Spectrophotometers measure the intensity of light at specific wavelengths to determine the concentration of compounds in solution. They consist of a light source, wavelength selector like a monochromator, cuvette to hold samples, photodetector like a photomultiplier tube, readout device, and data system. For accurate results, spectrophotometers must meet various performance specifications including wavelength accuracy checked using holmium oxide or didymium filters, low stray light verified with cutoff filters, linear detector response across concentration ranges, and photometric accuracy assessed using neutral density filters.

1. Spectrophotometer
Specification
And
performance

2. Introduction
 What is spectrophotometer?
A spectrophotometer is a machine which measures the
intensity of light at selective wavelength.
 In clinical chemistry laboratory, we use spectrophotometer to
detect the concentration of a compound of interest in a
solution with the help of Beer-Lambert law by absorption
spectrophotometry.

3. The Beer-Lambert law-
• The Beer-Lambert law states that, the concentration of a
substance is directly proportional to the amount of radiant
energy absorbed or inversely proportional to the logarithm of
transmitted radiant energy.

4. Types of spectrophotometers
 Single-beam spectrophotometers.
 Double-beam spectrophotometers
1.double beam in space spectrophotometer.
2.double-beam in time spectrophotometer

5. In broad heading these specifications on the part of the machine
are-
 The incident radiant energy which reaches the cuvette must
be monochromatic and specific, i.e. of a fixed wavelength.
 Radiant energy should not be transmitted by any other
mechanism, i.e. there should not be any stray light.
 The photometer i.e. the detector & the read out device must
detect the intensity of light accurately.

6. Diagrammatic representation of some
spectrophotometers

7. Schematic representations of Single-beam
spectrophotometers
⃝→→→¦→→ →→¦→→→→→→→
Source entrance exit slit
light slit
monochromator cuvet detector read out device

9. Schematic representations of Double-beam
spectrophotometers.

11. Specifications

12. Parts
 1.A light source.
 2.A device to isolate light of a desired wave-lengths.
 3.A cuvette.
 4.A photodetector.
 5.A readout device and
 6.A data system.

13. 1. Light source
Types of light sources used in spectrophotometer are-
 For visible wavelength range (380-750 nm) - like Tungsten
filament bulb, Argon fluoride LASER etc.
 For UV wavelength range i.e. <380 nm- like hydrogen lamp
and deuterium lamp, high pressure mercury lamp, mercury
arc lamp, gallium phosphate light-emitting diode etc.
 For Infra red wavelength range i.e. >750 nm –like CO2 laser,
Silicon light-emitting diode etc.

14. 2.A device to isolate light of a desired wave-lengths.
 These are a device used for isolating and excluding radiant
energy of a desired wavelengths from undesired wavelengths.
Commonly used are-
 Simple filters- these are thin layer of colored glass.
Monochromators – these are-
 1.Prisms- separates white light into a continuous spectrum
because shorter wavelengths are bent or retracted.
 2.Diffraction gratings- these are prepared by depositing thin
layer of aluminum-copper on the surface of a flat glass plate.
Then ruling many small parallel grooves into the metal
coatings.

15. 3.A cuvette.
 A cuvette is a small vessel used to hold a liquid sample to be
analyzed in the light path of a spectrophotometer.
 These are may be rounded, squared or rectangular shaped.
 Constructed from either glass or silica (quartz) or plastic.
 Most popular is 1 cm light path.

16. 4. photodetector
 A photodetector is a device that converts light into an electrical
signal that is proportional to the number of photons striking it’s
photosensitive surface. A photomultiplier tube (PMT) is commonly
used for measuring light.
photomultiplier tube (PMT)
 PNT are members of the class of vacuum tubes, and more
specifically vacuum phototubes, are extremely sensitive detectors of
light in the ultraviolet, visible, and near-infrared ranges of the
electromagnetic spectrum.
 These detectors multiply the current produced by incident light by as
much as 100 million times in multiple dynode stages, enabling (for
example) individual photons to be detected when the incident flux of
light is very low.

17. photomultiplier tube (PMT) cont…
It consists of
 A cathode or called as photocathode
 Multiple dynodes - made by light-sensitive metal like lead
selenide, gallium arsenide, Indium antimonide etc.
 Anode.
 The whole system is kept in a vacuum container

18. Schematic diagram of a photomultiplier tube (PMT)

19. Photodetector cont…
How to work PMT-
 Voltage is applied between the dynodes and each successive
stage.
 The normal incremental increase in voltage at each dynodes is
from 50-100 V larger than that of previous dynode.
 Typically , a conventional PMT tube has approximately 1500 V
applied to it.
 When 1st photon strikes into the 1st photocathode it releases
excited electron , which are then strikes 2nd photocathode and
release more electrons and again these electrons strikes next
dynodes. This way signal multiplication occurs.

20. Schematic diagram of a photomultiplier tube (PMT

22. 5.A readout device
 Electrical energy from a detector is displayed on some type of
meter or readout system
 Previously analog devices were used but now a days digital
readout devices which provide visual numeric display of
absorbance or converted value of concentrations.

23. 6.A data system.
 Data system is usually used a computer.
 Here data are analyzed and stored.

