Spectroscopy is the branch of chemistry concerned with the investigative measurements of spectrum. UV-VIS (ultraviolet-visible) spectroscopy or spectrophotometry is the study of the interaction of light with matter at electronic levels. It ranges from the vacuum level ultraviolet region i.e. 180nm to visible region i.e. 780nm. UV spectrum extends from 180nm to 400nm whereas the visible region ranges from 400nm to 780nm.
UV-VIS spectroscopy is an instrumental technique used for chemical analysis. It is used for qualitative as well as quantitative analysis. This technique has several applications for a large number of compounds. It is used to observe the optical behavior of chemical compounds, identification of various species, and quantification of specific analytes.
|Classical analytical techniques|
|Basics of spectroscopy|
|Interaction of radiation with matter|
In UV-VIS spectroscopy, the transition of electrons at various levels by absorption of radiation from ultraviolet to visible region is plotted in a graph. This line graph of various absorptivities on specific levels of radiations is because of the absorption capacities of compounds at certain levels. These levels are called regions of absorption and the compounds are termed as chromophores.
The chromophores are present in almost every compound. This can be deduced by the fact that almost all compounds and especially organic compounds can be identified and quantified by the uv-vis spectroscopy.
Working principle of UV-visible spectroscopy
When a chemical compound absorbs light, some excitation and de-excitation processes of electrons occur in atoms which result in the production of the distinct spectrum.
The electromagnetic spectrum is the division of electromagnetic radiation based on the energy, frequency, or wavelength of a photon.
Interaction of radiation and matter
In spectroscopic analysis, when radiations interact with a chemical species, they can cause transition at different energy levels. The type of transition depends upon the energy of the radiation and the detection mode.
The transition of electrons always occurs from the ground state of low energy HOMO (highest occupied molecular orbital) to a higher energy excited state LUMO (lowest unoccupied molecular orbital). The overlap of atomic orbitals forms three types of molecular orbitals. This whole concept arises from molecular orbital theory (MOT).
- Bonding molecular orbitals of lower energy.
- Non-bonding molecular orbitals of intermediate energy.
- Anti bonding molecular orbitals are of higher energy.
Energy order in electronic transitions
The electronic transitions that require the highest energy are σ-σ* electronic transitions, while the transitions requiring the least energy are n-π* electronic transitions.
The energy order of different types of electronic transitions is as under.
σ-σ* > n-σ* > π-π* > n-π*
This phenomenon is studied under Beer-Lambert law.
Lambert law implies that the absorbance ‘A’ of incident monochromatic light is directly proportional to the path length (cell length) ‘ℓ’. This means that equal portions of absorbing material absorb equal fractions of incident light.
A ∝ ℓ
Beer law implies that the absorbance ‘A’ of incident light or electromagnetic radiation is directly proportional to the concentration ‘c’ of solution. This law gives the quantitative relationship between the intensity of radiation and the concentrations of chemical species.
A ∝ c
Collectively Beer and Lambert’s laws state that the absorbance ‘A’ of an incident monochromatic beam is directly proportional to concentration ‘c’ of the solution and path length ‘ℓ’.
The rate of decrease in intensity of monochromatic light is proportional to the thickness of medium ‘ℓ’ and concentration ‘c’ of absorbing substance in dilution.
A ∝ c . ℓ
A = ε . c . ℓ
ε is the molar absorptivity coefficient constant.
ε is the absorbance for a solution of concentration 1mole/dm-3 and a path length of 1cm.
It is the ratio of the intensity of incident electromagnetic radiation from the source to that of refracted electromagnetic radiation detected by the detector.
A = Log ℓo / ℓ
A = ε . c . ℓ
Log ℓo / ℓ = ε . c . ℓ
It is the ratio of power of electromagnetic radiation leaving the sample Pt to that of the incident radiation on the sample from the source.
Mathematical representation in terms of transmittance
T= Pt / Po
% T = T × 100
% T = Pt / Po × 100
Conversion of absorbance to transmittance
A = – log T
A = – log Pt / Po (Since T= Pt / Po)
A = log Po / Pt
A = 2- log T %
% T = antilog (2 – A)
Limitations of lambert beer law
- The light source used must be monochromatic.
- This is not suitable for concentrated solutions i.e. It can only be applicable to dilute solutions.
- With an increase in dilution, dissociation of weak acids occurs. The weak acids reach equilibrium with their conjugate base. The acid (HA) and conjugate base (A–) cannot have the same absorbance. Hence this law is not completely applicable to weak acidic solutions.
