Spectrophotometer- Principle, Parts, Procedure, Types, Uses

A spectrophotometer is a piece of laboratory equipment designed by two scientists named Arnold J. Beckman and his students in 194 at the national technologies laboratory and named Beckman DU spectrophotometer.

Spectrophotometer
Spectrophotometer

It is a device that has applications in measuring electromagnetic energy at a particular wavelength of light transmitted. It has wide applications in knowing the concentration of known sample material by passing light through it and measuring the light that is absorbed by the material and the rest which is transmitted by it. Thus it utilizes the light and energy properties to differentiate between colors and determine the intensity of color by passing rays of light. 

Working principle of Spectrophotometer

A spectrophotometer works on the principle of Lambert-beer law, according to which the light beam incident on a homogenous solution reflects some amount of incident light while the rest of it gets absorbed and transmitted through the solution.

Principle of Spectrophotometer
Figure: Principle of Spectrophotometer. Image Source: Linquip.

The law is based on the principle of absorption of radiation. According to which they have given a mathematical equation.

In the mathematical equation, A= ECl

A= absorbance of light

Io= incident light intensity

It= transmitted light intensity

Epsilon= coefficient of absorption

C= absorbing material concentration

l= path length (cm)

thus, the amount of light transmitted through the solution is always inversely proportional to the light absorbed by the solution.

                                   Thus, A= log10 1/T

Absorbance can range from 0-10 as it is a non-dimensional quantity and since the relationship between absorbed and transmitted light is inversely proportional thus, with an increase in absorbance, the transmittance of light through the solution decreases or vice-versa.

The sequential event of light passage in spectrophotometer starts with falling of light source on dispersion device aka monochromator, which further produces a single source of light which falls onto the focusing wavelength selector. The focusing convex lens will allow the passage of only a small amount of light from the sample to the photocell detector which converts the light energy into electrical energy and thus the amplifier transmits this electric signal into the internal circuit, which is the final output being display by the digital meter.

Types of Spectrophotometer

  1. Single beam spectrophotometer:
  • A small amount of light generated from a diverging device is allowed to pass through the sample solution.
  • This light falls on the convex lens, which moves it to the diaphragm.
  • Diaphragm by making sure that 100 % light transmit, allow to fall it on monochromator. This allows the transmission of light on a focusing convex lens.
  • This lens will transmit the light of a set wavelength from the sample to the photocell detector, which converts light energy into electrical energy, and this goes to the internal circuit and is read by a digital meter.
  1. Double beam spectrophotometer:
  • The light coming from the monochromator splits into two beams. 
  • One falls on the reference sample and another on the test sample.
  • Similar to a single beam, only the dual mirrors divide the single beam into a team.
  • One light goes from test sample to photocell and another from reference to another photocell. This later gets displayed on a digital meter.
  1. UV spectrophotometer:
  • Specialized quartz is used in this type instead of normal glass cuvette, and the light source used is either hydrogen or deuterium lamps.
  • Hydrogen lamp emits various continuous or discontinuous spectral UV-light, which ranges between 200-450nm.
  • It estimates the OD or transmittance for fluids and even solutions.
  1. Visible spectrophotometer:
  • Either glass or plastic cuvettes can be used
  • Light source: tungsten halogen lamp
  • Tungsten lamps contain tungsten filament, which emits light of visible spectrum ranging between 330-900nm.
  • The life of the lamp is 1200h.
  • This type can estimate the color change intensity according to the change in concentration of moderately diluted solutions.
  1. Infrared spectrophotometer:
  • Conductive device: Nernst glowers
  • Have a long life
  • It helps in studying different vibrations generated by different molecules at a specific wavelength.
  • Near and mid-IR-rays cause rotational and harmonic vibrations
  1. Nuclear magnetic resonance spectrophotometer:
  • Used to determine the structure of organic compounds.
  • Gives entire structural information of the entire molecule as well as the dynamic information of organic reactions.
  1. Atomic absorption spectrophotometer:
  • Water in the sample is allowed to dissociate into ions after subjecting it to evaporation using a flame.
  • This dissociation leads to changes in the intensity of light as observed by the detector.
  • It helps in estimating the unknown concentration of the sample
  • High in precision.
  • Application in toxicology, testing of environmental samples, and quality control in laboratories.
  1. Mercury spectrophotometer/analyzer:
  • Measure the trace amount of mercury in water.
  1. Fluorometers: 
  • Measures the fluorescence release once the object being studied is exposed to a single wavelength of light.

