Chem 5486, Instrumental Analysis
Spring, 2005
• Text: Principles of Instrumental Analysis, 5th Ed.,
Skoog, Holler, Nieman, Harcourt Brace, 1998
• Lecture MW, 1:00 - 1:50 pm
• Lab MW, 2:00 pm - 4:50
• Office hours by appointment
• J. O’Brien, 321A Fondren Science
• jobrien@smu.edu
Lab Schedule
• Jan 24 Spectrophotometric Analysis of a Mixture of Absorbing
Substances
• Feb 23 High Throughput Screening Via Microplate Absorbance
Spectrophotometry (Dr. Hua)
• Feb 28 Caffeine Analysis by High Performance Liquid
Chromatography (A. Humason)
• March 21 Gas Chromatography/Mass Spectroscopy Applications
(A. Humason)
• March 28 Gel Permeation Chromatography (Dr. Wisian-Neilson)
• April 4&6 Thermogravimetric Analyzer TGA and Differential
Scanning Calorimetry (Dr. Son)
• April 20 Characterization of Semiconductors and Tour of
Engineering Cleanroom, Junkins Hall
Grading
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Four lab reports (100 points each)
400 pts
Three exams (100 points each)
300 pts
Final (200 points)
200 pts
Oral Presentation
100 pts
Total
1000 pts
Lab reports are due within two weeks of
completion of lab
Important Dates to Know
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Jan 17 University Holiday
Mar 12-20 Spring Break
Mar 25 University Holiday
Apr 4 Last day to drop a course
Course Objectives
• To learn about the theory, instrumentation, and
applications of instrumental analysis methods
• To gain experience acquiring, treating, and
interpreting data
• To gain experience reading and writing scientific
documents and presenting orally
• To gain exposure to a wide range of instrumental
techniques and the fields associated with them
Scope and Relevancy of
Analytical Chemistry/Instrumental
Analysis
• Approximately 66% of all products and
services delivered in the US rely on
chemical analyses of one sort or another
• Approximately 250,000,000 chemical
determinations are performed in the US
each day
– NIST, 1991, from Managing the Modern Laboratory, 1(1), 1995, 1-9.
Classification of Analytical Methods
• Classical
– Also called wet-chemical methods
– Separation of component of interest (analyte)
from the sample by precipitation, extraction,
or distillation
– Followed by gravimetric or titrimetric
measurement for quantitative analysis
• Instrumental
– Use of new methods for quantitative analysis
Instrumental Methods
• Involve interactions of analyte with EMR
– Radiant energy is either produced by the
analyte (eg., Auger) or changes in EMR are
brought about by its interaction with the
sample (eg., NMR)
• Other methods include measurement of
electrical properties (eg., potentiometry)
Instruments
• Converts information stored in the physical or
chemical characteristics of the analyte into
useful information
• Require a source of energy to stimulate
measurable response from analyte
• Data domains
– Methods of encoding information electrically
– Nonelectrical domains
– Electrical domains
• Analog, Time, Digital
• Detector
– Device that indicates a change in one variable in its
environment (eg., pressure, temp, particles)
– Can be mechanical, electrical, or chemical
• Sensor
– Analytical device capable of monitoring specific
chemical species continuously and reversibly
• Transducer
– Devices that convert information in nonelectrical
domains to electrical domains and the converse
Selecting an Analytical Method
• What accuracy is required
• How much sample is available
• What is the concentration range of the
analyte
• What components of the sample will cause
interference
• What are the physical and chemical
properties of the sample matrix
• How many samples are to be analyzed
Accuracy vs. Precision
• Accuracy
– Describes the correctness of an experimental result
– Absolute error
– Relative error
• Precision
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Describes the reproducibility of results
Standard deviation
Variance
CV
Figures of Merit
• Precision
– Degree of mutual agreement among data that
have been obtained in the same way
– A measure of the random, or indeterminate
error of an analysis
– FOM
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Absolute standard deviation
Relative standard deviation
Coefficient of variation
Variance
Bias
• A measure of the systematic, or
