UV-VISIBLE SPECTROSCOPY

*SPECTROSCOPYPRESENTED BY : ANJALI RARICHAN , 1st year MPHARM

*SPECTROSCOPY is the measurement and interpretation of Electro Magnetic Radiation (EMR) absorbed or emitted when the molecules or atoms or ions of a sample move from one energy state to another energy state.

*Electromagnetic Radiation

*This radiation may be regarded as energy propagated in a wave form. These radiations travel in space at enormous velocity. It consist of both electrical and magnetic vectors and hence it is named as electromagnetic radiation.

* Frequency (ν):It is defined as the number of wavespassing through a point per second.The unit for frequency is Hertz (Hz).

Wavelength (λ):It is the distance between the two successive peaks of waves, i.e. distance between two nearest crest or troughs.It is denoted by “lambda (λ).

*ELECTROMAGNETIC SPECTRUM* The arrangement of all types of electromagnetic radiations in order of their increasing wavelengths or decreasing frequencies is known as complete electromagnetic spectrum.

*PRINCIPLE OF UV- VISIBLE SPECTROSCOPY

*VISIBLE SPECTROSCOPY Colorimetry is concerned with the study of Absorption of visible radiation whose wavelength ranges from 400nm-800nm. Any Coloured substance will absorb radiation in this wavelength region. Coloured substances, absorb light of different wavelength in different manner and hence we get an absorption curve in a unique pattern for every coloured solution.

*UV SPECTROSCOPY*Ultraviolet spectroscopy is concerned with the study of absorption of UV radiation which ranges from 200 to 400nm. Compounds which are coloured, absorb radiation from 400-800nm. But compounds which are colourless absorb radiation in the UV region. In both UV as well as visible spectroscopy, only the valence electrons absorb the energy, thereby the molecule undergo transition from Ground state to excited state. Thus absorption is characteristic and depends on the nature of electrons present. The intensity of absorption depends on the concentration and path length.

*ABSORPTION CURVE AND CALIBRATION CURVE

*Coloured substances , absorb light of different wavelength in different manner and hence we get an absorption curve(absorbance vs wavelength) in a unique pattern for every coloured solution. In this absorption curve, the wavelength at which maximum absorption of radiation takes place is called as λmax. It is characteristic or unique for every coloured substance and this is a qualitative aspect, useful in identifying the substance.

* When we plot a graph of concentration Vs absorbance , we get a Calibration curve or Standard curve. This calibration curve is useful in determining the concentration or amount of a drug substance in the given sample solution or a formulation, by extrapolation or intrapolation and calculation.

Electronic Transitions

*The possible electronic transitions can graphically shown as:

• σ → σ* transition1• π → π* transition2• n → σ* transition3• n → π* transition4• σ → π* transition5• π → σ* transition6

* The possible electronic transitions are

• σ electron from orbital is excited to corresponding anti-bonding orbital σ*.

• The energy required is large for this transition.

• e.g. Methane (CH4) has C-H bond only and can undergo σ → σ* transition and shows absorbance maxima at 125 nm.

• σ → σ* transition1

• π electron in a bonding orbital is excited to corresponding anti-bonding orbital π*.

• Compounds containing multiple bonds like alkenes, alkynes, carbonyl, nitriles, aromatic compounds, etc undergo π → π* transitions.

• e.g. Alkenes generally absorb in the region 170 to 205 nm.

• π → π* transition2

• Saturated compounds containing atoms with lone pair of electrons like O, N, S and halogens are capable of n → σ* transition.

• These transitions usually requires less energy than σ → σ* transitions.

• The number of organic functional groups with n → σ* peaks in UV region is small (150 – 250 nm).

• n → σ* transition3

• An electron from non-bonding orbital is promoted to anti-bonding π* orbital.

• Compounds containing double bond involving hetero atoms (C=O, C≡N, N=O) undergo such transitions.

• n → π* transitions require minimum energy and show absorption at longer wavelength around 300 nm.

• n → π* transition4

•These electronic transitions are forbidden transitions & are only theoretically possible.•Thus, n → π* & π → π* electronic transitions show absorption in region above 200 nm which is accessible to UV-visible spectrophotometer.•The UV spectrum is of only a few broad of absorption.

• σ → π* transition5• π → σ* transition 6&

*LAWS GOVERNING

ABSORPTION OF RADIATION

BEER’S LAWBeer’s law states that “The intensity of a beam of monochromatic light decreases exponentially with increase in the concentration of absorbing species arithmetically”

-dI/dC α I

I=Intensity of incident light C=concentration

The rate of decrease of intensity of monochromatic light with the thickness of medium is directly proportional to the intensity of incident light.

i.e, -di/dt α I t=thickness or pathlength I=intensity of incident light

LAMBERT’S LAW

On combining these two laws A = Kct A = Єct A=absorbance or optical density or extinction co-efficient Є=molecular extinction co-efficientc=concentration of drugt=path length

These two laws are applicable under following conditions. I=Ia+It

• V

*COMMENT ON THERAPY