Chapter 4 Ultraviolet and visible spectroscopy Molecular Spectrophotometry

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1 Chapter 4 Ultraviolet and visible spectroscopy Molecular Spectrophotometry Properties of light Electromagnetic radiation and electromagnetic spectrum Absorption of light Beer s law Limitation of Beer s law Absorption of light by molecules Instrumentation: Spectrophotometer Applications: Individual species and mixtures in pharmaceutical analysis

2 Spectrophotometry It refers to the use of light to measure chemical concentrations. Mainly, the fundamental principles of absorption and emission of radiation by molecules and how these processes are used in quantitative analysis will be discussed.

3 Electromagnetic radiation Electromagnetic radiation or light, is a form of energy whose behavior is described by the properties of both waves and particles. The optical properties of electromagnetic radiation, such as diffraction and dispersion, are explained best by describing light as a wave. Many of the interactions between electromagnetic radiation and matter, such as absorption and emission are better described by treating light as a particle, or photon.

4 Wave Properties of EMR consists of oscillating electric and magnetic fields that propagate through space along a linear path and with a constant velocity Oscillations in the electric and magnetic fields are perpendicular to each other, and to the direction of the wave's propagation Plane polarized electromagnetic radiation showing the electric field, the magnetic field and the direction of propagation

5 In a vacuum, EMR travels at the speed of light, c, which is x 10 8 m/s. EMR moves through a medium other than a vacuum with a velocity, v, less than that of the speed of light in a vacuum. The difference between v and c is small enough (< 0.1%) that the speed of light to three significant figures, 3.00 x 10 8 m/s, is sufficiently accurate for most purposes.

6 The interaction of EMR with matter can be explained using either the electric field or the magnetic field. Only the electric field component will be used to discuss this matter A e is the electric field maximum amplitude Is the distance between successive maxima or successive minima

7 An electromagnetic wave is characterized by several fundamental properties, including its velocity, amplitude, frequency, phase angle, polarization, and direction of propagation. Frequency,, is the number of oscillations in the electric field per unit time. One oscillation/sec = one hertz (HZ) The wavelength of an electromagnetic wave,, is defined as the distance between successive maxima, or successive minima For ultraviolet and visible electromagnetic radiation the wavelength is usually expressed in nanometers (nm, 10-9 m) The wavelength for infrared radiation is given in microns (m, 10-6 m). Wavelength depends on the electromagnetic wave's velocity, where = c/ = v/ (in vacuum) = 1/ Wave number

8 Power and Intensity of light Power, P, and Intensity, I, of light give the flux of energy from a source of EMR P is the flux of energy per unit time I is the flux of energy per unit time per area

9 Particle Properties of Electromagnetic Radiation When a sample absorbs electromagnetic radiation it undergoes a change in energy. The interaction between the sample and the electromagnetic radiation is easiest to understand if we assume that electromagnetic radiation consists of a beam of energetic particles (packets of energy) called photons. When a photon is absorbed by a sample, it is "destroyed," and its energy acquired by the sample The energy of a photon, in joules, is related to its frequency, wavelength, or wavenumber by the following equations: E = h hc = = hc h is Planck's constant, which has a value of x J s.

10 Electromagnetic Spectrum The spectrum is the written records of the EMR EMR is divided into different regions based on the type of atomic or molecular transition that gives rise to the absorption or emission of photons The boundaries describing the electromagnetic spectrum are not rigid, and an overlap between spectral regions is possible.

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12 Colors of the visible light of maximum Color Color absorption (nm) absorbed observed Violet Green-yellow Violet-blue Yellow Blue Orange Blue-green Red Green Purple Yellow-green Violet Yellow Violet-blue Orange Blue Red Blue-green Purple Green

13 Measuring Photons as a Signal Spectroscopy is divided into two broad classes. 1. Energy is transferred between a photon of electromagnetic radiation and the analyte (Absorption or emission of radiation 2. Changes in electromagnetic radiation wave characteristics (changes in amplitude, phase angle, polarization, or direction of propagation. Class 1: Absorption of radiation In absorption spectroscopy the energy carried by a photon is absorbed by the analyte, promoting the analyte from a lower-energy state to a higher-energy, or excited, state Absorbing a photon of visible light causes a valence electron in the analyte to move to a higher-energy level. When an analyte absorbs infrared radiation one of its chemical bonds experiences a change in vibrational energy.

