Applications of spectrophotometry

 

What is spectrophotometry?

Spectrophotometry is a method to measure how much a chemical substance absorbs light by measuring the intensity of light as a beam of light passes through sample solution. The basic principle is that each compound absorbs or transmits light over a certain range of wavelength. This measurement can also be used to measure the amount of a known chemical substance. Spectrophotometry is one of the most useful methods of quantitative analysis in various fields such as chemistry, physics, biochemistry, material and chemical engineering and clinical applications.

Principle

The spectrophotometer technique is to measure light intensity as a function of wavelength. It does this by diffracting the light beam into a spectrum of wavelengths, detecting the intensities with a charge-coupled device, and displaying the results as a graph on the detector and then on the display device.

1.    In the spectrophotometer, a prism (or) grating is used to split the incident beam into different wavelengths.

2.    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.

3.    The spectrophotometer is useful for measuring the absorption spectrum of a compound, that is, the absorption of light by a solution at each wavelength.

 

 

 

 

 

 

 

 

Applications of Spectrophotometry

1. Concentration measurement

– 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

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.

 

3. Structure elucidation 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.

 

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.

 

Test sensitivity and specificity

 

• Sensitivity refers to the lower limit of detection, or the lowest concentration capable of being detected by a test method.

• Failure to detect small amounts of a substance in a test will result in a false-negative result.

• Specificity refers to the ability to detect only the substance for which the test is designed.

• Reaction with other substances (cross-reactivity ) decreases the specificity of the test and can cause false positive results .

Some General Applications

Ø  Detection of concentration of substances

Ø  Detection of impurities

Ø  Structure elucidation of organic compounds

Ø  Monitoring dissolved oxygen content in freshwater and marine ecosystems

Ø  Characterization of proteins

Ø  Detection of functional groups

Ø  Respiratory gas analysis in hospitals

Ø  Molecular weight determination of compounds

Ø  The visible and UV spectrophotometer may be used to identify classes of compounds in both the pure state and in biological preparations.

 

 Applications of Spectrophotometry in biological sciences

Ø  The theories and techniques of measuring the absolute values of the transmittance and reflectance of translucent biological materials with opal glass plates are presented. These methods were called opal glass transmission and reflection methods. They are simple and easy to practice with spectro-photometers commonly used for the measurement of transparent materials. From the transmission and reflection spectra of leaves observed by these methods, it was proved that they give us the exact values of the transmittance and reflectance.

 

Ø  From the results of the measurements by opal glass transmission and reflection methods, it was found that we need to define at least six quantities, which describe the optical properties of translucent or non-transparent biological materials.

 

Ø   The way and use of evaluating these properties are described, especially paying attention to the absolute measurement of the light absorbed by those samples.

Ø  By modifying the opal glass reflection method, we can observe the logarithm of the reciprocal of the relative value of reflectance, even more simply than by opal glass reflection method for absolute measurement. The measurements by the modified method showed clear absorption bands of biological non-transparent materials. As one of the application of the method, the state of carotenoids in vivo was studied. The results indicated that some carotenoids exists in their crystalline state in roots or fruits.

Spectrophotometry applications in Analysis of a biochemical mixture

Spectrophotometric analysis is essential for determining biomolecule concentration of a solution and is employed ubiquitously in biochemistry and molecular biology.

 The application of the Beer‐Lambert‐BouguerLawis routinely used to determine the concentration of DNA, RNA or protein.

There is however a significant difference in determining the concentration of a given species (RNA, DNA, protein) in isolation (a contrived circumstance) as opposed to determining that concentration in the presence of other species (a more realistic situation).

 

 To present the student with a more realistic laboratory experience and also to fill a hole that we believe exists in student experience prior to reaching a biochemistry course,

 We have devised a three week laboratory experience designed so that students learn to connect laboratory practice with theory, apply the Beer‐Lambert‐Bougert Law to biochemical analyses,

 Demonstrate the utility and limitations of example quantitative colorimetric assays, demonstrate the utility and limitations of UV analyses for biomolecules, develop strategies for analysis of a solution of unknown biomolecular composition,

Use digital micropipettors to make accurate and precise measurements, and apply graphing software.

Application in DNA and RNA Concentration

Spectrophotometry can be used to estimate DNA or RNA concentration and to analyze the purity of the preparation. Typical wavelengths for measurement are 260 nm and 280 nm. In addition measurements at 230 nm and 320 nm can provide further information. Purines and pyrimidines in nucleic acids naturally absorb light at 260 nm.

For pure samples it is well documented that for a pathlength of 10 mm, an absorption of 1A unit is equal to a concentration of 50 µg/ml DNA and 40 µg/ml for RNA. For oligonucleotides the concentration is around 33 µg/ml but this may vary with length and base sequence. So for DNA:

 Concentration (µg/ml) = Abs260 × 50.

These values are known as conversion factors. A number of other substances which also absorb light at 260 nm could interfere with DNA values, artificially increasing the result calculated from the absorption readings. To compensate for this a selection of ratios and background corrections have been developed to help eliminate false readings.

Wavelength scan for a pure DNA sample

There is a wide absorbance peak around 260 nm preceded by a ‘dip’ at 230 nm. Therefore to measure the DNA absorption, the 260 nm DNA peak must be distinguishable from the 230 nm reading. If the readings at 230 nm are too similar to those at 260 nm, DNA cannot be measured accurately.

Higher 230 nm readings can indicate contaminants in the sample. There should also be a rapid tail-off from 260 nm down to 320 nm. For this reason, 320 nm is often used to measure background (see background correction).