UNIT V

BIO-CHEMICAL MEASUREMENT

13
Colorimetry

Colorimetry is the field of determining the concentration of a coloured compound in a solution.

Colorimeter
A colorimeter, also known as a filter photometer, is an analytical machine that acts as the tool
quantify a solutions concentration by measuring the absorbance of a specific wavelength of light.

Applications of Colorimeters:
Colorimeters are used for a wide range of applications across the chemical and biological fields :
Specifically for
 analysis of blood, water, nutrients in soil and foodstuffs determining the concentration of
a solution
 determining the rates of reaction
 determining the growth of bacterial cultures and
 laboratory quality control.

working principle of Colorimeters:

When a wavelength of light is passed through a sample, some of the light is absorbed and some
passes through. It is the wavelengths of light that pass through that are detected. By knowing which
wavelengths have passed through, the detector can also work out which coloured wavelengths were
absorbed.

Beer-Lambert’s law
The Beer-Lambert law states that there is a linear relationship between the concentration and the absorbance
of the solution, which enables the concentration of a solution to be calculated by measuring its absorbance.
PHOTOMETERS AND COLORIMETERS:
The composition of blood serum is determined by specialized chemical techniques. The different
components of biological substances can be determined by measuring how they either absorb or emit
visible light. Colorimeters and photometers are used to measure the transmitted and absorbed light as it
passes through a sample. The colorimeter uses a light absorption to determine blood proteins and iron
levels. In order to enhance the colour of these substances in blood serum, it is necessary to mix it with
reagents. The basic principle behind these colorimeters is that many chemical compounds in solution

13
appear coloured with the saturation of the colour depending on the concentration of the compound. By
analysing the transmitted light through the sample or emitted light by the sample the concentration of the
substance can be determined.
Transmittance, T = I1 / Io
Where, I1 = Transmitted light intensity and
Io = Incident light intensity
Absorbance or optical density, A = - log (I1 / Io)
(or) A = log (1/T)
Thus A = αcL
Where, α = absorbtivity which depends on the absorbing substance and optical
wavelength at which the measurement is performed.
c = concentration of the absorbing substance and
L = pathlength of the cuvette

By measuring optical density or absorbance 'A', the concentration of given substance in the
sample can be determined.
1. FILTER PHOTOMETER (COLORIMETER):
Colorimeters can be either filter photometer or spectrophotometer. When an interference filter is used to
select a given wavelength, it is called filter photometer.

The schematic diagram of a simple filter photometer is as shown in the figure below. It is used to
measure transmittance.

Colorimeter
Light from a halogen lamp is made to incident on a filter, F. It transmits only a suitable
wavelength range, at which the measurement is performed. The divergent transmitted light is converted
into two parallel beams by an optical arrangement. One beam on a reference selenium photoelectric cell,
CR and other beam fall on a sample selenium photoelectric cell Cs after passing through sample in the
cuvette. Without the sample, the outputs from photoelectric cells are same. When the sample is placed in
the path, the output of the sample cell is reduced and hence the potentiometer is adjusted that both the
cells CR and Cs give the same output which is indicated by the null deflection in the galvanometer 'G'.
Since the potentiometer is calibrated in terms of transmittance, the concentration of the given substance
in the sample can be determined.

13
2. SPECTROPHOTOMETER:
When a diffraction grating or prism is used as a monochromator to get different spectral components or
wavelengths of interest in the colorimeter, then it is called as spectrophotometer. The spectrophotometer
allows the determination of the absorption of samples at various wavelengths.

The light output from a halogen lamp is passed through an entrance slit S1 and incident on a
concave reflector which focuses the light on a diffraction grating ‘g’ (or) a prism to disperse light. The
selective wavelength from the dispersed light is obtained by taking it at the given direction and then it is
allowed to incident on the reflector.

Spectrophotometer
From the reflector, the light beam is directed to the sample through a narrow exit slit S2. A
sensitive photodetector ‘D’ detects the transmitted light and gives an electrical output corresponding to
the intensity of the transmitted light.
The amplifier amplifies the output from the detector and finally the indicator indicates the
concentration of the substance. By rotating the grating, the measurements can be made at different
wavelengths.

