DEPARTMENT OF

CLINICAL CHEMISTRY

INSTRUMENTS &
PRINCIPLES

BY AZEEMA JAMIL
 INSTRUMENTS CAN BE DIVIDED ON THE BASIS OF THEIR
PRINCILPES.

A- SPECTROPHOTOMRTY
Following instrument based on spectrophotometer principle.
1- Genesis 2- Dimension 3- ELISA
4- Hitachi 5- Synchron 6- Micro lab
7- Advia 1800

B- ISE METHOD
Following instrument based on ISE principle.
1- Medica Easylyte
2- Synchron (CX3-portion)

C- CHEMILUMINESCENT
Following instrument based on CHEMILUMINESCENT principle.
1- ELECSYS 2010/ E-170
2- IMMULITE
D- DIFFERENT TECHNIQUES

Use to analyze single test. These instruments are following

1- Osmometer (freezing point method)
2- Electrophoresis
3- Atomic absorption
4- Competitive Label Immunoassay
5- Nephlometry
Spectrophotometer;

 Measure the amount of light that a sample absorbs. The

instrument operates by passing a beam of light through a sample
and measuring the intensity of light reaching a detector.

 deals with visible light, near-ultraviolet, and near-infrared.

 Spectrophotometry involves the use of a spectrophotometer.

 A spectrophotometer is a photometer (a device for measuring light

intensity) that can measure intensity as a function of the light
source wavelength.
 BEER’S LAW
Beer’s Law : Absorbance = (ε) (b) (c)
ε = molar absorptivity ( constant for each type of molecule )
b = length of the light path ( curvet )
c = concentration of the molecule absorbing the light

Hopefully, you will use the same size curvets for all your measurements,
so b remains the same for all measurements.

Also, since molar absorptivity does not change either, it remains the same
for all measurements.

The molar absorption coefficient, or molar absorptivity, is a measurement of how

strongly a chemical species absorbs light at a given wavelength.

Because Beer’s Law states that absorbance (A) is directly proportional to concentration. the
following mathematical relationships can be established

Aunk
Cunk  x Cstd
Astd 5
The following simulation illustrates the procedures for making spectrophotometric
measurements.
• First, the intensity of light (I0) passing through a blank is measured. The intensity is
the number of photons per second.

• The blank is a solution that is identical to the sample solution except that the
blank does not contain the solute that absorbs light. This measurement is
necessary, because the cell itself scatters some of the light.

• Second, the intensity of light (I) passing through the sample solution is measured.
• Third, the experimental data is used to calculate two quantities:
• the transmittance (T) and the absorbance (A).
T= I/IO
A = - log10 T
Transmittance; The fraction of light that passes through the sample and reaches the
detector. The remainder of the light is the fraction of the light absorbed by the
sample.
• Absorbance; A logarithmic measure of the amount of light absorbed (at
particular wavelength) as the light passes through a sample or substance.
There are two types of photometric assay:

 End Point Assay

 Rate Reaction Assay
END POINT ASSAY

Measurements taken after the reaction have stopped and makes a

colored complex.

The intensity of the colored product is an indicator of the sample

component’s concentration.

The colored formed should be read at a given wavelength.

Concentration = Abs of test – RB x Con of Std.
Abs of Std. – RB
Reagent Blank = Initial absorbance of any working
reagent.
Standard = A pure substance with known concentration.
RATE REACTION ASSAY

Found continuous and uniform change of absorbance

when add serum in reagent.

There is a continuous increase or decrease of

absorbance. Increase or decrease depending on the two
reaction method.

1- Oxidation method
2-Reduction method
 Reduction is the gain of an electron. Sometimes we also have H
ions along for the ride, so reduction also becomes the gain of H.

 Oxidation is the loss of an electron (or hydrogen). In

oxidation/reduction reactions, one chemical is oxidized, and its
electrons are passed (like a hot potato) to another (reduced,
then) chemical. Such coupled reactions are referred to as redox
reactions
Passage of electrons from compound A to compound B. When A loses its
electrons it is oxidized; when B gains the electrons it is reduced
ISE METHODLOGY

