UV VISIBLE SPECTROSCOPY.......INSTRUMENTATION

Introduction
Have a look at this schematic diagram of a double-beam UV-Vis. spectrophotometer;


Instruments for measuring the absorption of U.V. or visible radiation are made up of the following components;

1. Sources (UV and visible)
2. Wavelength selector (monochromator)
3. Sample containers
4. Detector
5. Signal processor and readout

Each of these components will be considered in turn.

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Instrumental components

Sources of UV radiation

It is important that the power of the radiation source does not change abruptly over it's wavelength range.
The electrical excitation of deuterium or hydrogen at low pressure produces a continuous UV spectrum. The mechanism for this involves formation of an excited molecular species, which breaks up to give two atomic species and an ultraviolet photon. This can be shown as;
D₂ + electrical energy ® D₂^* ® D' + D'' + hv

Both deuterium and hydrogen lamps emit radiation in the range 160 - 375 nm. Quartz windows must be used in these lamps, and quartz cuvettes must be used, because glass absorbs radiation of wavelengths less than 350 nm.

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Sources of visible radiation

The tungsten filament lamp is commonly employed as a source of visible light. This type of lamp is used in the wavelength range of 350 - 2500 nm. The energy emitted by a tungsten filament lamp is proportional to the fourth power of the operating voltage. This means that for the energy output to be stable, the voltage to the lamp must be very stable indeed. Electronic voltage regulators or constant-voltage transformers are used to ensure this stability.
Tungsten/halogen lamps contain a small amount of iodine in a quartz "envelope" which also contains the tungsten filament. The iodine reacts with gaseous tungsten, formed by sublimation, producing the volatile compound WI₂. When molecules of WI₂ hit the filament they decompose, redepositing tungsten back on the filament. The lifetime of a tungsten/halogen lamp is approximately double that of an ordinary tungsten filament lamp. Tungsten/halogen lamps are very efficient, and their output extends well into the ultra-violet. They are used in many modern spectrophotometers.

Wavelength selector (monochromator)

All monochromators contain the following component parts;

• An entrance slit
• A collimating lens
• A dispersing device (usually a prism or a grating)
• A focusing lens
• An exit slit

Polychromatic radiation (radiation of more than one wavelength) enters the monochromator through the entrance slit. The beam is collimated, and then strikes the dispersing element at an angle. The beam is split into its component wavelengths by the grating or prism. By moving the dispersing element or the exit slit, radiation of only a particular wavelength leaves the monochromator through the exit slit.
Czerney-Turner grating monochromator

Cuvettes

The containers for the sample and reference solution must be transparent to the radiation which will pass through them. Quartz or fused silica cuvettes are required for spectroscopy in the UV region. These cells are also transparent in the visible region. Silicate glasses can be used for the manufacture of cuvettes for use between 350 and 2000 nm.

Detectors

The photomultiplier tube is a commonly used detector in UV-Vis spectroscopy. It consists of a photoemissive cathode (a cathode which emits electrons when struck by photons of radiation), several dynodes(which emit several electrons for each electron striking them) and an anode.
A photon of radiation entering the tube strikes the cathode, causing the emission of several electrons. These electrons are accelerated towards the first dynode (which is 90V more positive than the cathode). The electrons strike the first dynode, causing the emission of several electrons for each incident electron. These electrons are then accelerated towards the second dynode, to produce more electrons which are accelerated towards dynode three and so on. Eventually, the electrons are collected at the anode. By this time, each original photon has produced 10⁶ - 10⁷ electrons. The resulting current is amplified and measured.
Photomultipliers are very sensitive to UV and visible radiation. They have fast response times. Intense light damages photomultipliers; they are limited to measuring low power radiation.

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Cross section of a photomultiplier tube

The linear photodiode array is an example of a multichannel photon detector. These detectors are capable of measuring all elements of a beam of dispersed radiation simultaneously.
A linear photodiode array comprises many small silicon photodiodes formed on a single silicon chip. There can be between 64 to 4096 sensor elements on a chip, the most common being 1024 photodiodes. For each diode, there is also a storage capacitor and a switch. The individual diode-capacitor circuits can be sequentially scanned.
In use, the photodiode array is positioned at the focal plane of the monochromator (after the dispersing element) such that the spectrum falls on the diode array. They are useful for recording UV-Vis. absorption spectra of samples that are rapidly passing through a sample flow cell, such as in an HPLC detector.
Charge-Coupled Devices (CCDs) are similar to diode array detectors, but instead of diodes, they consist of an array of photocapacitors.

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You should now have an understanding of the separate components which make up a spectrophotometer, and how they fit together. Have a look at this schematic of the Hitachi 100-60 manual double-beam spectrophotometer;


Do you understand what each component does?