determinate, error of an analytical method
• Bias = µ - xt
• In developing an analytical method,
sources of bias should be identified and
eliminated or corrected for with use of
blanks or instrument calibration
Standard Reference Materials
(SRM)
• Provided by National Institute of Standards and
Technology (NIST)
• Specifically prepared for validation of analytical
methods
• Concentration of constituents has been
determined by
– A previously validated reference method
– 2 or more independent, reliable measurement
methods
– Analyses from a network of cooperating labs
Sensitivity
• Of an instrument or method is its ability to
discriminate between small differences in
analyte concentration
• 2 factors limit sensitivity
– Slope of calibration curve
– Precision of measuring device
Detection Limit
• The minimum concentration or mass of
analyte that can be detected at a known
confidence level
• Sm = Sbl + ksbl
Dynamic Range
• Extends from the lowest concentration at
which quantitative measurements can be
made (LOQ), to the concentration at which
the calibration curve departs from linearity
(LOL)
• An analytical method should have a
dynamic range of at least 2 orders of
magnitude
Selectivity
• Of an analytical method refers to the
degree to which the method is free from
interference by other species contained in
the sample matrix
• No method is totally free from interference
from other species
Calibration of Instrumental Methods
• Analytical methods require calibration
• Process that relates the measured
analytical signal to the concentration of
analyte
• 3 common methods
– Calibration curve
– Standard addition method
– Internal standard method
Calibration Curve
• Standards containing known concentrations of the
analyte are introduced into the instrument
• Response is recorded
• Response is corrected for instrument output obtained
with a blank
– Blank contains all of the components of the original sample
except for the analyte
• Resulting data are then plotted to give a graph of
corrected instrument response vs. analyte concentration
• An equation is developed for the calibration curve by a
least-squares technique so that sample concentrations
can be computed directly
Standard Addition Method
• Usually involves adding one or more
increments of a standard solution to
sample aliquots of the same size (spiking)
Lab 1: Spectrophotometric Analysis
of a Mixture of Absorbing
Substances
• Purpose is to determine the individual
concentrations of a mixture of absorbing
substances
• Gain experience working with a UV-Vis
Spectrophotometer
• Practice several analytical techniques
• Understand absorbance and application of
the Beer-Lambert Law
Background: Absorption of
Radiation
• Absorption – A process in which
electromagnetic energy is transferred to the
atoms, ions, or molecules composing a sample
– Promotes particles from their normal room
temperature state (ground state) to one or more
higher-energy states.
• Atoms, molecules or ions have a limited number
of discrete energy levels
• For absorption to occur, the energy of the
exciting photon must exactly match the energy
difference between the ground state and an
excited state of the absorbing species
Absorption Methods
• Absorbance A of a medium is defined
A = -log10T = log10P0/P
• Beer-Lambert Law is defined
A = Єbc
b
P0
P
Absorbing solution of
concentration, c
Lab Report Write-up
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Introduction to spectroscopy, instrument basics, absorption principles and BeerLambert Law
Experimental section
– Specific instrumention (www.oceanoptics.com)
– Experimental procedures
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Results
– Abs vs. wavelength spectra
– Plots of concentration vs. absorbance, including equations of lines and R2
• Red at λ1 and at λ2
• Yellow at λ1 and at λ2
– Tables
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Dilutions
Red absorbance by concentration
Yellow absorbance by concentration
Є values
– Equations and unknown concentrations
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Conclusions
References
Spectrophotometric Analysis of a Mixture of
Absorbing Substances
Absorbance
1
0.9
0.8
0.7
0.6
0.5
Red 6 ppm
0.4
0.3
0.2
0.1
0
300
500
700
900
