Analytic Chem

Chem 155 Unit 1 Page 1 of 316
Chemistry 155
Introduction to Instrumental
Analytical Chemistry
Unit 1
Spring 2010
San Jose State University
Roger Terrill
Page 1 of 316
1 Overview and Review ........................................................................................ 7
2 Propagation of Error......................................................................................... 56
3 Introduction to Spectrometric Methods ............................................................ 65
4 Photometric Methods and Spectroscopic Instrumentation ............................... 86
5 Radiation Transducers (Light Detectors): ...................................................... 103
6 Monochromators for Atomic Spectroscopy: ................................................... 117
7 Photometric Issues in Atomic Spectroscopy .................................................. 138
8 Practical aspects of atomic spectroscopy: ..................................................... 152
9 Atomic Emission Spectroscopy ...................................................................... 163
10 Ultraviolet-Visible and Near Infrared Absorption .......................................... 178
11 UV-Visible Spectroscopy of Molecules ........................................................ 194
12 Intro to Fourier Transform Infrared Spectroscopy ........................................ 211
13 Infrared Spectrometry: ................................................................................. 234
14 Infrared Spectrometry - Applications ............................................................ 247
15 Raman Spectroscopy: .................................................................................. 259
16 Mass Spectrometry (MS) overview: ............................................................. 279
17 Chromatography .......................................................................................... 294
Page 2 of 316
1 Overview and Review ................................................................................ 7
1.1 Tools of Instrumental Analytical Chem. ............................................. 8
1.2 Instrumental vs. Classical Methods. ................................................ 12
1.3 Vocabulary: Basic Instrumental ....................................................... 13
1.4 Vocabulary: Basic Statistics Review................................................ 14
1.5 Statistics Review ............................................................................. 15
1.6 Calibration Curves and Sensitivity ................................................... 23
1.7 Vocabulary: Properties of Measurements ....................................... 24
1.8 Detection Limit ................................................................................ 25
1.9 Linear Regression ........................................................................... 31
1.10 Experimental Design: ................................................................... 35
1.11 Validation Assurance of Accuracy: ............................................ 43
1.12 Spike Recovery Validates Sample Prep. ..................................... 45
1.13 Reagent Blanks for High Accuracy: ............................................. 46
1.14 Standard additions fix matrix effects: ........................................... 47
1.15 Internal Standards ........................................................................ 52
2 Propagation of Error ................................................................................. 56
3 Introduction to Spectrometric Methods ..................................................... 65
3.1 Electromagnetic Radiation: ............................................................. 66
3.2 Energy Nomogram .......................................................................... 67
3.3 Diffraction ........................................................................................ 68
3.4 Properties of Electromagnetic Radiation: ........................................ 71
4 Photometric Methods and Spectroscopic Instrumentation ....................... 86
4.1 General Photometric Designs for the Quantitation of Chemical
Species .................................................................................................... 87
4.2 Block Diagrams ............................................................................... 88
4.3 Optical Materials ............................................................................. 89
4.4 Optical Sources ............................................................................... 90
4.5 Continuum Sources of Light: ........................................................... 91
4.6 Line Sources of Light:...................................................................... 92
4.7 Laser Sources of Light: ................................................................... 93
5 Radiation Transducers (Light Detectors): ............................................... 103
5.1 Desired Properties of a Detector: .................................................. 103
5.2 Photoelectric effect photometers ................................................... 104
5.3 Limitations to photoelectric detectors: ........................................... 106
5.4 Operation of the PMT detector: ..................................................... 107
5.5 PMT Gain Equation: ...................................................................... 108
5.6 Noise in PMTs and Single Photon Counting: ................................ 110
5.7 Semiconductor-Based Light Detectors: ......................................... 112
5.8 Charge Coupled Device Array Detectors: ..................................... 115
6 Monochromators for Atomic Spectroscopy:............................................ 117
6.1 Adjustable Wavelength Selectors .................................................. 118
6.2 Monochromator Designs: .............................................................. 119
6.3 The Grating Equation: ................................................................... 120
6.4 Dispersion ..................................................................................... 123
6.5 Angular dispersion:........................................................................ 124
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6.6 Effective bandwidth ....................................................................... 126
6.7 Bandwith and Atomic Spectroscopy .............................................. 127
6.8 Factors That Control EFF ............................................................ 128
6.9 Resolution Defined ........................................................................ 129
6.10 Grating Resolution ..................................................................... 130
6.11 Grating Resolution Exercise:...................................................... 131
6.12 High Resolution and Echelle Monochromators .......................... 133
7 Photometric Issues in Atomic Spectroscopy .......................................... 138
8 Practical aspects of atomic spectroscopy:.............................................. 152
8.1 Nebulization (sample introduction): ............................................... 153
8.2 Atomization ................................................................................... 157
8.3 Flame Chemistry and Matrix Effects.............................................. 158
8.4 Flame as sample holder: ............................................................. 159
8.5 Optimal observation height: ........................................................... 160
8.6 Flame Chemistry and Interferences: ............................................. 161
8.7 Matrix adjustments in atomic spectroscopy: .................................. 162
9 Atomic Emission Spectroscopy .............................................................. 163
9.1 AAS / AES Review: ....................................................................... 164
9.2 Types of AES: ............................................................................... 165
9.3 Inert-Gas Plasma Properties (ICP,DCP) ....................................... 166
9.4 Predominant Species are Ar, Ar+, and electrons ........................... 166
9.5 Inductively Coupled Plasma AES: ICP-AES .................................. 167
9.6 ICP Torches .................................................................................. 168
9.7 Atomization in Ar-ICP .................................................................... 169
9.8 Direct Current Plasma AES: DCP-AES ........................................ 170
9.9 Advantages of Emission Methods ................................................. 171
9.10 Accuracy and Precision in AES .................................................. 173
10 Ultraviolet-Visible and Near Infrared Absorption .................................... 178
10.1 Overview .................................................................................... 178
10.2 ........................................................................................................ 178
10.3 The Blank ................................................................................... 179
10.4 Theory of light absorbance:........................................................ 180
10.5 Extinction Cross Section Exercise: ............................................ 181
10.6 Limitations to Beers Law: .......................................................... 182
10.7 Noise in Absorbance Calculations: ............................................ 184
10.8 Deviations due to Shifting Equilibria: .......................................... 185
10.9 Monochromator Slit Convolution in UV-Vis: ............................... 188
10.10 UV-Vis Instrumentation: .......................................................... 190
10.11 Single vs. double-beam instruments: ...................................... 191
11 UV-Visible Spectroscopy of Molecules ................................................... 194
11.1 Spectral Assignments ................................................................ 195
11.2 Classification of Electronic Transitions ....................................... 196
11.3 Spectral Peak Broadening ......................................................... 197
11.4 Aromatic UV-Visible absorptions: ............................................... 201
11.5 UV-Visible Bands of Aqeuous Transition Metal Ions .................. 202
11.6 Charge-Transfer Complexes ...................................................... 205
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11.7 Lanthanide and Actinide Ions: .................................................... 206
11.8 Photometric Titration .................................................................. 207
11.9 Multi-component Analyses: ........................................................ 208
12 Intro to Fourier Transform Infrared Spectroscopy .................................. 211
12.1 Overview: ................................................................................... 212
1 molecular vibrations ............................................................................... 212
12.2 IR Spectroscopy is Difficult! ....................................................... 215
12.3 Monochromators Are Rarely Used in IR..................................... 216
12.4 Interferometers measure light field vs. time ............................... 217
12.5 The Michelson interferometer: ................................................... 218
12.6 How is interferometry performed? .............................................. 219
12.7 Signal Fluctuations for a Moving Mirror ...................................... 220
12.8 Mono and polychromatic response ............................................ 222
12.9 Interferograms are not informative: ............................................ 223
12.10 Transforming time frequency domain signals: .................... 224
12.11 The Centerburst: ..................................................................... 225
12.12 Time vs. frequency domain signals:........................................ 226
12.13 Advantages of Interferometry. ................................................ 227
12.14 Resolution in Interferometry .................................................... 228
12.15 Conclusions and Questions: ................................................... 232
12.16 Answers: ................................................................................. 233
13 Infrared Spectrometry: ........................................................................... 234
13.1 Absorbance Bands Seen in the Infrared: ................................... 235
13.2 IR Selection Rules ..................................................................... 236
13.3 Rotational Activity ...................................................................... 238
13.4 Normal Modes of Vibration:........................................................ 239
13.5 Group frequencies: a pleasant fiction! ........................................ 242
13.6 Summary:................................................................................... 246
14 Infrared Spectrometry - Applications ...................................................... 247
14.1 Strategies used to make IR spectrometry work - ....................... 248
14.2 Solvents for IR spectroscopy: .................................................... 249
14.3 Handling of neat (pure no solvent) liquids: .............................. 249
14.4 Handling of solids: pelletizing: .................................................... 250
14.5 Handling of Solids: mulling: ........................................................ 250
14.6 A general problem with pellets and mulls: .................................. 251
14.7 Group Frequencies Examples.................................................... 252
14.8 Fingerprint Examples ................................................................. 253
14.9 Diffuse Reflectance Methods: .................................................... 254
14.10 Quantitation of Diffuse Reflectance Spectra: .......................... 255
14.11 Attenuated Total Reflection Spectra: ...................................... 256
15 Raman Spectroscopy: ............................................................................ 259
15.1 What a Raman Spectrum Looks Like ......................................... 261
15.2 Quantum View of Raman Scattering. ......................................... 262
15.3 Classical View of Raman Scattering .......................................... 263
15.4 The classical model of Raman: .................................................. 265
15.5 The classical model: catastrophe! .............................................. 266
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15.6 Raman Activity: .......................................................................... 267
15.7 Some general points regarding Raman: .................................... 269
15.8 Resonance Raman .................................................................... 271
15.9 Raman Exercises ....................................................................... 272
16 Mass Spectrometry (MS) overview: ....................................................... 279
16.1 Example: of a GCMS instrument: ............................................... 279
16.2 Block diagram of MS instrument. ............................................... 280
16.3 Information from ion mass.......................................................... 281
16.4 Ionization Sources ..................................................................... 282
16.5 Mass Analyzers:......................................................................... 287
16.6 Mass Spec Questions: ............................................................... 292
17 Chromatography Chapter 26 ............................................................... 294
17.1 General Elution Problem / Gradient Elution ............................... 307
17.2 T-gradient example in GC of a complex mixture. ....................... 309
17.3 High Performance Liquid Chromatography ................................ 310
17.4 Types of Liquid Chromatography ............................................... 311
17.5 Normal Phase: ........................................................................... 311
17.6 HPLC System overview: ............................................................ 314
17.7 Example of Reverse-phase HPLC stationary phase: ................. 315
17.8 Ideal qualities of HPLC stationary phase: .................................. 316
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1 Overview and Review

Skoog Ch 1A,B,C (Lightly) 1D, 1E Emphasized
Analytical Chemistry is Measurement Science.
Simplistically, the Analytical Chemist answers the
following questions:
What chemicals are present in a sample?