 Synchron
 Cx3-portion (Na,K,Cl)
 MEDICA EASYLYTE
ION-SELECTIVE ELECTRODE (ISE)
This principle is used for the determination of sodium ion, potassium
ion, and chloride ion concentration by measuring electrolyte activity in
solution.
Sample is mixed with a buffered solution.
The buffered established a constant ionic strength and set a constant
activity co efficient for the electrode.
Sodium -> consist of glass electrode
Potassium -> consist of electrode with valinomycin membrane
Chloride -> Ag/ Agcl type.
Lithium -> Plastic tube electrode
When sample / buffer mixture contact with their specific electrode, the
ion exchange take place and a change in potential (voltage), is
developed at the face of the electrode and change in potential allows the
calculation of each analyte in a solution.
CHEMILUMINESCENT
PRINCIPLE
CHEMILUMINESCENCE

 Chemiluminescence as an analytical tool has three important

strengths:
 Sensitivity
 Selectivity
 A wide linear range

SENSITIVITY
Lower detection limits when compared to absorption or
fluorescence techniques in pursuit of the same analyte.
Selectivity
The analyte of interest generates its signal often in the presence of
normally interfering compounds and do not themselves produce light
until the chemiluminescent reagents are mixed together.

A wide linear range

Analytically useful because samples of larger concentration ranges
can be analyzed without dilution.

Reactions that produce light without heat are called

chemiluminescent reactions.
Chemoluminescence
Is the term for light that’s emitted as a product of chemical
reactions. Chemiluminescent reactions produce unstable products,
which then decay in order to form more stable products. In the
process, energy is emitted in the form of light.

Two chemicals react to form an excited (high-energy) intermediate,

which breaks down releasing some of its energy as photons of light
to reach its ground state.
A+ B AB* Products + Light
(Excited intermediate)
Photon Is a discrete bundle of electromagnetic (or light) energy.
Photons are always in motion and, in a vacuum, have a constant
speed of light to all.
The following is a brief primer on common Chemoluminescence
reagents.

 LOPHINE
 LUMINOL
 OXALATE ESTERS
 RUTHINIUM TRIS BIPYRIDINE
 LUCIFERIN
 H2O2 or organic peroxides are commonly used as the oxidants in
the solution phase in chemiluminescent techniques. Hydrogen
peroxide (H2O2) is the simplest peroxide and an oxidizer.

 The oxidizing capacity of hydrogen peroxide is so strong that it

is considered a highly reactive oxygen species.

 The bond between the two oxygen atoms is easily cleaved, and
releases a significant quantity of energy when broken.
 ELECTROCHEMILUMINESCENCE

 ECL is a means of converting electrical energy into light

(radiative energy).

 It involves the formation of electronically excited states by

electron transfer reactions.

 ECL is a process in which highly reactive species are

generated at the surface of an electrode. These highly
reactive species react with one another, producing light and
this emission of light initiated electrically, rather than
chemically.
ECL was generated in an electron transfer reaction between an
oxidized and reduced species, both are generated at an electrode by
alternate pulsing of the electrode potential.

 A + e_ → A__ (reduction at electrode) (1)

 D – e_ → D_+ (oxidation at electrode) (2)
 A_+ D_+ → A*+D excited state formation (3)
 A* → A + hí (light emission) (4)

It generally uses Ruthenium complexes, (which releases a photon at

~620 nm) regenerating with TPA (Tripropylamine) in liquid phase or
liquid–solid interface
OTHER TECHNIQUES
OSMOMETRY

 Osmometers are analytical instruments used to measure

the concentrations of total dissolved substances that
contribute to the osmotic pressure of solutions

 There are two common types of osmometers

 Freezing Point Depression analyzers

 Vapor Pressure analyzers

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FREEZING POINT
Temperature at which a liquid solidifies under a specified pressure .
The temperature at which the liquid and solid phases of a substance of
specified composition are in equilibrium at atmospheric pressure.

Concentration:- the amount of solute in a given amount of solvent. The

amount of solute is usually expressed in terms of moles.
Morality: Moles of solute per liter of solution
Molality Moles of solute per kilogram of pure solvent.