14 Energy level diagram showing absorption of a photon The intensity of photons passing through a sample containing the analyte is attenuated because of absorption. The measurement of this attenuation, which we call absorbance, The energy levels have well-defined values (i.e., they are quantized). Absorption only occurs when the photon's energy matches the difference in energy, E, between two energy levels. A plot of absorbance as a function of the photon's energy is called an absorbance spectrum

15 Ultraviolet/visible absorption spectrum for bromothymol blue

16 Class 1 Emission of Radiation Emission of a photon occurs when an analyte in a higher-energy state returns to a lower-energy state The higher-energy state can be achieved in several ways, including thermal energy, radiant energy from a photon, or by a chemical reaction. Emission following the absorption of a photon is also called photoluminescence, and that following a chemical reaction is called chemiluminescence.

17 Various spectroscopic techniques of class 1

18 Class 2 Changes in the EMR wave characteristics In this class of spectroscopy, the electromagnetic radiation undergoes a change in amplitude, phase angle, polarization, or direction of propagation as a result of its refraction, reflection, scattering, diffraction, or dispersion by the sample. Several representative spectroscopic techniques are listed in the following table

19 Various spectroscopic techniques of class 2

20 Emission (luminescence) Spectrum

21 Typical Emission Spectrum

22 Sources of Energy All forms of spectroscopy require a source of energy. In absorption and scattering spectroscopy this energy is supplied by photons (EMR or light). Emission and luminescence spectroscopy use thermal, radiant (photon), or chemical energy to promote the analyte to a less stable, higher energy state.

23 Common sources of EMR

24 Sources of Electromagnetic Radiation A source of electromagnetic radiation must provide an output that is both intense and stable in the desired region of the electromagnetic spectrum. Sources of electromagnetic radiation are classified as either continuum or line sources. A continuum source emits radiation over a wide range of wavelengths, with a relatively smooth variation in intensity as a function of wavelengths. Line sources emit radiation at a few selected, narrow wavelength ranges

25 Emission spectrum from a continuum emission source Emission spectrum fro ma typical line source

26 Absorbance of Electromagnetic Radiation In absorption spectroscopy a beam of electromagnetic radiation passes through a sample. Much of the radiation is transmitted without a loss in intensity. At selected wavelengths the radiation's intensity is attenuated. The process of attenuation is called absorption. Two general requirements must be met if an analyte is to absorb electromagnetic radiation. The first requirement is that there must be a mechanism by which the radiation's electric field or magnetic field interacts with the analyte. For ultraviolet and visible radiation, this interaction involves the electronic energy of valence electrons. A chemical bond's vibrational energy is altered by the absorbance of infrared radiation.

27 The second requirement is that the energy of the electromagnetic radiation must exactly equal the difference in energy, AE, between two of the analytes quantized energy states.

28 Molecular Absorption Molecules undergo three types of quantized transitions when excited by ultraviolet, and visible radiation. 1. electronic transition The transition of an electron between two orbitals (the energy by the photon must be exactly the same as the energy difference between the two orbital energies) and the absorption process is called electronic absorption

29 2. vibrational and rotational transitions Vibration of the atoms of the molecule with respect to one another Atoms and groups of atoms within molecules can undergo various types of vibrations and each requires a discrete amount of energy to initiate or maintain. Also molecules can rotate around their axes a matter that requires discrete amount of energy.

