Part I. Introduction to Spectroscopy Rodolfo J. Romañach, Ph.D.

1. Part I. Introduction to SpectroscopyRodolfo J. Romañach, Ph.D. Vibrational Spectroscopy for Pharmaceutical Analysis

2. c =νλ, where c is the speed of light – 3.0 x 108 m/s in vacuum. And ν is the frequency – number of oscillations per second. Figure 18-1 Exploring Chemical Analysis, page 375.

3. Energy and Frequency • Light can also be visualized as particles called photons that have energy E = hν, and have discrete energy levels. • E = hν, where h is Planck’s constant (=6.626 x 10-34 J-s).

4. Electromagnetic Spectrum Fig. 18-2, Exploring Chemical Analysis, 3rd Ed., page 376.

5. Spectroscopy • Spectroscopy – the study of the interaction between electromagnetic radiation and matter. • A guiding theory of analytical chemistry can be used to specify what information can be extracted from the data produced by an analytical instrument or method. In addition, it can be used to optimize existing analytical tools and direct researchers to construct more powerful analytical tools. K.S. Booksh and B.R. Kowalski, Theory of Analytical Chemistry, 1994, 66(15), 782 A – 791A.

6. Energy Diagram - Spectroscopy

7. Absorption of Radiation • When radiation passes through a layer of solid, liquid, or gas, the intensity of the radiation at certain frequencies may be reduced by absorption. • Atomic absorption occurs only at a few frequencies, electronic absorption. • Molecular absorption includes E = E Elec + E Vib + E rotational • The energy of the exciting photon must exactly match the energy difference between the ground state and one of the excited states of the absorbing species. SHN, page 134.

8. Absorption of Light A spectrophotometer measures transmission of light. A = - log T = - log P/P0 If P/P0 = 0.01, then A = - log (0.01) = -(-2) = 2.0 If P/P0 = 0.001, then A = - log (0.001) = - (-3) = 3.0

9. Beer’s Law • Absorbance is proportional to the concentration of light-absorbing molecules in the sample. • A = εbc • Where ε is the molar absorptivity, its has units M-1cm-1, because the product of εbc must be dimensionless. • Molar absorptivity tells how much light is absorbed at a particular wavelength. Exploring Chemical Analysis, section 18-2, 3rd Edition.

10. Optical Pathlength, Beer’s Law, and cells Define optical pathlength in A = εbc.

11. A = f(λ). The molar absorptivity coefficient, ε, changes as the wavelength changes; A =ελbc UV-Visible Spectrum, Harris, Exploring Chemical Analysis, page 382, Figure 18-6.

12. Application of Beer’s Law Developing mathematical relationship between absorbance (at one λ) and the concentration – univariate calibration.

13. Good Operating Techniques • Spectrophotometry is most accurate at intermediate absorbance levels of 0.4 – 0.9. • If too little light gets through the sample (high absorbance), the intensity is difficult to measure. • If too much light gets through (low absorbance), then its difficult to discriminate between intensity of sample and that of reference. • Make measurements at a peak in the spectrum and not in slope. • First record a base line spectrum with pure solvent in both cells. Absorbance should be zero; a good check on instrument.

14. Common Spectrometer Design Figure 19-1. Exploring Chemical Analysis, page 397.

15. Light Sources Used in UV-Vis Spectrophotometers These are called continuum sources.

16. Monochromator • A monochromator disperses light into its component wavelengths to pass through the sample. Used in most modern instruments. • Grating is a component with a series of ruled lines. When light is reflected or transmitted, each line acts a separate source of radiation dispersing the light. • The bending of light rays by a grating is called diffraction.

17. Monochromator and Components

18. Diffraction of Light by Grating There is a repeat distance between the parallel grooves of the grating. When light is reflected each groove acts as a source of radiation. If adjacent light rays are in phase, they reinforce one another. If they are out of phase, they can each other.

19. Resolution • Some spectra have closely spaced absorption bands, and a monochromator of high resolution is needed to differentiate between these bands. • Sometimes the spectral bands are wide and a monochromator of high resolution is not needed.

20. Monochromator Bandwidth and Spectrum Obtained Figure 19-7 – Exploring Chemical Analysis, page 402.

21. Stray Light • This is a phrase often encountered in many papers. • It refers to small accounts of scattered or stray radiation with wavelengths far different from the instrument setting. • Could come from reflection of the beam from various optical parts (mechanical imperfections of the monochromator). • Could also occur from scattering by dust particles or from surfaces of optical parts (sealed to avoid dust). The inner parts of the spectrometer are usually coated black to avoid reflection. SHN page 161

22. Dispersive Spectrophotometers • Harris talks about “dispersive spectrophotometers” – p. 403. • Dispersive means that light is dispersed using grating in monochromator and a spectrum is obtained as a sample is scanned (wavelength is varied through a certain time interval). • The UV spectrophotometer (Beckman DU-650) works by recording the absorbance at one wavelength at a time.

23. A photodiode array detector (PDA) can be used to detect all the wavelengths dispersed at the same time. A PDA consists of a series of detectors, a detector array. The PDA receives polychromatic radiation that is dispersed through its detector arrays. This type of instrument is called a multichannel analyzer. Photodiode Array Detector

24. Spectrophotometer with PDA

25. Photomultiplier Tube Figure 19-9, Exploring Chemical Analysis, page 403.

26. Photomultiplier Tubes • Offers a number of advantages over ordinary phototubes for measurement of low radiant power, may be used for fluorescence. • Cathode surface emits electrons when exposed to radiation. Cathode is at a high voltage of -500 to -1500 V. • Electrons are accelerated towards dynodes, and upon striking the dynode each photoelectron causes emission of several additional electrons. • This multiplicative effect creates 105 to 107 electrons for each photoelectron that is ejected from the photocathode. • Can be cooled to reduce dark current. SHN, pages 170 – 172, and http://www.chem.vt.edu/chem-dept/tissue/4114/, accessed Feb. 27, 2005

27. Relaxation Processes • Non-radiation relaxation – involves loss of energy in a series of small steps, the excitation energy being converted to kinetic energy by collision with other molecules. • Fluorescence and phosphorescence involve emission of a beam of electromagnetic radiation; as the excited species returns to the ground state. • Fluorescence occurs more rapidly and is generally complete in 10-5 seconds. SHN, page 137.

28. Emission & Luminescence • The power of the radiation emitted by an analyte after excitation is usually directly proportional to the analyte concentration. • S = k’c, where c is the concentration, and k’ is a constant that can be obtained with the evaluation of one or more standards to develop a calibration.