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Application of UV in Chemical Analysis

Application of UV in Chemical Analysis

Also called as Ultraviolet–visible spectroscopy, it is one of the methods of Instrumental analysis which is itself a part ofanalytical chemistry that investigates analytes using scientific instruments. For a better clarification, the keywords are further explained :

1. Instrumental Analysis: A part of analytical chemistry that investigates analytes using scientific instruments. It consists of process like Spectroscopy, Mass spectrometry,Crystallography,Electrochemical analysis, Calorimetry, etc

2. An analyte, or component is a substance or chemical constituent that is of interest in an analytical procedure.

3. Analytical chemistry is the study of the separation, identification, and quantification of the chemical components of natural and artificial materials.While qualitative analysis gives an indication of the identity of the chemical species in the sample, quantitative analysis determines the amount of one or more of these components. The separation of components is often performed prior to analysis.

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BLOCK DIAGRAM OF INSTRUMENTAL CHEMISTRY

UV Spectroscopy is defined as absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region. It utilizes light in the visible and adjacent (near-UV and near-infrared (NIR) ranges. The absorption or reflectance in the visible range directly affects the perceived color of the chemicals involved. In this region of the electromagnetic spectrum, molecules undergo electronic transitions, from ground state to excited state.

Working Principle : Molecules with π-electrons or non-bonding electrons (n-electrons) absorb the energy in the form of ultraviolet or visible light to excite these electrons to higher anti-bonding molecular orbitals.But the resulting structures are unstable and the electrons fall back to their ground state, releasing the extra energy absorbed in the form of radiations.

APPLICATIONS OF UV SPECTROSCOPY

1. Detection of Impurities
UV absorption spectroscopy is one of the best methods employed for determination of impurities in organic molecules. Additional peaks are observed due to impurities in the sample and it can be compared with that of standard raw material. By also measuring the absorbance at specific wavelength, the impurities can be detected.

2. Structure elucidation of organic compounds.
UV spectroscopy is useful in the structure elucidation of organic molecules, the presence or absence of unsaturation, the presence of hetero atoms. From the location of peaks and combination of peaks, it can be concluded that whether the compound is saturated or unsaturated, hetero atoms are present or not etc.

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3. Quantitative analysis
Also used for the quantitative determination of compounds that absorb UV radiation. This determination is based on Beer’s law which is as follows.
A = log I0 / It = log 1/ T = – log T = abc = εbc

Where ε is extinction co-efficient, c is concentration, and b is the length of the cell that is used in UV spectrophotometer.
4. Qualitative analysis
UV absorption spectroscopy can characterize those types of compounds which absorbs UV radiation. Identification is done by comparing the absorption spectrum with the spectra of known compounds. It is generally used for characterizing aromatic compounds and aromatic olefins.

5. It is also used to study the kinetics of the reaction by passing UV radiation through the reaction cell and observing the absorbance changes.

6. To identify the presence or absence of a functional group in a compound.

Some Related Questions

Q1. What is UV?

Ans. Ultraviolet (UV) light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, that is, in the range between 400 nm and 10 nm. It is so named as it lies just next to the visible spectra of violet color but remains invisible to the human eye.

Q2. What is UV Spectroscopy?

Ans. UV Spectroscopy is defined as absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region. It utilizes light in the visible and adjacent (near-UV and near-infrared (NIR) ranges. The absorption or reflectance in the visible range directly affects the perceived color of the chemicals involved.

Q3. What is Beer’s law ?

Ans. The law states that there is a logarithmic dependence between the transmission (or transmissivity),of light through a substance and the product of the absorption coefficient of the substance, and the distance the light travels through the material (i.e., the path length).

A = log I0 / It = log 1/ T = – log T = abc = εbc
Where ε is extinction co-efficient, c is concentration, and b is the length of the cell that is used in UV spectrophotometer.

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Chemical Deviations and Limitations to Beer-Lambert Law

Chemical Deviations and Limitations to Beer-Lambert Law

Chemical deviations occur due to chemical phenomenon involving the analyte molecules due to association, dissociation and interaction with the solvent to produce a product with different absorption characteristics. For example, phenol red undergoes a resonance transformation when moving from the acidic form (yellow) to the basic form (red). Due to this resonance, the electron distribution of the bonds of molecule changes with the pH of the solvent in which it is dissolved. Since UV-visible spectroscopy is an electron-related phenomenon, the absorption spectrum of the sample changes with the change in pH of the solvent.

