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UV-VISIBLE SPECTROSCOPYGroup Members:Sidra Rasheed Asma MuhammadRaheel Qamar ButtKamran HaiderMuzammil ZaidiContentsIntroduction and principleQuantitative measurment and electronic TransitionsInstrumentation and the experimentApplicationsLimitations What is Spectroscopy??Spectroscopyis the use of the absorption, emission, or scattering of electromagnetic radiation by matter to qualitatively or quantitatively study the matter or to study physical processes. The matter can be atoms, molecules, atomic or molecular ions, or solids.Types of spectroscopyEmission Spectroscopy: A method ofchemical analysisthat uses the intensity of light emitted from a flame or spark at a particular wavelength to determine the quantity of element in a sample.

What will the audience be able to do after this training is complete? Briefly describe each objective how the audience will benefit from this presentation.4Absorption spectroscopyItis the analytical technique based on measuring the amount of light absorbed by a sample at a given wavelength.1. Atomic absorption Spectroscopy2. Molecular Absorption SpectroscopyUV-Visible SpectroscopyIt is type of Molecular Absorption Spectroscopy in which UV and Visible Radiations are used for quantitative and qualitative analysis of compounds especially conjugated Pi systems.The wavelength range is from 200nm to almost 800nm.

Electromagnetic RadiationViolet400 - 420 nmYellow570 - 585 nmIndigo420 - 440 nmOrange585 - 620 nmBlue440 - 490 nmRed620 - 780 nmGreen490 - 570 nm

What will the audience be able to do after this training is complete? Briefly describe each objective how the audience will benefit from this presentation.7

Interaction with MatterInteraction with matterWhen Electromagnetic Radiations Interact with molecules, it imparts energy to the electrons, which get excited.Electrons will only be excited if the wavelength of incoming photon is comparable to the energy gap between two energy levels.Interaction of EMR with matterElectronic Energy Levels:At room temperature the molecules are in the lowest energy levels E0.

E = 35 to 71 kcal/mole

The absorption of energy excites the electron from the ground state to an electronic excited state What will the audience be able to do after this training is complete? Briefly describe each objective how the audience will benefit from this presentation.11Interaction of EMR with matterVibrational Energy Levels:A given electronic energy level has a number of vibrational energy levels in it.

The spacing between energy levels are relatively small i.e. 0.01 to 10 kcal/mole.What will the audience be able to do after this training is complete? Briefly describe each objective how the audience will benefit from this presentation.12Interaction of EMR with matterRotational Energy Levels:Each of the vibrational energy level has a number of rotational energy levels in it.

The spacing between energy levels are even smaller than vibrational energy levels.

Erotational < Evibrational < EelectronicWhat will the audience be able to do after this training is complete? Briefly describe each objective how the audience will benefit from this presentation.13Physics Behind the color of a material When white falls on a material, some portion of light is absorbed.The other portion is reflected back and this is what we see as the color of that substance.The excited state of an electron is unstable.It has a lifetime of about 10^-8sWhen electron gets de-excited it emits radiation(reflection) of specific frequency(color) in the visible Range.

Quantitative Analysis

Lamberts LawWhen a monochromatic radiation is passed through a solution, the decrease in the intensity of radiation with thickness of the solution is directly proportional to the intensity of the incident light.

Let I be the intensity of incident radiation.x be the thickness of the solution.

Give a brief overview of the presentation. Describe the major focus of the presentation and why it is important.Introduce each of the major topics.To provide a road map for the audience, you can repeat this Overview slide throughout the presentation, highlighting the particular topic you will discuss next.16Beers Law Includes concentrationA=.C.LWhere; A = absorbance= absorptivity or extinction co-efficientL= the path lengthC = Concentration of the absorbing specie

Electronic Transitions

The possible electronic transitions can be graphically shown as:

This is another option for an Overview slide.

Generally the possible Electronic Transitions are:This is another option for an Overview slide.

