RAFFLES INSTITUTION (Junior College) H3 CHEMISTRY 2009/2010 Lecture Notes – Analytical Techniques (I

)
(Basic Principles of Spectroscopy & UV/Vis Spectroscopy)
Prepared by: Ms Ng Xinyi

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A Lecture Outline 1. Analytical techniques in synthesis and drug development 2. Basic principles of spectroscopy 2.1 Electromagnetic spectrum 2.2 Energy and energy levels 2.3 Types of spectroscopy 3. UV/Vis Spectroscopy 3.1 Instrumentation 3.2 Molecular orbital theory 3.3 Analysis of UV/Vis spectra 3.3.1 Nature of electronic transitions 3.3.2 Chromophores and conjugation 3.3.3 Quantitative analysis using UV/vis spectroscopy: Beer-Lambert’s Law Learning outcomes Candidates should be able to: a) recognize the importance of analytical techniques in drug development, for example, in isolation of drugs, and in the determination of drug structure and purity b) understand qualitatively the range of wavelengths (frequencies, energies) with different types of radiation used in spectroscopy c) understand the concept of quantized energy levels [quantal formulae and selection rules are not required] d) explain that ultraviolet/visible absorption in organic molecules requires electronic transitions between energy levels in chromophores which contain a double or triple bond, a delocalized system or a lone pair of electrons [detailed theory of why chromophores have absorptions of appropriate energy is not required, nor is any consideration of molecular orbitals involved] e) predict whether a given organic molecule will absorb in the ultraviolet/visible region f) explain qualitatively how increasing conjugation in an organic molecule decreases the gap between energy levels and hence shifts the absorption towards longer wavelength g) use Beer’s Law, absorbance = lg(I0/I) = εcl, where ε is taken merely as a constant characteristic of the substance concerned, to calculate the concentration of a given species in solution h) apply uv/visible spectroscopy to quantitative analysis of a given species in solution

B

C

References & Websites 1. Introduction to Spectroscopy by Pavia, Lampman and Kinz 2. Thinking Straight about Pharmaceutical Chemistry by Wee et. al. 3. http://www.chemistryexplained.com/Ny-Pi/PharmaceuticalChemistry.html#ixzz0VJLCyCYW- What is Pharmaceutical chemistry about? 4. http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm#uv1 – A concise guide to the various aspects of UV/vis spectroscopy

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chemists are able to design new drugs with enhanced potency and decreased adverse side effects. schools of pharmacy trained pharmacists and physicians how to prepare medicinal remedies from natural organic products or inorganic materials. Greek. Pharmaceutical chemists are involved in the development and assessment of therapeutic compounds. energies) with different types of radiation used in spectroscopy understand the concept of quantized energy levels [quantal formulae and selection rules are not required] What is spectroscopy? Spectroscopy is the study of the _____________________________________________________. Roman.1. Basic Principles of Spectroscopy Candidates should be able to understand qualitatively the range of wavelengths (frequencies. 2. and Asian societies were administered without any knowledge of their biological mechanism of action. Impurities can also be detected using thin layer chromatography. a study of the various spectroscopic and separation techniques would be necessary in drug development to determine drug structure and purity. So how do we know if a compound that has been synthesized is the correct compound? How do we ensure that there are no impurities present in the compound synthesized? How can we tell the structure of an unknown compound that has poisoned many people? We can answer these questions by making use of various analytical techniques to elucidate the structure of the compound by using nuclear magnetic resonance (NMR) spectroscopy or check if there is a change in the functional group by using infra-red spectroscopy. To fully identify the structure of an unknown compound. The result of this interaction is an exchange or transfer of energy between the two. we can deduce the structure of the unknown compound. 2 . This absorption/emission of energy therefore enables us to detect these structural features. Drug development and synthesis is the core of pharmaceutical chemistry. and in the determination of drug structure and purity Prior to the nineteenth century. Analytical techniques in synthesis and drug development Candidates should be able to recognize the importance of analytical techniques in drug development. No single spectroscopic method can determine exactly the structure of a large compound by itself. Certain structural features/properties of compounds will undergo an absorption or emission of energy in the form of EM radiation. for example. This transfer of energy may involve either an _______________ or ______________ of energy in the form of EM radiation. Herbal medications and folk remedies dating back to ancient Egyptian. purification is needed and a combination of data by analyzing the purified compound using different spectroscopic methods is required. It was not until the early 1800s that scientists began extracting chemicals from plants with purported therapeutic properties to isolate the active components and identify them. By piecing together various features that different probes reveal. in isolation of drugs. Hence. By discovering and structurally characterizing compounds with medicinal activity.