24. Performance

25.  So it is very obvious from the discussions that , to measure the
exact concentration of a compound ,a spectrophotometer
must follow some specifications.
 So the sensitivity of response of a spectrophotometer results
from combined effects lamp output, efficiency of the filters /
monochromator in the transmissions of light and response of
the photometer detector.

26. Several analytical control checks should be performed
to certify that the machine is functioning within
desired specifications-
These includes-
 1.Wavelength accuracy
 2.Absence of stray light
 3.Linearity of the detector response.
 4.Photometric accuracy.
 5.multiple wavelength reading

27. 1.Wavelength accuracy
To ensure that a spectrophotometer uses accurate
wavelength, two specific measures are used.
These are-
 Wavelength calibration and
 Use of spectral bandwidth.

28.  Wavelength calibration
 In narrow spectral bandwidth instruments, it is done with a
holminum oxide glass.
 Holmium oxide glass shows very sharp absorbance peaks at defined
wavelengths over the range of 280-650 nm.
 Thus the operator may compare the wavelength scale readings that
produce maximum absorbance with established values.
 If compared values do not coincides , a calibration correction table
can be constructed to relate scale readings to true wavelengths.
 The absorbance peaks for holmium oxide glass are typically seen at
279.3, 287.6, 333.8, 360.8. 418.5, 536.4, 637.5nm.
 Solutions of holmium oxide in dilute perchloric acid have also been
recommended and may be used with any spectrophotometer.

29. Spectral transmittance curve of holmium oxide filter

30. Wavelength calibration cont…
In broader-bandpass instruments wavelength calibrations is done
by using a didymium filter. The spectral transmittance curve for a
didymium filter -example -

31. Wavelength calibration cont…
• Didymium has several absorption peaks, the setting should be
verified grossly by visual examination of transmitted light. This
light should appear green at 530 nm.

32.  Use of spectral bandwidth.
 It is the range of wavelengths that reaches the exit slit of a
monochromator.
 It is usually referred to as the range of wavelengths, transmitted at
a point equal to half the peak intensity transmitted.
 Spectral bandwidth may also be calculated from manufacturer’s
specifications.
 Interference filters with spectral bandwidths of 1 to 2 nm are
available and may be used to check those instruments with a
spectral band-width of 8nm or more.

33. 2.Absence of stray light
 Stray light is the radiant energy reaching the detector that consists
of wavelengths other than those defined by the filter /
monochromator.
 It is defined as a ratio or percentage to the total detected light.
 It is mainly due to scattering and diffraction within the
filter/monochromator.
 The effect of stray light on the performance of a
spectrophotometer is an absorbance error especially in the upper
end of absorbance range of the instrument.
 So in practice most of the spectrometer are equipped with stray
light filters.
 Thus a blue filter is used for wavelength setting below 400nm.

34. 2.Absence of stray light cont…
For example
 when the machine is set for 350nm, most of the light is of
wavelengths of visible range.
 Here the blue filter absorbs most of the visible lights but
transmits only UV portion of the spectrums.
 Red filter is used for wavelengths in the range of 650-800 nm.

35. 2.Absence of stray light cont…
Error due to other sources of unwanted light include-
 Light leaks from the cuvette and
 Fluorescence of the sample.
 Light leaks should be excluded by covering the cell
compartments.
 Light arising from fluorescence can increase the signal to the
detector and cause an apparent decrease in absorbance.
 These sources of light are not included in the usual definition
of stray light.

36. 2.Absence of stray light cont…
How to detect stray light?
 Cutoff filters are satisfactory for detection of stray light.
 These may be of glass which produces a sharp cut in the
spectrum with complete absorbance on one side and high
transmittance to the other side.
 Liquid cutoff filters are satisfactory and convenient in the UV
range, where stray light is usually more of a problem.
 50gm/L aqueous solution of sodium nitrate should shows 0%
transmittance when read against water over 300-385nm.
 Acetone read against water , should show 0% T over the range of
250-320nm.

37. 3.Linearity of the detector response.
 A properly functioning spectrophotometer must exhibit a
linear relationship between the radiant energy absorbed and
the instrument readout.
 Solid glass filters may be used to check the linearity of the
instrument.
 A very common method for certifying linearity of detector
response involve the use of solution of varying concentrations
of compounds which follow Beer’s law.
 Such compounds example ,p-nitrophenol at 450 nm,
oxyhaemoglobin at 415 nm, copper sulphate at 650 nm etc.

38. 4.Photometric accuracy.
 The photometer must be able to measure the intensity of
transmitted light accurately in order to measure the
absorbance perfectly.
 Neutral density filters (SRM 1930) available from NIST are
used to check an instrument’s photometric accuracy.
 In practice solutions of potassium dichromate (K2Cr2O7) are
used for overall checks of photometric accuracy.

39. 5.Multiple wavelength readings
 Background interference due to interfering chromogens can
often be eliminated or minimized by inclusion of blanks or by
reading by absorbance at two or three wavelengths.
 In one approach , termed bichromatic , absorbance is
measured at two wavelengths. One to peak absorbance and
another at a point near the base of the peak to serve as the
base line.
 The difference in absorbance at the two wavelengths is
related to concentrations.

40. Thank you