Biomedical Instrumentation

Ms. Divya B AP/BME

Clinical Laboratory Instruments
 • The analysis not only helps in the diagnosis of various ailments
but also in determining the progress of treatment and for making
a prognosis. Samples taken from the body are analysed in three
different areas within the clinical laboratory set u.
• Chemistry section which deals with the analysis of blood, urine,
cerebrospinal fluid (CSF) and other fluids to determine the
quantity of various important substances they contain.
• Haematology section which deals with the determinations of the
number and characteristics of the constituents of the blood,
particularly the blood cells.
• Microbiology section in which studies are performed on various
body tissues and fluids to determine the presence of pathological
micro-organism
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 Fundamentals of Spectrophotometry
Introduction

1.) Colorimetry
 An analytical technique in which the concentration of
an analyte is measured by its ability to produce or
change the color of a solution
- Changes the solution’s ability to absorb light
2.) Spectrophotometry
 Any technique that uses light to measure
chemical concentrations
 A colorimetric method where an instrument is
used to determine the amount of analyte in a
sample by the sample’s ability or inability to
absorb light at a certain wavelength.

Colorimetry

Instrumental Methods
4 (spectrophotometry)
Non-Instrumental Methods
 Spectrophotometric Analysis

• Spectrophotometric techniques are used

to measure the concentration of solutes in
solution by measuring the amount of light
that is absorbed by the solution in a cuvette
placed in the spectrophotometer.
• The spectrophotometer can measure the
amount of light or electromagnetic radiation
(of certain frequency) transmitted or absored
by the solution.
 Fundamentals of Spectrophotometry
Properties of Light

1.) Particles and Waves

 Light waves consist of perpendicular, oscillating electric and
magnetic fields
 Parameters used to describe light

- amplitude (A): height of wave’s electric vector

- Wavelength (l): distance (nm, cm, m) from peak to

peak

- Frequency (n): number of complete oscillations that

the waves makes each second
 Hertz (Hz): unit of frequency, second-1 (s-1)
 1 megahertz (MHz) = 106s-1 = 106Hz

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 Fundamentals of Spectrophotometry

Properties of Light

1.) Particles and Waves

 Parameters used to describe light

- Energy (E): the energy of one particle of light

(photon) is proportional to its frequency

E h
where: E = photon energy (Joules)
n = frequency (sec-1)
h = Planck’s constant (6.626x10-34J-s)

As frequency (n) increases, energy (E) of light increases

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 Fundamentals of Spectrophotometry
Properties of Light

1.) Particles and Waves

 Relationship between Frequency and Wavelength

  c   c / 
where: c = speed of light (3.0x108 m/s in vacuum))
n = frequency (sec )
-1

l) = wavelength (m)
 Relationship between Energy and Wavelength

hc ~
E   hc

where:
~ = (1/ l)) = wavenumber
8
As wavelength (l) decreases, energy (E) of light increases
 The radiation may be transmitted with
little absorption taking place, and
therefore, without much energy loss.

The direction of propagation of the

beam may be altered by reflection,
refraction, diffraction or scattering.

The radiant energy may be absorbed in

part or entirely by the substance.

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 Fundamentals of Spectrophotometry

Absorption of Light

1.) Colors of Visible Light

 Many Types of Chemicals Absorb Various
Forms of Light

 The Color of Light Absorbed and Observed

passing through the Compound are
Complimentary

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 Fundamentals of Spectrophotometry
Absorption of Light

2.) Ground and Excited State

 When a chemical absorbs light, it goes from a low energy state (ground
state) to a higher energy state (excited state)

Energy required of
photon to give this
transition:
DE = E1 - Eo

 Only photons with energies exactly equal to the energy difference

between the two electron states will be absorbed

 Since different chemicals have different electron shells which are filled,
they will each absorb their own particular type of light
- Different electron ground states and excited states
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 Fundamentals of Spectrophotometry
Absorption of Light