QUALITATIVE ANALYSIS
At what concentrations are they present?
QUANTITATIVE ANALYSIS
Additionally, Analytical Chemists are asked:

Where are the chemicals in the sample?
liver, kidney, brain
surface, bulk
What chemical forms are present?

Are metals complexed?
Are acids protonated?
Are polymers randomly coiled or crystalline?
Are aggregates present or are molecules in
solution dissociate?
At what temperature does this chemical
decompose?
Myriad questions about chemical states
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1.1 Tools of Instrumental Analytical Chem.

1.1.1 Spectroscopy w/ Electromagnetic (EM)
Radiation
Name of EM Wavelength Predominant Name of

regime: Excitation Spectroscopy
Gamma ray 0.1 nm Nuclear Mossbauer
X-Ray 0.1 to 10 nm Core x-ray absorption,

electron fluorescence, xps
Vacuum 10 - 180 nm Valence Vuv
Ultraviolet electron
Ultraviolet 180 - 400 Valence Uv or uv-vis
electron
Visible 400-800 Valence Vis or uv-vis
electron
Near Infrared 800-2,500 Vibration Near IR or NIR
(overtones)
Infrared 2.5-40 m Vibration IR or FTIR
Microwave 40 m 1 rotations Rotational or

mm microwave
Microwave 30 mm Electron spin ESR or EPR
in mag field
Radiowave 1m Nuclear spin NMR
in mag field
Page 8 of 316
1.1.2 Chromatography Chemical Separations
Different chemicals flow through separation

medium (column or capillary) at different speeds
plug of mixture goes in chemicals come out of
column one-by-one (ideally)
Gas Chromatography GC
Powerful but Suitable for Volatile chemicals only
Liquid Chromatography High Performance

(pressure), HPLC in its many forms
Electrophoresis -Liquids, pump with electric

current, capillary, gel, etc.
Chromatogram
absorbance
time / s
Page 9 of 316
1.1.3 Mass Spectrometry
Detection method where sample is:

volatilized,
injected into vacuum chamber,
ionized,
usually fragmented,
accelerated,
ions are weighed as M/z mass charge.
Often coupled to:
chromatograph
laser ablation
atmospheric sniffer.
Very sensitive (pg) quantitation

Powerful identification tool
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1.1.4 Electrochemistry
Simple, sensitive, limited to certain chemicals
Ion selective electrodes (ISEs):

e.g. pH, pCl, pO2 etc.
ISEs measure voltage across a selectively
permeable membrane (e.g. glass for pH)
E log[concentration]
ISEs have incredible dynamic range!
pH 4 pH 10
[H+] = 0.0001 0.0000000001 M
Dynamic electrochemistry
measure current (i) resulting from redox
reactions at an driven by a controlled voltage
at an electrode surface
i(E,t) [concentration]
1.1.5 Gravimetry
Precipitate and weigh products
very precise, very limited
1.1.6 Thermal Analysis
Thermogravimetric Analysis TGA

Mass loss during heating loss of waters of
hydration, or decomposition temperature
Differential Scanning Calorimetry DSC

Heat flow during heating or cooling
Page 11 of 316
Relax, you dont need
1.2 Instrumental vs. Classical Methods. to memorize this table
just humor Dr. Terrill
while he talks about it.
Methods of Analytical Chemistry
# Chemicals # Chemicals
Isolated / hr Isolated / hr
Classical Instrumental
and amount and amount
(g) (g)
High Performance
Extraction 1-2 g Liquid 10 ng
Chromatrography
Gas
Distillation 1-2 g 100 ng
Chromatography
Separation
Precipitation 1-2 g
Electrophoresis 50 pg
Crystallization 1-2 g
Estimated Number of uniquely identifiable molecules by method

Combination of UV-Vis 1,000s
Color / Smell
Melt / Boiling Infrared 100,000s
Qualitative Point,
100s
Speciation Solubility Mass Spectrometry > 106
Wetting
Density NMR
> 106
Hardness Spectroscopy
Best Quantitative Precision and Sensitivity
Optical
Titration 0.1
1 ppm Spectroscopy 0.1% 10-23 M
%
0.1%
Mass
Quantitation Gravimetry 0.01 amount
1 ppm Spectrometry 10-13 M
Precision % -4
10 %
mass
Colorimetry NMR 100
10% 1 ppm
Spectroscopy 1% pppm
What are the more precise measurements that you

have made and what were they?
Page 12 of 316
1.3 Vocabulary: Basic Instrumental
Analyte The chemical species that is

being measured.
Matrix The liquid, solid or mixed
material in which the analyte
must be determined.
Detector Device that records physical or
chemical quantity.
Transducer The sensitive part of a detector
that converts the chemical or
physical signal into an electrical
signal.
Sensor Device that reversibly monitors a
particular chemical e.g. pH
electrode
Analog signal A transducer output such as a
voltage, current or light intensity.
Digital signal When an analog signal has been
converted to a number, such
3022, it is referred to as a digital
signal.
Analog signals are susceptible to distortion, and so
are usually converted into digital signals (numbers)
promptly for storage, transmission or readout.
Page 13 of 316
1.4 Vocabulary: Basic Statistics Review
Precision If repeated measurements of the same thing are

all very close to one another, then a
measurement is precise. Note that precise
measurements may not be accurate (see below).
Random Error Random error is a measure of precision.
Random differences between sequential
measurements reflect the random error.
Accuracy If a measurement of something is correct, i.e.
close to the true value, then that measurement is
accurate.
Systematic Error Systematic error is the difference between the
mean of a population of measurements and the
(bias) true value.
Histogram A graph of the number or frequency of
occurences of a certain measurement versus the
measurement value.
Probability A theoretical curve of the probability of a certain
measured value occurring versus the measured
Distribution value.
A histogram of a set of data will often look like a Gaussian probability distribution.
Average The sum of the measured . xn
values divided by the number
n
of measurements. x MEAN
n
Median Half of the measurements fall above the median
value, and half fall below.
Variance ( 2) A measurement of
precision. The sum of the
x MEAN x n
2
2 n
squares of the random
measurement errors. n 1
Standard A widely accepted 2

x M EAN x n
standard measurement of
Deviation ( ) precision. The square root n
of the variance. n 1
Relative Standard The standard deviation divided by the mean, and

often expressed as : %RSD= /xMEAN100%.
Deviation
Propagaion of When the mean of a set of measurement (x) has
a random error ( x), it is reported as x x. If we
Error wish to report the result of a calculation y=f(x)
based on x, we propagate the error through the
calculation using a mathematical method.
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6 2.94
a 73.278
84.096
93.404
103.493
113.431
1.5 Statistics Review 123.458
133.337
142.478
1.5.1 Precision and Accuracy 153.035
Histogram of normally distribut ed event s
80
Mean
Number of times it was observed
60
Mean -
one Mean +
40 standard one
deviation standard
20
deviation
0
0 1 2 3 4 5 6
Value observed
His togram of 1024 events
1.5.2 Basic Formulae
2
xN
Population
lim xN Standard N
N x lim
N Deviation: N N
Mean :
N
2
xN xN xavg
Sample
N Standard s N
Average: xavg N Deviation: x N 1
Bias or absolute systematic error = xavg

s
Relative standard deviation =
xavg
Page 15 of 316
When you make real measurements of things you generally dont

know the true value of the thing that you are measuring. (Call this
the true mean, , for now. For the purposes of this discussion let us
assume that there is no systematic (accuracy) error (i.e. no bias).
1.5.3 Confidence Interval

WHAT DO YOU DO TO ENSURE THAT YOUR ANSWER IS AS
CLOSE AS POSSIBLE TO THE TRUTH?
TAKE THE AVERAGE xAVG
But, you still dont know the exact answer
SO WHAT DO YOU REALLY WANT TO SAY?
I am highly confident that the true mean lies within

this interval (e.g between 92 and 94 grams).
In fact, there is only a 1 in 20 chance that I am wrong!
How do you calculate what that interval is?

You need to know:
The average of the data set: x

The standard deviation: or s
The number of measurements (observations) made: N
This interval is called a confidence interval (CI). Which is better,a

bigger or a smaller CI?
Smaller is better
How can you improve your CI?
Make more measurements (N)
Page 16 of 316
Confidence Interval (continued).

If you know the standard deviation, , (less common case), then:
The x% confidence interval for = xAVG z / N
If you have only a rough

If you dont know the standard
estimate of xAVG, then you are
deviation, , (more commonly the
less confident that it is close to
case), then:
, hence you divide by N.
The x% confidence interval for = xAVG ts / N

In this case t is a function of N
This leaves only z and t what are they? These numbers represent
the multiple of one standard deviation ( or s) that correspond to the
confidence interval. In the second case, s is only an estimate of , so
the error in s needs to be taken into account, so t is a function of the
number of degrees of freedom. For our purposes, i.e. averaging
multiple identical measurements, the number of degrees of freedom
is simply N-1.
Page 17 of 316
An example:
Assume that we do our best to measure the concentration of basic
amines in a fish tank. Our answers are 4.2, 4.6, 4.0.
N= 3
XAVG = (4.2+4.6+4.0) / 3 = 4.27
s= (4.2-4.27)2+(4.6-4.27)2+(4.0-4.27)2 = 0.31
3-1
Number of degrees of freedom for confidence interval = 3-1=2
95% confidence limits for = 4.27 (4.3*0.31) / (31/2)

= 4.270.93 or
5.4 = 4.3 0.9 or
= 3.4 to 5.2
5.0
4.5
3.5
Page 18 of 316
1.5.4 Confidence Interval (CI) in Words

Consider this
experiment. You have
a camera device that
97.5, 96.0,
measures the 99.1 98.055
temperature of objects
from a distance by
measuring their
97.323
infrared light emission. 99.051
It is very convenient
but somewhat
imprecise. Assume for the moment that the camera is perfectly
accurate that is, if you measure the same object with it many times
the average temperature result will equal the true temperature.
In order to evaluate the precision of your camera thermometer, you

measure the temperature of each item three times. In each case you
get an average and a standard deviation.
PROBLEM: The average camera reading is sometimes higher than

the true value, and sometimes lower, but you dont know how to
evaluate this fluctuation. In just a few words, how can you
characterize this fluctuation?
SOLUTION: Calculate the sample standard deviation.
QUESTION: A series of experiments, each of three measurements

each yields a set of sample standard deviations that also different
each time! If you repeat the whole experiment, but this time you
measure each sample ten times, then the standard deviations are
much closer, but still not equal each time.
Why does the sample standard deviation calculation give a different

result each time? Assume for the moment that the camera
performance (precision) is not changing.
ANSWER:
Realize this fact: Sample standard deviations are
only estimates.
Page 19 of 316
1.5.5 CI PROBLEM:
This imperfect camera thermometer is going to be used to screen
passengers boarding an airliner. Passengers with a high temperature
may have avian flu.
Our criterion is this: if there is more than a 90% probability that a

given passengers temperature exceeds 102, then we will take him
aside and test him for bird flu.
We have only moments to acquire three measurements per

passenger, so precision is low. Also, the precision is not the same
each time. For three passengers we get the following results:
Passenger 1: 100.3, 101.1, 103.0.