Wavelength, nm
1100
Badger Red
0.6
0.5
Abs
0.4
abs at l1
0.3
abs at l2
0.2
0.1
0
0
2
4
6
Conc, ppm
8
10
12
Signals and Noise
• Analytical measurements consist of 2
components
– Signal
– Noise
• Signal to noise ratio
– S/N = x/s = mean / standard deviation
Sources of Noise in Instrumental
Analysis
• Chemical Noise
• Instrumental Noise
– Thermal noise
– Shot noise
– Flicker noise
– Environmental noise
Signal to Noise Enhancement
• Hardware
• Software
– Ensemble Averaging
– Boxcar Averaging
– Digital Averaging
• Fourier transformation
An Introduction to Spectrometric
Methods
• Spectroscopy
– Interactions of various types of radiation with
matter
• Electromagnetic radiation (light, X-Rays)
• Ions and electrons
Properties of EMR
• Described by means of sine wave
– Wavelength, frequency, velocity, amplitude
– Particle model of radiation is necessary
– Represented as electric and magnetic fields
that undergo sinusoidal oscillations at right
angles to each other and the direction of
propogation
• vi = n li
• Frequency is determined by source and remains
invariant
• Velocity depends on medium
• Velocity (air or vacuum) = c = 3.00 x 108 m/s = l n
Transmission of Radiation
• Refractive index
– A measure of the interaction of radiation with
the medium it travels through
hi = c/vi
Scattering of Radiation
• Small fraction of radiation is scattered as it
passes through a medium
– Rayleigh Scattering (elastic)
• Scattering by molecules with wavelengths smaller
than wavelength of radiation
• Its intensity is proportional to 1/l4
– Raman Scattering (inelastic)
Diffraction of Radiation
• All types of EMR exhibit diffraction
• Is a consequence of interference
• A parallel beam of radiation is bent as it
passes a barrier or slit
• nl = BC sin q (Bragg Equation)
The Photoelectric Effect
• Experiments revealed that a spark jumped
more readily between 2 charged spheres
when their surfaces were illuminated with
light
• EMR is a form of energy that releases
electrons from metallic surfaces
• Below a certain frequency, no additional
sparks (electrons) are observed
• E = hn (Einstein)
• eV0 = hn - w
• E = hn = eV0 + w
Emission of Radiation
• EMR is produced when excited particles
(atoms, ions, or molecules) relax to lower
energy levels by giving up their excess
energy as photons
• Excitation can be brought about by
– Bombardment with electrons
– Irradiation with a beam of EMR
• Radiation from an excited source is
characterized by an emission spectrum
– Plot of relative power of emitted radiation vs
wavelength or frequency
– Types of spectra
• Line
• Band
• Continuum
Absorption of Radiation
• In absorption, EM energy is transferred to
atoms, molecules comprising the sample
• Absorption promotes these particles from RT
state to a higher-energy excited state
• For absorption to occur, 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
Atomic Absorption
• Passage of radiation through a medium
that consists of monoatomic particles
results in absorption of a few frequencies
• Simplicity is due to small number of
possible energy states for the absorbing
particles
Molecular Absorption
• More complex because the number of
energy states is large compared to
isolated atoms
• The energy, E, associated with the
molecular bands:
E = Eelectronic + Evibrational + Erotational
Components of Optical Instruments
• Stable source of radiant energy
• Transparent sample container
• Device that isolates a restricted region of
the spectrum
• Radiation detector
• Signal processor and readout
Sources of Radiation
• Source must generate a beam of radiation
with sufficient power
• Output must be stable for reasonable
periods
• Radiant power of a source varies
exponentially with the voltage of its power
supply
– Continuum (tungsten)
– Line (lasers)
Laser Sources
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High intensity
Narrow bandwidth
Coherent nature of output
Components
– Lasing medium
– External source
– Mirrors
Laser Mechanism
• Pumping
– Excitation of active species
– Causes population inversion
• Spontaneous Emission
– Random process yielding incoherent monochromatic
radiation
• Stimulated Emission
– Emitted photon has same energy and is in phase with
photon that caused the emission
– Therefore is coherent
• Absorption
