Spectroscopic Methods the Electromagnetic Spectrum Lecture 11

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Spectroscopic methods The Electromagnetic Spectrum Lecture 11

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Frequency, wavelength, energy

Velocity of light, 3 . 108 m s-1

Wavelength (m) • Wavelength is defined as the distance between adjacent maximal values of waves and may be presented in meters, centimeters or frequency energy nanometers (10-9 meters). Planck’s constant, 6.6 .10-34 J s • Frequency is the number of wave cycles that travel past a fixed point per unit of time, and is usually given in cycles per second, or hertz (Hz).

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Quiz

1. The shortest length has: a) gamma rays; b) X-rays; c) UV; d) VIS; e) IR; f) microwave; f) radio wave 2. The smallest energy exhibits….

The energy associated with a given segment of the spectrum is proportional to its frequency.

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Quiz Quiz Calculate the energy of UV rays with λ = 340nm.

What is the wavelength of rays with energy 3.2 .10-16 J. A) 6.19 .10-20m; b) 6.19 .10-10m; c) 61.9 .nm; d) 619 nm

ΔE = 5.82 . 10-19 J

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decrease Photon

e nergy

UV-Vis spectroscopy

Physical Chemistry; Understanding our Chemical World. Paul Monk. Manchester Metropolitan University,

UK; John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, England 9 10

The energies of the UV - VIS range of spectrum are sufficient to promote or excite a molecular electron to a higher energy orbital. Consequently, absorption spectroscopy carried out Theoretical background in this region is sometimes called "electronic spectroscopy".

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LUMO

HOMO

Comparison of the transitions met most frequently with simple organic compounds. The four types of transition are united on a single energy diagram in order to situate them with respect to each other and to correlate them with the corresponding spectral ranges concerned.

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The graph of absorbance (A) versus wavelength the isoprene spectrum . Since isoprene is colorless, it does not absorb in the visible part of the spectrum and this region is not displayed on the graph.

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Chromophore Example Excitation λmax, nm ε Solvent Chromophores C=C Ethene π __> π* 171 15,000 hexane

C≡C 1-Hexyne π __> π* 180 10,000 hexane The functional groups of organic compounds n __> π* 290 15 Hexane C=O Ethanal (ketones, amines, nitrogen derivatives, etc.), π __> π* 180 10,000 hexane responsible for absorption in UV/Vis range of the spectrum are called chromophores. n __> π* 275 17 Ethanol N=O Nitromethane π __> π* 200 5,000 ethanol

Methyl bromide C-X X=Br n __> σ* 205 200 Hexane Methyl X=I n __> σ* 255 360 hexane Iodide

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Isolated chromophores - do not interact with each other because they are separated by at least two single bonds in the skeleton, then the overlapping of the effects of each individual chromophore is observed. Conjugated chromophore systems - interact with each other and this process cause displacement of the absorption spectrum towards longer wavelengths (bathochromic effect) and additionally enhancement of the absorption intensity (hyperchromic effect).

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Terminology for Absorption Shifts Bathochromic and hyperchromic effects Nature of Shift Descriptive Term

Change of To Longer Wavelength Bathochromic wavelength

To Shorter Wavelength Hypsochromic Hyperchromic Hyperchromic To Greater Absorbance Hyperchromic Change of light intensity To Lower Absorbance Hypochromic

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The comparison of systems conjugated chromophores in naphthalene, anthracene and tetracene.

Visible light Hyperchromic Hyperchromic

bathochromic 23 24

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Violet: 400 - 420 nm Indigo: 420 - 440 nm Blue: 440 - 490 nm Green: 490 - 570 nm Yellow: 570 - 585 nm Orange: 585 - 620 nm Red: 620 - 780 nm

Chlorophyll A ROY G BIV. Chlorophyll B carotenoids Violet: 400 - 420 nm Indigo: 420 - 440 nm Blue: 440 - 490 nm Green: 490 - 570 nm Yellow: 570 - 585 nm Orange: 585 - 620 nm Red: 620 - 780 nm

http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/Spectrpy/UV-Vis/spectrum.htm#uv2 25 26

Food colouring- Red3;

Because the λmax of 524 nm (the green region of the spectrum) the compound appears red to our eyes

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Factors affecting the absorption •pH

A common feature of all these coloured compounds, displayed below, is a system of extensively conjugated pi-electrons (chromophores).31 32

The pH of the solvent in which the solute is dissolved can have an important effect on its spectrum. Amongst the compounds that present this effect in a spectacular fashion are chemical indicator strips, whose change in color is used during acidimetric measurements.

