Applications of UVVis Spectroscopy 1 The Molar Absorptivity

Applications of UV-Vis Spectroscopy 1

The Molar Absorptivity As seen earlier: validation of Beer’s law is dependent on the nature of the molar absorptivity. Factors affect the molar absorptivity: 1. The wavelength of radiation. 2. The refractive index and is thus indirectly related to concentration. 3. Electrostatic interactions taking place in solution; and thus electronic distribution. 4. In rare cases, like methylene blue, the molar absorptivity is directly dependent on concentration. 2

For Beer’s law to be valid: The molar absorptivity is supposed to be constant. The molar absorptivity is a measure of the ability of an analyte to absorb light at a specified wavelength. Therefore: The value of the molar absorptivity is crucial for the sensitivity of an analysis. Molar absorptivity usual range: From zero (for a nonabsorbing species) to 105 (for highly absorbing species). For quantitative analysis: A value of at least 103 is necessary for a reasonable quantitative analytical determination of an analyte. 3

Absorbing Molecular Species v What happened if an incident beam of radiation having a suitable wavelength hits a molecule: Ø Absorption of a photon takes place and the molecule becomes excited. Ø Excited molecules will lose excitation energy as heat or photons (luminescence). v Absorption of UV-Vis radiation is capable of affecting the excitation of bonding electrons and other valence electrons. v Types of electrons that affected by excitation of electrons in chemical by bonds absorption of UV-Vis radiation of a suitable wavelength. (1) , and n electrons (mostly organics) (2) d and f electrons (inorganics/organometallics) 4 (3) charge-transfer (CT) electrons

Molecular UV-Vis Spectroscopy: Theory · Molecular energy levels and absorbance wavelength: v * and * transitions: high-energy, accessible in vacuum UV ( max <150 nm). Not usually observed in molecular UV-Vis. vn * and * transitions: non-bonding electrons (lone pairs), wavelength ( max) in the 150 -250 nm region. vn * and * transitions: v most common transitions observed in organic molecular UV-Vis, observed in compounds with lone pairs and multiple bonds with max = 200 -600 nm.

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Molecules Containing , , and n Electrons 1. Molecules with Bonds Only Let us start with a molecule like CH 4 and then expand our discussion to more complex molecules: Ø All bonds in methane are bonds. ØThe only transition possible is the - * transition. Ø However, the - * transition requires very high energy which occurs in vacuum UV (125 -185 nm). 7

It is not wise to think of doing UV measurements on molecular species in the vacuum UV region (125 -185 nm) for five important reasons: 1) The high energy required can cause rupture of the bonds and breakdown of the molecule. 2) Air components absorb in vacuum UV which limits the application of the method. 3) Working in vacuum UV requires special training and precautions which limit wide application of the method. 4) Special sources and detectors, other than those described earlier, must be used. 5) All solvents contain bonds. 8

2. Molecules with n Electrons in the valence shell that are not used up in chemical bonds are referred to as nonbonding electrons (n electrons). Consider a molecule like ammonia: The dots on nitrogen is a symbol for two nonbonding electrons. Now, the type of transitions observed in this molecule can be listed as: - * n- * We have seen earlier that the - * transition is not useful in practical UV-Vis spectroscopy. The other transition (n- *) is of lower energy and should be further discussed. 9

The absorption wavelength for a n- *transition occurs at about 185 nm. Unfortunately, most solvents absorb at this region. For example: The most important solvent is, undoubtedly, water: Has two pairs of nonbonding electrons that will strongly absorb as a result of the n-s *transitions. This precludes the use of this transition for studies in aqueous and other solvents with nonbonding electrons. In summary: It is also impractical to think of using UV-Vis absorption spectroscopy to determine analytes based on a n- * transition. 10

3. Molecules with Bonds. Absorption of radiation by an alkene, containing a double bond: Can result in - * or - * transitions. We have seen that a - * transition is not useful. The - * is useful since: ØIt requires reasonable energy. ØHas good absorptivity. A molecule having , , and n electrons can show all types of transitions possible in UV-Vis spectroscopy. For example: The aldehyde molecule shows all these transitions since it contains , , and two pairs of n electrons. Two transitions are possible for the n electrons: 11 n- *

n- * transition: It is not very useful due to absorbance from solvents and other frequently used additives which have n electrons. n- * transition: Requires very little energy and seem to be potentially useful. However, unfortunately, the molar absorptivity of this transition is very small which precludes its use for sensitive quantitative analysis. 12