Acid and Base forms of phenol red along with their UV spectra at different pH demonstrates chemical deviations of Beer-Lambert law in UV-Visible spectroscopy

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Instrumental Deviations and Limitations to Beer-Lambert Law

A] Due to Polychromatic Radiation (Also the reason why absorbance measurements are taken at the wavelength of maximum absorbance λ_max)

Beer-Lambert law is strictly followed when a monochromatic source of radiation exists. In practice, however, it is common to use a polychromatic source of radiation with continuous distribution of wavelengths along with a filter or a grating unit (monochromators) to create a monochromatic beam from this source. For example (see figure below), consider a molecule having molar absorptivities ε’ and ε” at wavelengths λ’ and λ”.  The absorbance (A_m) for such a species can be calculated as:

Equation to calculate absorbance of a sample with polychromatic light source.

When the molar absorptivities are the same at both wavelengths (i.e. ε’ = ε”) , the relationship between absorbance and concentration follows Beer-Lambert law to obtain a straight line. However, as the difference between ε’ and ε” increases, the deviations from linearity also increases.

Why absorption measurements are taken at wavelength of maximum absorbance λ_max?

If the band of wavelength selected on the spectrometer is such that the molar absorptivities of the analyte is essentially constant, deviations from Beer-Lambert law are minimal. However, if a band is chosen such that the molar absorptivity of the analyte at these wavelengths changes a lot, the absorbance of the analyte will not follow Beer-Lambert law. It is observed (as demonstrated in the figure below) that the deviations in absorbance over wavelengths is minimal when the wavelength observed is at the λ_max. Due to this reason absorption measurements are taken at wavelengths.

Figure A: Shows the difference in deviations in absorbance when values are obtained at maximum wavelength of absorbance (band A) vs other wavelengths of absorbance (band B). Figure B: shows the deviations in Beer-Lambert law due to observations made at wavelengths other than lambda max.

B] Due to Presence of Stray Radiation

Stray radiation or scattered radiation is defined as radiation from the instrument that is outside the nominal wavelength band selected. Usually the wavelength of the stray radiation is very different from the wavelength band selected. It is known that radiation exiting from a monochromator is often contaminated with minute quantities of scattered or stray radiation. Usually, this radiation is due to reflection and scattering by the surfaces of lenses, mirrors, gratings, filters and windows. If the analyte absorbs at the wavelength of the stray radiation, a deviation from Beer-Lambert law is observed similar to the deviation due to polychromatic radiation.

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C] Due to Mismatched Cells or Cuvettes

If the cells holding the analyte and the blank solutions are having different path-lengths, or unequal optical characteristics, it is obvious that there would be a deviation observed in Beer-Lambert law. In such cases when a plot of absorbance versus concentration is made, the curve will have an intercept k and the equation will be defined as:
A = εbc + k

In today’s instrument this problem is generally not observed, however if it is present, appropriate linear regression to quantify this deviation must be made.

Mass spectroscopy...........bromomethyl benzene (benzyl bromide)

Analysis: C₇H₁₂Br MW = 171.04


The spectrum shows two small peaks of equal intensity in the molecular ion region, strongly suggesting that the molecule contains bromine (equal concentrations of the ⁷⁹Br and ⁸¹Br isotopes). The base peak represents loss of this bromine to give the peak at m/e = 91, which is highly suggestive of a benzyl fragment.


Structure: 
IUPAC Name: bromomethyl benzene (benzyl bromide)
MS Fragments:


The spectrum shows two small peaks of equal intensity in the molecular ion region, strongly suggesting that the molecule contains bromine (equal concentrations of the ⁷⁹Br and ⁸¹Br isotopes ). The base peak represents loss of this bromine to give the peak at m/e = 91, which is highly suggestive of a benzyl fragment, which rearranges to form the tropylium cation.

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MASS SPECTRUM.......2-pentanol

Structure: 
IUPAC Name: 2-pentanol

Analysis: C₅H₁₂O MW = 88.15







The spectrum shows a small molecular ion and a small m-1 peak, suggesting the presence of an alcohol (it cannot be an aldehyde since there are no degrees of unsaturation). The m-15 peak represents loss of a methyl group and the m-17 is consistent with loss of a hydroxy radical. For an alcohol, the base peak is often formed by expulsion of an alkyl chain to give the simple oxonium ion R^'CR^''OH⁺; to generate the observed m/e = 45, R^' must be CH₃ and R^' a H.






MS Fragments:


The spectrum shows a small molecular ion and a small peak resulting from loss of a hydrogen atom from the alcohol. Loss of a methyl group gives the m-15 peak and loss of hydroxy radical gives the secondary carbocation at m-17. The base peak, not surprisingly, is formed by expulsion of the alkyl chain to give the simple oxonium ion at m/e = 45.

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