Instrumentation of UV-visible spectroscopy
The basic instrumentation of the UV-vis spectrometer comprises of
- Light source
- Diffraction grating
- Wavelength selector
- Sample container or cuvette
1. Light Source
Light sources that lie in the ultraviolet and visible region are used as UV-visible spectrometer sources.
- Hydrogen & deuterium lamps range 160-380nm
- Xenon arc lamps range 250-600nm
- Tungsten halogen lamps range 240-2500nm
2. Wavelength selector
UV-Vis spectroscopy requires a single wavelength for proper functioning whereas the ideal output of a single wavelength is not possible. This is so because no real wavelength selector is ideal. Although a single wavelength is not possible, a band of radiation could be used. So an instrument with narrow bandwidth would be better.
Types of the wavelength selectors
Filters are used to permit a certain band of wavelength. The simplest type of filter is the absorption filter. Most commonly colored glass filters are used. They absorb a broad portion of the spectrum (complementary colors) and transmit other portions (its own color).
- The filters are inexpensive.
- They possess technical simplicity.
- The filters are restricted to the visible region only.
- They are not very good wavelength selectors.
- Not useful for research purposes as they allow broad bandwidth. So there are more chances of deviation from Lambert beer’s law.
A monochromator is an optical device that is used to select a narrow band of a wavelength of light. It may be a quartz prism or grating.
Uses of monochromators
- Monochromators are used for spectral scanning i.e. varying wavelength of radiation over a range.
- They can be used for the UV-visible region.
Components of a monochromator
All monochromators are similar in mechanical construction. The essential components of a monochromator are:
3. Sample container/cells or cuvettes
Sample containers or cuvettes may be made up of
- Only quartz is transparent in both UV & visible regions (200-700nm range).
- Glass & plastic are suitable for the visible region only.
- Glass is not suitable for the UV region because it absorbs UV radiation i.e. it is not transparent in the UV region.
- Plastic cells are not used for organic solvents.
The most common cuvette size is 1 cm, although it can vary from 0.1-10 cm.
Detectors are devices that indicate the existence of some physical phenomenon. Some examples of simple detectors are
- Photographic films
- Mercury level in thermometers (temperature detector)
- Human eye
A transducer is a special type of detector that converts signals such as light intensity, pH, mass, and temperature, etc into electrical signals. This electrical signal is amplified and manipulated. The signal is represented in numbers (digital form).
Properties of transducers
- Transducers produce a fast response to low levels of radiant energy.
- Suitable for a wide range of wavelengths.
- Electrical signals produced by transducers should have low noise.
- The signal produced by the transducer is directly proportional to the beam of power.
Photodetectors are used as:
- Photo tubes
- Photomultiplier tubes
- Silicon diodes
- Photovoltaic cells
Photo tubes or photoemissive cells are a type of photodetector. They are concave surfaces of cathode and anode inside a glass bulb. The cathode is coated with photoemissive material. e.g. cerium, potassium oxide, silver oxide, etc. The anode is a metallic sheet or ring at high voltage by the battery. The interior of a bulb is filled with an inert gas at low pressure.
Photomultiplier tubes are structured as repeated dynodes at particular angles. The emitted electrons strike on different dynodes. Every dynode has a high voltage than the previous ones. This voltage difference accelerates the electrons. These fast electrons knock out more electrons upon striking the next dynodes.
The repetition of the above process causes up to 106 electrons collected for each photon, striking the first cathode.
Photomultiplier tube is sensitive and costly with respect to the phototubes.
Spectral analysis in UV-VIS spectroscopy
UV-VIS spectrum is a graph that shows absorption at different wavelengths. The relative maxima are known as lambda max (λmax). Spectra obtained by such techniques can be useful for extracting information such as purity and composition.
UV-Vis spectroscopic spectra are frequently utilized to check the presence of different organic and conjugated chemical species that have a wavelength in that range.
Chlorophyll-a absorbs the regions of violet and orange wavelength regions. This is the fact that makes plants unable to absorb green light making them appear green. The below graph shows the absorbance activity of chlorophyll-a in visible light and it proves the fact that it is most active in violet and orange regions.
Amoxicillin is a famous antibiotic that gives an identification peak at approximately 265nm on the wavelength scale.
The spectral analysis of uv-vis spectroscopy is also used for the quantitative analysis of analytes. The absorbance at a certain wavelength of light is directly proportional to the concentration by Beer-Lambert law.
Shifting of absorption band and change in intensity
The chromophore is an atom or group of atoms that are responsible for the absorption of UV-visible radiation.