Effect of Solvent Polarity on Absorption Wavelength The molar absorptivity for a n- * transition: Ø Rather small (10 -100 L mol-1 cm-1) Ø Energy required for transition is affected by solvent polarity. Effect of presence of a polar solvent on n- * : Ø Nonbonding electrons will interact with protic solvents to form hydrogen bonds. Ø The solvation of n electrons is the result. So: Ø lowers the energy of the orbitals holding the n electrons. Ø Partial stabilization of the polar * is also observed but to a much lower extent than the n electrons. 13

Overall Conclusion (hypsochromic shift or blue shift): Ø A net increase in energy required for a n- * transition is thus observed in protic solvents; like water or alcohols. Ø Therefore, an increase in energy will reflect a decrease in transition wavelength. 14

Effect of presence of a polar solvent on - * : Ø The - * transition is affected in an opposite manner with solvent polarity. Ø In presence of a polar solvent, the more polar * orbital will be more stabilized than the orbital leading to a net decrease in the transition energy. Overall Conclusion: (bathochromic shift (red shift)): This results in an increase in transition wavelength. 15

Spectral nomenclature of shifts 16

Molecular UV-Vis Spectroscopy: Terminology · UV-Vis Terminology – Chromophore: a UV-Visible absorbing functional group – Bathochromic shift (red shift): to longer wavelengths – Auxochrome: a substituent on a chromophore that causes a red shift – Hypsochromic shift (blue shift): to shorter wavelengths – Hyperchromic shift: to greater absorbance – Hypochromic shift: to lesser absorbance

Conclusions on Electronic Transitions A - * and a n- * transition : are not useful for reasons discussed earlier. The n- * transition: Ø Requires low energy but the molar absorptivity is also low and transition energy will increase in presence of polar solvents. Ø Is seldom used in quantitative UV-Vis spectroscopy. The - * transition: The most frequently used transition is for the following reasons: 1. The molar absorptivity for the - * transition is high allowing sensitive determinations. 2. The energy required is moderate, far less than dissociation energy. 3. In presence of the most convenient solvent (water), the energy required for a - * transition is usually smaller. It is therefore primitive that an analyte to be determined by UVVis absorption spectroscopy be of unsaturated nature (or!!). 18

Organic Chromophores: Molecules having unsaturated bonds or free nonbonding electrons that can absorb radiation of relatively low energy are called chromophores. Examples: Include alkenes, alkynes, ketones, aldehydes, phenyl and other aromatic species, etc. Auxochromes: Is a functional group that does not absorb by itself (like -NH 2 and –OH), but its presence in a molecule can increase absorption and usually results in a red-shift. 19

The human eye sees the complementary color to that which is absorbed 20

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Chromophore Example Excitation λmax , nm ε Solvent C=C Ethene π g π* 171 15, 000 hexane C≡C 1 -Hexyne π g π* 180 10, 000 hexane C=O Ethanal n g π* π g π* 290 180 15 10, 000 hexane C-X X=Br X=I Methyl bromide Methyl Iodide n g σ* 205 255 200 360 hexane 22

Effect of Conjugation of Chromophores: Ø As conjugation is increased in a molecule, more delocalization (stability) of the electrons results. Ø The effect of this delocalization is to decrease the * molecular orbital. Ø The result is a decrease in transition energy from - * and thus a red or bathochromic shift. Ø The molar absorptivity will increase in this case and better quantitative analysis will be achieved. Ø In cases of introduction of more unconjugated double bonds, the molar absorptivity will increase as well depending on the number of the double bonds. For example: Ø At 185 nm, 1 -hexene has a molar absorptivity of about 10, 000 L mol-1 cm-1 Ø But hexa-1, 4 -diene has a molar absorptivity of twice as much as 1 -hexene. Ø However, when the double bonds are conjugated as in hexa 1, 3 -diene the molar absorptivity is about 21, 000 L mol-1 cm-1. 23

Rule of thumb for conjugation If greater then one single bond apart: -e are relatively additive (hyperchromic shift) - constant CH 3 CH 2 CH=CH 2 max= 184 emax = ~10, 000 CH 2=CHCH 2 CH=CH 2 max=185 emax = ~20, 000 If conjugated - shifts to higher ’s (red shift) H 2 C=CHCH=CH 2 max=217 emax = ~21, 000 24