Passenger 2: 98.8, 98.5, 98.4
Passenger 3: 104.0, 103.9, 103.9
How do we answer the question: does this persons temperature

exceed our 90% / 102 criterion?
To answer this, we must accept the following: Assuming that

measurements are unbiased (accurate) we can state, for the 80% CI,
that there is a 10% probability that the true mean lies below the lower
limit of the CI, an 80% probability that the true mean lies within this
CI, and a 10% probability that the true mean lies above this CI.
So, there is a 90% probability that the true mean lies within or above
the 80% CI.
For example, if we took some measurements and then computed the

80% CI to be 101.8 to 102.6 then we could say that the probability
that the true temperature is 101.4 or higher is 90%.
101 102 103
10% chance 80% chance 10% chance

is 101.8 or lower 101.8 < < 102.6 is 102.6 or higher
Page 20 of 316
Use this table: Use this formula:

% confidence interval
ts
freedom 50 80 95 99 CI
1 1.00 3.08 12.71 63.66 N
2 0.82 1.89 4.30 9.92
3 0.76 1.64 3.18 5.84 Note the following:
4 0.74 1.53 2.78 4.60
5 0.73 1.48 2.57 4.03
6 0.72 1.44 2.45 3.71 For a straight average of N
7 0.71 1.41 2.36 3.50 points, the number of
8 0.71 1.40 2.31 3.36 degrees of freedom is N-1.
9 0.70 1.38 2.26 3.25
10 0.70 1.37 2.23 3.17
20 0.69 1.33 2.09 2.85
50 0.68 1.30 2.01 2.68
100 0.68 1.29 1.98 2.63
Complete the following table:
Lower Upper Is the probability that

Passenger TAVG(F) St boundary boundary the passengers
dev of 80% of 80% Temp is > 102
(F) CI CI 90% or more?
1 101.5 1.1
2 98.57 0.17
3 103.93 0.047
What is another way to word the conclusion above?
Assuming our equipment is accurate, then averaged over many

passengers, and using this criterion, our conclusion that the persons
temperature is > 102 will be wrong less than 10% of the time!
Page 21 of 316
1.5.6 tEXP PROBLEM:

Another way of approaching this type of problem is to calculate an
experimental value of t called tEXP.
In the example below, we will compare a measured result with an
exact one. The question one answers with tEXP is this: Am I confident
that the observed value (x) differs from the expected value ( )?
Our threshold temperature, exactly 102 was tested, so we can make
measurements and test the hypothesis that the true temperature is
greater than 102.
Given the following three measurements of a passengers
temperature: 103.76, 102.11, 105.38 calculate an experimental
value of the t statistic for this population relative to the true value of
102. Average = 103.75, std dev = 1.34
% confidence interval
freedom 50 80 95 99
xav N
1 1.00 3.08 12.71 63.66 texp
2 0.82 1.89 4.30 9.92 s
3 0.76 1.64 3.18 5.84 = tes t value
( 103.76 102) 3
t exp
1.34
t exp 2.275
Can you state with the given confidence that this persons
temperature differs from the expected value of 102?
99%? No
95%? No
80%? Yes
50%? Yes
Page 22 of 316
1.6 Calibration Curves and Sensitivity

A highly sensitive instrument can
discriminate between small differences in
Calibration Sensitvity: analyte concentration.
S = mC + Sb
S = instrument signal
C = analyte conc.
m = slope calibration
15
13.166
Sb = signal for blank
S
m = calibration =m
Instrument Signal (S)
sensitivity C
10
S S
i
S
5 But, can we
really
distinguish
C between small
changes in
0 0
0 20 40 60 80 100concentration?
0 C 100
i
Analyte Concentration (C)
= Analytical Sensitvity m 1 / = noise in

=m/ S and concentration
S C.
= ( S/ C) / S so
= 1/ C for the C So small is?

corresponding to S
Good
Bad
Page 23 of 316
1.7 Vocabulary: Properties of Measurements
Sensitivity A detector or instrument that responds to only a

small change in analyte concentration is
sensitive. Numerically, the sensitivity of an
instrument is the slope of the calibration curve in
untis of: signal / unit concentration often given
as m. Sometimes the detection limit (see below)
is called the instrument sensitivity but this is not
correct.
Selectivity The ratio of the sensitivity of an instrument to an
analyte to that of an interferant.
Specimen The material removed for analysis e.g. a given

tablet from an assembly line.
Sample The mixture that contains the chemical to be
measured e.g. the blood sample that we want
to measure iron in.
Analyte The chemical that is being measured e.g. the
iron in the blood sample.
Calibration Curve A linear or non-linear function relating instrument
response (signal) to analyte concentration.
Interferant Another chemical in the sample that either affects
the instruments sensitivity to the sample or gives
a signal of its own that may be indistinguishable
from the analyte signal.
Detection Limit CMIN or The minimum detectable concentration of
CM analyte. Usually defined as that concentration
that gives a signal of magnitude equal to three
times the standard deviation in the blank signal.
Limit of Quantitation The minimum concentration of analyte for which
(LOQ) an accurate determination of concentration can
be made. The LOQ is typically that concentration
for which the signal is 10x the standard deviation
of the blank signal.
Limit of Linearity (LOL) The largest concentration for which a calibration
curve remains linear.
Linear Dynamic Range The range of concentrations (or signal strengths)
(LDR) between the LOQ and the LOL. An instrument is
most useful within its LDR.
Page 24 of 316
1.8 Detection Limit

The detection limit is denoted CM
CM is the minimum concentration that can be
detected, or distinguished confidently from a blank.
Let us define the minimum detectable signal change

as: SM
Therefore: SM = mCM must be a multiple (n) of the

noise level in the blank: ( b).
SM 3 b = mCM
Minimum Signal due to Minimum

Detectable Signal blank detectable
concentration
By convention, m=3.
Derive a formula for CM based on B and m:
Derive a formula for CM based on :

1.8.1
Page 25 of 316
A Graphical Look at Detection Limit (CMIN)
Consider the minimum detectable signal in the

context of the confidence interval:
SMIN = 3 b + Sb
To what confidence interval does 3 correspond?
Assume N=2 (two replicate measurements of Sb)
Another way of saying this (crudely) is that we

consider a signal detected when it falls outside of the
boundaries corresponding to the 99% confidence
interval for the blank signal.
Page 26 of 316
1.8.2 Minimum Detectable Temperature Change?
Using the thinking that we developed for the general

case of signals with random error what do you think
is the probability that the following signal change is
due to random fluctuations?
Page 27 of 316
Dynamic Range
LOQ = Limit of Quantitation ( S/S 0.3)
LOL = Limit of Linearity
1.8.3
Between LOQ and LOL your instrument is most
useful!
This is called:
Linear Dynamic Range
Page 28 of 316
Selectivity
Sometimes another chemical species will add to or

subtract from your analyte signal
S = mACA + mBCB + mCCC + mDCD + + SB
Analyte Interferants Blank
Selectivity coefficients determine how serious an

interferant is to your determination of analyte CA:
kB,A = mB/mA, kC,A = mC/mA, etc
So:
S = mA(CA + kB,ACB + kC,ACC + ) + SB
Page 29 of 316
1.8.4 Direct Interference
In order to measure a concentration directly, without

corrections, kB,A, kC,A must be approximately:
If there is interference, it is necessary to know both:
and
before one can determine the desired quantity CA!
Page 30 of 316
1.9 Linear Regression
Least-Squares Assuming that a set of x,y data pairs are well

described by the linear function y = a+bx, and
Regression or assuming that most error is in y (x is more
Linear Regression precisely known) and the errors in y are not a
function of x, then the coefficients that minimize
the residual function:
2
= (yI (a + bxI))2
can be found from the following equations:
from Data Reduction and Error Analysis for the

Physical Sciences, Philip R. Bevington, cw. 1969
Mc Graw Hill.
Non-linear Methods for fitting arbitrary curves to data sets.
Regression
Standard Additions A nearly matrix-effect free form of analysis. A
standard additions plot is a linear plot of
Plot instrument signal versus quantity of a standard
analyte solution spiked or added to the unknown
analyte sample. The unknown analyte
concentration is derived from the concentration
axis-intercept of this plot.
Sample Matrix / The matrix is the solution, including solvent(s)
and all other solutes in which an analyte is
Standards Matrix dissolved or mixed
Matrix Effect A matrix effect refers to the case where the
instrumental sensitivity is different for the sample
and standards because of differences in the
matrix.
Internal Standards A calibration method in which fluctuation in the
instrument signals due to matrix effects are,
ideally, cancelled out by monitoring the
fluctuations in the instrument sensitivity to
chemicals, internal standards, that are chemically
similar to the analyte.
Page 31 of 316
1.9.1 Linear Least Squares Calibration

The most common method for determining the
concentration of an unknown analyte is the simple
calibration curve.
In the calibration curve method, one measures the
instrument signal for a range of analyte
concentrations (called standards) and develops an
approximate relationship (mathematically or
graphically) between some signal S and analyte
concentration C.
If the signal-concentration relationship is linear, then:
S = SB + mC y = a + bx
But, one can not just draw the line between any two
points because all the points have some error. So,
one mathematically attempts to minimize the
residuals.
2
= (yi (a + bxi))2
1.5
1.5
Instrument Signal (S)
S
i 1
S
i
F
i
F 0.5
i
0 0
0 2 4 6 8 10
0 C 10
i
Analyte Concentration (C)
Page 32 of 316
2
The values of a and b for which is a minimum are
the following:
The errors in the coefficients, a and b can be found,

similarly, using:
These quantities are best found using a computer

program. Modern versions of Microsoft Excel will
calculate a and b for you (use display equation
option on the trend line), and a and b if you have
the data analysis toolpack option installed. Excel is
also fairly well suited to doing the sums and formulas.
Page 33 of 316
See also, appendix a1C in Skoog, Holler and Nieman.

