H
125

I emits gamma rays while 3H emits beta particles

Gamma rays are detected by scintillation counter which is a large sodium iodide (NaI) crystal with thallium as
an activator.

Crystal is in close contact with photomultiplier tube

When gamma ray strikes a molecule of NaI, a photon of light is produced.

Light is amplified by photomultiplier tube & converted to pulse of electrical energy

Number of pulse is proportional to the quantity of radioactive material in the sample.

Beta particle are detected by liquid scintillation system

These particles are called fluors and are dissolved in special grade solvent such as toluene

Light signals are picked up by two photomultiplier tubes and converted to pulses

Application

Radioactivity is used for estimation of hormones, drugs & other substances present in minute quantities in
body fluids

Radio Immunoassay (RIA) is the method of choice

Gravimetric analysis

Gravimetric analysis describes the set of methods in analytical chemistry

This is for the quantitative determination of analyte based on mass of solid

Can be generalized into two types
 Precipitation
 Volatilization

Quantitative determination by precipitation involves isolation of an ion in solution by precipitation reaction,
filtering, washing the precipitate free of contaminants, conversion of precipitate to a product of known
composition & finally weighing the precipitate & determining the mass difference.

From the mass of unknown composition of precipitate, the amount of the original ion can be determined

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 Example
-

When Cl is precipitated out by adding Ag+, low solubility of AgCl is reduced still further by the excess of
Ag+ which is added pushing the equilibrium to the right

We can further decrease the solubility by decreasing the temperature of the solution by using an ice bath.

ADVANTAGES OF GRAVIMETRIC ANALYSIS

It provides exceedingly precise analysis

Provides very little room for instrumental error

Does not require a series of standard for calculation of unknown

Can be used to calibrate other instruments in view of reference standard

DISADVANTAGES OF GRAVIMETRIC ANALYSIS

Only provides for analysis of a single element or limited group of element at a time

Methods are often convoluted & a slim mis-tep in a procedure can often mean error for the analysis

Spectrophotometry methods are much more efficient than gravimetric analysis

Osmolarity vs. Osmolality

Measurements of osmolar concentration are often expressed in osmolarity and osmolality. Osmolarity is a measure
of the osmoles of solute per liter of solution. A capital letter M is used to abbreviate units of mol/L. Since the volume
of solution changes with the amount of solute added as well as with changes in temperature and pressure, osmolarity is
difficult to determine.

Osmolality is a measure of the moles (or osmoles) of solute per kilogram of solvent expressed as (mol/kg, molal, or
m). Since the amount of solvent will remain constant regardless of changes in temperature and pressure, osmolality is
easier to evaluate and is more commonly used, and often preferred, in practical osmometry. Most commercially
available osmometers report results using osmolality units mOsm/kg.

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