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Hydrangea in acidic soil Hydrangea in alkaline soil. Absorption spectra of bromocresol green at different stages of protonation

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Red cabbage juice as the pH indicator

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Solvent type

1- high pH value (strong base), 2 – base; 3- neutral pH; 4- weak acid; 5 – medium acidic; 6- strong acid (low pH)

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• Different compounds may have very different absorption maxima and absorbances. Intensely absorbing compounds must be examined in dilute solution, so that significant light energy is received by the detector, and this requires the use of completely transparent (non- absorbing) solvents.

• The most common solvents used in UV-VIS range of spectrum are: water, ethanol, hexane and cyclohexane.

• Solvents having double or triple bonds, or heavy atoms (e.g. S, Br & I) are generally avoided. 39 40

Vis spectrum of 1,2,4,5 tetrazine. Spectrum of solute in a) gaseous state b) in hexane (nonpolar solvent) c) in water (polar solvent)

From S.F. Mason, J. Chem Soc., 1959, 1265

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In spectroscopy, the transmittance T is a measure of the attenuation of a beam of monochromatic light. It is based upon the comparison between the intensities of the transmitted light (I) and the incident light I0. T is expressed as a fraction or a percentage:

T = I/I0 or

%T = I/I0 ×100 The absorbance (old name optical density) is defined by:

Transmittance vs. absorbance A=2−log %T ; A = log T

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• Absorption may be presented as transmittance or absorbance. If the sample Quiz

compound does not absorb light of a given wavelength, I = I0. Thus no Calculate the absorbance of the compound when its absorption has occurred, T = 1.0 and A= 0. transmittance is equal 50%.

• However, if the sample compound absorbs light then I is less than I0 and the transmittance or absorbance is observed. (T < 1; A > 0)

• Most spectrometers display absorbance on the vertical axis, and the commonly observed range is from 0 (100% transmittance) to 2 (1% transmittance).

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UV-VIS spectroscopy equipment

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• Light source.

More than one type of source can be used in the same instrument which automatically swaps lamps when scanning between the UV and visible regions:

• for the visible region of the spectrum, an incandescent lamp;

• for the UV region a deuterium arc lamp (<350nm);

• a xenon arc lamp can be used for routine apparatuses. http://devarchive.cnx.org/contents/02e7b3d6-cf47-4c92-a380-d011ce5658b1@1/basics-of-uv-visible-spectroscopy

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As the monochromator gratings the following tools may be used: (a) single concave spherical mirror Photodetectors (b) two spherical concave mirrors system (c) the concave grating • Photodiodes • Phototransistors • Semiconductors • photomultiplier

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Quantitative analysis

• The measurements are based upon the Lambert–Beer law which, under certain conditions, links the absorption of the light to the concentration of a compound in solution. UV –Vis analysis

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A= l C ε

• A is an absorbance,

• l is the thickness (in cm) of the solution through which the incident light is passed (optical path),

• C the molar concentration of solution Absorption of the light by a homogeneous material and representation −1 −1 • ε the molar absorption coefficient (molar absorptivity) L mol cm at of percentage transmittance as a function of the material’s thickness. The light reaching the sample can be reflected, diffused, transmitted wavelength , at which the measurement is made. It characterizes the or absorbed. Here only this last fraction is taken into account. compound being analyzed. 55 56

Rose Bengal (4,5,6,7- tetrachloro-2',4',5',7'- tetraiodofluorescein) UV-vis spectra of different concentrations of Rose Bengal

Calibration curve of Rose Bengal. Equation of line: y = 0.0977x – Illustration of the Lambert–Beer law. Spectra of aqueous solutions of 0.1492 (R2 = 0.996) increasing concentration in potassium permanganate. Graph of the corresponding absorbances measured at 525nm showing the linear growth of this parameter.

57 http://devarchive.cnx.org/contents/02e7b3d6-cf47-4c92-a380-d011ce5658b1@1/basics-of-uv-visible-spectroscopy 58

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This law applies when the following conditions are fulfilled: Quiz • the light used must be monochromatic Calculate the absorbance of an organic dye C =7×10−4 mol L−1, knowing that the • the concentrations must be low molar absorptivity ε= 650 L mol −1 cm−1 and that the length of the optical path of the cell used is 1cm. • the solution must be neither fluorescent or heterogeneous What would happen to the absorbance if the cell used was of triple its present thickness? • the solute must not undergo to photochemical transformations

• the solute must not undertake variable associations with the solvent.

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For all wavelengths, the absorbance of a mixture is equal to the sum of the absorbances of each component within the mixture (assuming the Additivity of absorbances same molar concentrations in the two experiments).

