INSTRUMENTATION

CLINICAL CHEMISTRY 1

PREPARED BY:
LGGRMT
➢ There are many instruments to choose from when doing a chemical
analysis of blood samples.
➢ They differ usually in their principles and applications.
➢ The most common instrument used in clinical chemistry involves the
principle of light absorption.
➢ COLORIMETRY:
The constituent measured is colored. (e.i., it absorbs light within the visible
spectrum (350-700 nm light)

Uses ordinary filters to screen light which will strike the solution.

Since filter resolution is not sharp enough, what is measured is light

intensity of multiple wavelength.

❖ 2 basic types of colorimetry:

a. Visual colorimetry
- Color intensity of solution is matched against a standard solution.
- Relies much on human eye
- Example: Dubowski (Duboscq) colorimeter
- Less precise, insensitive and subjective
b. Photoelectric colorimetry:

- Measurement of light intensity using photoelectric device or

detector
- Independent of wavelength

b.1 Filter Photometry

b.2 Spectrophotometry

STANDARD CURVE (STRAIGHT LINE)

- Used to define the limits of an assay
- Involves measuring the absorbance of several standards & calibrating
the results to establish the standard curve.
1.) Determine the range of values expected for both normal and
abnormal.
2.) Assess whether these ranges can be measured by the method you
chose.

3.) Prepare a number of standard solutions within the ranges determined.

4.) Assay the standards together with the samples

5.) Plot the Standard curve.

6.) Examine the curve for some limitations.

Example of standard
curves. Typical standard
curves for bovine serum
(BSA) and bovine gamma
globulin (BGG) in the
Pierce Coomasie Plus
Protein Assay. The assay
kit (Part No. 23236)
includes ampules of
Albumin standard.
LIMITATIONS in the CURVE

➢ 1. Upper limit of detection

- Identified when the upper portion of the curve (high
concentration range) deviates from the straight line pattern.

➢ How to deal?
b a.) Report as “greater than
a (upper limit)”
b.) Dilute the original sample
& re-assay, then compute w/
dilution factor.
concentration
➢ 2. Lower limit of detection
- Corresponds to lower portion of the curve that deviates from the
straight line
- 2 solutions of different concentrations give essentially same
absorbance readings, but at the next highest concentration in the
series follows a linear relationship between absorbance &
concentration
How to deal?

- No way to measure the analyte

under such prevailing conditions.
b - Report result as “ less than
a (lower limit)”
➢ Also take note of signs for
Concentration reagents’ deterioration
➢ Prepare new curve
Ratio of Standard to Unknown

Cu = Au x Cs
As
● Blanks (Correction)
- Sets the disturbance to zero
- Commonly used is distilled H₂O
- Ensures the accuracy of the results by eliminating interfering
substances inherent with the sample or the reagent
- Blanks and Standard = Reference Materials
- While controls are used to test & check the accuracy.
Two types of blanks:

1. Reagent blank
- Contains only 1 reagent (no sample)
- Used to correct for the absorbance readings due to other
materials in the reagents.

● Two ways of Reagent Blanking:

a. - Set the absorbance to zero
- commonly used is distilled H₂O
- take the absorbance of reagent blank & sample
b. - “Zero” the instrument using a reagent blank
- subtract the absorbance of the reagent blank from the sample
2.
2. Sample blank
- Contains only the sample (no reagent)
- Used to correctly non-specific absorbance of other substance in
the sample
e.g., hemoglobin, bilirubin, lipids; or if the product being
measured has the same absorbance as bilirubin or hemoglobin
- A separate blank is needed for every sample.
BEER-LAMBERT-BOUGUER’S LAW
- Concentration of substance is inversely proportional to the log of the
transmitted light or directly proportional to amount of light absorbed.
A = abc = log (100/%T) C= A
Ab
Where,
A = absorbance
a = absorptivity of compound
under standard condition
b = light path of the solution
c = concentration of the
compound
%T = percentage transmittance
Absorbance = is proportional to the inverse log of transmittance.

A = abc = 2-log%T
A = 2-log%T

Example if %T = 10.0 and log 10.0 = 1.0, then

A = 2-1 = 1.0
Absorptivity

- Unique to one particular substance, depends on wavelength

- Specified absorptivity assured only if accuracy of wavelength is
maintained
-
Purity of wavelength
Band pass - line BC
* smaller spectral band
width, the higher the
absorptivity, better analysis
BEER’S LAW

- May only be applied in accurate quantitative analysis by light

absorption, if the following requisites are met:

1. Incident radiation on the substance of interest is monochromatic

2. Solvent absorption is insignificant compared to sokute absorption
3. Solute concentration is within “linear limits”
4. A chemical reaction does not occur between the molecule interest and
another solute or solvent molecule (“quenching” phenomenon) - due
to inertness.