that have no mass. microwave.2. Since E α f. the visible region we perceive as light. The behavior of electromagnetic (EM) radiation depends on its wavelength. the speed of light (3 x 108m s-1). X-rays and gamma rays. h. and the constant of proportionality. They can thus be considered as packets of energy. but have associated with them a quantity of energy due to wave particle duality. h = 6. Relationship between photon energy and its frequency This simple relationship was first put forward by Max Planck. is called Planck’s constant. f = frequency in Hertz. All electromagnetic radiation travels at a constant speed.62 x 10-34 J The frequency and the wavelength of a particular radiation are related by an equation involving the speed of light. and the amount of energy associated with each photon is proportional to its frequency. c. [2] Combining [1] & [2] gives us the equation: [3] 3 . Photons are particles with considerable wave-like properties. ultraviolet. Higher frequencies have _________________ wavelengths. infrared. and is composed of photons.1 Electromagnetic spectrum The spectrum of electromagnetic radiation is classified by wavelength into radio. [1] where E = energy in Joules. and lower frequencies have ___________________ wavelengths.

31 x 10-19 J (ii) Calculate the energy per mole of photons of yellow light. In order for a molecule to acquire more of any of these forms of energy. E = hc/λ = (6. it has to absorb a packet of energy of a particular size.02 x 1023 = 200 kJ mol-1 Energy-per-mole-of-photons Area of spectrum Average frequency. which is determined by the size of the gap between the energy levels concerned. Transitions between energy states are therefore detected by spectroscopy. so no absorption takes place 4 .02 J mol-1 X-ray UV IR microwave radio waves 2. The particle that absorbed the EM radiation is promoted to a higher energy state(excited state). Energy difference between the ground state and 1st excited state is E1. E = 3.e. f/Hz 1 x 1017 1 x 1015 1 x 1013 1 x 1010 1 x 108 Average wavelength. we multiply this value by the Avogadro constant. To convert energy per photon into energy per mole of photons.2 Energy and energy Levels What happens when EM energy is absorbed? All energy is _________________. The promotion of electron requires a photon energy hf1 (electronic transition). i. the more energy is associated with it. λ/m 3 x 10-9 3 x 10-7 3 x 10-5 3 x 10-2 3 Average energy per mole 20.31 x 10-19 x 6. requiring photon energy hf1 so that E1=hf1 If a photon of lower energy(and frequency) hf2 irradiates the particle.000 kJ mol-1 200 kJ mol-1 2 kJ mol-1 2 J mol-1 0. the energy is insufficient to bring about a transition.62 x 10-34 x 3 x 108)/(600 x 10-9) ∴ E = 3.Thus we can see that the higher the frequency or the shorter the wavelength of the photon. Worked Example 1 (i) Calculate the energy of a photon of yellow light with a wavelength of 600 nm. Energy difference between the ground state and 1st excited state is E1. a particle such as a molecule or atom can only associate with a ___________________ amount and this energy is determined by quantum rules.

Molecule interacts with photons in the infrared region (λ ≈ 5 x 10-6m or 5000nm) molecule changing from one vibrational energy level to another. This is associated with the movement of electrons between electronic energy levels (or orbitals) in the molecule.What are the various types of energy a molecule can have? Restricting ourselves to molecules for the moment. 5 .10mm) molecule changing from one rotational energy level to another. 3. 1. vibrational energy . Energy level electronic energy levels vibrational energy levels Energy gap Several hundreds of kJ mol-1 About 22 kJ mol-1 rotational energy levels Less than 1 kJ mol-1 Molecule interacts with UV/vis light (λ=100nm . Molecule interacts with photons in the far-infrared to microwave region (λ= 0.700nm) an electron is likely to be changing from one electronic energy level to another. This is associated with the regular periodic stretching-and-contracting and the bending of bonds within the molecule.1mm . 2. electronic energy. nuclear magnetic energy. rotational energy. we can identify various types of energy associated with particles. 4. The following diagram illustrates the different energy levels a molecule can have. This is associated with the energy of interaction of a magnetic field with those nuclei that have magnetic moments. This is associated with the speed of rotation of the molecule around various axes.