Experiment: A1 = ε1C1 and A2=ε2C2 A=A1+A2

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+ + Both NAD and NADH absorb at 260 nm. However NADH, unlike NAD , has a second absorbance band with λmax = 340 nm -1 -1 and ε = 6290 L*mol *cm . The figure below shows the spectra of both compounds superimposed, with the NADH spectrum offset slightly on the y-axis: Mixture of red and blue dyes

Red dye By monitoring the absorbance of a reaction mixture at 340 nm, we can 'watch' NADH being formed as the reaction Blue dye proceeds, and calculate the rate of the reaction. http://chemwiki.ucdavis.edu/Organic_Chemistry/Organic_Chemistry_With_a_Biological_Emphasis/Chapter_04%3A_Structu re_Determination_I/Section_4.3%3A_Ultraviolet_and_visible_spectroscopy 63 64

Quiz

• What is the total absorbance at 450nm of the mixture of two components if the concentration of the first component is 1x10-4M (ε = 4000), the concentration of second compound is 1x10-6M (ε = 45000). • Both components obey Lamber-Beer law.

Infrared spectroscopy (IR)

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• Analytical infrared studies are based on the absorption (or reflection) of the electromagnetic radiation that lies between 780 nm - 1mm (1 and 1000 m-1 ).

• This spectral range is sub-divided into three smaller areas, the near infrared (near-IR, 1–25μm), the mid infrared (mid-IR, 25–50μm) and the

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Absorption of lower energy radiation (from 1 to 15 kcal/mole) causes vibrational and rotational excitation of groups of atoms within the molecule. Because of their characteristic absorptions identification of functional groups is easily accomplished.

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• Some General Trends: The complexity of infrared spectra in the 1450 to 600 cm-1 region makes it i) Stretching frequencies are higher than corresponding bending difficult to assign all the absorption bands, and because of the unique frequencies. (It is easier to bend a bond than to stretch or compress it). patterns found there, it is often called the fingerprint region. ii) Bonds to hydrogen have higher stretching frequencies than those to Absorption bands in the 4000 to 1450 cm-1 region are usually due to heavier atoms. stretching vibrations of diatomic units, and this is sometimes called the group iii) Triple bonds have higher stretching frequencies than corresponding frequency region. double bonds, which in turn have higher frequencies than single bonds. (Except for bonds to hydrogen).

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Gas Phase Infrared Spectrum of Formaldehyde, H2C=O

http://www.spectra- analysis.com/infraredspectra/controll ed.htm

Mid-IR spectrum of a polystyrene film Infrared spectrum of aspirin, i.e. 2-acetoxybenzoic acid. (aspirin)

Physical Chemistry;Understanding our Chemical World Paul Monk.Manchester Metropolitan University, UK; John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, England

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• Infrared spectra may be obtained from samples in all phases (liquid, solid and gaseous). Liquids are usually examined as a thin film sandwiched between two polished salt plates (note that glass absorbs infrared • If solvents are used to dissolve solids, care must be taken to radiation, whereas NaCl is transparent). avoid obscuring important spectral regions by solvent • Alternatively, solids may either be incorporated in a thin KBr disk, prepared under high pressure, or mixed with a little non-volatile liquid absorption. Perchlorinated solvents such as carbon and ground to a paste (or mull) that is smeared between salt plates. tetrachloride, chloroform and tetrachloroethene are commonly used.

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IR instruments can be divided into following categories: • the Fourier transform spectrometers, which undertake a simultaneous analysis of the whole spectral region from interferometric measurements, • specialized analysers Equipment • Dispersive-type spectrometers (for the near-IR).

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• Light sources: • They are either a lamp filament or a hollow rod, made of fused mixtures of zirconium oxide or rare earth oxides (Nernst source) heated by Joule effect by the means of an internal resistor (for example Globar™).

IR Detectors

 The principle relies upon the thermal effect of IR radiation. Sensors that measure radiation by means of the change of temperature of an absorbing material are classified as thermal detectors- thermistors, thermocouples, thermopiles and other sensors.

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• Some of the major applications of IR spectroscopy are as follows: Applications of IR  identification of functional group and structure elucidation entire IR region • In industry as well as in scientific research; quality control and identification of substances dynamic measurement. studying the progress of the reaction • Forensics analysis for civil and criminal analysis. detection of impurities quantitative analysis. The quantity of the substance can be determined either in pure form or as a mixture of two or more compounds.

