Spectrophotometer Instrumentation : Principle and Applications

What are the basic components of Spectrophotometer instrumentation? What is Electromagnetic radiation? Electromagnetic radiation has been put to many uses in our daily routine. The radio and television broadcasting, medical x-ray etc are some common examples. The use of electromagnetic radiation in analytical chemistry gained much importance during the last 50 years for characterization of materials. Electromagnetic radiation in the region of 200 to 700nm is generally termed as light. the eye can perceive radiation between 340 to 650 nm and can distinguish it as various (VIBGYOR).

What is VIBGYOR (or) ROYGBIV

ROYGBIV or Roy G. Biv is an acronym for the sequence of hues commonly described as making up a rainbow: Red, Orange, Yellow, Green, Blue, Indigo, and Violet.

A rainbow spans a continuous spectrum of colors; the distinct bands are an artifact of human color vision. In ROYGBIV, the colors are arranged in the order of decreasing wavelengths, with red being 650 nm and violet being about 400 nm.

VIBGYOR or ROYGBIV colors

A Spectrophotometer has all the basic components of a photoelectric colorimeter with more sophistication.

The instruments that are used to study the absorption (or) emission of electromagnetic radiation as a function of wavelength are called “SPECTROMETERS” or “SPECTROPHOTOMETERS”.

History:

For millions of years, light has defined the life of Homo sapiens. Through photosynthesis, light has given us food, energy, and atmosphere. And using light we communicate information, see the big objects far from us through the telescope and small objects through the microscope.

From where does light get this transcending power?

It took nearly a millennium until James Clark Maxwell in 1864 told the world that light is made of waves of disturbances of electric and magnetic fields.

Spectrophotometer Instrumentation device

Principle:

What is the Principle of spectrophotometer? The Spectrophotometer is a much more refined version of a colorimeter. In a colorimeter, filters are used which allow a broad range of wavelengths to pass through, whereas in the spectrophotometer a prism (or) grating is used to split the incident beam into different wavelengths. By suitable mechanisms, waves of specific wavelengths can be manipulated to fall on the test solution. The range of the wavelengths of the incident light can be as low as 1 to 2nm. The spectrophotometer is useful for measuring the absorption spectrum of a compound, that is, the absorption of light by a solution at each wavelength.  This is the basic Principle of spectrophotometry in biochemistry.

Spectrophotometer Instrumentation:

The essential components of a spectrophotometer instrumentation include:

Spectrophotometer instrumentation

  1. A Stable and cheap radiant energy source
  2. A monochromator, to break the polychromatic radiation into component wavelength (or) bands of wavelengths.
  3. Transport vessels (cuvettes), to hold the sample
  4. A Photosensitive detector and an associated readout system

1. Radiant Energy Sources:

Materials which can be excited to high energy states by a high voltage electric discharge (or) by electrical heating serve as excellent radiant energy sources.

  1. Sources of Ultraviolet radiation: Most commonly used sources of UV radiation are the hydrogen lamp and the deuterium lamp. Xenon lamp may also be used for UV radiation, but the radiation produced is not as stable as the hydrogen lamp.
  2. Sources of Visible radiation: “Tungsten filament” lamp is the most commonly used source for visible radiation. It is inexpensive and emails continuous radiation in the range between 350 and 2500nm. “Carbon arc” which provides more intense visible radiation is used in a small number of commercially available instruments.
  3. Sources of IR radiation: “Nernst Glower” and “Global” are the most satisfactory sources of IR radiation. Global is more stable than the nearest flower.

2. Wavelength selectors:

Wavelength sectors

Wavelength selectors are of two types.

  • Filters
  • Monochromators
  1. Filters: “Gelatin” filters are made of a layer of gelatin, colored with organic dyes and sealed between glass plates.
  2. Monochromators: A monochromator resolves polychromatic radiation into its individual wavelengths and isolates these wavelengths into very narrow bands. The essential components of a monochromator are.
    • Entrance slip-admits polychromatic light from the source
    • Collimating device – Collimates the polychromatic light onto the dispersion device.
    • Wavelength resolving device like a PRISM (or) a GRATING
    • A focusing lens (or) a mirror
    • An exit slip – allows the monochromatic beam to escape.

The kinds of the resolving element are of primary importance

  • PRISMS
  • GRATINGS

PRISMS:

A prism disperses polychromatic light from the source into its constituent wavelengths by virtue of its ability to reflect different wavelengths to a different extent;

The degree of dispersion by the prism depends on upon

  • The optical angle of the Prism (usually 600)
  • The material of which it is made

Two types of Prisms are usually employed in commercial instruments. Namely, 600 cornu quartz prism and 300 Littrow Prism.

GRATINGS:

Gratings are often used in the monochromators of spectrophotometers operating ultraviolet, visible and infrared regions.

3. Sample Containers:

Sample containers are also one of the parts of Spectrophotometer instrumentation. Samples to be studied in the ultraviolet (or) visible region are usually glasses (or) solutions and are put in cells known as “CUVETTES”. Cuvettes meant for the visible region are made up of either ordinary glass (or) sometimes Quartz. Most of the spectrophotometric studies are made in solutions, the solvents assume prime importance.