– Competes with stimulated emission
Wavelength Selectors
• Narrow bandwidth is required
• Filters
• Monochromators, consisting of
– Entrance slit
– Collimating lens (or mirror)
– Grating (or prism, historical)
– Focusing element
– Exit slit
Radiation Transducers
• Convert radiant energy into an electrical
signal
• Photon transducers
– Photomultiplier tube (PMT)
• Contain a photoemissive surface
• Emit a cascade of electrons when struck by
electrons
• Useful for measurement of low radiant power
Component Configuration for
Optical Absorption Spectroscopy
Source Lamp
Photoelectric
Transducer
Sample Holder
Wavelength
Selector
Signal Processor
and Readout
Atomic Absorption Spectrometry
• Most widely used method for determination
of single elements in analytical chemistry
• Quantification of energy absorbed from an
incident radiation source from the
promotion of elemental electrons from the
ground state
• Technique relies on a source of free
elemental atoms electronically excited by
monochromatic light
Sample Introduction in AAS
• Flame
– Method of supplying atom source
– Utilizes a nebulizer in conjunction with
air/acetylene flame
– Solvent evaporates
– Metal salt vaporizes and is reduced to
complete the atomization process
• Radiation source is a hollow cathode lamp
Graphite Furnace AAS
• Samples are atomized by electrothermal
atomization
• Provide an increase in sensitivity and
improved safety compared to Flame AAS
instruments
• Applications
Mass Spectrometry
• Relies on separating gaseous charged
ions according to their mass-to-charge
ratio (m/z)
• Widely used in conjunction with other
analytical techniques
Operating Principles
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Sample inlet
Sample ionization
Ion acceleration by an electric field
Ion dispersion according to m/z
Identification of ion mass
Mass to Charge Ratio
• Obtained by dividing the atomic or
molecular mass of an ion, m, by the
number of charges, z, of the ion
• Most ions are singly charged
Vacuum Systems
• Reduce gas molecules in analysis
chamber
– Ion detectors need low pressure environment
– Don’t want to detect gas in system
– Want to detect analyte before it can collide or
react with gas in system
– Surface analysis: a need for analyzing an
unoxidized surface
Vacuum Pump Selection
• Atmosphere to 10-2 Torr
– Roughing pumps
• 10-4 – 10-8 Torr
– Diffusion pumps
– Turbomolecular pumps
– Cryogenic pumps
• 10-9 – 10-12 Torr
– Ion pumps
Vacuum Regions
• Rough Vacuum
– 760-1 Torr
• Mid Vacuum
– 1 – 10-3 Torr
• High Vacuum
– 10-3 – 10-7 Torr
• Ultrahigh Vacuum (UHV)
– <10-7 Torr
Vacuum Characteristics at Room
Temperature
Pressure,
Fraction of
Atoms
Pressure, Torr Number
Density
Mean Free
Path, cm
Time for One
Monolayer, s
1/1,000
0.76
2.5 x 1016
0.0065
3 x 10-6
1/10,000
7.6 x 10-2
2.5 x 1015
0.065
3 x 10-5
1/100,000
7.6 x 10-3
2.5 x 1014
0.65
3 x 10-4
1/1,000,000
7.6 x 10-4
2.5 x 1013
6.5
3 x 10-3
1/10,000,000
7.6 x 10-5
2.5 x 1012
65
3 x 10-2
1/100,000,000 7.6 x 10-6
2.5 x 1011
650
3 x 10-1
Molecular Absorption
• Measurement of Transmission and
Absorption
• Limitations to Beer-Lambert Law
– Concentration
– Chemical deviations
– Polychromatic Radiation
Raman Spectroscopy, Chapter 18
March 2
• Review concepts from Chapter 6, Fig 6-20, and
Section 6C-5
– Relaxation processes
– Emission processes
• Fluorescence
• Phosphorescence
• Theory and mechanisms of Raman
Spectroscopy
– 18A1, 18A2, 18B1, 18C
– Homework: 18-2, 18-3
Fluorescence and Phosphorescence
• Following absorption
– Nonradiative relaxation
• Loss of energy in a series of small steps
• Energy of molecule is conserved
– Fluorescence Emission
• Excited State analyte molecule returns to the GS producing
radiative emission (a photon is emitted)
• ~10-5 s
– Phosphorescence Emission
• Similar to fluorescence but process is > 10-5 s
• Due to relaxation from an excited triplet state
Raman Scattering
• In addition to fluorescent and phosphorescent
emission, scattering of photons off analyte
molecule occurs
• Raman scattering
– Inelastic
– Scattered photons have a frequency that differs from
incident photons
• Rayleigh scattering
– Elastic
– Scattered photons have the same frequency as
incident photons
History
• Effect discovered by Indian physicist C.V.