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Bioluminescence, fluorescence and chemiluminescence Luminescence

• Many compounds, when excited by a light source in the visible or near ultraviolet regions, absorb energy which is then re-emitted in the form of radiation.

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Fluorescence Bioluminescence

Chlorophyll in VIS

An example of fluorescence. Tonic water is clear under normal light, but vividly fluorescent under ultraviolet Planktonic jellyfish with light, due to the presence of the bright green-fluorescent quinine tentacles. The red The fluorescence of the chlorophyll in UV fluorescent in the middle comes from chlorophill. Glow- worm, fire- fly

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Chemo luminescence Phosphorescence The-glow-in-the dark toys stickers Paint clock dials

Phosphorescent materials: zinc sulfide; strontium aluminate

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Theoretical background

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According to Stokes’ law, the maximum of the spectral emission band is located at a longer wavelength than that of the original excitation light. After excitation, the light intensity decays extremely rapidly according to an exponential law.

Expression below links the intensity of fluorescence It and the time passed t since the excitation:

The Jablonsky diagram illustrating the process involved in the creation of an excited  I =I -kt singlet state by optical absorption and subsequent emission of the fluorescence. t 0

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Representation on the same graph of the absorbance and fluorescence spectra of an ethlyenic compound.. Example extracted from Jacobs H.et al., Tetrahedron 1993, 6045.

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Relative position of absorption, Relative position of absorption and fluorescence and fluorescence bands of anthracene. Fluorescing aromatic compounds. The names are followed by their fluorescence phosphorescence bands of quantum yield Φ, for which the values are obtained by comparison with chrysene. compounds of known fluorescence. The measurements are made at 77 K. 8-Hydroxyquinoline is representative of various molecules forming fluorescent chelates with certain metal ions.

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• The extremely rapid extinction of the light intensity when excitation ceases is the object of analysis. By contrast, phosphorescence is characterized by a more gradual diminution during time. • The intensity of the fluorescence is proportional to the concentration of the analyte and the Fluorescence is equally employed as the basis of detectors used in measurements are made with the aid of fluorometers liquid chromatography. or spectrofluorometers • Although of different origin, chemiluminescence which comprises the emission of light during certain chemical reactions, has also received several applications in analytical chemistry.

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Intensity of fluorescence and concentration. A maximum of fluorescence is observed beyond Equipment which, with a continuing increase in concentration, it diminishes. After the maximum the more concentrated the solution then the weaker is the fluorescence – a kind of roll-over or self- quenching. The illustrations correspond to three recordings, at the same scale of biacetyl tetrachloromethane. The curve records the light collected from a small volume situated at the centre of the solution 104 105

A block diagram of a spectrofluorometer with a xenon arc lamp. The fluorescence is measured, in contrast to studies on fluorescence dynamics, under ‘steady state’ conditions by maintaining the primary excitation source. Below right, a schematic of a xenon arc lamp. The pressure of xenon in the lamp is around 1 MPa. These arc lamps made from a quartz envelope and without a filament, Comparison, following a chromatographic separation, of UV and fluorescence detection. Aflatoxins, are sources of ‘white light’. The cathode is the finer of the two electrodes. which are carcinogenic the subject of analysis by HPLC. (reproduced courtesy of a document from Agilent Technologies).

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Application of fluorescence

• Analysis of biological samples: • Oligonucleotides • Gens • Antibodies • Liquid chromatography detectors DyLight 680/800 labeled antibodies used in two-color Western blot. Proteins were separated in 4-20% Precise Protein Gels and transferred to Low-Fluorescence PVDF Transfer Membrane. The membrane was blocked overnight in SEA BLOCK and target proteins were detected following the recommended protocol. Membranes were imaged with the LI-COR Odyssey Infrared Imaging System. Tubulin was detected from the indicated quantity of HeLa cell lysate. Purified TNFα was detected at the indicated quantity.

http://www.piercenet.com/browse.cfm?fldID=5A409573-5056-8A76-4E35-8EE0AA401E7B 108 109

Quiz • Which of the following statements best defines luminescence? A. The emission of light by a substance after absorption of excitation energy B. Emission of light due to non-thermal process, a chemical reaction, or the absorption of ionizing radiation Absorption and emission methods, C. This light is absorbed by the ground state atoms atomic methods D. Emission of light requiring a light source

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• Atomic absorption spectroscopy (AAS) and flame emission spectroscopy (FES; flame photometry) are two analytical measurement methods relying on the spectroscopic processes of excitation and emission. Methods of quantitative analysis only, they are used to measure of

around seventy elements (metal or nonmetal). Flame emission 589 nm Na There exists a broad range of applications, as concentrations to the ppb level can be accessed for certain elements. Atomic absorption