The ultraviolet (UV) region scanned is normally from 200 to 400 nm. we will only be looking at 3 different areas of EM spectrum absorbed by molecules and what knowledge of structures of molecules we can gain from the frequencies of absorption. As a beam of radiation is absorbed in its passage through a sample. Infra-red (IR) spectroscopy • Transmittance against frequency (or wavenumber) spectra 3. 3. and the visible portion is from 400 to 800 nm.1 Instrumentation A diagram of the components of a typical spectrometer is shown in the above diagram. as we “sweep” a range of frequencies. I = intensity of emerging beam Io = intensity of incident beam T = 100 x (I/Io) T = transmittance The absorbance of a sample is defined as the negative logarithm of (I/Io) ratio The relationship between absorbance and transmittance is illustrated in the following table.2. UV/Vis spectroscopy UV-visible spectroscopy is the study of the ________________ of ultraviolet and visible radiation by molecules. the spectrometer automatically scans all the component wavelengths in the manner described. Nuclear Magnetic Resonance (NMR) spectroscopy • Absorbance against change of frequency in parts per million (δ) 3. 3 types of spectroscopy 1. Ultraviolet/visible (UV/vis) spectroscopy • Absorbance against wavelength (λ) spectra 2. The __________________ is defined as the ratio of the intensities of the emerging beam and the incident beam.3 Types of spectroscopy For our syllabus. expressed as a percentage. 6 . Absorption spectroscopy involves the measurement of how much radiation is absorbed at each frequency. Over a short period of time. the intensity of radiation decreases.

as shown in the figure above. if we have a molecular that is involved in π bonding. Electrons are added to molecular orbitals. Example: H2 molecule One of the molecular orbitals in this molecule is constructed by adding the mathematical functions for the two 1s atomic orbitals that come together to form this molecule. the H2 molecule is more stable than a pair of isolated atoms. As a result.2 Molecular orbital theory (Not in syllabus) There is a second major theory of chemical bonding whose basic ideas are distinct from those employed in valence bond theory. Example: O2 molecule Similarly. one at a time. or sigma star ( *). This function reflects the probability of finding an electron in any specific region. starting with the lowest energy molecular orbital. One of these orbitals is called a bonding molecular orbital because electrons in this orbital spend most of their time in the region directly between the two nuclei. molecular orbital. or bonding. This diagram suggests that the energy of an H2 molecule is lower than that of a pair of isolated atoms. Figure : Molecular orbital of O2 molecule 7 . in accordance with the Pauli Exclusion Principle. Another orbital is formed by subtracting one of these functions from the other. The Schrödinger wave equation is a mathematical function to study the wave-like behavior of an electron in a molecule. Electrons placed in the other orbital spend most of their time away from the region between the two nuclei. which was found to provide the key to the understanding of the electronic structure of atoms. The theory applies the orbital concept. molecular orbital. It is called a sigma ( ) molecular orbital.3. a π and π* orbital is created. Molecular orbitals are obtained by combining the atomic orbitals on the atoms in the molecule. This orbital is therefore an antibonding. The total number of molecular orbitals is always equal to the total number of atomic orbitals contributed by the atoms. The two electrons associated with a pair of hydrogen atoms are placed in the lowest energy. to molecular systems. as shown in the figure below. This alternative approach to the study of the electronic structure of molecules is called molecular orbital theory.