Parts/components of Spectrophotometer

  1. Light source:
  • Its function is to provide sufficient light for measuring samples.
  • It generates a high yield polychromatic light with a wide range of the spectrum.
  • Tungsten lamp: 
    • most commonly used light source.
    • Consist of tungsten filament with a wavelength ranging from 330-900nm
    • Long life of 1200h
    • Used for moderately diluted samples those having various color change intensities.
  • Hydrogen/deuterium lamp:
    • For UV spectrophotometer
    • Range= 200-450nm
    • Deuterium lamps are generally more stable
    • Generate continuous or discontinuous spectral
  • Xenon flash lamp:
    • Range= 190-1000nm
    • Emit UV and visible range
    • Long life
    • Prevent overheating of equipment
    • Reduce warm-up time
Parts of Spectrophotometer
Figure: Parts of Spectrophotometer. Image Source: Dr. Eby Bassiri.
  1. Dispersion device/monochromator:
  • It helps in the dispersion of light of different wavelength
  • Accept polychromatic input light from the lamp and output monochromatic light
  • Consists of an entrance slit, exit slit, and dispersion device.
  • Prism:
    • Used to differentiate light of different wavelength
    • Glass prisms are used for visible spectrophotometers and quartz prisms for UV spectrophotometers.
  • Filter:
    • Separate different parts of EM waves by absorbing or reflecting a certain wavelength.
    • They are of two types; absorption and interference filters.
    • An example of absorption is cut on color filter and an example of interference is band pass.
  • Diffraction grating:
    • It is an optical component used to diffract light into various beams traveling in various directions
  1. Optical mirror: mirror material changes according to the type of rays.
  • X-rays / UV: aluminum mirror
  • Visible rays: aluminum mirror
  • Near-infrared: gold mirror
  • Infrared: copper or gold mirror
  1. Lens: lens material also changes with the type of rays.
  • X-rays: fused silica, sapphire
  • Visible rays: glass lens
  • Infrared: glass lens
  1. Absorption cells (cuvette): It’s a small square tube, closed at one end, made up of glass, plastic, or quartz, and specially designed to hold the sample material to be studied for spectroscopy. The cuvette is specific for spectroscopy because of its transparency in the spectral wavelength of interest.
  2. Detectors: these are photosensitive devices such as photocell detectors, which produce the current directly proportional to the light incident on them.
  • Silicon PIN photodiodes photovoltaic V-series: 
    • Range: 350-1100nm
    • Work on low noise
    • Used for chemical and analytical measurements, low light level measurement, etc.
  • Gallium nitride  (GaN) UV detectors:
    • Range: 200-365nm
    • Ideal for UVA and UVB
  1. Display device: it displays the reading after the photocell detector converts light energy into electrical energy and contains a digital display, analog meter, etc.

Procedure for running Spectrophotometer

  1. Firstly, turn the device ON and keep it warm up for some time. This will ensure better performance.
  2. Set the required wavelength according to the sample.
  3. Transmittance should be kept at zero at the start of the experiment, and it could be done later by using the dial built on the left.
  4. Cleaning of apparatus should be ensured using distilled water from inside the chamber. 
  5. Wipe the cuvette from outside using distilled water to wipe off the fingerprints, if any. 
  6. Transfer the blank in both the cuvettes and keep them in their respective compartments.
  7. Press ZEROING button on the software and let it process.
  8. After this, remove the blank from the cuvette in the front, clean it properly using distilled water and wipe it off carefully.
  9. Now, let the other cuvette containing blank as it is and transfer the sample to the cleaned cuvette and keep it in the front compartment.
  10. Now, keep the transmittance at 100% by using the dial to the right.
  11. Everything is now set; close the lid and take the reading by clicking RUN on the software and note down the reading. 
  12. Remove the cuvette containing the sample and clean it with distilled water and let it dry or take another reading with a different sample.

