Spectrophotometer | Instrument of Spectrophotometer | Color Measurement Procedures by Spectrophotometer

Spectrophotometer is a photometric device that measures spectral transmittance, spectral reflectance relative spectral emitance. It compares light leaving from the object with that incident on it at each wavelength. According to Beer's law, the amount of light absorbed by a medium is proportional to the concentration of the absorbing material or solute present. Thus the concentration of a colored solute in a solution may be determined in the lab by measuring the absorbency of light at a given wavelength. Wavelength (often abbreviated as lambda) is measured in nm. The spectrophotometer allows selection of a wavelength pass through the solution. Usually, the wavelength chosen which corresponds to the absorption maximum of the solute .
Spectrophotometer
Absorption Spectroscopic methods of analysis rank among the most widespread and powerful tools for quantitative analysis. The use of a spectrophotometer to determine the extent of absorption of various wavelengths of visible light by a given solution is commonly known as colorimetry. This method is used to determine concentrations of various chemicals which can give colors either directly or after addition of some other chemicals.

As an example, in the analysis of phosphate, a reaction with orthophosphate is made, to form the highly coloured molybdenum blue compound. The light absorption of this compound can then be measured in a spectrophotometer. Some compounds absorb light in other than the visible range of the spectrum. For example, nitrates absorb radiation of 220 nm wave length in the UV region.

Absorption Spectroscopic methods of analysis are based upon the fact that compounds ABSORB light radiation of a specific wavelength. In the analysis, the amount of light radiation absorbed by a sample is measured. The light absorption is directly related to the concentration of the colored compound in the sample.

The wavelength (l) of Maximum Absorption is known for different compounds. For example, the colored compound formed for analysis of Phosphate (molybdenum blue) has maximum light absorption at l= 640 nm. Conversely, a minimum amount of light is transmitted through the compound at l= 640 nm.

The  Instrument of
Spectrophotometer:
All spectrophotometer instruments designed to measure the absorption of radiant energy have the basic components as follows :
  1. A stable source of radiant energy (Light);
  2. A wavelength selector to isolate a desired wavelength from the source (filter or monochromator);
  3. Transparent container (cuvette) for the sample and the blank;
  4. A radiation detector (phototube) to convert the radiant energy received to a measurable signal; and a readout device that displays the signal from the detector.
Components of a spectrophotometer
 The energy source is to provide a stable source of light radiation, whereas the wavelength selector permits separation of radiation of the desired wavelength from other radiation. Light radiation passes through a glass container with sample. The detector measures the energy after it has passed through the sample. The readout device calculates the amount of light absorbed by the sample displays the signal from the detector as absorbance or transmission.

The spectrophotometers which are used for such measurements may vary from simple and relatively inexpensive colorimeters to highly sophisticated and expensive instruments that automatically scan the ability of a solution to absorb radiation over a wide range of wavelengths and record the results of these measurements.

One instrument cannot be used to measure absorbance at all wavelengths because a given energy source and energy detector is suitable for use over only a limited range of wavelengths.

True linearity between absorbance and concentration according to Beer-Lambert Law requires the use of monochromatic light. In addition, a narrow band of light ensures a greater selectivity since substance with absorption peaks in other close by wavelengths are less likely to interfere. Further, it increases sensitivity as there is a greatest change in absorbance per increment of change in concentration.

Both filters and monochromators are used to restrict the radiation wavelength. Photometers make use of filters, which function by absorbing large protions of the spectrum while transmitting relatively limited wavelength regions. Spectrophotometers are instruments equipped with monochromators that permit the continuous variation and selection of waveiength. The effective bandwidth of a monochromator that is satisfactory for most applications is about from 1 to 5 nm.

The sample containers, cells or cuvettes, must be fabricated from material that is transparent to radiation in the spectral region of interest. The commonly used materials for different wave length regions are:

  • Quartz or fused silica: UV to 2 mm in I R
  • Silicate glass: Above 350 nm to 2 mm in I R
  • Plastic: visible region
  • Polished NaCI or AgCI: Wave lengths longer than 2mm
Cuvettes or cells are provided in pairs that have been carefully matched to make possible the transmission through the solvent and the sample. Accurate spectrophotometric analysis requires the use of good quality, matched cells. These should be regularly checked against one another to detect differences that can arise from scratches, etching and wear. The most common cell path for UV-visible region is 1 cm. For reasons of economy, cylindrical cells are frequently used. Care must be taken to duplicate the position of such cells with respect to the light path; otherwise, variations in path length and in reflection losses will introduce errors.

General Measurement Procedures :
As explained above, the Beer-Lambert Law forms the basis of the measurement procedure. The amount of light radiation absorbed by a compound is directly related to the concentration of the compound.

The general measurement procedure consists of 5 steps:

  1. Prepare samples to make colored compound
  2. Make series of standard solutions of known concentrations and treat them in the same manner as the sample for making colored compounds
  3. Set spectrophotometer to l of maximum light absorption
  4. Measure light absorbance of standards
  5. Plot standard curve: Absorbance vs. Concentration, 
Uses of  Spectrophotometer:
The use of spectrophotometers spans various scientific fields, such as physics, materials science, chemistry, biochemistry, and molecular biology. They are widely used in many industries including semiconductors, laser and optical manufacturing, printing and forensic examination, as well in laboratories for the study of chemical substances. Ultimately, a spectrophotometer is able to determine, depending on the control or calibration, what substances are present in a target and exactly how much through calculations of observed wavelengths.

Note: Minolta, Datacolor, Spectroflash, SF-300, F-600 are the examples of some Spectrophotometers. Portable Spectrophotometers are used in finishing dept.

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