30 Molecular Absorptions Each molecular energy state is comprised of an electronic, vibrational and rotational component such that: E molecule = E electronic + E vibrational + E rotational Our Focus E electronic (UV/Vis) E vibrational (IR)

31 Energy of a Molecule E electronic --> kj/mole --> UV-Vis UV-Vis range: nm E vibrational --> kj/mole --> IR Near IR: nm (5000 nm) Mid-IR : 5000 nm - 25,000 nm (5 microns - 25 microns) E rotational --> 10 kj/mole --> microwaves

32 Molecular Orbital Theory Molecular orbitals (MOs) are mathematical equations that describe the regions in a molecule where there is a high probability of finding electrons Atomic orbitals of atoms are combined to give a new set of molecular orbitals characteristic of the molecule as a whole The molecular orbitals are spread out over the entire molecule. Electrons are now in orbitals that belong to the molecule as a whole. The number of atomic orbitals combined equals the number of molecular orbitals formed. (Two s-orbitals Two molecular orbitals)

33 Molecular orbitals Two atomic orbitals combine to form a bonding molecular orbital and an anti-bonding MO*. Electrons in bonding MO s stabilize a molecule Electrons in anti-bonding MO s destabilize a molecule For the orbitals to combine, they must be of comparable energies. e.g., 1s(H) with 2s(Li) is not allowed The molecular orbitals are arranged in order of increasing energy. The electronic structure of a molecule is derived by feeding electrons to the molecular orbitals according to same rule applied for atomic orbitals

34 Formation of molecular orbitals by combination of 1s orbitals The hydrogen molecule Destructive interference 1s * H B H A Antibonding MO 2 = region of diminished electron density Electron density is concentrated away from internuclear region Bonding MO 1 = enhanced region of electron density Electron density is concentrated between nuclei 1s Constructive interference

35 Energy level diagram in hydrogen (H 2 ). Bonding molecular orbital has lower energy and greater stability than the atomic orbitals from which it was formed. antibonding molecular orbital has higher energy and lower stability than the atomic orbitals from which it was formed.

36 Molecular orbitals diagram Each molecular orbital can hold a maximum of two electrons with opposite spins Electrons go into the lowest energy molecular orbital available Hund s rule is obeyed Molecular orbital model will be applied only to the diatomic molecules of the elements of the first two periods of the Periodic Table

37 Pi (p) molecular orbitals Wave functions representing p orbitals combine in two different ways yielding either orbitals or p orbitals. End-to-end combination yields sigma () orbitals antibonding orbital difference * p ± - + 2p z 2p z sum p Atomic orbitals bonding orbital Molecular orbitals

38 Sideways combination yields pi (p) orbitals + - 2p y ± + - 2p y difference sum * p p 2 p 2p - ± p x 2p x Atomic orbitals difference sum + Molecular orbitals - * p p 2 p 2p

39 Molecular Orbital (MO)Theory Review MO Theory: Electrons in atoms exist in atomic orbitals while electrons in molecules exist in molecular orbitals. Bonding MO: A MO where electrons have a lower energy than they would in isolated atomic orbitals Anitbonding MO: A MO in which electrons have a higher energy than they would in isolated atomic orbitals. Ground State: Refers to the state of lowest energy. Electrons can be promoted from a ground state to a higher excited state by input Of energy. Excited State: Any electronic state other than the ground state.

40 Types of molecular Transitions Types of molecular orbitals in formaldehyde

41 Origin of absorption in relation to molecular orbitals In common organic compounds the observed molecular transitions involve, p and non-bonding n electrons. Each transition is characterized by its wavelength

42 Various types of molecular transitions Valence electrons are the only ones whose energies permit them to be excited by UV/visible radiation The most common transitions in organic molecules are shown below * (anti-bonding) p* (anti-bonding) Four types of transitions * n (non-bonding) p (bonding) pp* n* np* (bonding)

43 Types of Transitions -----> * Far (vacuum) UV Saturated hydrocarbons like hexane or cyclohexane that contain only bonds are transparent in the conventional UV Hexane absorbs and cyclohexane absorb at less than 200 nm Consequently they are used as solvents

44 UV Spectra UV spectra for typical organic compounds

45 Ultraviolet absorption spectra for 1,2,4,5- tetrazine (a.) in the vapor phase, (b.) in hexane solution, and (c.) in aqueous solution.