Types of chromophores
There are two types of chromophores:
- Chromophores that can only contain π electrons. They undergo π-π* transitions only e.g. ethylenic group (C＝C) and acetylenic group (C ≡ C) etc.
- Chromophores that contain π as well as n (nonbonding) electrons. This type of chromophore contains lone pair(s) of electrons. So they are responsible for two types of transitions i.e. n-π* and π-π* e.g. nitro group (-NO2 ), azo group (-N＝N-), nitro group (-NO3 ), carbonyl group ( >C＝O), nitrite group (-ONO).
An auxochrome is an atom or group of atoms that do not give rise to an absorption band of its own but change the absorption characteristics of a chromophore. Auxochromes may change both intensity and wavelength of chromophore when added to it. It is also called a color-enhancing group. The replacement of hydrogen on a basic chromophore changes its absorption characteristics. i.e. Methyl (-CH3,), chloride (Cl–), hydroxyl (OH–), amino (-NH2), alkoxy (CH3O–), etc.
There exist four types of shifts corresponding to auxochromes:
- Bathochromic shift (redshift)
- Hypsochromic shift (blueshift)
- Hyperchromic shift
- Hypochromic shift
Bathochromic shift or redshift
The bathochromic shift is the change of position of the absorption band towards the longer wavelength. This change occurs in the presence of auxochrome or change in the solvent.
Hypsochromic shift or blue shift
The hypsochromic shift is the change in the position of the spectral band towards a shorter wavelength. It occurs due to the removal of conjugation or change in polarity of the solvent.
Hyperchromic shift is the increase in the intensity of the absorption band i.e. εmax. A hyperchromic shift occurs due to the presence of an auxochrome.
A hypochromic shift is a decrease in the intensity of the absorption i.e. εmax band.
Factors affecting change in the absorption and wavelength
These are the factors that result in changes in absorption and wavelength:
Effect of conjugation
According to MOT, as the number of pi electrons increases, delocalization increases. Due to this increase in delocalization, the molecules of the sample get stabilized and hence reach a state of lower energy. This lowering of energy causes a change in wavelength towards a higher wavelength known as redshift.
The maximum absorption of 1,2-butadiene is at 210nm but for 1,3-butadiene is at 217nm due to conjugation of double bonds. This wavelength increases further to 260nm for 1,3,5-hexatriene because of conjugation at two points.
Effect of additive characteristics
When a molecule contains two or more chromophores separated by more than one single bond the total absorption is equal to the sum of absorption characteristics of each chromophore.
For example, ethylene and 1,5 hexadiene absorb the same wavelength. But the amount of radiation absorbed for similar concentrations is almost doubled for 1,5 hexadiene than ethylene.
Effect of the aromatic ring
The aromatic ring especially when two or more rings in conjugation (polycyclic compounds) absorbs a higher wavelength in the visible region, it alters the spectrum of absorption.
Naphthalene (C6H6 ) absorbs at 268nm
Anthracene absorbs at 311nm
Tetracene absorbs at 476nm
Effect of substitution of auxochrome
Benzene is a less effective chromophore, the substitution of a polar group to it causes an increase in λmax in the visible region and hence εmax value increase.
Effect of the polarity of the solvent
Polarity causes a pronounced effect on the position and intensity of absorption bands. This increase is due to the n-π* and π-π* transitions. In the presence of polar hydrolytic solvent (i.e. water ) hydrogen bonds form with the lone pair of electrons of auxochrome. As a result, the auxochrome’s energy lowers to an equal amount of the bond formation energy, and hence the energy gap between HOMO and LUMO increases, so, a hypsochromic shift is observed for n-π* transition.
|n-π* Transitions||Solvent||Absorption wavelength|
While for π-π* transition the π* orbital is more polar than π orbital therefore it is stabilized to a greater extent in the presence of a polar solvent. This will cause a bathochromic shift because the energy gap between π-π* is reduced due to the stability of the π* orbital.
|π-π* Transitions||Solvent||Absorption wavelength|
Types of UV-Vis spectrometers
There are two types of UV-vis spectrophotometers
- Single beam UV-Vis spectrometer
- Double beam UV-Vis spectrometer
Single beam UV-Visible spectrophotometer
Single beam uv-vis spectrophotometer has a single beam as the name indicates. The incident light coming from the source is passed through a monochromator then that incident monochromatic light moves through a slit. Then it passes through the sample solution. Where some of the incident light is absorbed by the sample while other is transmitted. That transmitted light is detected by the detector. The detected light is then amplified, recorded, and then displayed on a suitable readout device. Spectrum is plotted and the λmax is located.