3.) Beer’s Law

 The relative amount of a certain wavelength of light absorbed (A)
that passes through a sample is dependent on:
- distance the light must pass through the sample (cell path length
- b)
- amount of absorbing chemicals in the sample (analyte
concentration – c)
- ability of the sample to absorb light (molar absorptivity - e)

Increasing [Fe2+]

12
Absorbance is directly proportional to concentration of Fe +2
 Fundamentals of Spectrophotometry

Absorption of Light

3.) Beer’s Law

 The relative amount of light making it through
the sample (P/Po) is known as the transmittance
(T)
P
T
Po

 P
%T 100 
Percent transmittance
 Po 
13
T has a range of 0 to 1, %T has a range of 0 to 100%
 Fundamentals of Spectrophotometry
Absorption of Light

3.) Beer’s Law

 Absorbance (A) is the relative amount of light absorbed
by the sample and is related to transmittance (T)
- Absorbance is sometimes called optical density
(OD)

P 
A log   log(T)  log(%T / 100)
 Po 

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A has a range of 0 to infinity
 Fundamentals of Spectrophotometry

Absorption of Light

3.) Beer’s Law

 Absorbance is useful since it is directly related
to the analyte concentration, cell path length and
molar absorptivity.
 This relationship is known as Beer’s Law

Abc
where: A = absorbance (no units)
e = molar absorptivity (L/mole-cm)
b = cell pathlength (cm)
c = concentration of analyte (mol/L)

Beer’s Law allows

compounds to be
quantified by their
ability to absorb light,
15 Relates directly to
concentration (c)
 Fundamentals of Spectrophotometry
Absorption of Light

4.) Absorption Spectrum

 Different chemicals have different energy
levels
- different ground vs. excited electron
states

- will have different abilities to absorb

light at any given wavelength

 Absorption Spectrum – plot of absorbance

(or e) vs. wavelength for a compound

 The greater the absorbance of a compound

at a given wavelength (high e), the easier it
will be to detect at low concentrations

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 Fundamentals of
Spectrophotometry
Absorption of Light

4.) Absorption Spectrum

 By choosing different wavelengths of light (lA
vs. lB) different compounds can be measured

lA lB

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 The absorption process
How does matter absorb radiation

When polychromatic light (white light), which

contains the whole spectrum of wavelengths in
visible region, is passed though an object will absorb
certain of the wavelengths, leaving the unabsorbed
wavelengths to be transmitted. These residual
transmitted wavelengths will be seen as a color. This
color is complementary to the absorbed colors.
Absorption is a process in which chemical species (atom, ion or molecule) in a
transparent medium selectively attenuate certain frequencies of EMR.
Absorption spectrum is a plot of the amount of light absorbed by a sample as a
function of wavelength.
At room temperature most substance are in their lowest energy or ground state.
When an atom, molecule or ion absorbs EMR it is promoted to higher energy
states or excited states.
The excited state is a transition one and the species soon looses the energy it
gained and returns to its ground state by relaxation process either as heat of
collision or sometimes emits radiation of specific wavelength.
 Second Excitation state
E2
hv2

E1 First Excitation state

hv1

E0 Ground state

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•

•When a molecule interacts with photons of UV or VIS radiation excitation of electrons

takes place to higher electronic energy level at any of its vibrational level.
• Eex-Eg= hn of the photon absorbed.

• UV / VIS radiation cause electronic transition which is accompanied by vibrational and

rotational.
• If the compound subjected to IR radiation vibrational and rotational transitions in ground
state occure.
• Rotational transitions alone can be brought about by microwave.
•Ultraviolt and visible radiations have sufficient energy to cause trnsitions of the
outermost or valence electrons.
• If large amount of energy is absorbed by certain substance, bonds may be ruptured
and new compounds are formed photolysis.