46 n* Transitions Still rather high in energy. between 150 and 250 nm. Not many molecules with n* transitions in UV/vis region max max H2O CH 3 OH CH 3 Cl CH 3 I (CH 3 ) 2 S (CH 3 ) 2 O CH 3 NH 2 (CH 3 ) 3 N

47 np* and pp* Transitions Most UV/vis spectra involve these transitions. pp* are generally more intense than np*. max max type C 6 H 13 CH=CH pp* C 5 H 11 CC CH pp* O CH 3 CCH n* O CH 3 COH np* CH 3 NO np* CH 3 N=NCH np*

48 Relative Energies of Molecular Orbitals Energy sigma* π* n π sigma Compounds containing only sigma bonds have absorptions only in the ultraviolet. These transitions correspond to sigma-sigma* n-sigma* transitions are common Compare the energy of n-sigma* vs a sigma-sigma*

49 Example of Electronic Transitions: Absorptions H O C H Formaldehyde Contains both π and nonbonding electrons (n)

50 Chromophores They are small groups of atoms responsible for characteristic absorptions Chromophore is responsible for electronic transition Characteristic chromophores of several nitroge containing groups. Name Chromophore Amine -NH, 195 oxime -NOH 190 nitro -NO, 210 Nitrite -ONO 230 Nitrate -ONOZ 270 nitroso -N=O 300

51 Absorption Characteristics of Some Common Chromophores

52 Effect of Multichromophores on Absorption

53 Auxochrome Substitutent groups which are not themselves optically active in this energy range, but which do interact with other chromophores to shift both intensity and wavelength. Absorption Characteristics of Pyridine Derivatives Derivative max max Pyridine CH CH CH F Cl I OH

54 Solvent Effects - Shifts Solvents can interact with the analyte molecules and shift absorbance peaks and intensities. Red Shift (Bathochromic) Peaks shift to longer wavelength. Blue Shift (Hypsochromic) Peaks shift to shorter wavelength. np* generally blue shifted; solvation of and hydrogen bonding to the lone pair. Large shifts (up to 30 nm). Both np* and pp* red shifted; attractive polarization forces, increase with increasing solvent polarity. Small shifts (less than 5 nm).

55 Solvent Effects - Intensity Solvents can also induce significant changes in the intensity of peaks. Hyperchromic Increase in absorption intensity. Hypochromic Decrease in absorption intensity. Absorption characteristics of 2-methylpyridine Solvent max max Hexane Chloroform Ethanol Water Ethanol - HCl (1:1)

56 Absorption of Light Beer s Law-1 P 0 P T P P 0

57 Beer s Law-2 P 0 = 10,000 P = 5,000 -b- T P P

58 Beer s Law- 3 P 0 = 10,000 P = 2, b-- T P P

59 Beer s Law-4 P 0 = 10,000 P = 1, b---- T P P

60 Beer s Law-5 P 0 = 10,000 P = b T P P

61 Transmittance Relationship between transmittance and cell thickness Thickness, b Transmittance, T Thickness, multiples of b T P P 0

62 Absorbance Relationship between absorbance and cell thickness Thickness, b Transmittance, T A = -log T Thickness, multiples of b A A log T abc log P P 0 A a absorbance absorptivity b thickness c concentration

63 Relation between Absorbance and Transmittance Calculation of absorbance from transmittance T %T A %T A Cell B3=100*A3 Cell C3=-log(A3) Transmittance / Nanometers File # 1 : UVSIN204 Paged Z-Zoom CURSOR Res=None A log T abc

64 Absorbance and Transmittance Spectra %T 60 A Transmittance / Nanometers File # 1 : UVSIN204 Paged Z-Zoom CURSOR Res=None Absorbance / Nanometers File # 1 : UVSIN204 Paged Z-Zoom CURSOR Res=None % Transmission Spectrum Absorbance Spectrum A log T abc

65 Absorbance Spectra and Concentration 1 conc A A.2 0 conc B Absorbance Spectra A log T abc

66 Absorbance and Concentration: Beer's Law When monochromatic EMR passes through an infinitesimally thin layer of sample, of thickness dx, it experiences a decrease in power of dp. The fractional decrease in power is proportional to the sample's thickness and the analyte's concentration, C

67 Thus, where P is the power incident on the thin layer of sample, and is a proportionality constant. Integrating the left side of equation from P = P o to P = P T, and the right side from x = 0 to x = b, where b is the sample's overall thickness, gives

68 Converting from ln to log and substituting log p o /p T by A (absorbance) gives A = abc Where a is tha anlayte absorptivity with units of cm -1 conc -1. When concentration is expressed using molarity the absorptivity is replaced by molar absorptivity The absorptivity and molar absorptivity give, in effect, the probability that the analyte will absorb a photon of given energy. As a result, values for both a and depend on the wavelength of electromagnetic radiation.