Possible Transitions in UV-VisAllowed transitions and forbidden transitionsThus, n * & * electronic transitions show absorption in region above 200 nm which is accessible to UV-visible spectrophotometer.

Instrumentation

UV-Visible SpectrometerSpectrometer consists of following parts:Radiation sourceMonochromatorSample containerdetectorRadiation SourceHydrogen or deuterium Lamps are used as UV radiation. The mechanism for this involves formation of an excited molecular species, which breaks up to give two atomic species and an ultraviolet photon. wavelength range 160 - 375 nmThe tungsten filament lamp is commonly employed as a source of visible light.wavelength range of 350 - 2500 nm

MonochromatorAll monochromators contain the following component parts;An entrance slitA collimating lensA dispersing device (usually a prism or a grating)A focusing lensAn exit slit

Monochromator

Cuvettes/Sample containerThe 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.

DetectorThe photomultiplier tubeis a commonly used detector in UV-Vis spectroscopy. It consists of aphoto-emissive cathode, severaldynodes(which emit several electrons for each electron striking them) and ananode.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.

Sample preparationSample must always be clear solutionsSample cell should not be touched by handsAbsorbance due to solvent should be less than 1A.The dilution Factor must be estimatedBe sure there are not air bubbles inside the cell.

The Experiment

Reference cell A measurement of the absorbance of the reference cell ( not containing the absorbing specie) is made.The exact value of the absorption of the sample is obtained by subtracting the absorbance of the reference cell from that of the whole sample.Chlorophyll

Applications of U.V. Spectroscopy:Detection of ImpuritiesUV absorptionspectroscopyis one of the bestmethodsfor determination of impurities in organic molecules. Additional peaks can be 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 clarification of organic compounds.

UVspectroscopyis useful in the structure elucidation of organic molecules,the presenceor absence of unsaturation,the presenceof 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.3.ChemicalkineticsKinetics of reaction can also be studied using UVspectroscopy. The UV radiation is passed through the reaction cell and the absorbance changes can be observed.4.Detection of Functional GroupsThis technique is used to detect the presence or absence of functional group in the compound

Absence of a band at particular wavelength regarded as an evidence for absence of particular group

5. Quantitative analysis of pharmaceutical substancesMany drugs are either in the form of raw material or in the form of formulation. They can be assayed by making a suitablesolutionof the drug in a solvent and measuring the absorbance at specific wavelength.

Diazepam tablet can be analyzed by 0.5% H2SO4 in methanol at the wavelength 284 nm.LIMITATIONS ON UV-VIS SPECTROSCOPYThere are three types of deviations from usual behaviourReal Deviations These are fundamental deviations due to the limitations of the law itself.Chemical Deviations These are deviations observed due to specific chemical species of the sample which is being analyzed.Instrument Deviations These are deviations which occur due to how the absorbance measurements are made.

Real Limitation and Deviation of Beer-Lambert Law

Beer law and Lambert law is capable of describing absorption behavior of solutions containing relatively low amounts of solutes dissolved in it (10mM), the analyte begins to behave differently due to interactions with the solvent and other solute molecules and at times even due to hydrogen bonding interactions.At high concentrations, solute molecules can cause different charge distribution on their neighboring species in the solution. Since UV-visible absorption is an electronic phenomenon, high concentrations would possibly result in a shift in the absorption wavelength of the analyte. At times, even electrolyte concentrations (such as those present in buffers) play an important role in altering the charge distributions and affecting UV-visible absorbance. Some large ions or molecules show deviations even at very low concentrations. For e.g. methylene blue absorptivity at 436 nm fails to observe Beer Lambert law even at concentrations as low as 10M.High analyte concentrations can also possibly alter the refractive index () of the solution which in turn could affect the absorbance obtained. If the addition of solute causes a significant change in the refractive index of the solution a correction to the Beer Lambert formula can be placed as:A = bc (2+ 2)2

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

Instrumental Deviations and Limitations to Beer-Lambert Law

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

TESEKKUR EDIRIM (Thank you very much)