2 Introduction of Spectrometric Analyses The study of how the chemical compound interacts with different wavelengths 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 AbsorptionThe fundamental principle 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 THE NATURE OF LIGHTLight is composed of electric and magnetic fields, which are mutually perpendicular and which radiate out from a sourceIt is therefore a form of electromagnetic radiation.Electric and magnetic fields are propagated through space as wave functions which may be characterized by wavelength, λ (the distance from one part of the wave to the corresponding position on the next wave) and frequency, ν (the number of times a wave passes through a fixed point in space every second).
7 This relationship can be rearranged ; λ = c / ν , this relationship means that the frequency and wave length are inversely related ( higher frequency means shorter wave length).thus a red light color of wave length of 650nm and a green color of 540nm wave length , the red light has a higher wave length thus it has a lower frequency) .As for energy: the light with the highest energy will be the one with the highest frequency - that will be the one with the smallest wavelength.Light of each color has a different wavelength - blue light has a shorter wavelength than red light. Blue light therefore has a larger number of peaks per unit of length thus it has higher frequency and larger energy.
9 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.
12 Electronic Molecular Energy Levels The higher energy transitions ( *) occur a shorter wavelength and the low energy transitions (*, n *) occur at longer wavelength.
13 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
14 SpectrophotometerAn instrument which can measure the absorbance of a sample at any wavelength.
15 The essential components of spectrophotometer CollimatorCuvette sample containerLight’s bandλ1λ2λ3λ4Photometer or detectorLight sourceMonochromatorPrismPhotocellSlitWavelength selector
16 Components of a Spectrophotometer 1-Light Source Deuterium Lamps－a truly continuous spectrum in the ultraviolet region (160nm~375nm)Tungsten Filament Lamps－the most common source of visible and near infrared radiation.
17 Components of a Spectrophotometer 2-Monochromator Used as a filter: the monochromator will select a narrow portion of the spectrum (the bandpass) of a given sourceUsed in analysis: the monochromator will sequentially select for the detector to record the different components (spectrum) of any source or sample emitting light
18 Single and Double Beam Spectrometer Single-Beam: There is only one light beam or optical path from the source through to the detector.Double-Beam: The light from the source, after passing through the monochromator, is split into two separate beams-one for the sample and the other for the reference.```````````
19 Single Beam Spectrophotometer DetectorLampLenscuvette
22 The Beer-Lambert lawWhen a beam of radiation (light) passes through a substance or a solution, some of the light may be absorbed and the remainder transmitted through the sample.The ratio of the intensity of the light entering the sample (Io) to that exiting the sample (I1) at a particular wavelength is defined as the transmittance (T).z% T = (Io / I1 ) x 100 A = - log (T)
23 The Beer-Lambert lawε = molar absorptivity or extinction coefficient of the chromophore at wavelength λ (the optical density of a 1-cm thick sample of a 1 M solution). ε is a property of the material and the solvent.L = sample pathlength in centimetersc = concentration of the compound in the sample, in molarity (mol L-1)AbsorbanceA= -log(I1/I0)=ε·c·l
24 TransmittanceThe ratio of the intensities of the transmitted and incident light gives transmittance.T = I/I0I0 is the intensity of incident radiationI is the intensity of transmitted radiationA 100% value of T represents a transparent substance whereas a zero value represents a totally opaque.
25 Deviation of the Beer’s law At high concentrations many molecules form dimers or higher polymers which have a spectrum that differs than their monomeric form , which could lead to either positive or negative deviation.Also at high concentrations aggregation can occur which frequently leads to scattering of light thus decreasing the amount of light transmitted and thus causing positive deviations.Deviations in absorptive coefficients at high concentrations (>0.01M) due to electrostatic interactions between molecules in close proximity.Stray light ; it is the quantity of light that reaches the detector that is of a wavelength other than that selected. Therefore, stray light causes the measured transmittance to be high , thus leading to negative deviation
27 Factors affecting absorption properties of a chromophore; The spectrum of a chromophore is primarily determined by the chemical structure of the molecule.However a large number of environmental factors can cause detectable changes in λmax and ε these factors are ;pH ; The pH of the solvent determines the ionization state of the chromophore.The polarity of the solvent or or neighboring molecules. For polar chromophores the value of λmax is affected , for example the λmaxFor tyrosine is less in polar solvents.Orientation effects; Geometric features have strong effects on λmaxand εThe best example of such an effect is the hypochromism of nucleic acids .That is the absorption coefficient of a nucleotide decreases when the nucleotide is contained in a single-stranded polynucleotide in which the bases are in close proximity .There is a further decrease with a double stranded polynucleotide because the bases are arranged in an even more stacked array.
28 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 = ECLWavelength (nm)
29 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?
30 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?