clinical chemistry

1. SPECTROPHOTOMETRY
BASIC CONCEPTS,
INSTRUMENTATION AND
APPLICATION
By
Dr. Basil, B – MBBS (Nigeria),
Department of Chemical Pathology/Metabolic Medicine,
Benue State University Teaching Hospital, Makurdi.
September 2014.
1

2. INTRODUCTION:
• Photometry is the measurement of the amount
of luminous light (Luminous Intensity) falling on a
surface from a source.
• Spectrophotometry is the measurement of the
intensity of light at selected wavelengths.
• The method depends on the light absorbing
property of either the substance or a derivative
of the substance being analyzed
• Spectrophotometers use prisms and gratings for
isolating wavelengths while devices that require
filters for this purpose are called Filter
Photometers.
2

3. NATURE OF LIGHT:
• Electromagnetic waves are characterized by their
frequency and wavelength.
• Light is a spectrum of different wavelengths which the
eye recognizes as “white” but can be isolated into
discrete portions and measured.
• Human eye responds to radiant energy btw 380 and
750nm, but modern instruments can measure shorter
wavelengths (UV) and longer (IR) ones.
• Wavelength describes a position within a spectrum. It is
the distance btw 2peaks as the light travels in a wave-like
manner
• Light also is composed of discrete energy packs called
photons whose energy is inversely proportional to the
wavelength
3

4. BASIC CONCEPTS:
• When light passes through a solution, a certain fraction
is being absorbed.
• This fraction is detected, measured and used to relate
the light absorbed or transmitted to the concentration
of the substance.
• This enables both qualitative and quantitative analyses
of substances.
• The spectrophotometric technique is used to measure
light intensity as a function of wavelength. It does this
by:
– Diffracting the light beam into a spectrum of wavelengths
– Direct it to an object
– Receiving the light reflected or returned from the object
– Detecting the intensities with a charge-coupled device
– Displaying the results as a graph on the detector and then
the display device 4

5. • The light absorption is directly related to the concentration of
the compound in the sample.
• As Concentration increases, light Absorption increases linearly
and light Transmission decreases, exponentially
5

6. Transmittance and Absorbance:
• When a sample is illuminated, it absorbs some
of the light and transmits the rest.
• The transmitted light (Is) is of lower intensity
than the incident light (Io), and the
transmitted light is defined as:
T = Is / Io
6

7. • To ensure accuracy (by eliminating effects of
reflection by surface of the cell, absorption by the
cell wall and by solvent) an identical reference cell
without the compound of interest is also used.
• Thus, the amount of light absorbed (A) as the
incident light passes through the sample is
equivalent to:
A = - log Is / IR = - log T
• In practice, the Reference cell is inserted and the
instrument adjusted to an arbitrary scale
corresponding to 100% transmittance, after which
the percentage transmittance reading is made on the
sample 7

8. Beer’s Law:
• This states that the concentration of a substance if directly
proportional to the amount of light absorbed or inversely
proportional to the logarithm of transmitted light.
A = abc
Where:
A = Absorbance
a = proportionality constant defined as absorptivity
b = light path in centimeters
c = concentration in g/L of the absorbing compound
NB: - Absorbance (A) has no units, so the unit for a =
reciprocals those for b and c.
• When b is 1cm and c is expressed in mol/L, ἐ (epsilon) is
substituted for the constant, a.
• The value of ἐ is a constant for a given compound at a given
wavelength under prescribed conditions of solvent,
temperature, pH, etc., and is called the Molar absorptivity
(ἐ).
8

9. Spectrometry Nomenclature:
Application of Beer’s Law:
• In practice, a direct proportionality between
absorbance and concentration must be established
for a given instrument under specific conditions.
• A linear relationship exists up to a certain
concentration or absorbance beyond which the
solution is said to no longer obey Beer’s Law
• Within this limitation, a calibration constant (K) may
be derived and used to calculate the concentration of
unknown solutions by comparison 9

10. Recall, a = A/bc
Thus, A1 / b1c1 = A2 / b2c2
Where 1 and 2 refers to the calibrating (c) and unknown
(u) solutions respectively
• But because the Light path b remains constant,
b1 = b2
• Then, A1/c1 = A2 /c2 or Ac/cc = Au/cu
• Hence, concentration of the unknown:
cu = Au/Ac x cc
And, cu = Au x cc / Ac = Au x K
Where, K = cc / Ac
10