Deviations from Beer’s Law

1. Simultaneous absorption at multiple wavelength
2. Absorption of light by other species
3. Transmission of light by other mechanisms

To measure background interference:

- Allen Correction or Dual Wavelength method

Acorr = Apeak absorbance - (AAnalyte-free serum absorbance; lower & higher)

2
➢ Among possible causes of this deviation from Beer’s Law are:
1. Absorbance of solution is too strong for the instrument to
measure
2. More analyte present than the detecting reagent

➢ Note: it is never valid to simply extend the line all the way to zero.
Always determine the limits of the curve.
➢ SPECTROPHOTOMETRY

- Monochromators, e.g., prisms & diffraction gratings are used,

instead of low-resolution filters.
- Allows measurement of light intensity in a much narrow
wavelength range
- Suitable for both colored and colorless solutions
- Both visible and UV spectrophotometers, depending on the
absorbance peak of the analyte
● A solution transmits light corresponding in wavelength to its color, and usually
absorbs light of wavelength complementary to its color:
Wavelength Color absorbed Complementary color

350-430 Violet Yellow-blue

430-475 Blue Yellow

475-495 Green-blue Orange

495-505 Blue-green Red

505-555 Green Purple

555-575 Yellow-green Violet

575-600 Yellow Blue

600-650 Orange Green-blue

650-700 Red Blue-green

➢ SPECTROPHOTOMETRY
- Based on the measurement of radiant energy absorbed or
transmitted under controlled conditions.

> Electromagnetic radiation (EMR) is the flow of energy through space

at the speed of light. EMR exists as Maxwell’s waves and as streams of
particles called photons.

➢ Photons of radiant energy are exchanged whenever electrically

charged subatomic particles interact.
➢ When these electrons move from one orbit to another, some
energy is either absorbed or released.
➢ The wavelength of light is the distance between successive peaks.
➢ Frequency is the number of waves that pass an observation point in a
unit of time.
➢ The wavelength is inversely related to frequency and energy, that is,
the shorter the wavelength, the higher the frequency and energy and
vice versa
➢ Photometer is often used in its generic sense as any instrument that
measures light intensity
➢ Spectrophotometer measure the absorption of monochromatic light
produced by a grating monochromator.
➢ A flame photometer measures the light emitted by single atoms
burned in a flame.
➢ An atomic absorption photometer measures the light absorbed by
atoms dissociated by heat
➢ A fluorometer measures the light of a specific wavelength emitted by
a molecule after it has been excited by electromagnetic radiation of a
given energy.
➢ In an electrolytic cell, the half-cell where reduction takes place is
known as cathode.
➢ When the pH-sensitive glass electrode is not actively in use, it should
be kept in the medium recommended by the manufacturer
➢ The main advantages of fluorometric over spectroscopic methods of
analysis are:
= increased specificity and increased sensitivity
● Basic component of a spectrophotometer consist of the exciter lamp,
the entrance slit, the monochromator, the analytical cell or cuvette
and photodetector.
● Tungsten and halogen quartz lamp are good source of radiant energy
● For ultraviolet range, hydrogen lamp is widely used.
● Function of the entrance slit is to reduce stray light and prevent
scattered light from entering the monochromator.
● Types of monochromator include prisms, diffraction gratings, and
interference filters.
● Prisms are wedge-shaped pieces of glass, quartz, or sodium chloride
● Diffraction grating are made by cutting tiny grooves or slits into an
aluminized surface of a flat piece of crown glass.
● Cuvettes are made of soft glass, borosilicate glass, quartz, or plastic
● Photodetectors include barrier-layer cell, phototube, photomultiplier
tube (PMT), and variety of semiconductor photodetectors.
● All of these devices use photosensitive materials in their cathodes that
release electrons when they are exposed to light energy.
● In a double-beam system, monochromatic light from either a single or
two identical monochromators pass through both a reference and a
sample compartment.
● The intensity of these two light beams is then measured by one or two
photodetectors.
● In a double-beam in time spectrophotometer, the light beam is split
with a rotating chopper that alternately presents a mirror and an
opening.
● When performing spectrophotometer quality control checks, the
holmium oxide glass filter is used to assess wavelength accuracy.
A feature that distinguishes a
double-beam from a single-beam
spectrophotometer is that the
double-beam compares sample and
reagent blank absorbances
simultaneously.
➢ ATOMIC EMISSION (FLAME PHOTOMETRY)
- Are no longer commonly used in clinical laboratories
- A flame photometer must have an atomizer as a component.

