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How to Use a Spectrophotometer to Determine Absorbance?

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Background concepts

The spectrophotometry of absorption in the ultraviolet and visible regions of the electromagnetic spectrum is, possibly, the most used in the practice of the quantitative analysis of all spectroscopic techniques.
It is based on the absorption of ultraviolet radiation and visible by the analyte, as a result of which an activated state is created that later removes its excess energy in the form of heat.
The term spectrophotometry is usually applied when the radiation used extends to the ultraviolet and infrared regions, and also uses monochromators instead of filters, as well as more visible detection systems, such as photomultiplier tubes.

Law of the absorption Radiation

When a beam of monochromatic radiation of a certain wavelength passes through a layer of a solution containing an absorbent species, the power (energy per unit time and unit area).
TRANSMITTANCE, T, is defined as the fraction of the incident radiation that the sample manages to pass through. It varies from 0 to 1 and can also be expressed as a percentage:

T = P / Po; T = P / Po (100)

A parameter of greater practical utility is ABSORBANCE, defined as:

A = -log = log Po / P

When there is no radiation absorption P = Poy then, A = 0, while if 99% is absorbed, only 1% is transmitted, with which A = 2.

BEER’S LAW: ” ABSORBANCE IS DIRECTLY PROPORTIONAL TO THE CONCENTRATION AND LENGTH OF THE STEP OF THE LIGHT

                                    A = Ebc
A = Absorbance
E = Molar extinction (absorption coefficient
b = light path length
c = concentration of the absorber

Spectrophotometer

A more sophisticated instrument that has a MONOCHROME instead of filters. Also, the detection system is usually a PHOTOMULTIPLIER, more visible than a photocell.
It allows comparing the radiation absorbed or transmitted by a solution containing an unknown quantity of solute, and one containing a known quantity of the same substance.
All substances can absorb radiant energy, even glass that appears to be completely transparent absorbs wavelengths that belong to the visible spectrum, water absorbs strongly in the infrared region.
When light passes through a substance, part of the energy is absorbed, the radiant energy can not produce any effect without being absorbed.
The color of the substances is because they absorb certain wavelengths of white light that impinge on them and only let these non-absorbed wavelengths pass to our eyes.
It can handle a beam of electromagnetic radiation commonly called light, separating it into facilitating the identification, qualification and quantification of energy.
When a particle is in a state of rest, it interacts with a beam of light, absorbs it and transforms it into a particle in an excited state.
Each absorbent species has a specific light spectrum.

Components

MONOCHROME: It is characterized by producing a beam of radiation of great spectral purity and allowing for varying, continuously and over a wide range, the wavelength of radiation.

Its basic components are – an input slit, which selects a beam of incoming polychromatic radiation, a dispersing element, prism or network, which scatters the radiation at its wavelengths, and an output slit, which isolates the spectral band desired.

The dispersion of radiation by a PRISMA is based on the phenomenon of refraction, that is, the change of direction that a radiation beam experiences when passing from one medium to another with a different refractive index.

The degree of deviation depends on the wavelength, so the blue ones deviate more than the red ones.

SOURCE OF LIGHT: Provides radiant energy in the form of visible or non-visible light, must comply with stability, directionality, distribution of spectral energy.

Types of lamps

– Tungsten filament lamps: are used for wavelengths of the visible spectrum and the near ultraviolet. They are sources of a continuous spectrum of radiant energy between 360-950 nm.
– Tungsten halide filament lamps: they are longer lasting and emit radiant energy of greater intensity.
– Hydrogen and Deuterium lamps: produce a continuous spectrum in the ultraviolet region between 220-360 nm.
– L sunlamps Mercury vapor that emits a discontinuous spectrum or spectrum lines that are used for wavelength calibration, they are used only for spectrophotometers and HPLC.
· Precautions:
– Sudden highs and lows of tension produce suffering of the lamp and changes in the readings of Absorbance
– The lamp has a limited vitality and must be monitored so that the device works properly.

CUBETA: Used to place liquids in the beam of the spectrometer.

DETECTORS: Translators that convert radiant energy into an electrical signal.

METER: They are reading systemsenergyelectrical that collects the detector and that can be direct reading (a photovoltaic cell is used) or can be signal amplifiers as in the case of the phototube multiplier. The current devices incorporate digital reading and automatic calculations of concentrations in relation to calibration curves.

Reading Errors

Indeterminate errors in the reading of transmittance or absorbance are errors that are potentially always present and must be taken into account. Sometimes small errors can cause large errors in concentration.

Personal Errors

The biggest mistakes are usually made by the inadequate use of the absorption cuvettes. The following recommendations are useful:

* It is necessary that the buckets are perfectly clean, not scratched and exceed traces to
Adhesions in the walls through which the radiation has to pass.

* The glass and quartz covers can be cleaned with nitric acid or with regia cold water, but not with the chromic mixture.

* Once the buckets are clean, they should be cleaned with distilled water and with various proportions of the solution to be measured.

* They should not be dried internally, while on the outside it should be with a soft handkerchief, also checking that once filled with the problem solution, it does not contain air bubbles.

Calibration

Before using the spectrophotometer or photocell, it is essential to perform basic calibration routines to ensure that the device provides reliable data and readings.
Before using your equipment, you should do the following:
* Cleaning the surface of the instrument.
* Cleaning of filters and a light source (lamp and condenser).
* Verify electrical installations .
– The steps to test the operability of the team are the following:

* The equipment is turned on and allowed to warm up for at least 15 minutes (if the device is automatic, it will give a signal when it is ready to operate).
* The desired wavelength is selected (this depends on the sample to be read and the reagent used).
* The absorbance function is selected or
transmittance.
* The device is set to zero with distilled water. If the apparatus to be used has the two scales (absorbance and transmittance), the readings are adjusted to zero absorbance and 100% transmittance using the coarse and fine vacuum controls.
* A standard of known concentration is read and the device is adjusted to that concentration. If the device to be used does not have standard control, it is used to obtain the calibration factor,
dividing
the concentration of the standard between its reading.

Applications

  • It is used in the standardization of the colors of materials, as in the case of plastics and paints, they allow them to detect absolutely all levels of air and water pollution.
  • Determination of impurities that normally appear in reagents and food. APPLICATIONS
  • It is used in the standardization of the colors of materials, as in the case of plastics and paints, they allow them to detect absolutely all levels of air and water pollution.
  • Determination of impurities that normally appear in reagents and food.

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