11. • The constant cannot be used once Beer’s Law
have been violated
• A non-linear calibration curve can be used if a
sufficient number if calibrators of varying
concentration is included to cover the entire
range encountered for reading on the unknowns
• Published constants should be used only if the
method is followed in detail and readings are
made on a spectrophotometer capable of
providing light of high spectral purity at a verified
wavelength.
• Use of broader band light leads to some decrease
in absorbance
11

12. • Beer’s Law is followed only if the following
conditions are met:
– Incident radiation on the substance of interest is
monochromatic
– Solvent absorption is insignificant compared to
the solute absorbance
– Solute concentration is within given limits
– Optical interferant is not present
– Chemical reaction does not occur between the
molecule of interest and another solute or solvent
molecule.
12

13. INSTRUMENTATION: The Spectrophotometer
13

14. • The basic components of a spectrophotometer
include: a light source, a means to isolate light of
desired wavelength, fiber optics, cuvets, a
photodetector, a readout device, a recorder and a
computer.
• Three different types of the device available are:
The Single Beam Spectrophotometer:
14

15. Double-beam-in-space Spectrophotometer:
Double-beam-in-time Spectrophotometer:
15

16. Light Sources:
• This provides a sufficient amount of light which is
suitable for making a measurement.
• The light source typically yields a high output of
polychromatic light over a wide range of the
spectrum.
Electromagnetic spectrum:
• Types of light sources used in spectrophotometers
include: Incandescent lamps and lasers. 16

17. Incandescent Lamps:
• Tungsten Filament Lamp: The most common source of visible and near
infrared radiation ( at wavelength 320 to 2500 nm)
• Deuterium lamp: Continuous spectrum in the ultraviolet region is
produced by electrical excitation of deuterium at low pressure. (160nm-
375nm)
• Hydrogen Gas Lamp and Mercury Lamp, Xenon (wavelengths from 200
to 800 nm): high-pressure mecury and xenon arc lamps are commonly
used in UV absorption measurements as well as visible light.
• Globar (silicon carbide rod): Infra-Red Radiation at wavelengths: 1200 -
40000 nm
• NiChrome wire (750 nm to 20000 nm); ZrO2 (400 nm to 20000 nm): for
IR Region 17

18. Laser Sources:
• These devices transform light of various frequencies
into an extremely intense, focused, and nearly non-divergent
beam of monochromatic light
• Through selection of different materials, different
wavelengths of light emitted by the laser are obtained.
• Used when high intensity line source is required
• Unique properties of laser sources include:
– Spatial coherence: a property that allows beam diameters in
the range of several microns
– Production of monochromatic light
– Have pulse widths that vary from microseconds to (flash
lamp-pulsed lasers) to nanoseconds (nitrogen lasers), to
picoseconds or less (mode-locked lasers) 18

19. Spectral Isolation:
• A system for isolating radiant energy of a desired
wavelength and excluding that of other wavelength is
called a Monochromator.
• Monochromator consists of these parts:
– Entrance slit
– Collimating lens or mirror
– Dispersion element: A special plate with hundreds of
parallel grooved lines. The grooved lines act to separate the
white light into the visible light spectrum
– .
19

20. • Focusing lens or mirror: Combinations of lenses, slits, and
mirrors which relays and focuses light through the
instrument
• Exit slit
20

21. • Devices used for spectral isolation include: Filters,
Prisms, and Diffraction gratings.
• Variable slits are also used to permit adjustments
in total radiant energy reaching the photocell
21

22. • The spectral purity of a monochromator is usually
described as its spectral bandwidth – width, in nm, of
the spectral transmittance curve at a point equal to
half the peak transmittance
• Filters:
– Simplest type is a thin layer of coloured glass which is not a
true monochromator because it transmits light over a
relatively wide range of wavelength 22

23. – Commonly used glass filters have spectral bandwidth of
about 50nm and are refered to as wide-bandpass filters
– a cut-off filter shows a sharp rise in transmittance over
a narrow portion of the spectrum and is used to
eliminate light below a given wavelength
– Narrow-bandpass filter is constructed by combining
two filters like shown above, however, the availability of
high intensity light sources now favours the use of
narrow bandpass interference filters.
– Alternatively, narrow bandpass filter can be constructed
by use of dielectric material of controlled thickness
sandwiched btw two silvered pieces glass
• Prisms and Gratings:
– Prisms seperates white light into a continous spectrum
by refraction with shorter wavelengths being bent or
refracted more than longer ones 23