69 Absorbance Predicting Concentrations from Absorbance Spectra unknown Regression equation slope Intercept Conc of unknown Conc, Micro-M

70 Absorption Spectra of Mixtures Containing n components n n n bc a bc a bc a bc a A bc a bc a bc a bc a A bc a bc a bc a bc a A n n n n n

71 Absorption Spectra of Mixtures Containing n components Constant pathlength n n n c k c k c k c k A c k c k c k c k A c k c k c k c k A n n n n n

72 Limitations to Beer s Law Ideally, according to Beer's law, a calibration curve of absorbance versus the concentration of analyte in a series of standard solutions should be a straight line with an intercept of 0 and a slope of ab or b. In many cases, calibration curves are found to be nonlinear. Deviations from linearity are divided into three categories: fundamental, chemical, and instrumental.

73 Fundamental Limitations to Beers Law Beer's law Beer s law is a limiting law that is valid only for low concentrations of analyte. 1. At higher concentrations the individual particles of analyte no longer behave independently of one another. The resulting interaction between particles of analyte may change the value of a or. 2. The absorptivity, a, and molar absorptivity,, depend on the sample's refractive index. Since the refractive index varies with the analyte's concentration, the values of a and will change. For sufficiently low concentrations of analyte, the refractive index remains essentially constant, and the calibration curve is linear.

74 Chemical Limitations to Beer's Law Chemical deviations from Beer's law can occur when the absorbing species is involved in an equilibrium reaction. Consider, as an example, the weak acid, HA. To construct a Beer's law calibration curve, several standards containing known total concentrations of HA, C tot, are prepared and the absorbance of each is measured at the same wavelength. Since HA is a weak acid, it exists in equilibrium with its conjugate weak base, A -

75 If both HA and A - absorb at the selected wavelength, then Beer s law is written as where C HA and C A are the equilibrium concentrations of HA and A -. Since the weak acid's total concentration, C tot, is C tot = C HA + C A The concentration of HA and A - can be written as Where HA is the fraction of week acid present as HA

76 Thus, Because values of HA may depend on the concentration of HA, equation may not be linear. A Beer's law calibration curve of A versus C tot will be linear if one of two conditions is met. 1. If the wavelength is chosen such that HA and A are equal, then equation simplifies to A = b C tot and a linear curve is realized

77 2. Alternatively, if HA is held constant for all standards, then equation will be a straight line at all wavelengths. Because HA is a weak acid, values of HA change with ph. To maintain a constant value for HA, therefore, we need to buffer each standard solution to the same ph. Depending on the relative values of HA and A, the calibration curve will show a positive or negative deviation from Beer's law if the standards are not buffered to the same ph.

78 Instrumental Limitations to Beer's Law There are two principal instrumental limitations to Beer's law. 1. Beer s law is strictly valid for purely monochromatic radiation; that is, for radiation consisting of only one wavelength. even the best wavelength selector passes radiation with a small, but finite effective bandwidth. Using polychromatic radiation always gives a negative deviation from Beer's law, but is minimized if the value of is essentially constant over the wavelength range passed by the wavelength selector. For this reason, it is preferable to make absorbance measurements at a broad absorption peak.

79 Effect of wavelength on the linearity of a Beer s law calibration curve

80 2. Stray Radiation Stray radiation arises from imperfections within the wavelength selector that allows extraneous light to "leak" into the instrument. Stray radiation adds an additional contribution, P stray, to the radiant power reaching the detector; thus For small concentrations of analyte, P stray is significantly smaller than P o and P T, and the absorbance is unaffected by the stray radiation. At higher concentrations of analyte, P stray is no longer significantly smaller than P T and the absorbance is smaller than expected. The result is a negative deviation from Beer's law.