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Effect of Aromaticity of Chromophores Aaromaticity results in extraordinarily high degree of delocalization of electrons and thus stabilization of the *. Ø If we assume a molar absorptivity of about 10, 000 L mol-1 cm-1 for each double bond. Ø We expect the sum of the three double bonds in benzene to be just above 30, 000 L mol-1 cm-1 (at 185 nm). Ø But actually the value is about 60, 000 L mol-1 cm-1 due to increased delocalization as a result of aromaticity. It is therefore advantageous to use UV-Vis absorption spectroscopy for determination of compounds having aromatic character. 26

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Absorption by Inorganic Groups Inorganic groups containing double bonds absorb in the UV-Vis region. The most transitions are a result of n- * transitions: ØIn nitrate (313 nm), ØCarbonate (217 nm), ØNitrite (280 and 360 nm) ØAzide (230 nm) 28

Absorption Involving d and f Orbitals: Ø Many transition metals have colored solutions and are also colored in the solid state. Ø The transition metals have some of their d orbitals empty where a d-d transition can occur. Ø The d-d transitions require excitation energy in the UV-Vis region. Ø The direct interaction of the d electrons with ligands around the transition metal results in a spectrum of broad band nature. Ø On the other hand, inner transition elements show transitions by absorption of UV-Vis radiation (f-f transitions). Ø Since the electrons in the f orbitals are far inside the metal orbitals and are screened by electrons in orbitals of higher principal quantum numbers, f-f electronic transitions will not be affected by the nature of ligands or solvent around the inner transition metals. Ø Therefore, the spectra of inner transition metals have narrow bands. 29

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The crystal field theory: Ø Usually used to explain splitting of the d orbital energy so that a transition from a lower energy d orbital electron can be excited to a higher energy d orbital. Ø The theory will be described for a transition metal with six ligands or molecules of water around it. Ø An octahedral (only this case will be discussed) arrangement of these ligands is most appropriate where ligands will be located at the z axis and at the x and y axis (will repel electronic cloud in the dz 2 and dx-2 y 2 orbitals and thus will make these two orbitals to have higher energies). Ø The other four ligands will be arranged in between axis) dxy , dxz , and dyz (which will increase their energies but to a lower extent. Ø The net result is the splitting of the degenerate d orbital into two groups of d orbitals of different energies (the energy difference is referred to as D). 31

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Transition metal ions 33

Charge Transfer Absorption: Ø When a ligand permanently donates an electron to a metal ion, a charge transfer is said to take place. Ø The net outcome of the process is an oxidation reduction phenomenon occurring within the complex. Ø An example is the reaction of Fe 3+ with thiocyanate where the product is an excited species with neutral thiocyanate and Fe 3+. Ø In less common situations, the transfer of electrons can take place from the electron deficient metal ion to the ligand. Ø An example is the Fe 2+ or Cu+ complexes with 1, 10 phenanthroline where Fe 2+ and Cu+ metal ions donate electrons to 1, 10 -phenanthroline. Ø The complex will then have Fe 3+ and Cu 2+ ions. Charge transfer complexes are of special interest their molar absorptivities are unusually high; allowing very sensitive determinations. 34

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Applications of Absorbance Measurement to Qualitative Analysis As seen earlier, the broad band absorption spectra obtained in UV-Vis absorption spectroscopy is usually featureless and lacks details that can be used in qualitative analysis. Therefore, this technique is mainly a quantitative technique. Plotting Spectral Data A plot of either the absorbance or %transmittance against wavelength can be made. However, the most common practice is to plot the absorbance versus wavelength. 36

Solvents Ø Solvent polarity affects the absorptivity of the analyte molecules: ØDue to change in transition energies. ØUsually, polar solvents are used when possible. ØHowever, polar solvents like water or alcohol tend to remove the fine spectral details. ØSo, a non polar solvent like hexane should be used: Ø To obtain the fine spectral details which really needed (as in qualitative analysis). Addition conditions of solvents: 1. The solvent must be optically clear (does not absorb incident radiation). 2. Well dissolve the sample. 3. Chemically pure. 37

38 Cut off wavelength

Quantitative Analysis The basis for quantitative analysis in the UV-Vis relies on Beer’s law. Several characteristics of quantitative measurements using UV-Vis absorption spectroscopy can be rationalized: 1. Applicability to all types of analytes as far as they absorb in the UV-Vis region. 2. Moderate sensitivities in the range from 10 -4 to 10 -6 with possibility to extend this range under certain conditions. 3. The relative standard deviation occurs within 1 -3% which reflects good precision. 4. Easy to perform and convenient. 5. Can be used for quantitative analysis in liquid chromatographic separations (as detector). 6. Non absorbing species can also be determined if they are derivatized with an absorbing species as the case of metal ions when complexed to ligands. 39