24. – Diffraction gratings are prepared by depositing a thin
layer of aluminuin-copper alloy on the surface of a flat
glass plate, then ruling many small parallel grooves into
the metal coating
Selection of a Monochromator:
– Narrow spectral bandwidth – for resolving and
identifying sharp absorption peaks that are closely
adjacent.
– Lack of agreement with beer’s law will occur when a
part of the spectral energy transmitted by the
monochromator is not absorbed by the analyte as
commonly observed with wide-bandpass instruments
– Increase in absorbance and improved linearity with
concentration is usually observed with instruments that
operate at narrower bandwidths of light 24

25. Cuvets:
• This is a small vessel used to hold a liquid sample to
be analyzed in the light path of a
spectrophotometer.
– May be round, square or rectangular and are constructed
from glass, silica (optical grade quartz) or plastic.
– It should be without impunities that may affect
spectrophotometric readings
– Reference solution must be transparent to the radiation
which will pass through them.
– Quartz or fused crystalline silica cuvettes for UV
spectroscopy.
– Glass cuvettes for Visible Spectrophotometer.
– NaCl and KBr Crystals for IR wavelengths.
25

26. Photodetectors:
• These are devices that convert light into an
electric signal that is proportional to the number
of photons striking its photosensitive surface.
• The photocell and phototube are the simplest
photodetectors, producing current proportional
to the intensity of the light striking them
• The Photomultiplier tube (PMT) is a commonly
used photodetector for measuring light intensity
in the UV and Visible region of the spectrum.
They are extremely rapid, very sensitive and slow
to fatigue.
26

27. • The PMT consists of:
– A photoemissive cathode (a cathode which emits
electrons when struck by photons)
– Several dynodes (which emit several electrons for
each electron striking them)
– An anode – Produces an electric signal proportional
to the radiation intensity
– Signal is amplified and made available for direct
display
– A sensitivity control amplifies the signal
– Examples: Phototube (UV); Photomultiplier tube
(UV-Vis); Thermocouple (IR); Thermister (IR)
27

28. • Other photodetectors include: Barrier layer cells (photovoltaic
cells), Photodiodes,
• Photodiodes are made of photosensitive semi-conductor
materials like silicon, gallium, arsenide etc which absorb light
over a characteristic wavelength range e.g 250nm to 1100nm
for silicon. They are capable of measuring light at a multitude
of wavelengths. 28

29. Display or Readout Devices:
• Electrical energy from the detector is
displayed on a meter or readout system such
as an analog meter (obsolete), a light beam
reflected on a scale, or a digital display, or LCD
• Digital readout devices operate on the
principle of selective illumination of portions
of a blank of light emitting diodes (LEDs),
controlled by the voltage signal generated.
• Compared to meters, digital read out devices
have faster response and are easier to read
29

30. Computers:
• Using the computer;
– output from calibrator is digitally stored
– digital signals from blanks are subtracted from
calibrators and unknowns, and
– the concentration of unknowns is automatically
calculated
• Data from multiple calibrators often are used to:
– store a complete calibration curve
– display or print out the curve for visible inspection
– calculate result of unknowns based on the curves or
some mathematical transformation of the data
• Computers are also used to convert kinetic data
into concentration or enzyme activity. 30

31. Recorders:
• Spectrophotometers may be equipped with
recorders in addition to or instead of a digital
display
• They are synchronized to provide line traces of
transmittance or absorbance as a function of either
time or wavelength.
• When a continuous tracing of absorbance versus
wavelength is recorded, the resultant figure is called
an absorption spectrum. If a substance absorbs
light, distinct peaks of absorbance will be observed.
• Measuring the absorption spectra of an unknown
sample and comparing them with spectra from
known compounds is very useful for qualitative
purposes. 31