Single beam uv-vis spectrophotometer comprises of:
- Light source
- Wavelength selector
- Sample container/cuvette
- Digital meter/Recorder
Double beam UV-Visible spectrophotometer
The instrumentation of single and double beam spectrophotometers is almost the same. The basic difference from a single beam UV-Vis spectrophotometer is that the beam of incident light is passing simultaneously from the sample and the reference cells.
The incident light splits and is directed towards both the reference and sample cuvette. The refracted or transmitted beam is detected by the detectors. A double beam UV-vis spectrophotometer needs two detectors that detect electron ratio to measure or calculate absorbance in a test sample. It also requires a stabilized voltage supply.
Applications of UV-Vis Spectroscopy
- UV-visible spectroscopy is used to identify organic and inorganic species present in a solution.
- It is used to find the concentration of the unknown solution.
- For the determination of structure along with other data such as bands and intensities of functional groups uv-visible spectroscopy is used.
- It is also used in the study of chemical kinetics i.e. disappearance of one functional group and appearance of another functional group.
- UV-visible spectroscopy is used to study isomers, i.e. in geometric isomerism, the trans-species absorb a high wavelength with a large molar absorptivity ‘ε’ value than the cis-species.
- It helps in the detection or presence of conjugation.
- UV-vis spectroscopy is used in clinics and hospitals for drug analysis.
- It is used in petrochemical industries.
- UV-visible spectroscopy is used in water quality control labs.
- It is used in forensic labs.
- Another important application of UV-visible spectroscopy is in the field of chemical and biological plants.
- For qualitative analysis maximum absorption is used and for quantitative analysis, Lambert beer’s law is used.
What is a UV-Vis spectrophotometer?
Uv-vis spectrophotometer is an instrument in which the radiation from ultraviolet and visible regions interacts with the sample solution. The electrons get excited when absorbing some of the incident radiation and transmitting the other. This transmitted radiation is detected, amplified, recorded, and displayed on a readout device in the form of percent transmittance.
What information can be obtained from UV-Vis spectra?
UV-visible spectra explain the radiation absorbance region of a compound. When UV-visible light strikes with the matter, some of the light is absorbed within the molecules of the compound while the rest will be transmitted through it. This transmitted light shows how much light is being absorbed by the sample (absent from the refracted radiation).
What is the principle of UV visible spectroscopy?
The basic principle of UV-visible spectroscopy is based on the absorption of light (ranges from -nm) by different chemical compounds. It is the interaction of ultraviolet and visible light with matter. Every chemical compound has a particular or distinct spectrum as it only absorbs a specific wavelength of light (radiation).
How is beer Lambert law used in spectroscopy?
Lambert beer’s law is used to determine the transmittance ‘T’ and absorbance ‘A’ of a solution in a transparent cell with path length ‘ℓ’. According to this law, the concentration of the absorbing analyte is directly proportional to the absorbance and path length of the cell.
A = – log T = log Po / P = ε . c . ℓ
What is UV visible spectroscopy used for?
UV-visible spectroscopy is used to identify functional groups, water analysis, and measure an analyte’s concentration using Lambert Beer’s law. It is also used for quantitative measurements of DNA, RNA, and proteins in the sample solution. UV visible is widely used in forensic, chemicals, environmental, and clinical laboratories. UV-visible spectrometer is used as a detector for various analytical techniques i.e. chromatography.
What are the applications of UV-visible spectroscopy?
UV-visible spectroscopy has various applications in different fields, such as clinical analysis, food industries, petrochemical industries, forensic laboratories, research, educational laboratories, pharmaceutical industries, and quality control purposes. It is also used to detect and identify different organic and inorganic species, functional groups, isomeric studies, and the presence of conjugation, etc.
Why is quartz cuvette used in UV-visible spectroscopy?
Fused silica and quartz cuvettes are most commonly used in ultraviolet spectroscopy as they are transparent in the ultraviolet region i.e. quartz can not absorb ultraviolet light so are used in ultraviolet spectrophotometers. Plastic and glass materials absorb ultraviolet light which interferes with the results.
Which detectors are used in UV visible spectroscopy?
The most commonly used detector in UV visible spectroscopy is a photomultiplier tube. Repetition of the dynode is structured with a slight potential difference at a particular angle. The incoming photon strikes the cathode, after knocking out several electrons from the dynodes every time.