3. SODIUM POTASSIUM ANALYSER (FLAME PHOTOMETER):

A flame photometer is used to analyze urine or blood in order to determine concentration of
potassium (K), sodium (Na), calcium (Ca) and lithium (Li). Lithium is used as a calibration substance in
the analysis of the other three substances. A known amount of lithium is added to the sample and the
emitted light intensity of the sample under analysis is measured relative to that of the lithium.

13
 Using an atomizer, the liquid sample is sprayed into fine droplets by passing oxygen or air
through the opening in it. A combustible gas, like acetylene is also added with air. The sample-air
mixture is burnt out and the light emitted in the flame is passed through a narrow slit and then to
diffraction grating. The diffracted colours are incident on various photodiodes as shown in the figure.

The variations in the intensity of light due to changes in the flow rate of the air or the changes in
the flow of fuel gas are eliminated by proper electronic monitoring circuit. The concentration of
potassium ions is detected by observing the peak height of the spectral line corresponding to it. For
potassium, the wavelength is 4047Å (violet). For sodium, the wavelength is 5890Å (yellow). For lithium
it is 6708 Å (red). Separate photodetector is used for each channel.

The photodetector circuit consists of a reverse biased diode in which current flow increases as the

13
intensity of light incident upon it increases. Calibration potentiometer in each channel is used to calibrate
the instrument with standard solution whose concentration is known.

4. AUTOANALYSER:
The autoanalyzer sequentially measures blood chemistry and displays this on a graphic readout.
As shown in Figure below, this is accomplished by mixing, reagent reaction, and colorimetric mea-
surement in a continuous stream. The system includes the following elements.
1. Sampler - aspirates samples, standards, and wash solutions to the autoanalyzer system.
2. Proportioning pump and manifold - introduces (mixes) samples with reagents to effect the
proper chemical color reaction to be read by the colorimeter. It also pumps fluids at precise flow
rates to other modules, as proper color development depends on reaction time and temperature.
3. Dialyzer - separates interfacing substances from the sample material by permitting selective
passage of sample components through a semipermeable membrane.

AutoAnalyzer
4. Heating bath - heats fluids continuously to exact temperature (typically 37°C incubation,
equivalent to body temperature). Temperature is critical to color development.
5. Colorimeter - monitors the changes in optical density of the fluid stream flowing through a
tubular flow cell. Color intensities (optical densities) proportional to substance concentrations are
converted to equivalent electrical voltages
6. Recorder – Converts optical density electrical signal from the colorimeter into a graphic display
on a moving chart.

13
 The heart of the autoanalyzer system is the proportioning pump. This consists of a peristaltic
pump. Air segmentation in the mixing tube separates the sample / reagent mixture from the cleaning fluid
and other samples. As these air-separated fluids traverse the coil of the mixing tube, effective mixing
action is achieved.
One problem with automatic analyzer is certain identification of samples. Patient data can be
intermixed with that of other patients if care is not taken.
Sterilization is also needed for samples, glassware and equipment parts that are contaminated with
disease. Diseases such as hepatitis or other communicable infections can be spread to equipment
operators.
5. BLOOD CELL COUNTER:
The blood cells have important functions in our body. The red blood cell is used for the transport
of oxygen and carbon dioxide. The white blood cells are part of the body’s defenses against infections
and foreign substances. The platelets are involved in the clotting of blood. The red blood cells in the
blood consist of hemoglobin.
When the body produces too many red blood cells, the amount of hemoglobin in the blood
increases and a chronic disease called polycythemia is produced. When the hemoglobin in the blood
decreases, anemia is produced. The anemia produces headache and giddiness. The amount of
hemoglobin is normally 130-170 g/l for men and 120-160 g/l for women.
When the whole blood is centrifuged, the blood cells sediment and form a packed column at the
bottom of the test tube. Most of this column consists of the red blood cells, with the other cells forming a
thin, buffy layer on top of the red cells. The volume of the packed red cells is called the hematocrit. If
the number of red blood cells is known, this number and the hematocrit can be used to calculate the mean
cell volume.

