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Jitender Kumar Experiment No-710001033

UV SPECTROPHOTOMETER

(UV Spectrophotometer-SHIMADZU UV-1800, DOUBLE BEAM DOUBLE DETECTOR CONFIGURATION)

Aim: To find out the concentration of the given sample using UV spectrophotometer.

Apparatus: UV Spectrophotometer Distilled water Potassium permanganate(KMnO4) solution Cells (couvettes) and Pipette, Beaker, Burette

Procedure:

1. Switch on the UV Spectrophotometer by operating the switch given at the bottom. Wait for fewminutes till the system becomes ready.

2. Select Photometric mode and wait for few minutes.3. Set the required wavelength by assigning an appropriate value of wave length (for KMnO4 set

the wavelength of 450-550 nm).

4. Take the couvette of Reference cell. Hold it carefully in such a way that you dont touc h theplain surface of the couvette. Wash it with distilled water. And then fill it (approximately 90% ofits volume) with the distilled water and keep it back in its position. DO NOT TOUCH THEREFERENCE CELL COUVETTE TILL THE EXPERIMENT IS OVER.

5. Take th e couvette of Sample cell and repeat step 4.6. Press AUTOZERO and wait. Note down the Absorptivity (Ab s). Now you are done with your

Initial process and instrument is ready for testing the samples.7. Take out the couvette from Sample cell and carefully fill it with KMnO4 of known concentration

and note down the Absorptivity (Abs). Repeat the same for 4-5 known concentration of KMnO 4. 8. Plot the graph of concentration vs absorbance(Abs)

Theory:Absorption of photons in the ultraviolet/visible range (UV/VIS) is a result of excitation of ground statevalence or bonding electrons into higher energy orbitals. UV/VIS spectrophotometry takes advantageof these electronic transitions to identify and quantify chemical substances. Chemical identification ispossible by recording a spectrum and matching peak location and shape with known spectra.When light is irradiated on a substance, the light of certain wavelengths is absorbed according to themolecule structure of that substance. This happens as the result of the fact that the electrons existingat the ground state of the molecule absorb light energy and a transition to excitation state occurs. The

amount of absorption differs depending on wavelength, and so the absorption spectrum (the curvemeasuring absorption when monochromatic light is irradiated to a substance with varyingwavelengths) becomes unique to that substance.

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Jitender Kumar Experiment No-710001033

D2: Deuterium lamp; WI: Halogen lamp; F: Filter; GR: Grating; S1: Entrance slit; S2: Exit slit; W: Window PlateL.M.: Light-source switching mirror; M1~M5: Mirrors; B.S.: Beam splitter; L: Lens; Sam: Sample cell; Ref: Reference cell

P.D.: Photodiodes

A spectrophotometer is an instrument for measuring spectra, i.e., for determining absorbance as afunction of wavelength. It does so by determining the fraction of an incident beam of light that istransmitted by a sample at each wavelength. In general, spectrophotometer consists of the basiccomponents shown in Figure 1. A source, which may be a tungsten/halogen lamp (WI) or Deuteriumlamp (D2) or both radiates light over a broad range of wavelengths. This light is reflected by mirror

(LM) and enters monochromator. The monochromator slit aperture (S1, S2) is fixed at 1nm. Then thelight passes through various mirrors and stray light cutter filter (F).The light that passes through filterreaches beam splitter (B.S) and gets split into the sample side beam and reference side beam whichthen passes through their respective cells and strike the detectors-photodiodes (P.D). The initialintensity is abbreviated as Io If the sample absorbs light of that wavelength, the intensity leaving the sample or is I a fraction of what went in. A detector, such as a photodiode, measures I and sends aproportional signal to a device which indicates how much light is being transmitted. Or it can be saidthat the signal tells how much light has been absorbed. Various mirrors and lenses are used to directthe light through these various components so the instruments can be made quite compact.

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Jitender Kumar Experiment No-710001033

Calculation Table:

Obs No. Conc of KMnO4(c)(M)

Absorption (Abs)(nm)

Molar extinctioncoefficient

()mol -1 cm -1

1 0.00068 2.42 14702 0.00034 1.167 29403 0.00017 0.705 58804 8.5E-05 0.35 11760

RESULT:

Average molar extinction coefficient for wave length 450-550 nm ( ) for the given sample ofconcentrations = 5512.5 Mol -1cm -1 Concentration of the unknown sample (Actual) c5 = 0.0125 g/lConcentration of the unknown sample (from calibration curve/graph) = 0.012401 g/lConcentration of the unknown sample (shown on UV-Spec screen) = 0.0125 g/l

Sample Calculation:For 1 st set of readingUnknown concentration from calibration curve (c5)As we know that absorptivity and concentration has linear relationship so we get a linear equationi.e.y = 11.66x + 0.0674 ; where y = absorptivity and

x = concentrationSo for given unknown concentration sample y = 0.212

Unknown concentration c5 = (0.212-0.0674)/11.66C5 = 0.012401 g/l

Abs = cL

And hence,=Abs/(c .L)Since we know that in our case l = 1 cmSo,=Abs/(c ) = 2.42/0.00068 = 1470

Conclusion:It is clear from the calibration curve that Concentration is directly proportional to the absorptivity sobeer lambert law is verified.Concentration of unknown sample from calibration curve (0.0124 g/l) is approximately equal to theconcentration from UV spectrometer (0.0125 g/l).