The schematics for the optical set-up (collimator,objective), have been simplified 112 for reasons of clarity. 113

• To measure an element by AAS or FES methods, it must be in the form of free atoms. Thus the sample is heated to a temperature of at least 2000oC, in order to dissociate all chemical combinations in which the element under study is engaged, including the rest of the sample. • This pyrolysis leads to the total concentration of the element without distinguishing the different chemical structures in which the element was possibly bound in the original sample (this is therefore opposite to a speciation analysis). Equipment •

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Atomizers The diverse components of a single beam atomic absorption apparatus. Model IL 157(Thermo Jarrell Ash) constructed during the 1980s. 1, source (spectral lamp); 2, flame burner which provides the atomic aerosol; 3, monochromator grating; and 4, detector (photomultiplier). The source illuminates a slit situated at the entrance the dispersive system. The exit slit, is close to the detector window. It determines a narrow bandwidth of the spectrum, ( of 0.2 to 1 nm), which must not be confused with either the width of the exit slit or with the image of the entrance slit. 116 117

The pyrolysis process can be performed with: • Bunsen burner (flame atomizer), • graphite furnace (electrothermal atomizer) • Glow-discharge (GD, Special atomizer) • device for hydride formation (special atomizer) • Cold vapour atomization (special atomizer) –determination of mercury

Radiation sources

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• An alternative to HCL are electrodeless discharge lamps (EDL) whose light intensity is about 10–100 times greater but are not as stable as HCL. They are made of a sealed quartz tube that contains a salt of the element of interest along with an inert gas. These lamps are in general reserved for elements such as As, Hg, Sb, Bi and P.

Typical model of a hollow cathode lamp. The cathode is a hollow cylinder whose central axis corresponds to that of the optical axis of the lamp.

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Elements measured by AAS and FES.

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• In AAS, as in FES, the measurement of light intensity is carried out • The absorbance (or emision) of the element in the flame depends at a wavelength specific to each element being analysed. upon the number of ground state atoms. • In AAS, the concentration can be deduced from the measurement • Measurements are made by comparing the unknown to the standard solutions. of light absorption by the atoms remaining in the ground state when they are irradiated by an appropriate source of excitation. • A=k ·C • where A is absorbance, C is concentration of element and k is a • In FES, conversely, the concentration can be deduced from the specific coefficient to each element at the given wavelength. intensity of the radiation emitted by the fraction of atoms that have passed into excited states.

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Application • Atomic Absorption Spectroscopy can be used to measure the concentration of metals in : • mining operations and in the production of alloys as a test for purity • contaminated water, especially heavy metal contamination in industrial waste water • organisms, such as mercury in fish • air, e.g. lead Examples of calibration graphs in AAS. Left, a straight calibration line at sub-ppb concentrations • food obtained with an instrument equipped with a Zeeman effect device for the quantification of sodium. Right, a quadratic curve for the measurement for zinc at concentrations in the ppm range with a burner type instrument. This second graph reveals that when concentrations increase, the absorbance is no longer linear. The quantitative analysis software for AAS provides several types of calibration curves. 126 127

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• Most applications of FES: • determination of trace metals, especially in liquid samples (including Sources 1. Physical Chemistry;Understanding our Chemical World the alkali and alkaline earths, as well as several transition metals such as Paul Monk. Manchester Metropolitan University, UK; John Wiley & Sons Ltd, The Atrium, Southern Gate, Fe, Mn, Cu, and Zn; nonmetals: H, B, C, N, P, As, O, S, Se, Te, halogens, Chichester, and noble gases). FES detectors for P and S are commercially available West Sussex PO19 8SQ, England for use in gas chromatography. 2. http://ull.chemistry.uakron.edu/analytical/ 3. • in agricultural and environmental analysis, http://search.conduit.com/Results.aspx?q=spectrophotometry&start=10&hl=pl&SearchSource=13&SelfSearch= 1&SearchType=SearchWeb&ctid=CT2475029&octid=CT2475029&FollowOn=True • industrial analyses of ferrous metals and alloys as well as glasses and 4. http://cfcc.edu/faculty/jjenkins/courses/msc180/lectures/Spe ceramic materials, 5. http://www.shsu.edu/~chm_tgc/primers/pdf/spect.pdf • clinical analyses of body fluids. • FES can be easily automated to handle a large number of samples. Array detectors interfaced to a microcomputer system permit simultaneous analyses of several elements in a single sample.

http://www.tau.ac.il/~chemlaba/Files/Flame%20supplement.pdf 128 129