Procedural Details 1. Selection of Wavelength The first step in a successful determination is to find the suitable wavelength for the analysis. This is accomplished by plotting the absorbance/wavelength curve. However, the following points should also be considered: 1. If more than an absorption maximum is available, the wavelength far from the instrument extremes should be preferred 2. A wavelength at the maximum of a broad peak should be preferred to another of a sharp peak 3. The peak with a maximum peak height is preferred 4. If interferences are present, the wavelength that is far away from interferences should be selected 5. Working in the visible region should be preferred 40

2. Cleaning and Handling the Cell. Ø First, one should appreciate the use of good quality matched cells that are free from wearing, etching, and scratches. Ø In addition, cleaning procedures of external and internal cell surfaces are also important. Ø A suggested cleaning procedure involves moistening a lens paper with methanol and wiping the external surface, then leaving the cell to evaporate. Ø The interior of the cell is first washed with water followed by methanol and the solvent is also allowed to evaporate. Ø Disposable polypropylene cuvettes are incompatible with non polar solvents and formulations having these solvents should be avoided; or large errors will be encountered. 41

3. Calibration Curves. Ø Usually, a plot of the absorbance of a series of standards is plotted versus the concentration. Ø The absorbance of the unknown is then determined and the prepared calibration plot is used for the determination of the analyte concentration. Ø If the absorbance of the analyte was located outside the calibration plot, more standars should be made or the analyte concentration must be adjusted to occur on the calibration plot. Ø We have seen earlier that it is not allowed to theoretically extrapolate or interpolate a calibration plot. Ø It should also be appreciated that the composition of standard solutions must approximate that of the sample solution. Ø In cases where the sample composition is not clear, the method of standard addition should be used. Ø The slope of the linear calibration plot is the molar absorptivity when the path length is 1. 00 cm. Larger slopes mean higher sensitivities. 42

4. Standard Addition method Ø The use of this method provides better correlations between absorbances of standards and sample due to constant matrix effects. Ø The method involves addition of the sample volume to a set of tubes or containers. Ø Variable volumes of a standard are added to the tube set followed by completion to a specific volume. Ø Now, all tubes contain the same amount of sample but different concentrations of analyte. Ø A plot is then made for the volume of standard and absorbance. Ø This plot will have an intercept (b) with the y axis and a slope equals m. Ø The concentration of the analyte can be determined by the relation: Cx = b. Cs/m. Vx Where, Cx and Vx are concentration and volume of analyte and Cs is the concentration of standard. 43

Conc. Of Un (if Ab. is taken versus conc. of standard 44 Cx = b. Cs/m. Vx

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Two points calibrations (Using two solutions): One can only use two points to get the analyte concentration using the relation: Cx = Ax. Cs. Vs/(At –Ax)Vx Where: Ax is the absorbance of the analyte solution brought to a final volume Vt. At is the total absorbance of the solution (final volume is Vt) containing same amount of analyte and a volume of the standard (Vs). Ø However, this procedure assumes that both points occur at the linear portion of the calibration plot. Ø In case where this is not true, an error will be encountered. 46

5. Analysis of Mixtures of Absorbing Substances When the sample solution contains more than one absorbing species, the absorbance of the solution will be the sum of all absorbances: At = A 1 + A 2 + A 3 + …. The different constituents can be determined if we build equations equal to the number of unknowns. However, this procedure, if manually performed, is impractical due to lengthy and difficult math involved especially when more than two components are present. Remember that we need to calculate n 2 values for e where n is the number of components. For two unknowns y and X: (4 e values are needed) A ’ = ex’bcx + ey’bcy (1) A ” = ex”bcx + ey”bcy (2) ex’, ex”, ey’, ey” can be determined from standards of analytes x and y at ’, ” and values obtained are inserted in equations 1 and 2 where two equations in two unknowns can be easily solved. 47

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6. Derivative UV-Vis Absorption Spectroscopy In this technique: Ø DA/D is plotted against the wavelength. Ø The derivative spectrum contains sharper peaks where better location of peaks and wavelengths maxima can be achieved. Ø Not only the first derivative of the absorbance spectrum can be obtained but up to the fourth derivative is possible. Ø However, as the differentiation order increases, the noise increases as well and if a lower derivative is fine, going to higher derivatives is a waste of time and effort. Ø Derivative spectroscopy is excellent for determination of multi components in a sample, if they can be resolved. 49