ATOMIC ABSORPTION SPECTROPHOTOMETRY

- Are now almost limited to analysis of metals such as lead in specialized
toxicology laboratories.
- Most atomic absorption spectrophotometers incorporate a beam
chopper and a tuned amplifier to avoid errors caused by measurement
of light specific wavelength emitted by analyte.
ATOMIC SPECTROPHOTOMETER
➢ MOLECULAR LUMINESCENCE SPECTROSCOPY
(FLUOROMETRY)
- Luminescence (bioluminescence) is based on an energy exchange
princess that occurs when certain compounds absorb
electromagnetic radiation, become excited, and return to an energy
level higher than or equal to their original level.
- Because some energy is lost before emission from the excited
state by collision with the solvent or other molecules, the
wavelength of the emitted light is longer than that of the exciting
light.
- The light emission from a single excited state is called
fluorescence
● If the excitation energy comes from a chemical or electrochemical
reaction and not from photolumination.
● Chemiluminescence involves the oxidation of an organic compound
(luminal, isoluminol, acridinium ester, or luciferin) by an oxidant.
● Fluorescence, is the more common of these processes in clinical
laboratory application.
● Basic components: a light source, an excitation (primary
monochromator), a cuvette, an emission (secondary) monochromator,
and a photodetector.
● An advantage of fluorescence is its extremely high sensitivity, which
approximately 100 to 1000 times that absorption measurements.

Spectrophotofluorometer

● Fluorometers are designed so that

the path of the exciting light is at a
right angle to the path of the
emitted light

To prevent emitted fluorescent light from reaching the detector.

➢ NEPHELOMETRY & TURBIDIMETRY
Two useful methods available for measuring the concentration of a
solution that contains particles too large for absorption spectroscopy are
nephelometry and turbidimetry.

These methods may be suitable for quantitative assays using

antigen-antibody complexes or measuring the amount of proteins in fluids.

When a collimated light beam strikes a particle in suspension, portions

of the light are absorbed, reflected, scattered, and transmitted.
*Nephelometry is the measurement of the light scattered by a particulate
solution.

*Three types of light scatters occur based on the relative size of the light
wavelength (Gauldie, 1981)

*If the wavelength (𝞴) of light is much larger than the diameter (d) of the
particle, where d > 10𝝺, the light scatters forward owing to the destructive
out-of-phase back-scatter, as described by the Mie theory.
* If the wavelength of light is approximately the same as the particle size,
more light scatters in the forward direction than in other directions, as
defined by the Rayleigh-Debye Theory.

* a common application of nephelometry is the measurement of

antigen-antibody reactions.

* A typical nephelometer consists of a light source, a collimator, a

monochromator, a sample cuvette, stray light trap, and a photodetector
*For macromolecules with size close to
or larger than the light wavelength,
measurement of the forward light
scatter increases the sensitivity of
nephelometry.

* Light sources include a. mercury-arc

lamp, a tungsten-filament lamp, a
light-emitting diode, and a laser

NEPHELOMETER
 ● Larger particles
produce light
scattering that is
more intense but
irregular, with the
scattering being
greater in the
direction of the
incident light beam

Figure 1. Angular patterns of scattered intensity from particles of three

sizes. (A) small particles, (B) large particles, (C)larger particles. From
brumberger, et. al, “Light Scattering” Science and Technology,
November, 1968, page 38.
➢ TURBIDIMETRY
- Is the measurement of the reduction in light transmission caused by
particle formation. Light transmitted in the forward direction is
detected.
- The amount of light absorbed by a suspension of particles depends
on the specimen concentration and on the particle size.
- Many clinical applications exist for turbidimetry.
- Various microbiology analyzer measures turbidity of samples to
detect bacterial growth in broth cultures.
- Turbidimetry is routinely used to measure the antibiotic sensitivity
from such cultures.
- In coagulation analyzers, turbidimetric measurements detect clot
formation in the sample cuvettes.
- Turbidimetric assays have long been available in clinical chemistry to
quantify protein concentration in biological fluids, such as urine and
cerebrospinal fluid (CSF).
➢ REFRACTOMETRY
- Refractometry is based on light refraction.
- When light passes from one medium into another, the light beam
changes its direction at the boundary surface if its speed in the second
medium is different from that in the first. The ability of a substance to
bend light is called refractivity.
- The refractivity of a liquid depends on the wavelength of the incident
light, the temperature, the nature of the liquid medium, and the
concentration of the solute dissolved in the medium. If the first three
factors are held constant, the refractivity of a solution is an indirect
measurement of total solute concentration.
- Refractometry has been applied to various measurements, for example
total serum protein concentration, specific gravity of urine, and column
effluent of high-performance liquid chromatography analysis.

REFRACTOMETER
➢ OSMOMETRY
- Osmometry is the measurement of the osmolality of an aqueous
solution such as serum, plasma, or urine.
- As osmotically active particles are added to a solution causing its
abnormality to increase, four other properties of the solution are also
affected.
- These properties are osmotic pressure, boiling point, freezing point,
and vapor pressure. They are called colligative properties of the
solution because they can be related each other and to the
osmolality.
➢ OSMOMETRY
- As the osmolality of a solution increases, (1) the osmotic pressure
increases, (2) the boiling point elevated, (3) the freezing point is
depressed, and (4) the vapor pressure is depressed.
- Osmometry is based on measuring changes in the colligative
properties of solutions that occur owing to variations in particle
concentration.
- Freezing-point depression osmometry is the most commonly used
method for measuring the changes in colligative properties of
solution.