What is the need of UV spectroscopy –
1.To make research procedure easy and accurate.
2 .To determine the concentration of drug, compounds in the media.
3 .To determine impurity.
4.For quantitative analysis of compounds.
5. To determine geometrical isomer.
Some terms –
Wavelength In physics, the wavelength of a sinusoidal wave is the spatial period of the wave – the distance over which the wave's shape repeats. Wavelength is commonly designated by the Greek letter lambda (λ).
A chromophore is part (or moiety) of a molecule responsible for its color.
Auxochrome - this is a group of atoms attached to a chromophore, which modifies the ability of that chromophore to absorb light. Example: -OH , - NH2 , Aldehydes.
Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or UV/Vis) refers to absorption spectroscopy in the UV-visible spectral region(200-400 nm and 400 –700mn). This means it uses light in the visible and adjacent ranges. The absorption in the visible range directly affects the perceived color of the chemicals involved. In this region of the electromagnetic spectrum, molecules undergo electronic transitions.
Many compounds absorb ultraviolet (UV) or visible (Vis.) light. A beam of monochromatic radiation of radiant power P0, directed at a sample solution. Absorption takes place and the beam of radiation leaving the sample has radiant power P.
The amount of radiation absorbed may be measured in a number of ways:
Transmittance, T = P / P0 % Transmittance, %T = 100 T
The Beer-Lambert Law
The Beer-Lambert law states that the absorbance of a solution is directly proportional to the concentration of the absorbing species in the solution and the path length. Thus, for a fixed path length, UV/VIS spectroscopy can be used to determine the concentration of the absorber in a solution. It is necessary to know how quickly the absorbance changes with concentration. This can be taken from references, or more accurately, determined from a calibration curve.
Now let us look at the Beer-Lambert law and explore it's significance. This is important because people who use the law often don't understand it - even though the equation representing the law is so straightforward:
A=ebc
Where A is absorbance (no units, since A = log10 P0 / P ) e is the molar absorbtivity with units of L mol-1 cm-1 b is the path length of the sample - that is, the path length of the cuvette in which the sample is contained. We will express this measurement in centimetres. c is the concentration of the compound in solution, expressed in mol L-1
Electronic transitions
The absorption of UV or visible radiation corresponds to the excitation of outer electrons. There are three types of electronic transition which can be considered;
Transitions involving (, (, and n electrons
Transitions involving charge-transfer electrons
When an atom or molecule absorbs energy, electrons are promoted from their ground state to an excited state. In a molecule, the atoms can rotate and vibrate with respect to each other. These vibrations and rotations also have discrete energy levels, which can be considered as being packed on top of each electronic level.
Absorbing species containing (, (, and n electrons
Absorption of ultraviolet and visible radiation in organic molecules is restricted to certain functional groups (chromophores) that contain valence electrons of low excitation energy. The spectrum of a molecule containing these chromophores is complex. This is because the superposition of rotational and vibrational transitions on the electronic transitions gives a combination of overlapping lines. This appears as a continuous absorption band.
Possible electronic transitions of (, (, and n electrons are;
( to (* Transitions
These transitions can occur in compound in which all the electron are involve in single bonds and there are no lone pair of electron
Example –In saturated hydrocarbons without lone pairs of electron.
An electron in a bonding ( orbital is excited to the corresponding antibonding orbital. The energy required is large. For example, methane (which has only C-H bonds, and can only undergo ( to (* transitions) shows an absorbance maximum at 125 nm.
n to (* Transitions
Saturated compounds containing atoms with lone pairs (non-bonding electrons) are capable of n to (* transitions. These transitions usually need less energy than ( to (* transitions. They can be initiated by light whose wavelength is in the range 150 - 250 nm. The number of organic functional groups with n to (* peaks in the UV region is small.
. Example –Saturated alkyl halides .
n to (* and ( to (* Transitions
Most absorption spectroscopy of organic compounds is based on transitions of n or ( electrons to the (* excited state. This is because the absorption peaks for these transitions fall in an experimentally convenient region of the spectrum (200 - 700 nm). These transitions need an unsaturated group in the molecule to provide the ( electrons.
Example –Unsaturated molecule containing atoms such as oxygen ,nitrogen and sulphur.
Absorption and intensity shift
Hypsochromic shift is a change of spectral band position in the absorption, reflectance, transmittance, or emission spectrum of a molecule to a shorter wavelength (higher frequency). Because the blue color in the visible spectrum has a lower wavelength than most other colors, this effect is also commonly called a blue shift.
Bathochromic shift is a change of spectral band position in the absorption, reflectance, transmittance, or emission spectrum of a molecule to a longer wavelength (lower frequency). this effect is also commonly called a red shift.
Solvent selection criteria
A suitable solvent for ultraviolet spectroscopy should meet the following requirements –
1.It should not itself absorb radiation in the region under investigation.
2.It should be less polar so that it has minimum interaction with the solute molecule.