Hematocrit determination

13
 The hematocrit can be determined by aspirating a blood sample into a capillary tube and closing
one end of the tube with a plastic sealing material. The tube is then spun for 3 to 5 minutes in a high-
speed centrifuge to separate the blood cells from the plasma. From the capillary tube, the blood and the
packed cell volume can be compared by measuring the length of the columns. The packed cell volume is
the ratio between the height of the packed cells and the height of the blood in the tube. This is made with
the help of nomogram (graph to show the direct reading of the hematocrit). Normal range of packed cell
volume for men is 42% - 54% and for women is 37% - 47%.

Manual Method:
Blood cell counts by manual method are performed by a microscope. At first the blood is diluted
in the ratio of 1:100 or 1:200 for counting RBC’s and 1:10 or 1:20 for counting WBC’s. The diluted
blood is then brought to the counting chamber of 0.1 mm deep which is divided into a number of squares.

Conductivity Method (Coulter Method):

Simple count of blood cells can be performed by coulter method or conductivity method. Here
blood cells have lower electrical conductivity than the solution in which they are suspended. The beaker
contains diluted blood solution and a closed glass tube with a very small orifice is inserted into the
beaker. The conductance between the solution in the glass tube and the solution in the beaker is
determined with the help of electrodes. The conductance can be determined by the diameter of the orifice.
The glass tube is connected to a suction pump through a U-tube filled with mercury. Due to the negative
pressure, the solution from the beaker flows into the glass tube through the orifice.

13
 Conductivity method
When each blood cell passes through the orifice into the glass tube, it temporarily blocks the
electrical path and causes a drop in the conductance measured between the electrodes. This change in the
conductance gives a pulse at the output of conductivity meter. The amplitude of the pulse is proportional
to the volume of the cell. A threshold circuit lets only those pulses pass that exceed certain amplitude.

The pulses that pass this circuit are fed to a pulse counter through a pulse gate. The gate opens when the
mercury column reaches a first contact and closes when it reaches the second contact, thus counting the
number of cells contained in a given volume of the solution passing through the orifice.
A count is completed in less than 20 seconds. With counts of up to 1,00,000 the result is
statistically accurate. Great care must be taken to keep the aperture from blockage. This method not only
determines WBC and RBC counts but also the hematocrit and the hemoglobin concentration.

Laser based Cell counting:

This modern technique is used to determine the number of RBC, WBC and platelets. The cell
volume of the red blood cell and the haemoglobin concentration can also be determined by this method.
The principle used in this laser based blood cell counting is “the angle of scattered light is
different from different sized blood cells”. The blood is diluted and passed through the capillary tube.
The laser light is passed through the glass tube and the blood cells in the tube scatter the light. The
scattering angles of platelets and RBC are different. They are detected by two different photo-detectors.
The detectors are given the digital voltmeter which gives the density of blood cells and platelets. Lysing
agent is used to destroy the RBC’s and the WBC number can be determined. The haemoglobin
concentration in the RBC’s can also be measured by this method.

13
 Laser based cell counting

6. BLOOD GAS ANALYZERS

6.1 Blood pH Measurement:
The most important indicator of chemical balance in the body is the pH of the blood and other
body fluids. The pH is directly related to the hydrogen ion concentration in a fluid. Specifically, it is the
logarithm of the reciprocal of the H+ ion concentration.

In equation form,

The pH is the measure of the acid-base balance of a fluid. A neutral solution (neither acidic nor
base) has a pH of 7. Lower pH numbers indicate acidity, whereas higher pH values define a basic
solution. Most human body fluids are slightly basic. The pH of normal arterial blood ranges between 7.38
and 7.42. The pH of venous blood is 7.35, because of the extra CO2.