But, be advised that there is a typo in older editions.
Equation a1-32 should read:
m = SXY/SXX
Also it is a somewhat more subtle problem to

calculate the error in a concentration determined from
a calibration curve of signal (y) versus concentration
(x).
You will need to follow Skoog appendix-a calculations

to deal with this problem in your lab reports where you
determine an unknown concentration. M = the
number of replicate analyses, N = the number of data
points.
2
Equation a1-37 sy 1 1 y cavg yavb
sc
Skoog 5th edition. m M N 2
m Sxx
Note: when computing a confidence interval using this

sC value, the degrees of freedom are N-2. (See for
example Salter C., Error Analysis Using the
Variance-Covariance Matrix J. Chem. Ed. 2000, 77,
1239.
Page 34 of 316
1.10 Experimental Design:
Designing an experiment involves planning how you

will: make calibration and validation standards;
prepare the sample for analysis; and perform the
measurements.
1.10.1 Making a set of calibration standards.
1. You need to know the dynamic range of your

instrument.
2. You need to know the sample size
requirement of your instrument.
3. You need to know the estimated expected
concentration of your sample.
4. You need to prepare and dilute your sample
until it is a. within the dynamic range of your
instrument and b. such that there is enough
solution to measure.
5. You need to choose target standard
concentrations that bracket the expected
sample concentration generously e.g. by a
factor of 2 to 3. For example, if your expected
concentration is 5.3 ppm, you may wish to make
a calibration set that consists of standards that
are about 2,4,8,10 and 12 ppm.
Page 35 of 316
6. You need to make a primary stock solution,

the concentration of which you know accurately
and precisely. This solution will usually be
more than twice as concentrated as your most
concentrated calibration standard. You will
dilute this primary stock solution to make the
calibration standards. You need to have enough
to make all of your calibration standards.
7. You need to choose the pipets and volumetric
flasks that you will use to perform the dilutions.
This means that you plan the preparation of
each standard. This takes some planning and
compromising and many choices there are
many ways to do this correctly there is more
than one right answer!
8. Decide on and record a labeling system in
your notebook, collect the glassware, and do
the work. I have a labeling system for your
caffeine, benzoic acid, iron and zinc standards
I need you to use these labels so that we can
sort things out in the class.
Page 36 of 316
1.10.2 Exercise in planning an analysis:
Assume that you will be analyzing sucrose in corn

syrup sweetened ketchup packets. The packets are
thought (i.e. expected) to contain about 0.8 grams of
corn syrup that is about 70% sucrose by weight. The
HPLC instrument that you will be using can detect
sucrose by refractive index in the 0.1-20 parts per
thousand (ppth) range (this is the instruments
dynamic range for sucrose). You have pure sucrose
for standards, and will be making five calibration
standard solutions. The instrument requires between
250 and 1000 L of sample.
You have the following glassware at your disposal:

Pipets Volumetric Flasks
Volume Relative Volume Relative
Precision Precision
20-200 L 5-1% 1 mL 1%
1 mL 1% 5 mL 1%
5 mL 1% 10 mL 1%
10 mL 1% 25 mL 1%
15 mL 1% 50 mL 1%
20 mL 1% 100 mL 0.5%
25 mL 0.5% 250 mL 0.5%
50 mL 0.5% 1000 mL 0.25%
Page 37 of 316
1.10.3 Plan the analysis! (start with guesses)
1. If you dissolve the packet in water, dilute it to

100.0 mL and filter it, what will the approximate
sucrose concentration be in ppth (parts per
thousand)?
0.8 g 0.7 g 1000 mg 1 mL

syrup sucrose 1 sucrose water = 5.6 ppth
100 mL 1g
g syrup water g sucrose water
Is this within the dynamic range?

Would it be better to use 10, 25 or 50 mL of
water?
2. You need to prepare a stock solution of sucrose
to make the calibration standards. What
concentration should this stock solution be?
1, 10, 100 or 1000 ppth?
3. How much sucrose would be required to make:
1, 10, 100 or 1000 mL of this solution?
1.0 100 mg 100 mg 0.100 g

mL sucrose = sucrose = sucrose
1 mL
soution
1.00 g
10 mL => sucrose
100 10.0 g
mL => sucrose
Page 38 of 316
1.10.4 How to make primary stock solution:
1. Weigh out the desired quantity of pure (e.g. dry,

or oxide-free) analyte material.
2. Dissolve this amount quantitatively, i.e. without
any loss, in the desired solvent.
3. Transfer this liquid quantitatively into the
desired volumetric flask.
4. Dilute to volume with the desired solvent this
process is important! It is often poor practice to
add 5 mL of a to 5 mL of b and anticipate that
the final volume will be exactly 10 mL!
Remember the words DILUTE TO VOLUME!
Page 39 of 316
4. What concentrations bracket the 5.6 ppth

target?
For example: Do you need to make

standards on an even
5.2, 5.4, 5.6, 5.8, 6.0 ppth spacing?
--- or ---
1, 2, 4, 6, 8 ppth No but calibration
--- or --- points above and
1, 2, 5, 10, 15 ppth below the std. are
--- or --- needed.
0.050, 0.50, 5.0, 50, 500 ppth
Does the analyte No, but it
have to fall right in the minimizes
middle of the error!
calibration standards?
5. What volumes of standards should you prepare?

How much is needed by the instrument?
What is the smallest volume that you can
conveniently and precisely measure?
How expensive is the analyte and solvent?
How expensive is it to dispose of the waste?
0.1 or 1 or 10 or 100 or 1000 mL
Page 40 of 316
6. How do you prepare the calibration series from

the primary stock solution?
Primary Stock (1) is diluted to make

Calibration Standard (2)
C1V1 = C2V2
First calibration standard:

Make 10 mL of 1 ppth sucrose from 100 ppth
stock solution.
C1 = 100 ppth
C2 = 1 ppth
V2 = 10.0 mL
V1 = ? = volume to pipet over
V1 = C2V2/C1
Page 41 of 316
7. How to plan a set of standard preparations in the

MS Excel spreadsheet program:
Calibration Standard Preparation:
Target Conc: Stock Conc: Final Volume: Volume to Pipet:
C2 / ppt C1 / ppt V2 / mL V1=C2*V2/C1
1.00 100.0 10.00 0.100 mL
2.00 100.0 10.00 0.200 mL
5.00 100.0 10.00 0.500 mL
10.00 100.0 10.00 1.000 mL
15.00 100.0 10.00 1.500 mL
Calibration Standard Preparation:

Target Conc: Stock Conc: Final Volume: Volume to Pipet:
C2 / ppt C1 / ppt V2 / mL V1=C2*V2/C1
1 100 10 =A4*C4/B4 mL
2 100 10 =A5*C5/B5 mL
5 100 10 =A6*C6/B6 mL
10 100 10 =A7*C7/B7 mL
15 100 10 =A8*C8/B8 mL
These are formulas that you type into

Excel normally only the result of the
formula calculation is displayed.
Page 42 of 316
1.11 Validation Assurance of Accuracy:
Calibration and linear regression optimizes the

precision of a calibration curve determination but a
calibration curve can only be said to be accurate if:
the analysis has been validated

Another way
of saying this is that a method is valid if:
all significant sources of bias
have been removed
Validation addresses the various aspects of an
analysis that can go wrong and give you a wrong
answer.
The table below lists some of the aspects of an
analysis that can be invalid, and suggests ways to
validate them.
Source of bias: Possible Solution(s) or Diagnostic:
Analyst erratic Analyst can repeat experiment
Error in analyst Different analyst does same analysis and
technique gets same result.
Calibration Entire analysis Independent standard
standards are in repeated with measured periodically -
error (are not what indpendent called validation standard
they say the are) calibration or QC standard (quality
standard set control)
Calibration Different calibration method used (standard
method additions)
Instrument Different instrument used (can be a different
function erratic kind of instrument)
Instrument drift Periodically measure validation standard or
internal standard
Page 43 of 316
In general validation of an analysis is done using

some kind of independent analysis of the sample.
For the purposes of this class, if the two methods
agree (t-test does not show a difference) then the
method is said to be validated.
If the two methods disagree (i.e. there is
evidence of bias), then there is evidence for
systematic error like a matrix effect, an
interferant, or a mistake in the preparation of
standards or samples.
If an analysis is repeated as described below, what

aspects of the analysis are validated (i.e. what must
have been good for the answers to have come out
the same)?
Validation Approach Aspect(s) validated

Completely independent Calibration method,
measurements of the including matrix effects.
same sample using a
different instrument or Instrument, including
technique. distortion and drift.
You! The analyst, did it
right
Use the same method, You! The analyst made no
but measure mistakes in the
independently prepared implementation of the
standards as samples. procedure.
Page 44 of 316
1.12 Spike Recovery Validates Sample Prep.
To do a spike recovery analysis, one takes replicate

samples and to a subset of them adds a spike of
analyte before the sample prep begins. So, for
example, one could take four vitamin tablets, and
divide them into two groups of two. To one group one
could add some Fe, say half the amount originally
expected. For example, if there is supposed to be 15
mg of iron in the tablet, one could spike two samples
each with 5 mg of iron and leave two unspiked.
Acids etc.
Dilute to Analyze
100 mL 140
digest filter volume ppm 14.0 mg
found
Sample
Dilute to Analyze
100 mL 187
found
Sample+ 5.00 18.7-14.0 = 4.7 mg of spike found: spike

mg spike recovery percent = 4.7 / 5.00 94%
What does this say about We may be losing about

our sample preparation 6% of the analyte during
method? sample prep.
Page 45 of 316
1.13 Reagent Blanks for High Accuracy:
A reagent blank is a blank that is made by doing

everything for the sample prep etc. but without the
sample:
Acids etc used
in sample prep. Dilute to Analyze
100 mL 2.3
Fe found
No Sample sample.
Ultrapure
water blank
In this example the reagent blank is analyzed against

ultrapure water. The 0.23 mg Fe found in the reagent
blank may be due to Fe impurities in the acids, but it
also may be a matrix effect. In either case, it
suggests that we should do what in order to arrive at
a more accurate result?
Either: a. analyze all standards and samples
against reagent blanks or
b. obtain higher purity acids
c. subtract the signal from the reagent
blank (with regulatory approval)
Page 46 of 316
1.14 Standard additions fix matrix effects:

Use the method of standard additions when matrix
effects degrade the accuracy of the calibration curves.
There is one important assumption built into the

calibration curve idea. That assumption is the
following:
the sensitivity of the instrument to the analyte in the standards is
EQUAL TO
the sensitivity of the instrument to the analyte in the sample matrix
Why would the sensitivity be different?
1. Many Instruments are sensitive to things like:

1. pH 2. ionic strength 3.organic
components of the solvent matrix
2. Sometimes other chemicals (interferants) can

change the calibration sensitivity by:
chemically binding to or interacting with the
analyte atom/molecule
These two things are examples of:

Matrix Effects
Page 47 of 316
1.14.1 How does one deal with matrix effects?
1. Make the sample and standard matrix as nearly

identical as possible:
matrix matching
But matrix matching requires that you already know

a lot about your unknown often not the case. So,
the matrix-immune alternative to the calibration curve
is to:
2. Use the method of: standard additions:
In other words, the assumption built into the

calibration curve method is that the matrix effects are
negligible or identical for standards and samples. If
this can not be assumed, one must match the sample
and standard matrices.
The way that the method of standards additions does

this is to dilute both standards and samples:
in the same matrix (solution)
Page 48 of 316
1.14.2 An Example of Standard Additions:

1. The analyte sample is split up into e.g. 6 aliquots of
identical, known volume e.g. 1.00 ml.
2. To each of these, a known quantity of standard
(known as a spike) is added e.g.
0, 0.1, 0.2, 0.3, 0.4, 0.5 ml standard is dissolved
in known matrix like water or 0.1M pH 7 phosphate
etc.
3. Each aliquot is diluted to a total volume.
1. Add sample
2. Add standard
3. Dilute to volume,
and mix mix mix!
Note: you split and dilute

your sample how does It may decrease it
this impact the precision of If dilution is too great!.
your measurement?
How does this process

impact the accuracy of It increases it!
your measurement?
Page 49 of 316
1.14.3 Calculating Conc. w/ Standard Additions:
a'
b'
a
One way of analyzing this uses similar triangles:
a/b= a/b
The y-axis absorbance signal (S) is proportional to the moles of

analyte (VXCX) and standard (VSCS).
The x-axis is simply the standard spike volume.
The x-intercept is the hypothetical spike volume (VS)0 containing the
same amount of analyte as the sample.
a is proportional to moles of analyte in the sample = VXCX
a is proportional to moles of std added = VSCS
b is (VS)0 the x-intercept of the graph
b is VS the spike volume VXCX VSCS
Substitute for a,a,b,b: =
(VS)0 VS
Solve for CX: CX = CS(VS)0/VX
Page 50 of 316
1.14.4 Standard Additions by Linear Regression:

Let:
VX = volume of unknown analyte solution added to each flask
CX = concentration of unknown solution
VT = final, diluted volume
VS = volume of spike added to unknown soln. before dilution
CS = concentration of analyte in spike solution
Dilution Calc 1: (V1C1 = VTCT CT = V1C1/VT)

Contribution to concentration of analyte from sample:
VXCX/VT
Dilution Calc 2:
Contribution to concentration of analyte from spike:
VSCS/VT
Total Signal (sensitivity = k) given that x variable is VS.
S=k VXCX/VT +k VSCS/VT
Slope = m = kCS/VT
intercept = b = kVXCX/VT
we can get b and m from linear regression
kVXCX/VT
and we want CX so b/m = = VXCX/CS
kCS/VT
bCS
so : CX = mVX
Page 51 of 316
1.15 Internal Standards

Internal standards can correct for sampling, injection, optical path
length and other instrument sensitivity variations.
An internal standard is a substance added (or simply present) in

constant concentration in all samples, and standards.
When something unexpected decreases the sensitivity of the

instrument (m) so the signal drops (S = mC + SB) it can be
impossible to distinguish this from a change in analyte concentration
without an internal standard.
Consider the ratio of the blank corrected analyte ( S' AN S AN sB ) to the

S ' AN m AN C AN C AN
internal standard (IS) signals (S): k
S ' IS mIS C IS C IS
Assumes for the moment that k is a constant, i.e. invariant to factors

affecting overall instrumental sensitivity. As an example, lets
consider k to be a correction for injection volume in a
chromatographic system. It is perfectly reasonable to assume that an
accidentally low or high injection volume would affect the internal
standard and analyte signals identically e.g. if a given injection were
6% high, then both analyte and internal standard peaks would be 6%
larger than expected.
If k is invariant to instrument fluctuations, then the true analyte

concentration can always be derived from the ratio of the corrected
signals so long as the internal standard concentration remains
constant. Anlalyte conc. in a sample.
S ' AN C IS C IS
C AN where is easily derived from a previously measured
S ' IS k k
calibration standard for which the analyte signal ( S' A STD ) and
concentration ( C A STD ) of the analyte and internal standard (
S ' A STD C IS m AN
S ' IS STD , CIS STD ) are known: k STD
S ' IS STD C A STD mIS
From a calibration standard.
Page 52 of 316
In other words, if CIS is held constant in all experiments, then the

ratio of the analyte to internal standard signals will be
independent of instrument sensitivity.
When to use an internal standard?
When substantial influence on instrument sensitivity is expected due

to variation in things like:
Sample matrix Temperature
Detector sensitivity Injection volume
Amplifier (electronics) drift Flow rate
An internal standard must:

a. not interfere with your analyte
b. ideally have the same dependence on the chemical matrix,
temperature (or other troublesome variable) as the analyte.
Consider the ubiquitous salt plate IR sampling method. A drop of

analyte is sandwiched between two salt plates and this is placed in
the IR beam. The path-length, b, is highly variable from
experiment to experiment. The signal A = bC where is
characteristic of the molecule, b is pathlength and C is concentration.
If you are doing an experiment to measure the increase in amide
formation versus time by the intensity of the amide bands near 1700
cm-1. You could take samples and measure them periodically, but
the variability of the pathlength would distort the results.
On the other hand, if all the samples were spiked with the same
amount of acetonitrile then the sharp nitrile stretch at 2250 cm-1 could
be used as an internal standard.
Assuming you always spiked with the same amount of benzonitril,

what simple metric, based on A1700 and A2250 would always be
proportional to the amide concentration? In other words, simply
by taking the ratio of the analyte to the internal standard peaks while
holding the internal standard concentration constant, one can cancel
a variable such as path-length!
Page 53 of 316
Internal Standards Example:

In HPLC the sample is injected into a flowing stream, and the signal
is a peak in a plot of absorbance versus time (a chromatogram).
Often the volume of the injection will vary from injection to injection,
so the peaks will vary in size!
Peak 1 is the Peaks 2, 3 and 4 are other
internal standard. ingredients in the sample.
100 ppm NaNO3,
mAu.s
480All Peak 5 is the
1
analyte, a caffeine
1 standard, 25 ppm,
530 mAu.s
absorbance
0.5
Chromatogram
0
1 is a standard.
0 400 800 1200 1600 2000 2400 2800 3200 3600 4000
time / s
Did the caffeine
1
concentration
increase from
2 chromatogram
absorbance
1 to 2?
0.5
Did the caffeine

concentration
0
increase in
0 400 800 1200 1600 2000 2400 2800 3200 3600 4000 chromatogram
time / s
1 to 3?
100 ppm NaNO3,
520 mAu.s 1
3 Unknown
caffeine conc.
absorbance
0.5 630 mAu.s
0
0 400 800 1200 1600 2000 2400 2800 3200 3600 4000
time / s
Page 54 of 316
1.15.1 Internal Standards Calculation

How about calculating the actual concentration of the sample in 3
above?
S' A STD 530 mAu.s

CA STD 25 ppm
Standard
S' IS STD 480 mAu.s
CIS STD 100 ppm
S ' A STD C IS STD m AN 530 100
k 4.417 unitles s
S ' IS STD C A STD mIS 480 25
S' AN 630 mAu.s

S' IS 520 mAu.s
Sample
C IS 100 ppm
S ' AN C IS 630 100
C AN 27.4 ppm
S ' IS k 520 4.417
Page 55 of 316
2 Propagation of Error
Skoog Chapters Covered:
Appendix a1B-4 a1B-5 and eqn. a1-28 table a1-5
There is a general problem in experimental science and engineering:
How to estimate the error in calculated results that are based on

measurements that have error?
Lets consider the sum of two measurements a and b that both have
some fluctuation sa = 0.5 lb and sb = 0.5 lb.
Lets also pretend that we know the true values of a and b.
True value of: a = 5.0 lb

b = 5.0 lb
Lets say that we are weighing a and b and putting them into a box for
shipment. We need to know the total weight. Our scale is really bad
(poor precision, lots of fluctuation), and it cant weigh both a and b at
the same time because it has a limited capacity. So, we have to first
weigh a, then b and then calculate the total weight. But we know that
there is a problem with fluctuations, so we repeatedly weigh the same
items a and b and do the following experiment:
Characteristics of numbers in Characteristics of sum

sum a and b c
Trial # Weight of: Deviation: Total Deviation
a b da db weight: from avg:
1 4.5 4.5 -0.5 -0.5 9.0 -1
2 5.5 4.5 +0.5 -0.5 10.0 0
3 4.5 5.5 -0.5 +0.5 10.0 0
4 5.5 5.5 +0.5 +0.5 11.0 +1
average 5.0 5.0 0.5 0.5 10 0.5
This is somewhat artificial and is not quite right, but it gives you the
general idea. Errors in a and b propagate into c.
Page 56 of 316
From this example above, one would conclude that the fluctuation in
the sum is equal to the fluctuation in the numbers summed but this
is not right.
Which is bigger?
a. the fluctuations in the individual numbers summed
b. the fluctuations in the sum
Graphically we consider here a similar case, for clarity we let a have

a slightly larger fluctuation than b.
a = 5 2, b = 3 1
a+sa+b+sb
theoretical
a+sa+b-sb
c
a-sa+b-sb
theoretical
a+sa a-sa+b+sb
a
distribution
a-sa
Real
Page 57 of 316
error propagation through sums :
a rnorm( 10000 3 0.5) b rnorm( 10000 3 0.5) c a b
0 0 0
0 2.781 0 3.554 0 6.334
1 2.66 1 3.331 1 5.991
a b c
2 2.763 2 1.98 2 4.744
3 2.524 3 3.482 3 6.006
4 2.157 4 3.848 4 6.005
mean ( a) 3.004 mean ( b ) 2.996 mean ( c) 6

stdev ( a) 0.498 stdev ( b ) 0.504 stdev ( c) 0.712
a_dist histogram ( 40 a)
2 2
0.5 0.5 0.707
b_dist histogram ( 40 b )
c_dist histogram ( 40 c)
1000
a_dist j 1
b_dist j 1 500
c_dist j 1
0
0 2 4 6 8 10
a_dist j 0 b_dist j 0 c_dist j 0
Page 58 of 316
error propagation through products :
c a b
i i i mean ( c) 9.003
stdev ( c) 2.156
0
0 9.881 a_dist hist ogram ( 40 a)
1 8.861 b_dist hist ogram ( 40 b )
2 5.473
c_dist hist ogram ( 40 c)
3 8.789
4 8.301
1000
5 8.057
6 7.561
c 7 9.909 a_dist j 1
8 13.589 b_dist j 1 500