Ultraviolet-visible spectrophotometer
The instrument used in ultraviolet-visible spectroscopy is called a UV/vis spectrophotometer. It measures the intensity of light passing through a sample (I), and compares it to the intensity of light before it passes through the sample (Io). The ratio I / Io is called the transmittance, and is usually expressed as a percentage (%T). The absorbance, A, is based on the transmittance:
A = − log(%T / 100%)
Instrumentation -
1.Radiation source –In UV Spectrophotometer the most commonly used radiation source are Hydrogen lamp, Deuterium lamp, Xenon discharge lamp and mercury arc.
Requirement of Radiation source -
a.It must be stable
b.It must supply continuous radiation over the entire wavelength region in which it is used.
c.It must be of sufficient intensity for the transmitted energy to be detected at the end of the optical path.
Hydrogen Discharge lamp – In these lamps hydrogen gas is stored under relatively high pressure, when the electric discharge is passed through the lamp excited hydrogen molecule will be produced which emit UV radiation .
It cover the range –120nm to 350nm.
Deuterium lamp –If deuterium is used in place of hydrogen the intensity of radition emitted is 3 to 5 times the intensity of hydrogen lamp .Its more expensive then hydrogen lamp.
Xenon discharge lamp-In this lamp xenon gas is stored under pressure in the range of 10 to 30 atmospheres .It has two tungsten electrode separated by about 8mm .When a intense arc is formed between two tungsten by applying a low voltage the UV light is produced.
The intensity of Xenon discharge lamp is much greater then Hydrogen lamp
Mercury arc-In this mercury vapor is under high pressure and excitation of mercury atom is done by electric discharge.
Monochromator-A monochromator is an optical device that transmits a mechanically selectable narrow band of wavelengths of light or other radiation chosen from a wider range of wavelengths available at the input.
The essential element of a monochrometer are an enterance slit ,a despersing element and an exit slit .
Enterance slit sharply define the incoming beam of heterochromatic radiation .The dispersing element disperse the hetrochromatic radiation in to its component wavelenght whereas exit slit the nominal wavelenght together with a band of wavelength on either side of it .
The dispersing element may be a prism or grating.
Detector-There are three common type of detector which widely used in UV spectrophotometer .
1.Barrier layer cell IT consist of semiconductor such as selenium which is deposited on a stron metal base such as iron .The a very thin layer of silver or gold sputtered over the surface of semiconductor to act as a semiconductor electrode .
The radiation falling on the produces electron at the seleniun silver interface .A barrier exist between the selenium and Iron which preven the electron from flowing in to iron .the electron are therefore accumulated on the silver surface.This accumulation on the silver surface produces electrical voltage difference between the silver surface and the base of the cell .now a photo current will flow which is directly proportional to the intensity of the incident beam.
2 Photo cell –it consist of high sensitivity cathode in the form of half cylinder which is contained in a evacuated tube .The anode is also present in the tube which is fixed more or less along the axis of the tube.The inside surface of the tube is coated with the light sensitive layer.
When the light is incident on the photocell .the surface coating emits electron they are attracted and collected by the anode .The current which is created between cathode and anode is regarded as a measure of radiation falling on detector.
3 Photomultiplier tube –IT is a combination of photo diode and electron multiplying amplifier. It is a evacuated tube which contain one photo cathode and 9-16 electrodes known as dynodes .
The radiation falls on a metal surface of the photocathode, it emits electrons, the electron are attracted toward first dynode which is kept at a positive voltage. When it strikes on first dynodes more electrons are emitted by the surface of dynodes these are then taken by second dynodes and similar type of electron emission takes place .the number of electron reaching the collector is a measure of intensity of light falling on the detector.
Recording system-IT is done by recorder pen
Sample cell –the most commonly used cells are made of quartz. These are readily available in matched pair.
Power supply –Electric power supply –it smoothes out any ripple which may occur in the line voltage in order to deliver a constant voltage to the source lamp and instrument,
A spectrophotometer can be either single beam or double beam.
Single beam instrument.
In a single beam instrument all of the light passes through the sample cell. Io must be measured by removing the sample. This was the earliest design, but is still in common use in both teaching and industrial labs.
Double-beam instrument.
In a double-beam instrument, the light is split into two beams before it reaches the sample. One beam is used as the reference; the other beam passes through the sample. Some double-beam instruments have two detectors (photodiodes), and the sample and reference beam are measured at the same time. In other instruments, the two beams pass through a beam chopper, which blocks one beam at a time. The detector alternates between measuring the sample beam and the reference beam.
Application of UV Spectroscopy –
1 Application of UV Spectroscopy to organic compound -
a Detection of Conjugation .
b Detection of Geometrical isomer-Trans Isomer exhibit longer wavelength and extinction coefficient then the cis form.
Example –Stilbenes trans isomer shows wavelength 294nm and extinction coefficient 24000 while cis isomer has wavelength 278 nm and extinction coefficient 9350.
2 Detection of impurity –The band due to impurity are very intense
eg the common impurity in cyclohexane is benzene .its presence can be easily detected by its absorption at 255nm .
3 Quantitative analysis –Its based on beers law which is as follows –
A=ecl
Small e = extinction coffecient c= concentration l is the length of the cell
Extinction coffecient is constant at any given wavelength for a given species and A is absorbance.
We can determind C by putting the value of Extinction coefficient ,l and A.
4 By calibration curve we can determine the concentration of unknown sample