Spectrophotometer

Specification And

performance

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.

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.

Types of spectrophotometers

Single-beam spectrophotometers. Double-beam spectrophotometers 1.double beam in space spectrophotometer. 2.double-beam in time spectrophotometer

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.

Diagrammatic representation of some spectrophotometers

Schematic representations of Single-beam spectrophotometers

⃝�→→→¦→→ →→¦→→→→→→→ Source entrance exit slit light slit monochromator cuvet detector read out device

Schematic representations of Double-beam spectrophotometers.

Specifications

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.

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.

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.

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.

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.

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

Schematic diagram of a photomultiplier tube (PMT)

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.

Schematic diagram of a photomultiplier tube (PMT

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.

6.A data system.

Data system is usually used a computer. Here data are analyzed and stored.

Performance

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.

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

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.

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.

Spectral transmittance curve of holmium oxide filter

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 -

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.

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.

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.

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.

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.

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.

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.

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.

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.

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