2 Introduction of Spectrometric Analyses The study how the chemical compound interacts with different wavelenghts in a given region of electromagnetic radiation is called spectroscopy or spectrochemical analysis.The collection of measurements signals (absorbance) of the compound as a function of electromagnetic radiation is called a spectrum.
3 Energy Absorption The mechanism of absorption energy is different in the Ultraviolet, Infrared, and Nuclear magneticresonance regions. However, the fundamentalprocess is the absorption of certain amount of energy.The energy required for the transition from a state of lower energy to a state of higher energy is directlyrelated to the frequency of electromagnetic radiationthat causes the transition.
4 Wave Number (cycles/cm) Spectral Distribution of Radiant EnergyWave Number (cycles/cm)X-RayUVVisibleIRMicrowave200nm400nm800nmWavelength (nm)
5 Electromagnetic Radiation V = Wave Number (cm-1)l = Wave LengthC = Velocity of Radiation (constant) = 3 x 1010 cm/sec.u = Frequency of Radiation (cycles/sec)The energy of photon:h (Planck's constant) = 6.62 x (Ergsec)C = u
7 Spectral Properties, Application and Interactions of Electromagnetic Radiation TypeRadiationTypespectroscopyTypeQuantum TransitionEnergyWaveNumber VWavelengthλFrequencyυKcal/moleVcm-1cmHz9.4 x 1074.9 x 1063.3 x 10103 x 10-1110219.4 x 1034.9 x 1023.3 x 1063 x 10-710179.4 x 1014.9 x 1003.3 x 1043 x 10-510159.4 x 10-14.9 x 10-23.3 x 1023 x 10-310139.4 x 10-34.9 x 10-43.3 x 1003 x 10-110119.4 x 10-74.9 x 10-83.3 x 10-43 x 103107Gamma rayGamma ray emissionNuclearX-ray absorption, emissionElectronic (inner shell)X-rayUltra violetUV absorptionElectronic (outer shell)VisibleInfraredIR absorptionMolecular vibrationMolecular rotationMicro-waveMicrowave absorptionMagnetically induced spin statesNuclear magnetic resonanceRadio
10 Dispersion of Polymagnetic Light with a Prism Prism - Spray out the spectrum and choose the certain wavelength(l) that you want by slit.
11 Ultra Violet Spectrometry The absorption of ultraviolet radiation by molecules is dependent upon the electronic structure of the molecule.So the ultraviolet spectrum is called electronic spectrum.
12 Electronic Excitation The absorption of light energy by organic compounds in the visible and ultraviolet region involves the promotion of electrons in , , and n-orbitals from the ground state to higher energy states. This is also called energy transition. These higher energy states are molecular orbitals called antibonding.
14 Electronic Molecular Energy Levels The higher energy transitions ( *) occur a shorter wavelength and the low energy transitions (*, n *) occur at longer wavelength.
15 Chromophore is a functional group which absorbs a characteristic ultraviolet or visible region. UV210 nm Double Bonds233 nm Conjugated Diene268 nm Conjugated Triene315 nm Conjugated Tetraene
16 SpectrophotometerAn instrument which can measure the absorbance of a sample at any wavelength.
17 FluorometerInstrument to measures the intensity of fluorescent light emitted by a sample exposed to UV light under specific conditions.Emit fluorescent lightAntibondingas energy decreasess'p'Antibondingn->s'n->p'nNonbondingp ->pGround state'pBondingEnergys ->s'sBondingElectron's molecular energy levelsUV Light SourceDetectorMonochromatorMonochromator90CSample
19 Chromophore is a functional group which absorbs a characteristic ultraviolet or visible region. UV210 nm Double Bonds233 nm Conjugated Diene268 nm Conjugated Triene315 nm Conjugated Tetraene
20 Beer – Lambert LawAs the cell thickness increases, the transmitted intensity of light of I decreases.
21 R- TransmittanceR = I0 - Original light intensityI- Transmitted light intensity% Transmittance = 100 xAbsorbance (A) = Log= Log = 2 - Log%TLog is proportional to C (concentration of solution) and is also proportional to L (length of light path through the solution).II0II01TI0III0
22 A CL = ECL by definition and it is called the Beer - Lambert Law. A = ECLE = Molar Extinction Coefficient ---- Extinction Coefficient of a solution containing 1g molecule of solute per 1 liter of solution
23 UNITS A = ECLA = No unit (numerical number only)
25 Steps in Developing a Spectrometric Analytical Method Run the sample for spectrum2. Obtain a monochromatic wavelength for the maximum absorption wavelength.3. Calculate the concentration of your sample using Beer Lambert Equation: A = ECL2.0Absorbance0.0200250300350400450Wavelength (nm)
27 There is some A vs. C where graph is linear. xnm1.0xA at 2800.5x12345Concentration (mg/ml)There is some A vs. C where graph is linear.NEVER extrapolate beyond point known where becomes non-linear.
28 Spectrometric Analysis Using Standard Curve 1.20.8A at 540 nm0.41234Concentration (g/l) glucoseAvoid very high or low absorbencies when drawing a standard curve. The best results are obtained with 0.1 < A < 1. Plot the Absorbance vs. Concentration to get a straight line
36 Practice Examples1. Calculate the Molar Extinction Coefficient E at 351 nm for aquocobalamin in 0.1 M phosphate buffer. pH = 7.0 from the following data which were obtained in 1 Cm cell.Solution C x 105 M Io IAB2. The molar extinction coefficient (E) of compound riboflavin is 3 x 103 Liter/Cm x Mole. If the absorbance reading (A) at 350 nm is 0.9 using a cell of 1 Cm, what is the concentration of compound riboflavin in sample?
37 3. The concentration of compound Y was 2 x 10-4 moles/liter and 3. The concentration of compound Y was 2 x 10-4 moles/liter and the absorption of the solution at 300 nm using 1 Cm quartz cell was What is the molar extinction coefficient of compound Y?4. Calculate the molar extinction coefficient E at 351 nm for aquocobalamin in 0.1 M phosphate buffer. pH =7.0 from the following data which were obtained in 1 Cm cell.Solution C x 105 M I0 IA
38 Spectroscopy Homework A substance absorbs at 600 nm and 4000 nm. What type of energy transition most likely accounts for each of these absorption processes?2. Complete the following table. [X](M) Absorbance Transmittance(%) E(L/mole-cm) L(cm)2.5 x4.0 x2.0 x [X](M) = Concentration in Mole/L
39 3. The molar absorptivity of a pigment (molecular weight 300) is 30,000 at 550 nm. What is the absorptivity in L/g-cm. 4. The iron complex of o-phenanthroline (Molecular weight 236) has molar absorptivity of 10,000 at 525 nm. If the absorbance of 0.01 is the lowest detectable signal, what concentration in part per million can be detected in a 1-cm cell?