9 7.471 c_dist j 1
10 9.206
11 6.054
1211.944 0
0 5 10 15 20 25
1310.643 a_dist j 0 b_dist j 0 c_dist j 0
14 5.773
15 8.27
Page 59 of 316
For a simple example if you construct a simple calibration curve of

instrument signal versus concentration, and then use a real (i.e.
noisy) signal to determine concentration.
Errors propagate from S to C according to the sensitivity.
Page 60 of 316
Lets consider an example wherein the relationship between the

measured signal and the desired quantity is non-linear :
Absorbance
A = -log (P/Po)
Absorbance is a nonlinear function of

light power - the measured quantity in a P
A log
spectrophotometric experiment. Pl
1.5
A 1
i
0.5
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
P
i
Obviously the same error in P can give rise to different errors in A!
This is a propagation of error problem. How can you calculate the

error in A that should result from a particular error in P?
Page 61 of 316
Obviously, the answer has to do with the way that the dependent
variable (A) in this case changes as a function of the independent
variable (P).
Consider a calculated value S that depends on the measured

quantites, e.g. instrument signals, a,b,c
S = f(a,b,c)
In fact the variance in S is proportional to the variance in a,b,c

and the proportionality is the partial derivative squared:
(Skoog appendix a1B-4 has a derivation if you are curious)
for: S f a a b b c c
2 2 2
2 S 2 S 2 S 2
S a b c
a b c
So lets take an example: S = a+b-c
find S = f(S,a, a, b, b,c, c)
Note that the larger terms dominate!
Page 62 of 316
Exercise: Prove the multiplication / division rule

Show that for S = a*b/c
2 2 2 2
S a b c
2 2 2 2
S a b c
Page 63 of 316
The propagation of error rules for special cases:
Page 64 of 316
3 Introduction to Spectrometric Methods

Skoog Chapter 6 all sections
Electromagnetic Radiation (EMR) is:

Light!
EMR described as a classical, electromagnetic wave:
distance
In vacuum (air): n=1 In matter: n>1
=c MEDIUM = cMEDIUM
= wavelength
MEDIUM = VAC /
= frequency
c = light speed cMEDIUM = cVAC /
Note: When the refractive index ( ) changes
Stays same
___________
___________
changes
Page 65 of 316
3.1 Electromagnetic Radiation:

A vast range of energies covers many physical
processes. These processes are the basis of
spectroscopy.
Page 66 of 316
Energy Nomogram
Energy nomogram -
Hard UV 100 nm to Far IR 1mm
102 105 103
101 105
1015 102
103 104 100 102 104

1014 101
104 103 10-1 101 103

13 100
10
105 102 10-2 100 102

10-1
1012
106 101 10-3 10-1 101

nm cm-1 s E / eV E / kCal E / kJ T/K
/mol /mol
Page 67 of 316
3.2 Diffraction
Diffraction is the basis of wavelength selection in most
spectrometers:
Page 68 of 316
3.2.1 Diffraction Exercises:
1. Derive a formula for the path difference , as a

function of the spacing d, and the angle of
incidence . (Use the diagram on the following page.)
first note that =

sin( ) = /d
= dsin( )
2. Under what conditions is the light intensity at the

detector high or bright?
=n
n = -2,-1,0,1,2 i.e. any integer
3. Calculate the wavelengths of light that are

diffracted at 48 for d = 1 micrometer (1 ).
Page 69 of 316
Diagram for deriving a formula for the path length

difference between diffracted rays:
Page 70 of 316
3.3 Properties of Electromagnetic Radiation:
Most EM Radiation is polychromatic, i.e. the beam is

a mixture of rays of different:
frequencies phases
E.g. incandescent light sources are polychromatic.

Another term for polychromatic light: is white light.
EM Radiation is monochromatic if all rays have

identical:
frequency
E.g. Na-atomic emission lamps are nearly

monochromatic because nearly all of the light is from
a single atomic transition that emits at 590nm.
White light can be filtered so that it is nearly
monochromatic.
Coherence:
EM Radiation is coherent if all rays have identical:
frequency phase
Coherent radiation comes from lasers and exotic light

sources called synchrotrons.
Page 71 of 316
3.3.1 Polarization:
Most light is unpolarized

Electric vector randomly oriented
Special filters, called polarizers, can remove all

E-field components except those falling in a given
plane. The result is plane polarized light:
All E-field lies in one plane
Elliptically polarized light:
E-field vector rotates around direction of

propagation.
Page 72 of 316
3.3.2 Refractive index:
ratio of speed of light in vacuum to speed of light in

medium.
= cVAC / cMEDIUM
=c/ 1 2.5
Refractive Wavelength Frequency s-1 Energy
J
index,
1.00 500 nm 3x108 ms-1 / 6x1015 s-1 x
500x10-9 m = 6.626x10-34 Js
6x1015 s-1 =
4x10-20 J
1.50 500/1.5 = 6x1015 s-1 4x10-20 J
333 nm
Page 73 of 316
3.3.3 Refraction and Snells Law:
Index = f(wavelength)
Blue
Blue Red Red
Page 74 of 316
3.3.4 Reflection:
Fresnel Equation for a special case:

Normal Incidence
No Absorption
Incident beam Reflected beam
Transmitted beam
Calculate the total reflection loss due to the two

reflections (air | glass and glass | water) when a light
beam passes through one side of a cuvette ( = 1.5),
containing water ( = 1.3).
Page 75 of 316
3.3.5 Scattering:
Incident Transmitted
Scattered
Scattering Scatterer Features

Process
Raleigh (elastic) atoms and weak, favors
molecules blue
Tyndall (Mie) Colloids Strong (easy to
Elastic normally (bigger / nm) image)
Raman (visible) Molecules very weak,
vibrational spec inelastic
Page 76 of 316
3.3.6 The Photoelectric Effect and the Photon:
Electrons are emitted from metal surfaces that are

irradiated with light of sufficiently high frequency.
Max Planck and Albert Einstein discovered that

Kinetic energy of the emitted electrons depends on:
Light frequency Type of Metal
and
Kinetic energy of emitted electrons is independent of:
Light intensity - more intensity means more

electrons, but not more energetic electrons!
Light
current
electrons
Metal Slope =
Free e-
Electron Energy
h
(KE)
EF
Page 77 of 316 Light Frequency

This leads to the concept of the light as a particle and

an expression for the energy of a photon.
Electron energy relationship to light frequency:
= Work function of metal

EF = Fermi energy of metal
= Light frequency (s-1)
E=h - Formula of line

h = slope = Plancks constant
Also the photoelectric effect is the basis of many

light detectors because it converts light energy into:
Electricity current or single-photon pulses
Page 78 of 316
The discovery of photon energy correlated with the

theory of atomic and molecular orbital energy.
Phase gas phase molecular molecular

atom liquid solid
Quantum
electronic electronic electronic
States
vibrational
ctronic
vibrational phonon
rotational
Resulting lines bands broad

Spectra
Page 79 of 316
3.3.7 Spectra typical of gas, liquid and solid.
Absorber Phase Notes /

transition
types
Atom Gas Extremely
narrow lines
/ electronic
Molecule Gas Fine

structure
due to
electronic+
rotational+
vibrational
Molecule Solution Broad
/ small bands +
some fine
structure /
electronic +
vibrational
Molecule Solution Broad

/ larger bands
electronic
only
resolved
Page 80 of 316
3.3.8 Energy levels and photon absorption and

emission.
Page 81 of 316
3.3.9 Typical fluorphore Jablonski Diagram.

3.3.10 Photophysical Processes
Page 82 of 316
3.3.11 Typical organic electronic spectrum.
Page 83 of 316
3.3.12 Energy Level Diagram on its Side

Line spectra, band spectra and continuum spectra of
atoms, molecules and solids: The relationship
between energy states, photon energies and spectra.
Absorbance
Emission or
Light Frequency
Energy
Absorbance
Emission or
Light Frequency
Energy
Heated Mat.
Emission or
Light Frequency
Page 84 of 316
Energy
Quantitation by interaction with light:
Page 85 of 316
Chem. 155 Unit 4 Page 86 of 316
4 Photometric Methods and Spectroscopic

Instrumentation
Review: Quantitation in Absorbance and Emission
7A Optical Designs Absorbance Emission
7A Optical Materials (lightly!)
7B Light Sources Continuum and Line
7B Lasers!
Page 86 of 316
4.1 General Photometric Designs for the Quantitation

of Chemical Species
4.1.1 Generalized Detector Response:
S = kP + SDARK
S = Signal
k = proportionality
P = light power
SDARK = detector response in absence of light
All absorbance methods!

4.1.2 Absorbance IR, VIS, UV, Xray!
More Analyte
Less Signal
4.1.3 Quantitation = molar absorptivity

b = pathlength (cm)
A = bc = -log(P/P0) c = concentration in moles/L
P = Light Power at Detector
PO = Light Power for Blank
c = concentration in moles/L
All light emission methods!
4.1.4 Emission and Fluorescence Fluorescence (Xray - UV),
More Analyte Scattering, Luminescence,
even NMR!
More Signal
4.1.5 Quantitation
P = PO + mC P = Light Power at Detector
PO = Light Power for Blank
c = concentration in any unit
Page 87 of 316
4.2 Block Diagrams

Instruments for Analytical Spectrometry:
1. Absorbance Cuvette Photomultiplier

Tube,
Flame
Photodiode
Gas Cell
Light beam is absorbed by analytes, not re-emitted
Amount of Absorption Amount of Analyte
2.1 Fluorescence
2.2 Raman
Scattering
Light beam stimulates analytes to emit light

Emitted light is collected at right angles to stimulating
Amount of Emission Amount of Analyte
3. Chemiluminescence
Analyte reacted with chemical that makes it emit light

Emitted light is collected
Amount of Emission Amount of Analyte
Page 88 of 316
4.3 Optical Materials

Cost Trade Off
$$ Water Soluble!
LiF
Sapphire Al2O3
Crystal $$$$ Hardness!
$$ UV-Vis
$ vis only
$ Water Soluble!
$$$$ Red and IR only
$$$$ IR Only
$$ non-linear dispersion
$$- $$$$ most common tool

order-overlap
$$ low-res short range
$ only one per filter
$-$$ very low resolution
$$$$ hard to optimize for both

Interferometers visible and IR at once
Time
Domain
Page 89 of 316
4.4 Optical Sources
Cost Trade Off
$$ VUV Only?
$$ Short Lifetimes
$$ Popular UV-source used

with W-Halogen for UV-Vis
$ W-Halogen
Visible only, long lifetimes
$$ NIR and IR Only
$ IR only
$ IR Only
$$ Low intensity, limited s
$$- $$$$ Exellent Intensity, limited

s
$ - $$ very high resolution, poor
dynamic range
$$-$$$ PMT v. fast, v.v. sensitive,
delicate, limited in IR
$-$$ low sensitvity, limited in IR
but cheap
$ - $$$ sensitive and fast, much
tougher than PMT
$$-$$$$ v. sensitive, slow, tough,
$$-$$$ detector of choice most IR
More exotic energy detectors dont
know much about these
Page 90 of 316
4.5 Continuum Sources of Light:

Broadband or White Light Sources
Also called:
1. Simplest Design: Blackbody Sources
a. Tungsten Visible / Near IR / IR

b. Quartz-Tungsten-Halogen
c. Nernst Glower IR / Far IR
2. Gas Emission Designs:

a. H2 / D2 UV Only
H2 + e- H2* H + H + h
Kinetic Energy of H atoms = Continuum
Therefore h can be: Continuum
UV-Vis-Near IR
b. Ar, Xe, Hg
Heavy (High-Z) atoms Many atomic states

+
High Pressure (extensive broadening of lines)
Quasi-Continuum
Page 91 of 316
4.6 Line Sources of Light:

1. Low Pressure Gas Emission
a. Hg Many Lines can be filtered to
b. Ar emit only one predominant line.
c. Xe
d. Na Na-D Line Predominates
589.00 and 589.59 nm doublet
2. Hollow Cathode Lamps:

a. Metals (Cathode!)
b. Used in atomic spectroscopy (absorbance
and fluorescence)
300V DC Electrical Discharge

Sputtering
+
+ Ne + Fe* Fe +h
Ne
Atomic Emission
Fe Metal Cathode
Page 92 of 316
4.7 Laser Sources of Light:
An Introduction to Lasers:
L Light
LASER is an acronym for A Amplification by
a light amplification S Stimulated
process:
E Emission of
Partially
transmitting R Radiation
mirror
Fully
reflective
mirror
Pumping Energy Gain Medium atoms or

Source: molecules that undergo
Intense Light lasing transitions
Electrical Discharge
What happens in the Gain Medium?
1.
relatively
long lived
Page 93 of 316
What happens in the Gain Medium?
2.
3.
4.
Stimulated Emission is: Coherent
Monochromatic
Page 94 of 316
4.7.1 A laser is a light amplifier

Some of the above processes degrade the light in the cavity
Spontaneous Emission
Absorption
Some of the above processes amplify the light in the cavity
Pumping
losses
Stimulated Emission
absorption,
gain! stimulated spontaneous
emission pump
emission
power in
beam out
2 Two common laser

configurations:
Fast Decay
1
Pump
Lasing!
0 3-state
3
Fast Decay
2
Lasing!
Pump 1
0
Fast Decay 4-state (or more)
Page 95 of 316
4.7.2 Polulation Inversion and laser amplification
absorption
population in state Ex
lasing amplification
population in state Ey
Roughly speaking lasing is possible when:
population in upper state 1 is greater than the

population in lower state 2
This is called a population inversion.
Page 96 of 316
4.7.3 Necessity of 3 or more states
Why are three or more levels (states) necessary for

lasing?
E E = ENERGY DIFFERENCE us-ls

recall:
N EXCITED kT k = Boltzmann const. 1.3x10-23 J/K
e
N GROUND T = temperature (K)
NEXCITED = population (concentration) US
NGROUND = population (concentration) LS
What happens when T infinity? NEXCITED NGROUND
Page 97 of 316
4.7.4 Common lasers categorized by lasing medium.
1. Solid Red - Near Infrared

1.1. Lanthanide-and transition metal ion lasers
1.1.1. Nd-YAG Neodymium ions in a crystal called a garnet
made of yttrium oxide and aluminum oxide. Other
lanthanide ions are substituted for other wavelengths in the
near IR
1.1.2. Ho-ZBLA Holmium (Ho+3) doped glasses made from
ZrF3, BaF3, La F3, Al F3 can be fabricated into optical fibers
that lase and can amplify optical signals of certain
wavelengths
1.2. Ti-sapphire tunable (650-1100 nm) CW or pulsed
1.2.1. Mode locked: 5-2000 fs, (MHz repetition)
1.2.2. Chirped-pulse amplification: 10-100 fs, 5, mJ, (kHz
rep) gW power densities
1.2.3. Fundamental or second harmonic can be used in
OPA optical parametric amplifier lasers that are freely
tunable, normally pulsed
1.3. Semiconductor Lasers

1.3.1. Silicon,
1.3.2. gallium arsenide and other light-emitting diodes can be
made to lase when many electrons are promoted to excited
states within microfabricated cavities in the semiconductor
crystal. Blue (very new) Near Infrared
2. Liquid dye-lasers (blue-red) are made from solutions of many
different fluorescent dye molecules. The dye molecules have
multiple excited-states that can be induced to lase usually by
pumping with other lasers (Ar-ion). Tunable!
3. Gas-Phase lasers (UV-near infrared) are very common, and

typically pumped by electrical discharge. Examples include:
3.1. He-Ne lasers (633 nm)
3.2. Ar-ion, Kr-ion (514, 488, 325 nm)
3.3. N2 (337 nm)
3.4. CO2 (10,600nm and many other IR wavelengths)
3.5. XeF (351) and KrF (248) and ArF (193) excimer lasers
Page 98 of 316
375 nm - excitation of Hoechst stain, Calcium Blue, and

other fluorescent dyes in fluorescence microscopy
405 nm - InGaN blue-violet laser, in Blu-ray Disc and HD
DVD drives
473 nm - Bright blue laser pointers, still very expensive, output of
DPSS systems
485 nm - excitation of GFP and other fluorescent dyes
532 nm - AlGaAs Bright green laser pointers, frequency doubled
1064 nm IR lasers (SHG)
593 nm - Yellow-Orange laser pointers, DPSS
635 nm - AlGaInP better red laser pointers, same power
subjectively 5 times as bright as 670 nm one
640 nm -
650 nm - AlGaInP DVD drives, laser pointers
660 nm -
670 nm - AlGaInP cheap red laser pointers
785 nm - GaAlAs Compact Disc drives
808 nm - GaAlAs pumps in DPSS Nd:YAG lasers (e.g. in green
laser pointers or as arrays in higher-powered lasers)
848 nm - laser mice
980 nm - InGaAs pump for optical amplifiers, for Yb:YAG DPSS
lasers
1064 nm - AlGaAs fiber-optic communication
1310 nm - InGaAsP fiber-optic communication
1480 nm - InGaAsP pump for optical amplifiers
1550 nm - InGaAsP fiber-optic communication
1625 nm - InGaAsP fiber-optic communication, service channel
Page 99 of 316
4.7.5 Excimer Lasers:
Kr* + F
KrF* Excimer!
KrF What is unusual

Kr + F about this
molecule?
Because of fast dissociation,
[KrF*] > or < [KrF]
This favors:
population inversion!
Page 100 of 316

4.7.6 Some laser pointers.
LS = lower state
US = upper state
GS = ground state
stimulated
h + US 2h + LS gain
emission
h + LS US absorption loss
spontaneous
US LS + h loss
emisson
Based on this, should the LS be:

short lived or
long lived?
Based on this, should the US be:

short lived or
long lived?
Page 101 of 316

Laser Questions:
Indicate all that apply:
1. For a laser gain medium with three or more states, a population

inversion is / means:
a. More electrons in the upper than lower states.
b. More molecules, atoms or ions in a given excited than
ground state.
c. Excited state energy is greater than ground state energy.
d. Concentration of molecules / atoms or ions in upper state
is greater than concentration in lower state.
e. Concentration in ground state is zero.
f. Concentration in upper state is greater than half of the
total.
2. For efficient laser gain:

a. Upper state should be long-lived.
b. Lower state should be short lived.
c. Ground state should be unstable.
d. Ground state should not be lower state.
e. Lower state should be long lived.
f. Upper state should be short lived.
Contributes to: Proportional To:

Phenomenon Gain Loss [upper states] [lower states]
Spontaneous
emission
Absorption
Stimulated
Emission
3. For gain to occur in a laser [upper states] must be _____

relative to [lower states]:
a. >
b. <
c. =
Page 102 of 316

5 Radiation Transducers (Light Detectors):

Chapter 7, Skoog Holler & Nieman
7E Photomultipliers
Photodiodes
Charge-Coupled Device Arrays
5.1 Desired Properties of a Detector:
High Quantum Efficiency:

A large fraction of photons that strike it
result in a response few are lost.
High Gain:
For each photon that strikes the
detector, a large signal is generated.
Low Noise:
Constant light flux gives a constant

signal - for measurement is low.
Low Dark Count:
Low or no signal is present in the

dark.
Page 103 of 316

5.2 Photoelectric effect photometers

Phototube:
High UV- Evacuated tube in

transparency fused which electrons can
silica window. travel if emitted from
cathode.
+ -
Readout
Amplifier
Photocathode Anode positive

transducer - emits bias, collects
electrons via the photocurrent.
photoelectric effect
Low work function
metal: Na, K, GaAs
Page 104 of 316

What is the maximum gain of a

phototube expressed as electrons per 1!
incident photon?
What is a typical gain for a phototube

expressed as electrons per incident 0.01
photon?
What limitation is built in to all

photoelectric detectors? h must be
Page 105 of 316

5.3 Limitations to photoelectric detectors:
Recall Einstein et al. used the photoelectric effect to

discover what particle: The photon!
Slope of
Photoelectron energy / j
this line =
Planks Constant:
h = 6.626x10-34 Js
Light frequency / s-1
Peak quantum efficiency:
or 1 photoelectron
About 10%
10 photons
Page 106 of 316

5.4 Operation of the PMT detector:
Faceplate material:
UV-transparent
Silica
Photocathode potential:
From www.hamamatsu.com pmtconstruct.pdf -100 to 1000V
A typical PMT may have 12 dynodes, each of which

gives off somewhere between 4 and 12 secondary
electrons depending on the applied voltage.
Question:
Derive a formula for PMT gain:
How many electrons are collected for each incident

photon? (Use outline on next page)
Page 107 of 316

5.5 PMT Gain Equation:
1 Photon Photocathode
Quantuum
Efficiency -
How many photoelectrons
per incident photon are
emitted? Secondary
electron yield -
How many secondary Dynode 1
electrons?
Dynode 2
Dynode 12
n
PMT Gain = G =
= Quantum efficiency
= Secondary electron yield
n= Number of dynodes
Page 108 of 316

How many dynodes do you want or need?
Ultimate sensitivity =
Detect a single photon!
Consider the arrival packet of electrons:
h e- at e- strikes n- n
electrons
photocathode dynodes strike anode
3x10-9s
What is the average current during the 10ns

pulse? Assume = 4, n = 10
F = 96485 coulombs/mole e-
Mol = 6.023x1023
Current (amperes) = # coulombs / s
410 = 1048576
Current (amperes) = # coulombs / s

i = 1048576 e- * 96485 coulombs / 6.023e23 e- / 10-8 s
= 1.7x10-5 amperes easily measureable
Page 109 of 316

5.6 Noise in PMTs and Single Photon Counting:

Two major sources:
Thermal:
Individual electrons are thermionically emitted
from photocathode or one of the dynodes.
Cosmic Ray Background:
Energetic particles ( and particles, -rays

etc. from space) strike photocathode or
dynodes and cause a large current spike.
Reject: cosmic
current / microamperes
ray e- cascade.
Accept: photon-
electron from
photocathode
Reject:
time / s Thermal e- from
dynodes
Muon: created by very high energy cosmic ray interaction with upper atmosphere, similar to
electron (-1 charge, spin 1/2) but about 200 times heavier.
About 10,000 muons reach every square meter of the earth's surface a minute; these charged
particles form as by-products of cosmic rays colliding with molecules in the upper atmosphere.
Traveling at relativistic speeds, muons can penetrate tens of meters into rocks and other matter
before attenuating as a result of absorption or deflection by other atoms.
Mark Wolvertron, science writer, Scientific American magazine, September 2007, page 26 "Muons for
Peace"
Page 110 of 316

In single photon counting:
Accept only those pulses originating at the

photocathode.
Retain signal
noise
Reject
Recall: Detection limit
SMIN = SBLANK + 3 BLANK
Large and small pulses are all noise!