Raman in 1928
• Nobel Prize in 1931
• Not widely used until 1960s when lasers
became available
– Raman lines are 0.001% of intensity of source
Raman vs. Infrared Spectroscopy
• Techniques are complementary
• Raman spectra can be obtained from
aqueous solutions
– Main advantage over IR
• More detail on Raman vs. IR next week
Mechanism of Raman Scattering
• Spectral acquisition
– Powerful laser source
• Visible or near-IR monochromatic radiation
• Emitted radiation (Figure 18-2)
– Stokes scattering: E = hn – DE
– anti-Stokes scattering: E = hn + DE
– Rayleigh scattering: E = hn
• Wavelength is equal to that of excitation source
Units
• Wavenumbers (cm-1) are convention
– Easy to convert between wavelength and
frequency
Infrared Spectroscopy, Chapter 16
March 10, 2005
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Theory of Infrared Spectroscopy
Components
Read Sections 16A, 16B
Homework: 16-2
IR Spectral Regions, Table 16-1
Region
Wavelength Range,
mm
Wavenumber
Range, cm-1
Frequency Range,
Hz
Near
0.78-2.5
12,800-4,000
3.8E14 – 1.2E14
Middle
2.5-50
4,000-200
1.2E14-6.0E12
Far
50-1,000
200-10
6.0E12-3.0E11
Most used
2.5-15
4,000-670
1.2E14-2.0E13
Dipole Changes During Molecular Vibrations
• IR radiation is not energetic enough to cause
electronic transitions
• To absorb IR radiation, a molecule must undergo
a net change in dipole moment due to its
vibrational (or rotational) motion
• If n of EMR matches a vibrational frequency of
the molecule, a net transfer of energy occurs
– Results in change in amplitude of vibration
– Absorption of radiation occurs
Types of Molecular Vibrations
• Stretching
– Continuous change in interatomic distance
along axis of atomic bond
• Bending
– Characterized by a change in angle between
2 bonds
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Scissoring
Rocking
Wagging
Twisting
Simple Harmonic Oscillator
• Model which approximates atomic stretching
vibrations
• Vibration of a single mass attached to a spring
hung from an immovable object (Figure 16-3a) :
F = -ky
Vibrational Frequency
1
vm 
2
k
m
1
vm 
2
k
1

m 2
1
v
2c
k
m
m1m2
m
m1  m2
(16-7)
k (m1  m2 )
m1m2
 5.3 x10
12
k
m
(16-9)
(16-14)
(16-8)
Vibrational Modes
• Linear molecules
3N-5 (number of possible molecular vibrations)
• Polyatomic molecules
3N-6 (number of possible molecular vibrations)
Infrared Sources
• Inert solid electrically heated to 1500-2200K
• Nernst Glower
– Rare earth oxides formed into a cylinder
– Formed into a resistive heating element, 1200-2200K
• Globar Source
– Silicon carbide rod, also electrically heated, 13001500K
– Greater output than Nernst Glower below 5 mm
• Tungsten Filament Lamp
– Used in near-IR region of 4,000-12,800 cm-1
• Infrared lasers
Chromatographic Separations, Chapter 26
March 30, 2005
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26 A, General Description
26 B, Migration Rates
26 C, Zone Broadening and Column Efficiency
26 D, Optimization of Column Performance
– Through 26 D-5, General Elution Problem
• Example 26-1, Homework 26-16 and 26-17
• Chapter 28 G through 28 G-3
General Description
• In all chromatographic separations, the sample
is transported in a mobile phase
– Gas, liquid, or supercritical fluid
– fundamental classification
• Mobile phase is forced through an immiscible
stationary phase
– Column or solid surface
• As a consequence of differences in mobility,
sample separates into bands or zones
Chromatograms
• Plot of analyte concentration vs. time
• Positions of peaks on time axis identify
components of sample
• Areas under peaks provide quantitative
measure of amount of each component
• Figure 26-4
Migration Rates of Solutes
• Distribution constant
Amobile ↔ Astationary
cs
K
cm
• Retention Factor
tR  tM
k A 
tM
Chromatographic Peak Shape
• Similar to normal error or Gaussian curve
• Attributed to additive combination of
random motions of solute molecules in
chromatographic zone
• Peak represents behavior of average
molecule
• Breadth of band is directly related to
residence time in column and inversely
related to mobile phase velocity
Column Efficiency
• Plate height
2
LW
H
2
16t R
• Plate count, N = L/H
• Maximum efficiency occurs at minimum H
Column Resolution
• Resolution, Rs, provides a quantitative
measure of its ability to separate analytes
2[(t R ) B  (t R ) A ]
Rs 
WA  WB