This may occur upon absorption of far Ultraviolt as its energy is sufficiently high to
exceed the energy of formation of certain bonds.
• The total energy of a molecule is given by

Etotal=Eelectronic+Evibrational +Erotational
 Fundamentals of
Spectrophotometry
Spectrophotometer

1.) Basic Design

 An instrument used to make absorbance or
transmittance measurements is known as a
spectrophotometer

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 Fundamentals of
Spectrophotometry
Spectrophotometer

1.) Basic Design

 Wavelength Selector (monochromator):
used to select a given wavelength of light
from the light source
- Prism:

- Filter:

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 Fundamentals of
Spectrophotometry
Spectrophotometer

1.) Basic Design

 Wavelength Selector (monochromator): used to
select a given wavelength of light from the light
source
- Reflection or Diffraction Grating:

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 Fundamentals of Spectrophotometry

Spectrophotometer

1.) Basic Design

 Sample Cell: sample container of fixed length (b).
- Usually round or square cuvet
- Made of material that does not absorb
light in the wavelength range of interest

1. Glass – visible region

2. Quartz – ultraviolet

3. NaCl, KBr – Infrared region

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 Fundamentals of Spectrophotometry
Spectrophotometer

1.) Basic Design

 Light Detector: measures the amount of light
passing through the sample.
- Usually works by converting light signal
into electrical signal
Photomultiplier tube Process:
a) light hits photoemissive cathode and e- is emitted.
b) an emitted e- is attracted to electrode #1
(dynode 1), which is 90V more positive.
Causes several more e- to be emitted.
c) these e- are attracted to dynode 2, which is
90V more positive then dynode 1, emitting
more e-.
d) process continues until e- are collected at
anode after amplification at 9 dynodes.
e) overall voltage between anode and cathode
is 900V.
f) one photon produces 106 – 107 electrons.
g) current is amplified and measured
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 Fundamentals of
Spectrophotometry
Spectrophotometer

2.) Types of Spectrophotometers

 Single-Beam Instrument: sample and blank
are alternatively measured in same sample
chamber.

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 Fundamentals of
Spectrophotometry
Spectrophotometer

2.) Types of Spectrophotometers

 Double-Beam Instrument
- Continuously compares
sample and blank
- Automatically corrects for
changes in electronic
signal or light intensity of
source

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 Fundamentals of Spectrophotometry
Chemical Analysis

1.) Calibration
 To measure the absorbance of a sample, it is
necessary to measure Po and P ratio
- Po – the amount of light passing through the
system with no sample present
- P – the intensity of light when the sample is
present

 Po is measured with a blank cuvet

- Cuvet contains all components in the
sample solution except the analyte of
interest

 P is measured by placing the sample in the cuvet.

 To accurately measure an unknown concentration,

obtain a calibration curve using a range of known
concentrations for the analyte
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 Fundamentals of
Spectrophotometry
Chemical Analysis

2.) Limitations in Beer’s Law

 Results in non-linear calibration
curve
 At high concentrations, solute
molecules influence one another
because of their proximity
- Molar absorptivity changes
- Affect on equilibrium, (HA
and A- have difference
absorption)

 Analyte properties change in

different solvents

 Errors in reproducible positioning

of cuvet
- Also problems with dirt &
31 fingerprints
Keep A in range of 0.1 – 1.5 absorbance
 Fundamentals of
Spectrophotometry
Chemical Analysis

3.) Precautions in Quantitative Absorbance Measurements

 Choice of Wavelength
- Choose a wavelength at an absorption
maximum
 Minimizes deviations from Beer’s law,
which assumes e is constant
- Pick peak in absorption spectrum where
analyte is only compound absorbing light
- Or choose a wavelength where the analyte has
the largest difference in its absorbance relative
to other sample components

Bad choice for either

Best choice
compound (a) or (b)
compound (b)
(a)

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 Multi channel calorimeter (photometer)

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 Microprocessor based spectrophotometer

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 Fundamentals of
Spectrophotometry
Chemical Analysis

4.) Example:

A 3.96x10-4 M solution of compound A exhibited an

absorbance of 0.624 at 238 nm in a 1.000 cm cuvet. A blank
had an absorbance of 0.029. The absorbance of an
unknown solution of compound A was 0.375.

Find the concentration of A in the unknown.

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 Thank you

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