The molecular orbitals in molecules are of three types. an electron is excited from an occupied orbital of a lower energy (LUMO) to an unoccupied orbital of a high energy (HOMO). respectively. Recall that a lower energy gap requires lower frequency/longer wavelength of EM radiation. Hence. or in the non-bonding orbitals if they are lone pairs on atoms such as nitrogen or oxygen. In methanol (formaldehyde) CH2=O. i. Electronic energy levels and transitions Possible transitions in some organic molecule In the above electronic transitions between energy levels. Specifically. only molecules with n or π electrons give characteristic UV/vis spectra. nor is any consideration of molecular orbitals involved] explain qualitatively how increasing conjugation in an organic molecule decreases the gap between energy levels and hence shifts the absorption towards longer wavelength predict whether a given organic molecule will absorb in the ultraviolet/visible region 3. the two electronic transitions absorbing UV radiation are shown below: σ π ∗ ∗ λ = 300 nm λ = 190 nm n π σ 8 . When a photon is absorbed by a molecule an electron gains the photon’s energy and is promoted to a higher energy orbital. The electrons are normally resident in the bonding orbitals. bonding (σ or π).3 Analysis of UV/vis spectra Candidates should be able to explain that ultraviolet/visible absorption in organic molecules requires electronic transitions between energy levels in chromophores which contain a double or triple bond. The highest occupied molecular orbital and lowest unoccupied molecular orbital are often referred to as the HOMO and LUMO. The higher energy orbital to which they are promoted is almost invariably an anti-bonding orbital.3. the energy involved in each transition decreases from left to right.3. non-bonding (n) and anti-bonding (σ* or π*).e.1 Nature of electronic transitions UV/vis spectroscopy involves the excitation of a particle in an electronic ground state to an electronic excited state. transitions of π → π*. n → π* and n → σ* normally produce absorption in the UV/visible region. a delocalized system or a lone pair of electrons [detailed theory of why chromophores have absorptions of appropriate energy is not required.

Even with a mixture of compounds.3. Different chromophores absorb strongly at different wavelengths. A typical UV spectrum is shown in on the left. the UV spectrometer could selectively detect the presence of a particular compound. The three absorbances are due to three different electronic transitions (n → π* or π → π*). but it is not possible to allocate a specific transition to each wavelength.3.2 Chromophores and conjugation A system containing the electrons responsible for the absorption of radiation is called a _______________________. 9 . if its optimum UV absorption wavelength is sufficiently different from that of the other compounds.

434 nm (20) values in brackets after the wavelengths are the molar extinction coefficients (see text) As can be seen from the above table. R C R R CH CH CH CH R O Absorption wavelengths 190 nm (>10. 281 nm (400).000) (benzene) 184 nm (60. The position of the UV/VIS absorption is therefore inadequate in the identification of compounds.500) O O (quinone) 242 nm (24. many different chromophores absorb at nearly the same wavelength.000). 203 nm (7400). 254 nm (204) O O 300 nm (5000) HN N O NH 2 NH 2 (cytosine) 267 nm (6130) N N (adenine) N N H 260 nm (13. 10 . 300 nm (30) 230 nm (15.000) R CH CH CH O 230 nm (12.000).The following table summarises the wavelengths at which some chromophores absorb radiation.000).

relative to propanone.This chromophores system is either an isolated bond. with which diphenylmethanone will absorb energy. Predict and explain the wavelength. shown below. since violet absorbed) N N Worked example 2 Diphenylmethanone. or more usually a conjugated system of double bonds. 11 . we observe beta carotene to have a red-orange colour Effect of lone pair on absorbance The incorporation of an atom with a lone pair of electrons can cause a major shift of absorbance to ____________________ wavelengths. highly conjugated π systems will appear coloured. since the n → π * energy gap is smaller than the π → π * gap. absorbs in the UV/vis region. and hence shifts the absorption towards longer wavelength: π → π* energy gap/kJ mol-1 R-CH=CH-R 690 RCH=CH-CH=CH-R 520 R-CH=CH-CH=CH-CH=CH-R 235 λ/nm 190 230 275 Note that with increased conjugation. Effect of increasing conjugation on the energy gap between π to π* More examples of conjugated systems: 1. Absorption λ/ nm C H C H 296nm (colourless) 400nm (orange. weaker absorption at 200 nm and a group of much weaker bands at 254 nm region due to various π to π* transitions 2. Since conjugated π systems will reduce energy gap and absorb at longer wavelengths. so lower energy) Since blue light is absorbed. β-carotene has a highly conjugated π electron system absorbs visible blue light at 480nm (lower frequency than UV. Benzene absorbs far UV light very strongly at 180nm. there is more than one possible π π* transition. Conjugation of π bonds reduces the energy gap between the π and the π* orbitals. hence multiple absorption bands will be observed for the UV spectrum of a conjugated system.