The most important factor in choosing the solvent is that the solvent should not absorb (optically transparent) in the same region as the solute.

4. Detection Devices:

Most detectors depend on the photoelectric effect. The current is then proportional to the light intensity and therefore a measure of it. Important requirements for a detector include

  • High sensitivity to allow the detection of low levels of radiant energy
  • Short response time
  • Long term stability
  • An electric signal which easily amplified for typical readout apparatus.

5. Amplification and Readout:

Radiation detectors generate electronic signals which are proportional to the transmitter light. These signals need to be translated into a form that is easy to interpret. This is accomplished by using amplifiers, Ammeters, Potentiometers and Potentiometric recorders.

The above 5 major parts are the major part of Spectrophotometer instrumentation. Now let us see the Applications of Spectrophotometer.

Spectrophotometer applications

How to use the spectrophotometer?  There are some uses of spectrophotometry in biochemistry which are listed below:

1. Qualitative Analysis:

The visible and UV spectrophotometer may be used to identify classes of compounds in both the pure state and in biological preparations. This is done by plotting absorption spectrum curves. Absorption by a compound in different regions gives some hints to its structure.

Absorption Range (nm) Structure (or) Type of compounds
220 to 280nm Aliphatic (or) alicyclic hydrocarbons (or) their derivatives
220 to 250 nm The compounds contain two unsaturated linkages in conjugation. Also, be due to “Benzene derivatives”
250 to 330 nm Presence of more than two conjugated double bonds usually gives rise to absorption.
450 to 500nm Beta-carotene, a precursor of Vitamin A has eleven double bonds in a conjugated system and appears yellow.
250 to 330 nm

(249nm; 260nm and 325nm)

Vitamin K1

(Due to the presence of “NAPTHAQUINONE”)

2. Quantitative Analysis: 

Spectrophotometer uses in the Quantitative analysis of Biochemistry practicals. Quantitative analysis method developing for determining an unknown concentration of a given species by absorption spectrometry. Most of the organic compounds of biological interest absorb in the UV-visible range of the spectrum. Thus, a number of important classes of biological compounds may be measured semi-quantitatively using the UV-visible spectrophotometer. Nucleic acids at 254nm protein at 280nm provide good examples of such use. The absorbance at 280nm by proteins depends on their “Tyrosine” and “Tryptophan” content.

3. Enzyme Assay:

This is the basic application of spectrophotometry. This assay is carried out most quickly and conveniently when the substrate (or) the product is color (or) absorbs light in the UV range.

Eg 1: Lactate Dehydrogenase (LDH)

Lactate + NAD +  ↔ Pyruvate + NADH + H+

  • The LDH is engaged in the transfer of electrons from lactate to NAD+.
  • The products of the reaction are pyruvate, NAD, and a proton
  • One of the products, NADH, absorbs radiation in the UV range at 340 nm while its oxidized counterpart, NAD+ does not.
  • The reaction in the forward direction can be followed by measuring the increment in the light absorption of the system at 540nm in a spectrophotometer.

Eg 2: Pyruvate Kinase

Phosphoenolpyruvate + ADP ↔ Pyruvate + ATP

Pyruvate + NADH + H+ ↔ Lactate + NAD +

We have added a large excess of NADH to the system, the system now absorbs at 340nm. According to the above-given reactions, each molecule of Pyruvate formed in the reaction, a molecule of NADH is oxidized to NAD+ in the second reaction when the system converts pyruvate to locate.

Since NAD+ does not absorb at 340nm the absorbance goes on decreasing with increased pyruvate generation. Such measurements are known as “Coupled assays”.

Sample enzymatic assays:

4. Molecular Weight determination:

Molecular weights of amine picrates, sugars and many aldehyde and ketone compounds have been determined by this method. Molecular weights of only small molecules may be determined by this method.

  1. Study of Cis-Trans Isomerism:  Geometrical isomers differ in the spatial arrangement of groups about a plane, the absorption spectra of the isomers also differs. The trans-isomer is usually more elongated than its cis counterpart. Absorption spectrometry can be utilized to study Cis-Trans isomerism.
  2. Control of Purification: Impurities in a compound can be detected very easily by spectrophotometric studies. “Carbon disulfide” impurity in carbon tetrachloride can be detected easily by measuring absorbance at 318nm where carbon sulfide absorbs. A lot many commercial solutions are routinely tested for purity spectroscopically.

5. Other Physiochemical Studies:

Spectrophotometry (UV-VIS) has been used to study the following physiochemical phenomena:

  • Heats of formation of molecular addition compound and complexes in solution
  • Determination of empirical formulae
  • Formation constants of complexes in solution
  • Hydration equilibrium of carbonyl compounds
  • Association constants of weak acids and bases in organic solvents
  • Protein-dye interactions
  • Chlorophyll-Protein complexes.
  • Vitamin-A aldehyde – Protein complex
  • Determination of reaction rates
  • Dissociation constants of acids and bases
  • Association of cyanine dyes

These are the basic spectrophotometer instrumentation and its applications.

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