Transcript Document

Vibrational Spectroscopy for
Pharmaceutical Analysis
Part I. Introduction to Spectroscopy
Rodolfo J. Romañach, Ph.D.
ENGINEERING RESEARCH CENTER FOR
STRUCTURED ORGANIC PARTICULATE SYSTEMS
RUTGERS UNIVERSITY
PURDUE UNIVERSITY
NEW JERSEY INSTITUTE OF TECHNOLOGY
UNIVERSITY OF PUERTO RICO AT MAYAGÜEZ
10/11/2005
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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.
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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).
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Electromagnetic Spectrum
Fig. 18-2, Exploring Chemical Analysis, 3rd Ed., page 376.
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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.
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Energy Diagram - Spectroscopy
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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.
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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
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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 M1cm-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.
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Optical Pathlength, Beer’s Law, and cells
Define optical pathlength in A = εbc.
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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.
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Application of Beer’s Law
Developing mathematical relationship between absorbance (at one λ) and
the concentration – univariate calibration.
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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.
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Common Spectrometer Design
Figure 19-1. Exploring Chemical Analysis, page 397.
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Light Sources Used in UV-Vis
Spectrophotometers
These are called continuum sources.
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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.
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Monochromator and Components
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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.
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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.
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Monochromator Bandwidth and Spectrum Obtained
Figure 19-7 – Exploring Chemical Analysis, page 402.
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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
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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.
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Photodiode Array Detector
• 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.
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Spectrophotometer with PDA
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Photomultiplier Tube
Figure 19-9, Exploring Chemical Analysis, page 403.
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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/chemdept/tissue/4114/, accessed Feb. 27, 2005
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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.
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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.
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