H H3C H3C CH2 C OH H2 O H3 C C H Cl 3. so l = 1. 2 different samples of the same concentration have different absorbance at a given wavelength (provided both compounds absorb at this wavelength). Circle the molecule(s) which will show more than one absorption peak in the UV/vis region.0 cm) The molar extinction coefficient (units: _____________________) is how much light of a particular wavelength is absorbed by 1. absorbance = lg(I0/I) = εcl. 12 .e.3. to calculate the concentration of a given species in solution apply uv/visible spectroscopy to quantitative analysis of a given species in solution Beyond detection of the presence of a particular compound. Hence.Worked example 3 Study each of the molecules drawn below. how well a compound absorbs at that wavelength. It is important to note that molar absorptivity is wavelength and sample specific.3 Quantitative analysis using UV/vis spectroscopy: Beer-Lambert’s Law Candidates should be able to use Beer’s Law.0 cm of a 1 mol dm–3 solution of a particular chemical i. the main use of UV/VIS spectroscopy is to analyse quantitatively how much of a particular compound is present in a solution. This makes use of Beer’s Law: A = log10(Io/I) = εcl A = absorbance Io = intensity of incident radiation I = intensity of emerging radiation ε = the molar extinction coefficient c = concentration of compound (in mol dm-3) l = path length of the absorbing solution (in cm) (the cells used in spectrophotometry are usually 1 cm2 in cross section. where ε is taken merely as a constant characteristic of the substance concerned.

Step 5. Step 6. What is the concentration of the solution? ** Do note that the deviations from Beer’s law can arise and the application of Beer’s law to a mixture containing 2 independent components is further elaborated in Appendix.How do we carry out a quantitative analysis using Beer’s Law? Step 1. of the compound of interest Using a UV/VIS spectrophotometer (capable of measuring absorbance highly accurately at a particular wavelength). Prepare standard solutions of the compound in question. Plot a calibration curve of absorbance against concentration. Step 4. to cover the range of concentrations expected. Worked Example 4 A solution of quinone (ε242nm = 24 000) had an absorbance of 0. Step 3. run these prepared solutions at the same wavelength to record their absorbances. Read the concentration of the compound in question from the calibration curve. Run the UV/VIS spectrum of the compound in question. eg. Place the sample of unknown concentration in the spectrophotometer. 13 . If the molar extinction coefficient.0 cm cell at 242 nm.574 in a 1. Step 7. Step 2. is known. and measure the absorbance. standard solutions 1 – 4 each contain a different known conc. and of other compounds that may be present in the mixture. Choose a wavelength where there is a large absorption of the compound in question but only a small absorption of the other compounds present.ε. the concentration can be calculated directly from the absorbance using the Beer’s Law equation.