One osmole is defined as Avagadro number of single particles. Common

unit of osmoles are

Osmolarity osmole of solute per liter of solution

Osmolaltiy osmole of solute particles per kilogram of pure solvent.
Freezing-point depression

Describes the phenomenon in which the freezing point of a liquid (a

solvent) is depressed when another compound is added, meaning that a
solution has a lower freezing point than a pure solvent.
 Freezing point depression is a colligative property of matter.
Colligative properties depend on the number of particles present, not
on the type of particles or their mass. Properties of solutions
determined by the number of dissolved molecules
 Freezing point depression can be calculated using the Clausius-
Clapeyron equation and Raoult's law. In a dilute ideal solution the
freezing point is:
 Freezing Pointtotal = Freezing Pointsolvent - ΔTf

 where ΔTf = molality * Kf * i

 Kf = cryoscopic constant (1.86°C kg/mol for the freezing point of
water) 25
Freezing point principle:- The quickest and most accurate way to measure the
freezing point of a solution is to super cool the solution several degree below its
freezing point and then vibrate it internally

Fast Cool:- The thermistor probe (sensing the temperature of the sample) and the stir
–wire (vibrate the sample) dip in the sample, the sample begin cooling rapidly.

Slow Cool:- The temperature of the sample reaches O0C, the cooling rate of the
sample slow.

Freeze:- The sample has been sufficiently super cooled and sample become freeze.

Heat of fusion:-The heat of fusion escape the sample and the temperature rises to
the actual freezing point of the sample

Plateau:- The instrument follows the plateau of sample

Read out:- As the plateau is constant it locks the reading onto the display.
ELECTROPHORESIS

Movement of charged particles because of an external field is called

electrophoresis.

Charged molecules can be separated if they have different velocities in an

electric field.

Therefore electrophoresis is a separation technique.

 Molecules from patient specimens are treated with buffer solutions
to give them electrical charge and are placed onto the surfaces of
semi-solid supporting media

 When electrical current flows through the media, electrically

charged molecules “migrate”, or move along the supporting media

 The rate at which different molecules move along the media will
vary depending on the physical characteristics of the molecules.

 After migration, the strip is removed and stained with an

appropriate stain - “bands” of stained molecules will be visible

28
ATOMIC ABSORPTION

 Atomic absorption spectroscopy is a technique for determining the

concentration of a particular metal element in a sample.

 The technique can be used to analyze the concentration of over 70

different metals in a solution

 A cathode tube emits a light wavelength unique for that metal.

Common metals are lead and copper.

 The patient specimen is vaporized into an acetylene flame and is

dispersed into the light path.
 Metal atoms in the ground state from patient specimen
absorb the light energy from the cathode tube.

 The chopper breaks up the signal from the cathode tube.

 The photo detector measures the difference in the signals to

measure the light emitted from the flame only.

 Atomic Absorption is only suited for metallic substances that

are not destroyed by the flame - Everything else gets “fried”.

30
Competitive Immunoassays (RIA,MEIA,FPIA)
 Competition between tagged and un-tagged antigen for limited
antibody
 Tagged antigen Reagent
 Untagged antigen Patient antigen we want to measure
 Specific antibody Reagent
 Let the competition begin !!!
 Mix the three components together
 Allow the antigens to compete for the limited antibody
 Antibody will bind with tagged or un-tagged antigen ( it
doesn’t care )
 Separation Step : Antibody-Antigen complexes are
separated from free antigen
 Tagged antibody-antigen complex is measured
31
 The tagged antigen and antibody from the reagent kit are
constant.
 The only variable is the concentration of the patient antigen
( the thing we want to measure )
 A standard curve can be constructed with known antigen
concentrations giving the following general results

 High concentrations of patient antigen means that more of

the antibody-antigen complexes are untagged

 Low concentrations of patient antigen means that more of

the antibody-antigen complexes are tagged

 There is an inverse relationship between patient antigen

concentration and tag activity after the separation process 32
 Nephelometry
 In nephelometry the measurement is made by measuring the light
passed through a sample at an angle. (usually at 90 degrees).
 It is based on the principle that a dilute suspension of small
particles will scatter light (usually a laser) passed through it
rather than simply absorbing it.
 Antibody and the antigen are mixed in a ratio that only small
aggregates are formed.
 That do not quickly settle to the bottom.
 The amount of light scatter is measured.
 Compared to the amount of scatter from known mixtures.
 The amount of the unknown is determined from a standard curve.
 What happens inside the flow cell?

Analyte-antibody complexing

Too small to detect optically

Now big enough to scatter light

Analyte Antibody Other stuff

 Scatter Signal Versus Time
Nephelometry

3 Adding antibody causes a large

increase in light scattering
Scatter

2
Adding sample increases scatter slightly
1
Fluid in the cell has causes little scatter

Time
1. Buffer addition 2. Sample Addition 3. Antibody addition
 Nephelometry can be used to detect either antigen or antibody.

 It is usually run with antibody as the reagent and the patient

antigen as the unknown.