Which lamp is used in UV-vis spectroscopy?
Hydrogen & deuterium lamps (ranging 160nm-380nm) with a tungsten halogen lamp (ranging 240nm-2500nm) are commonly used for UV-vis spectroscopy.
What is the range of UV Visible Spectroscopy?
The range of UV-vis is from 180nm to 780nm. The ultraviolet region comprises 180nm-400nm while the visible region extends from 400nm-780nm.
How do you read the UV-VIS spectroscopy spectrum?
To read the UV-vis spectrum the graph is plotted between the wavelength and the absorption. The wavelength at which maximum absorption occurs is called the λmax.
What is maximum absorbance?
The characteristic wavelength at which a chemical species shows the maximum absorption is called maximum absorption. It is represented by λmax.
What are chromophores?
Chromophores are atoms or groups of atoms that are responsible for the absorption of incident radiation (UV-visible radiation mainly).
What is the application of UV visible spectroscopy in natural products?
Uv-vis spectroscopy is used in the analysis of many natural products. It is used to identify organic and inorganic species present in a solution. It is also used for quantitative measurements of different components in natural compounds, for example, to find the concentration of vitamin C in orange juice, etc.
What are the advantages and disadvantages of UV visible spectroscopy?
UV-Vis spectroscopy gives accurate results. The instrument is easy to use and handle. Besides this, it is a time-consuming technique, its preparation is difficult and effort is required because external light and small vibration can cause interference with results.
Why is methanol a good solvent for IR and UV spectroscopy?
Methanol is transparent in both ultraviolet and visible regions. It has a cutoff point below these two regions hence it cannot cause any interference in results. So, it is a good solvent for UV and IR spectroscopies.
Why are most absorption bands in the visible UV spectra very broad?
Many electronic transitions occur by various molecules, each of which slightly differs from one another. A large number of closely spaced lines are difficult for the instrument to detect or resolve individually hence it gives a broad spectrum.
What is the use of a UV-Vis spectrophotometer?
UV-Vis spectrophotometer is used for the quantitative determination and measurement of different analytes i.e. organic, inorganic, biological molecules, etc.
Why the UV spectrum is broader than the IR spectrum?
In the ultraviolet spectrum, changes in all the energy levels (i.e. rotational, vibrational, and electronic energy levels) are observed while in IR only vibrational energy levels are observed. So, a UV spectrum is broader than IR one.
What is the difference between an absorbance, emission, and excitation spectrum for UV visible spectroscopy?
The main difference between absorbance, excitation, and the emission spectrum is the type of radiation understudy to analyze the respective chemical compounds. In absorption spectra, the light being absorbed is studied with transmitted light. That transmitted light shows the absorbed or missing light as the difference in the blank and sample transmittance. While in the emission spectrum, such as fluorescence spectrum the fluorescence intensity is taken as a function of excitation wavelength. The energy that is first absorbed by the electron of an atom is emitted back. As the energy is quantized so electrons that excite, gain a certain amount of energy and release the same when going back to their original state.
Why is ethanol a good solvent for UV measurement but not for IR?
Ethanol is good for UV measurement because its cutoff point is 210 nm which is near the lower limits of UV (190 nm- 400nm) so it does not interfere with the results of ultraviolet regions. Whereas, ethanol has a dipole moment, which means it is IR active, so it interferes with the results.
What is the difference between colorimetry and spectrophotometry?
Colorimetry is only used for colored compounds while spectrophotometry is used for various compounds either colored or colorless. Spectrophotometry comprises a wide range of wavelengths i.e ultraviolet, visible, and IR.
Why UV absorption value goes negative?
The negative value of absorption indicates that the sample is having an impurity in it, which causes interference with the result. The fluorescence caused by the impurity can enhance the value of transmitted radiation as compared to incident radiation. That is the reason it gives a negative absorption value.
Why are solid forms of the sample not suitable for UV vis spectroscopy?
For UV-vis spectroscopy, the analyte must be in solution form because the interaction of radiation is effective this way. The light interacts with all the molecules of the analyte in solution form and there are very low chances for losses as in solid form.
What is the difference between zero and baseline in UV Vis spectroscopy?
The zero in UV spectroscopy indicates the total transmittance while baseline is the amount of radiation absorbed by the cuvette and the sample solution.
- Introduction to spectroscopy by Pavia, Lampman, Kriz, Vyvyan Fourth Edition
- Modern analytical chemistry by David Harvey
- Analytical chemistry instrumental techniques by Mahinder Singh.