analyte concentration is so high such that it causes a change in the refractive index of the medium and affects the ability of molecules to absorb radiation 3.502 at 475nm and (b) 0. εX and εY are known at both wavelengths. cX = 2. These 2 components behave independently of one another and obey Beer’s Law.X = 8990 mol dm-3 cm-1 ….25) cY – [1] – [2] Solving by simultaneous equations. for same unit path length l. Worked Example When measured with a 1.912 at 700nm.50 x 10-5 mol dm-3 solution of species X gave absorbances of 0. where absorbance at any wavelength.X (8. Under the same circumstances.794 at 475 and 700nm respectively.25)cX + (1780)(1. At λ = 475nm.X = 1520 mol dm-3 cm-1 At λ = 700nm.X (8.50 x 10-5)(1. is. For species X.25)cX + (12200)(1.00) ε475. we make use of the following standard addition method.X ε475. 8.34 x 10-5 mol dm-3 and cX = 7.64 x 10-5 mol dm-3 14 .Appendix More on Beer’s Law… Deviations from Beer’s law may occur under the following circumstances: 1.65 x 10-5 mol dm-3 solution of species Y gave absorbances of 0.Y first. complex formation or polymerization of species resulting in the conversion of one chemical species to another causes change in measured absorbances 2. 0.912 = (8990)(1. 0. ε700. ε475. 0.Y ε700.50 x 10-5)(1.X ε700.567 and 0. consider a mixture in solution containing two components (X & Y) at concentrations cX and cY.25cm cell: (a) 0. chemical factors such as dissociation. Calculate the concentration of X and Y in the solutions that yielded the following absorbance data in a 1.00cm cell. We need to calculate ε475.25) cY At λ = 700nm.Y = 1780 mol dm-3 cm-1 At λ = 475nm.00) ε700.764 = ε700. 0.129 and 0. 4. given by A(λ) = εX(λ)cXl + εY(λ)cYl Therefore.0863 at 475 and 700nm respectively. stray light striking the detector decreases measured absorbances For a more complicated example.129 = ε475. association. detected radiation is not monochromatic but polychromatic radiation instead 4. measurements at two wavelengths yield two equations in two unknowns and will suffice to determine the concentrations cX and cY as long as the molar absorbances of the two components.Y = 12200 mol dm-3 cm-1. λ.502 = (1520)(1. To make quantitative determination of the individual constituents of the mixture.. For species Y.

ii.950 1.56 at 275 nm.0 24.630 0. The iron(II)-1. Calculate the range of acetone concentrations that can be used if the transmittance is to be greater than 10.15 nor greater than 0.0 i.0 32. A510 0. A 1. What is the molar extinction coefficient of guanine at this wavelength? 3) The molar absorptivity for the complex formed between bismuth(III) and thiourea is 9.00 10.470.0% with a 1.580 Sketch a calibration curve from these data. what experimental variables must be controlled to assure reproducible absorbance data? 2) A solution of guanine of concentration 2 x 10-4 mol dm-3 in a 1.75 at 366nm.390 0.10-phenanthroline complex was developed and the solution diluted to 50.160 0.260 1.0 mL of water.00 cm path length cell has an absorbance of 1.00-cm cells) were recorded at 510 nm: Original Fe(II) conc. 5) A standard solution was put through appropriate dilutions to give the concentrations of iron shown below.0 40.0 mL. The absorbance of this solution at 510nm in 1. after which iron(II)-1.0 16.10-phenanthroline complex was then developed in 25. 4) The logarithm of the molar absorptivity of acetone in ethanol is 2. Determine the percentage mass of iron in the food product.0% and less than 90.00-cm cell was found to be 1. The following absorbances (1.00-cm cells. ppm 4.50-cm cell.80 when the measurements are made in 1. Calculate the range of permissible concentration for the complex if the absorbance is to be no less than 0.0 g sample of a food product was crushed and extracted with 25.32 x 103 L cm─1 mol─1 at 470 nm.0 mL aliquots of these solutions.0 mL. following which each was diluted to 50.RAFFLES INSTITUTION (Junior College) H3 CHEMISTRY 2009/2010 Tutorial 1: UV/vis Spectroscopy Prepared by: Ms Ng Xinyi ________________________________________________________________________ 1) In UV spectroscopy. 15 .

a) Suggest suitable wavelengths to use for the estimation of the concentrations of each of these two drugs in solution.85 at 245nm and 0.6) The UV spectra of paracetamol and caffeine are shown in figures 2. A solution made up of such a pill had measured absorbances of 0.31. 16 .30 and 2. Peak 245nm in paracetamol spectrum 275nm in caffeine spectrum Molar Extinction coefficient (ε) 15 100 18 200 c) *(Refer to appendix) Paracetamol pills sometimes have small quantities of caffeine added in order to speed up the rate of absorption of the drug through the gut wall. calculate the concentration of each drug in the solution used for its spectrum. b) Given the following molar extinction coefficient for relevant peaks.45 at 275nm. Calculate the ratio of the molar amounts of paracetamol and caffeine in the pill.

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