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General Agriculture

Techniques Involved In Separation and Characterization of Components Involving the Use of Spectrometry and Colorimetry

Various techniques utilize the light wave to determine some characteristics of interest in a known solution. They include colorimetry and spectrometry.

These techniques, colorimetry, and spectrometry, have devices or apparatus that can be used to determine the values of interest in the laboratory.

The colorimeter is the device used for taking colorimetric measurements while the spectrometer, spectrophotometer, or spectrograph are the devices used in spectrometry.

Principles and Application of Spectrometry

Spectrometry is a method/technique used to obtain a quantitative measure of a spectrum. It is a spectroscopic technique that can assess the quantity or concentration of a given chemical (atomic, molecular, or ionic) species.

The main forms of spectrometry; are Rutherford backscattering spectrometry and neutron triple spectrometry, ion-mobility spectrometry, and mass spectrometry. However, the most common form used is mass spectrometry.

The spectrometer is the device used in spectrometry technique, however, the type of spectrometer depends on either of the four forms of spectrometry.

Mass spectrometry is the plot between intensity (i.e. number of incident particles) and the mass of the particle. A mass spectrometer creates charged particles (known as ions) from molecules of a substance. The analyses of these ions by the mass spectrometer will provide information about the molecular weight and chemical structure of the substance.

All mass spectrometers have three distinct components;

  • 1. Ionizer
  • 2. Ion Analyser
  • 3. Detector

1. Ionizer

In the ionizer, the charged particles (ions) required for mass analysis are created by Electron Impact (EI) Ionization. EI Ionization normally produces single-charged ions that have one unpaired electron.

The energy imparted by the electron impact causes instability in a molecular ion which will make that ion fragment (break into small pieces) E.g., The reaction of methanol in the ionizing region:

CH3OH + 1 electron CH3OH+ + 2 electrons

(Note: the symbol is an. indication that a radical cation has been formed)

The methanol ion may fragment in different ways, where one fragment will be charged and the other fragment remain uncharged. For example:

CH3OH+.(molecular ion) CH2OH+(fragment ion) + H(or)

CH3OH+.(molecular ion) CH3+(fragment ion) + .OH

2. Ion Analyzer

The acceleration of molecular ions and fragment ions through the mass spectrometer is by manipulation of the charged particles. The uncharged molecules and fragments will be pumped away. Ions travel down the path based on their mass-to-charge ratio (m/z).

EI ionization produces singly charged particles, so the charge (z) is one, hence, an ion’s path will depend on its mass.

3. Detector

Detectors in mass spectrometers work by producing an electronic signal when struck by an ion. Timing mechanisms incorporate these signals with the scanning voltages and allow the device to report which m/z struck detector.

The mass analyzer will then arrange the ions according to the m/z and the detector records the abundance of each m/z.

Techniques Involved In Separation and Characterization of Components Involving the Use of Spectrometry and Colorimetry

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Principles and Application of Colorimetry

Colorimetry is a technique that is used to measure the quantity or concentration of a known solute in a particular solution by analyzing the intensity of light. The technique is based on the application of Beer-Lambert law which states that ‘the concentration of a solute is proportional to the absorbance’.

The colorimeter is a device that measures the absorbance of a particular wavelength of light by a specific solution. This is to determine the concentration of the specific solution by analyzing its color intensity.

The protocol for measuring the concentration of a specific solution with the aid of a colorimeter is as follows;

1. The calorimeter should be calibrated by placing a blank in the cuvette slot and the lid of the colorimeter closed. The blank is a cuvette and is usually distilled water.

2. The blank acts as a control, which is used to measure the small amount of light that is absorbed by the solvent and by the sides of the cuvette.

3. Fill the cuvette up to the 2.2 to 3.5 ml mark with the sample solution.

4. Place the standard disposable cuvettes (1cm x 1cm cuvettes) containing the sample solution in the colorimeter. Ensure to place the cuvette appropriately in the colorimeter.

5. This is two opposite sides of the cuvette are ribbed and do not transmit light from the LED while the two smooth surfaces transmit light.

6. Monochromatic light from the LED light source will pass through the cuvette containing the sample solution to the photodiode

7. The monochromatic light will be absorbed by the solution. The light that has a lower intensity than was beamed will strike a photodiode.

Calculating Absorbance

The amount of light that penetrates a solution is called transmittance and is expressed as that ration of the intensity of the transmitted light, and the initial intensity of the light beam I0 as expressed by the formula: T = I t / I0

The transmittance of the sample is determined by the cuvette width d and the concentration of the solution c. The relation between T, d, c is:

ea*d*c (where a is a constant that depends on the solution and uses the light wavelength).

The formula for absorbance derived from equations (i and ii) is;

= -10log() =10log(1/) =10log(e) *a*d*e*d*c (where e is the molar absorptivity of the solution)

For a given solution contained in a cuvette with a constant cell width, the Absorbance is proportional to the concentration: (Beer’s law)

*c Where is the rate of reaction (k).

In conclusion, colorimetry and spectrometry use light waves to measure the concentration of particular substances in a solution. Spectrometry can measure the molecular weight and chemical structure of a substance in solution using lightwave technology.

There are four (4) main forms of spectrometry; Rutherford backscattering spectrometry and neutron triple spectrometry, ion-mobility spectrometry, and mass spectrometry. The instrument used for spectrometry is a spectrometer but the type of spectrometer depends on which form of spectrometry is used for analysis.

A typical spectrometer has three (3) basic components; ionizer, ion analyzer, and detector. Colorimetry determines the concentration of a particular solute in a solution by analyzing the intensity of light.

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Benadine Nonye is an agricultural consultant and a writer with over 12 years of professional experience in the agriculture industry. - National Diploma in Agricultural Technology - Bachelor's Degree in Agricultural Science - Master's Degree in Science Education - PhD Student in Agricultural Economics and Environmental Policy... Visit My Websites On: 1. - Your Comprehensive Practical Agricultural Knowledge and Farmer’s Guide Website! 2. - For Effective Environmental Management through Proper Waste Management and Recycling Practices! Join Me On: Twitter: @benadinenonye - Instagram: benadinenonye - LinkedIn: benadinenonye - YouTube: Agric4Profits TV and WealthInWastes TV - Pinterest: BenadineNonye4u - Facebook: BenadineNonye

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