Directly proprtional to conc. max 50

y 51 X

Zero order spectra 52

1 st derivetive: height related to conc. 53

Obtaining a Derivative Spectrum Ø Derivative spectra can either be recorded on time or by manipulation of data obtained in an absorbance/wavelength spectrum. Ø The later is easy and can be made by simple electronic or mathematic operations; by taking DA for a fixed D (few nm) and plotting the data versus wavelength. Ø However, the most common on line instrumental recording of derivative spectra involves the use of a dual wavelength instrument with two monochromators operated at the same speed but with a lag of few nm from each other (by Δ ). Ø A chopper will sequentially pass the beams from both monochromators and thus their difference divided by the constant nm lag value is recorded versus the average wavelength. Ø The following schematic is helpful to visualize how this is accomplished: 54

55 at 1 gives A 1 at 2 gives A 2 =( 2 - 1) is constant, A = (A 2 -A 1) so deriv. = A/

Applications of Derivative Spectra 1. Better qualitative analysis and identification of the number of absorbing species in a sample 2. Accurate determination of max 3. Obtaining spectra in solutions with high scattering was possible using dual wavelength instruments 4. Spectral resolution of multi component systems by measurement at two wavelengths; where the interferent has identical molar absorptivity while the analyte does not, can result in good exclusion of interferences. 56

Photometric Titrations ØIn cases where an analyte reacts with a reagent. ØSo that the analyte, the reagent or the product absorbs UV-Vis radiation. ØThe technique can be used for determination of the analyte by a photometric titration reaction. Ø Photometric titrations are similar to conventional visual titrations but following the course of a photometric titration occurs with the aid of a UV-Vis detector, rather than the naked eye. 57

Photometric Titration Curves: Ø A plot of absorbance versus titrant volume is called a photometric titration curve. Ø The titration curve is supposed to consist of two linear lines intersecting in a point corresponding to the end point of the reaction. Ø The absorbance reading should be corrected for volume where as a titrant is added to the reaction mixture the absorbance will change. Ø Several shapes of photometric titration curves can be identified depending on the reaction. 58

Instrumentation v. A conventional photometer or spectrophotometer can be adapted to performing photometric titrations where the analyte is placed in the sample cell which contains a small magnet and is located on the top of a magnetic stirrer. v. The wavelength is selected and the titrant is added, from a dark burette, gradually and the absorbance is recorded. 59

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Advantages and Applications of Photometric Titrations 1. Usually, photometric titrations are more accurate than visual titrations. 2. Photometric titrations are faster than visual titrations as only few points at the beginning and end of the titration is necessary. Extrapolation of the straight lines will intersect at the end point. 3. Titration reactions that are slow at the end point can not be performed by visual titrations but are well suited for photometric titrations. Only few points at the beginning and end of the titration, well away from the equivalence point where the reaction is slow, are necessary. Extrapolation of the straight lines will intersect at the end point. Therefore, dilute solutions or weak asids and bases can be also titrated photometrically. 4. Mixtures can be easily titrated using photometric titrations if absorbance is measured at the absorption wavelength of one of the two analytes. In addition, if both analytes give colored products but with different molar absorptivities, they can be determined photometrically. 63

For example: ØFigure a below shows the EDTA titration of Bi+3 and Cu+3 at the wavelength of the Cu-EDTA where the Bi-EDTA does not absorb. ØFigure b shows a hypothetical reaction where two products absorb incident radiation. ØThe first substance (x) reacts first and followed by the second one which has a lower molar absorptivity. 64

Photoacoustic Spectroscopy (PAS) Ø UV-Vis spectra of solid, semisolid, and turbid samples can be obtained by photoacoustic spectroscopy, although PAS is more important in infrared absorption spectroscopy. Ø The technique of photoacoustic spectroscopy involves subjecting the sample in a photoacoustic cell (containing a gas) to a chopped beam of radiation. Ø If the sample absorbs the incident wavelength, as the chopper rotates, the sample emits the absorbed energy as heat. Ø Periodic absorption and reemission causes periodic fluctuations in the gas temperature and thus pressure. Ø If the chopper rotates in a rate that occurs in the acoustical range, the fluctuations in pressure can be detected by a sensitive microphone. 65

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Applications of Photoacoustic Spectroscopy 1. Recording UV-Vis spectra of solids and turbid solutions. Samples as complex as whole blood were tested by PAS. 2. Study of the absorption characteristics of polymers, paints archeological samples. 3. Spectra of samples on surfaces, like silica thin layer chromatographic plates, can be recorded. 3. In the IR region, qualitative analysis is possible to a variety of samples. 67