Digital pH meter
13
 To pH meter consists of a pH electrode which consists of a glass (active) electrode terminal and
reference terminal. The glass electrode consists of a thin glass membrane that allows only hydrogen ions
to pass through it in the form of H3O+ (Hydronium ions). Inside the glass bulb a highly buffered solution
is present. The calomel or silver-silver chloride electrode is acting as a reference terminal. A salt bridge
consisting of a fiber wick saturated with KCl is at the tip of the reference electrode and keeps the
reference terminal potential essentially the same regardless of the solution under test. The active terminal
is sealed with common glass except for a tip made of pH sensitive glass which consists of a hydrated
gelatinous glass layer. Its membrane potential is proportional to the log [H+] and therefore is proportional
to the pH of the solution under test. Both the electrodes are kept in a single glass enclosure.
Eventhough the output from the pH electrode is high enough to deflect the voltmeter needle, to
increase the sensitivity and accuracy, the electronic circuitry is adopted. There is an external reference
voltage, to compensate the various errors and is also added with the output of the pH electrode. Further to
determine the pH at different temperature, a voltage from the temperature regulator circuit corresponding
to a given temperature is also added with the output from pH electrode. The operational amplifiers
amplify these voltages in the required manner and the final output is given to a digital voltmeter. In the
digital voltmeter, the display is obtained interms of pH as discrete numerals. Further the digital output
may be used for further processing of signals.

6.2 Blood pO2 Measurement:

The platinum wire, which is an active electrode, is embedded in glass for insulation and only the
tip is exposed. It is kept in the electrolyte solution in which the oxygen is allowed to diffuse. The
reference electrode is made up of silver-silver chloride (Ag/AgCl). A voltage of 0.7 V is applied between
the platinum wire and the reference electrode. The negative terminal is connected to the active electrode
through a micro ammeter and the positive terminal is given to the reference electrode.
Due to the negative terminal, the oxygen reduction takes place at the platinum cathode. Finally the
oxidation-reduction current proportional to the partial pressure of oxygen diffused into the electrolyte can
be measured in the micro-ammeter. The electrolyte is generally sealed into the electrode chamber that
holds the platinum wire and the reference electrode by means of a membrane across which the dissolved
oxygen can diffuse from the blood.

13
 pO2 electrode
The platinum cathode and the reference electrode can be integrated into a single unit (the clark
electrode). This electrode is used for in vitro and in vivo measurements.
In Vitro measurements:
In this method, the blood sample is taken and the measurement for oxygen saturation is made in
the laboratory. The electrode is placed in the sample blood solution and the pO2 value is determined.
In Vivo measurements:
In this method, the oxygen saturation is determined while the blood is flowing in the circulatory
system. A micro version of clark electrode can be placed at the tip of the catheter for insertion into
various parts of the heart or vascular system.

13
6.3 Blood pCO2 Measurement:
The pH electrode is used as the component of a pCO2 electrode to measure the partial pressure of
pCO2 by the arrangement as shown in the figure.
The pCO2 electrode consists of a standard glass pH electrode covered with a rubber membrane
stretched over it permeable to CO2. Between the glass surface and the membrane there is a thin layer of
water. For laboratory usage in some pCO2 electrodes, the water layer was replaced by a thin film of an
aqueous sodium bicarbonate (NaHCO3) solution. Rubber membrane can also be replaced by a thin Teflon
membrane which is permeable to CO2 but not to any other ions which might alter the pH of the
bicarbonate solution.

CO2 meter
The solution under test which contains dissolved CO2 enters a sample chamber and comes in
contact with a Teflon or silicon rubber membrane. This membrane separates the fluid from a sodium
solution but is permeable to CO2 in the solution.
The CO2 in the solution combines with water so as to produce free hydrogen ions in the sodium
solution. This changes the solution pH in propagation to the partial pressure of CO2 in the blood.

In the above equation, CO2 is directly proportional to the hydrogen ions, H+. After equilibrium, the pH of
the aqueous film is measured by the glass electrode and interpreted interms of PCO2.

13