Uses/applications of Spectrophotometer

  1. It is used in quantitative analysis of biological compounds such as RNA, DNA, and proteins.
  2. It is used to estimate the purity of biological molecules such as RNA, DNA, and proteins.
  3. It is used to determine the ion concentration of an unknown sample.
  4. It has applications in enzyme assay.
  5. It can quantify the substance concentration.
  6. Used to estimate the molecular weight of the given sample.
  7. Structure elucidation of organic compounds.
  8. Protein characterization.
  9. In the hospital, it is used to analyze respiratory gases.
  10. Estimate the dissolved oxygen content in a water body
  11. Detection of functional groups in the sample
  12. Compound classification.

Advantages of Spectrophotometer

  • Quick and easy interpretation of results
  • Easy operation
  • Cost-effective
  • Space efficient
  1. Single beam type: 
  • Cheaper due to lesser parts
  • High throughput
  • Sensitivity is higher
  • Complications are less
  1. Double beam type: 
  • Highly stable due to parallel measurement of reference and sample
  1. Split beam type:
  • Stability is higher
  • Good noise

Disadvantages of Spectrophotometer

  • Electricity dependent
  • Lower sensitivity and specificity
  • Further analysis of sample such as protein sample cannot be done as the colorimetric assay, destroys proteins.
  1. Single beam type: 
  • Too much time is required between two events when the measurements of reference and sample are to be measured, thus it creates problems
  • This problem can be solved by modern electronic spectrophotometer designs.
  1. Double beam type:
  • Cost inefficient
  • Poor sensitivity due to poor light throughput
  • Greater complexity makes it less reliable
  1. Split beam type:
  • poorer stability than double beam because of the presence of two detectors.
  • noise is not good as a single beam since it has splitted light thus less than 100% light pass through the sample.

Precautions

  1. Lamps and electronics should be kept for 10-15 mins before taking reading for warm-up.
  2. The correct wavelength should be set for different samples, such as 260nm for DNA and 280nm for proteins.
  3. Outside of cuvette should be carefully wiped off to remove any split sample or fingerprints before measuring; otherwise, it will deviate from the results.
  4. Correct ordering of set procedure should be followed.
  5. Before reading the result, the lid to the cuvette compartment should be closed.
  6. The inside of the cuvette compartment should be cleaned properly before taking readings.
  7. To make sure that %T or transmission is used as appropriate.
  8. The placing of the cuvette should be done in such a way that the opaque side should be away from the light source. Otherwise, the light will not pass through it.
  9. Proper cleaning of the cuvette with distilled water should be done after every reading.
  10. Homogenization of samples should be ensured during the colorimetric assay.
  11. Zeroing using blank in both the cuvette compartment should be ensured before starting taking the readings.
  12. The Blank should be set properly.

References

  1. https://laboratoryinfo.com/spectrophotometer/
  2. https://microbiologynote.com/spectrophotometer-principle/
  3. https://www.slideshare.net/AMASM/spectrophotometer
  4. https://paramedicsworld.com/biochemistry-practicals/demonstration-of-spectrophotometer-principle-components-working-applications/medical-paramedical-studynotes
  5. http://simulab.ltt.com.au/5/Laboratory/StudyNotes/snIntrodSpectrophotoM.htm
  6. https://biologyreader.com/spectrophotometer.html
  7. https://www.acttr.com/en/en-faq/en-faq-uv-vis/134-en-faq-uv-vis-advantage-disadvantage.html
  8. https://www.test-and-measurement-world.com/Terminology/Advantages-and-Disadvantages-of-Spectrophotometer.html

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