32. PERFORMANCE PARAMETERS:
Parameters tested to verify that the spectrophotometer
is performing satisfactorily include:
• Wavelength Accuracy:
– Holmium oxide in dilute perchloric acid is used for
calibration of Narrow-spectral-bandwidth instruments
– sharp absorbance peaks are obtained at defined
wavelengths which can be compared with established
values.
– Didymuin filter is used in broader-bandpass instruments
• Spectral Bandwidth:
• Width of and emission band at half-peak height
– Maybe calculated from the manufacturer’s specifications
– Interference filters maybe used to check the instrument32

33. • Stray Light:
• radiation of wavelengths outside the narrow band nominally
transmitted by the monochromator that hits the detector
– Defined as a ratio or percent of the total detected light
– Causes an absorbance error and can be minimized by an inbuilt
stray light filter
– Cutoff filters are used for the detection of stray light which they
are capable of absorbing with 0% transmission. In the UV range,
liquid cutoff filters are very effective
– Emanates from scattering and diffraction inside the
monochromator, light leaks, and fluorescence of the sample
• Photometric Accuracy:
• Neutral density filters are used to check an instrument’s
photometric accuracy.
– Solutions of potassium dichromate may be used for overall
checks on photometric accuracy which is compared with
literature values. 33

34. APPLICATIONS:
1. Measurement of Concentration:
– Prepare samples
– Make series of standard solutions of known concentrations
– Set spectrophotometer to the λ of maximum light
absorption
– Measure the absorption of the unknown, and from the
standard plot, read the related concentration
34

35. 2. Detection of impurities:
– UV absorption spectroscopy is one of the
best methods for determination of impurities in organic
molecules
– Additional peaks can be observed due to impurities in
the sample and it can be compared with that of
standard raw material
35

36. 3. Elucidation of the structure of Organic
Compounds:
• From the location of peaks and combination of
peaks UV spectroscopy elucidate structure of
organic molecules:
– the presence or absence of unsaturation,
– the presence of hetero atoms
4. Chemical Kinetics:
– Kinetics of reaction can also be studied using
UV spectroscopy. The UV radiation is passed through
the reaction cell and the absorbance changes can be
observed
36

37. 5. Detection of Functional Groups:
– Absence of a band at particular wavelength regarded as
an evidence for absence of particular group
6. Molecular weight determination:
– Molecular weights of compounds can be measured
spectrophotometrically by preparing the suitable
derivatives of these compounds.
– For example, if we want to determine the molecular
weight of amine then it is converted in to amine picrate
37

38. REFLECTANCE PHOTOMETRY
• In Reflectance photometry, diffused light illuminates a reaction
mixture in a carrier and the reflected light is measured.
– Alternatively, the carrier is illuminated and the reaction
mixture generates a diffuse reflected light which is measured.
• The intensity of the reflected light from the reagent carrier is
compared with the intensity of light reflected from a reference
surface.
• The intensity of reflected light is non-linear in relation to the
concentration of analyte,
– The data is first converted to linear format using the Kubelka-
Munk equation or the Clapper-Williams transformation.
• Reflectance photometry is used as the measurement method with
dry-film chemistry systems.
• The Electro-optical components used in reflectance photometry
are essentially the same as that used in absorbance photometry. 38

39. FLAME EMISSION SPECTROPHOTOMETRY
• This is based on the characteristic emission of light by atoms
of many metallic elements when given sufficient energy,
such as that supplied by a hot flame
• The wavelength to be used in the measurement of an
element depends on the selection of a line of sufficient
intensity to provide adequate sensitivity and freedom from
other interfering lines at or near the selected wavelength
(e.g lithium – red, sodium – yellow, potassium – violet,
rubidium – red, magnesium – blue)
• These colours are characteristic of the metal of the metal
atoms present as cations in solution.
• Under constant and controlled conditions, the intensity of
the characteristic wavelength produced by each of the
atoms is directly proportional to the number of atoms that
are emitting energy, which in turn is directly proportional to
the concentration of the substance of interest in the
sample. 39

40. REFERENCES:
• Tietz Textbook of Clinical Chemistry
• The principles of use of a spectrophotometer
and its application in the measurement of
dental shades[2003]
• Fundamentals of UV-visible spectroscopy,
Tony Owen, 1996
• Spectrophotometry FUNDAMENTALS
(Chapters 17, 19, 20), Dr. G. Van Biesen,
Win2011
• http://www.bio.davidson.edu
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41. THANK YOU FOR
LISTENING
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