These contribute to both SBLANK and BLANK!
Limitations to the PMT and to single photon counting:

1. Wavelength limitations:
Same as phototube - h
2. Intensity limitations:
In general, the PMT is used in
LOW LIGHT ONLY ambient
light will destroy the tube.
In Single Photon Counting,

light levels must be low
enough that light pulses do not
pile up or overlap.
Page 111 of 316

5.7 Semiconductor-Based Light Detectors:

Photodiodes:
p-type Si p-n junction n-type Si
+
+ P
-
B
- +
B P
+ -
The only carriers of The only carriers of

current in p-type Si are: current in n-type Si are:
Holes! Electrons!
Why does B (or Al) become (-) and P become (+)?

Both B and P are forced to become tetravalent
by the bounding atoms in the lattice this leaves
a curious formal charge!
B Mobile P Mobile
(p-dopant) holes. (n-dopant) electrons
Page 112 of 316

Depletion, anhiliation, injection and rectification:

1. Forward Bias:
+
p-n
junction
s
+
-
+ +
- -
Injection of Anhiliation of Injection of

holes holes and electrons
electrons
2. Reverse Bias:
+
+ -
-
+
+ -
Removal of Depletion of Removal of

holes holes and electrons
electrons
Under reverse bias: No current flows:

Rectification
Page 113 of 316
Photodiode:
+
+ -
-
+
+ -
Steps in Photoconduction:
1. Photon absorbed by Si, promotes e- into

concution band.
2. This leaves a mobile hole and electron.
Hole and electron are swept apart by the

3. applied field and:
h current transduction
Properties of the photodiode:
1. Gain: = 1 but is often near unity (1)
2. Dynamic Range: Largest measureable signal >>

smallest Excellent!
3. Ease of fabrication: Can be made very small.
Can be made into 1D and 2D
Page 114 of 316 arrays
5.8 Charge Coupled Device Array Detectors:

-10V -10V -10V
Al contact
SiO2
- - -
- - -
n-Si - - -
- - - - - -
1. e- are 2. h strikes 3. h+
repelled depletion accumulate
from Al region at Al
contact contact
-10V
+ + + + + +
-10V
-10V
+ + + + + +
-10V
+ + + + + +
Page 115 of 316

CCD Array detectors are very useful for

spectroscopy.
1. Maximum gain per pixel 1
Not as sensitive as a PMT.
2. Readout is relatively slow:

Ca. 100 frames per second 10-2 s.
3. But, conventional spectrometers measure the

light spectrum (power versus wavelength) work
by limiting the light incident on the detector to
one wavelength at a time. With a CCD Array
detector:
You can have a different detector for every
wavelength CCDs can acquire an entire spectrum
much more quickly than a PMT-based system.
4. The light spectrum falls in a 2-dimensional image

or picture.
5. Thermal noise is lower than in PMT, but cannot
be completely rejected.
6. Cosmic ray background can be dealt with
statistically:
If there is a single particular pixel (spot on the
picture) that is anomalously bright (i.e has an
unusually high reading) it can be rejected as
cosmic radiation noise using statistical analysis.
Page 116 of 316
6 Monochromators for Atomic Spectroscopy:

Chapter 7, Skoog Holler & Nieman
7C-2 Monochromators Dispersion
Resolution Speed
7C-3 Monochromator Slits and Spectral Resolution
Effective Bandwidth
Line Convolution (Effect of Slit Width on Resolution)
Czerny-Turner and Echelle designs
Monochromatic
Why do we care about monochromators? light is useful!
An analysis of the wavelength dependence of the

absorbance or emission of light is called: Spectroscopy
This technique is used to:

analyze the structure of atoms and molecules
identify the atoms and molecules
observe interactions between atoms and molecules
Measurement of the amount of monochromatic light

absorbed or emitted by atoms or molecules is called:
Spectrometry
This technique can tell you:
the concentration of atoms or molecules in a sample
Page 117 of 316

6.1 Adjustable Wavelength Selectors

Adjustable wavelength selectors are called:
Monochromators
Light Input Light Output

Polychromatic Monochromatic
White Narrow-band
Broadband
Wavelength / nm
455.3
Two important characteristics of monochromators are:

The amount of light that throughput
makes it through at a given
wavelength:
The range of wavelengths
bandwidth -
that exit the
monochromator:
Page 118 of 316

6.2 Monochromator Designs:
Czerney-Turner
blue
red
Prism
Wavelength
Dispersion:
Linear
Nonlinear with
UV-Absorption
Nonlinear
Page 119 of 316

6.3 The Grating Equation:
Grooves or blazes:
Lines or rulings etched into grating that scatter or
diffract the light.
Light emitted from the grating surface is called:
Diffracted light
For a single, polychromatic input beam:

Infinite monochrmatic output beams differing in
angle
For a particular wavelength ( ) to be diffracted at a

particular angle, the corresponding pathlength
difference (DBC) must be equal to:
n must = DBC where n is any integer
Page 120 of 316

The Grating Equation (cont):
For outgoing rays 1 and 2 to interfere constructively:

pathlength difference DBC must = n
i. b+i=? 90
ii. b=? 90 - i
iii. a+b+90 = ? 180
iv. substitute ii into iii
a + (90 i) + 90 = 180
a i +180 = 180
a=i
Page 121 of 316

The Grating Equation (cont):
For a particular wavelength to appear bright at

incident angle i, the following must be true:
n = DB + BC
We know that angle a = angle i. We can measure i,

so we use it in calculations.
sin(i) = sin(a) = opposite / hypotenuse = BC / AB = BC / d
sin(r) = sin(a) = opposite / hypotenuse = DB / AB = DB / d
BC + DB = d sin(i) + d sin (r) = d ( sin(i) + sin(r) )
For constructive interference (i.e. a bright condition):

n = d ( sin(i) + sin(r) )
Page 122 of 316
6.4 Dispersion
Dispersion characterizes the extent to which a
monochromator separates (disperses) the different
wavelengths of light.
Angular dispersion:
For a given incident angle, i, how quickly does the

color change as you change the viewing angle r?
In other words what is the derivative of

with respect to r?
d ( sin(i) + sin(r) )
n =
d ( sin(i) + sin(r) )/ n
(r) =
d/dr[dsin(i)] /n + d/dr[dsin(r)] / n
d / dr =
d / dr d cos(r) / n
Page 123 of 316

6.5 Angular dispersion:

order
dr n
d d cos(r) n=
= Groove
d= spacing on
Reciprocal linear dispersion: dy Fdr
grating
Angle of
r=
diffraction
wavelength
=
sin(r) r
sin(r) y/F
d d
dy Fdr
D-1 =
d cos(r) / nF
D-1 =
Page 124 of 316

For monochromators the important factor is:

Reciprocal Linear Dispersion
D-1 = d / dy = d cos(r) / nF
What is dispersion?
Consider a typical monochromator:
d = 1000 lines / mm r = 45 n = 1 F = 1m
First lets get the units straight!
d= d = 1/1000 mm = 10-3 mm = 1 m = 1000 nm
F= F = 1m = 1000 mm
D-1 = 1000 nm x 0.7 / 1 x 1000 mm Focal Plane

D-1 = 0.700 nm / mm
Exit Slit
0 100 200 300 400

y / mm
300 370 440 510 580

Page 125 of 316 / nm
6.6 Effective bandwidth
The combination of exit slit width and dispersion

determine EFF the effective bandwidth of the
monochromator. EFF is a number, in units of nm,
equal to the range of wavelengths that exit the
monochromator at any one time.
-1 W = slit width in mm
EFF = wD D-1 = dispersion in nm / mm
EFF is controlled in part by: the exit slit width - w
W = slit width in mm
D-1 = dispersion in nm / mm
EFF 15 nm
300 400 500 600 700
EFF 2 nm
300 400 500 600 700

/ nm
Page 126 of 316
6.7 Bandwith and Atomic Spectroscopy
Consider the case of a Ni atomic spectroscopy

experiment. Ni atoms emit at 231.7, 232.0 and
232.2 nm. However, Ni atoms only absorb light at
232.0 nm. (Two emission lines are non-resonant.)
So, it is important in atomic absorption spectroscopy
to remove Ni emission at 231.7 and 232.2, but
efficiently transmit light at 232.0 nm.
Page 127 of 316

6.8 Factors That Control EFF
In general, one wants a monochromator with as small

an effective bandwidth is practical. Such a
monochormator is called: high resolution
To achieve low effective bandwith / high resolution
EFF should be: small
EFF = wD-1 = wd cos(r) / nF ,
so d should be: small
F should be: large
w should be: small
The essential tradeoff is that very small EFF usually

equate to low light levels. For example, achieving
very small EFF by making w very small is often
unsuccessful because the source is imaged onto the
focal plane. Unless the source is a very bright, point
source, then making w is smaller than the source will
often cause light levels to drop dramatically.
Page 128 of 316

6.9 Resolution Defined
Resolution is defined as R /
Resolution is the ability to just separate two adjacent

spectral lines. For example, consider the output of a
monochromator with 0.1 nm versus 1 nm resolution.
In this case two purely monochromatic sources of 501
and 502 nm are focused into the entrance slit.
Grating Res olution Limted Throughput
Low resolution
(1 nm)
Light throughput
500 500.5 501 501.5 502 502.5 503

Wavelength / nm
R=0.1 nm
R=1 nm x 10
Note that the low resolution case has much lower

intensity as well.
Page 129 of 316

6.10 Grating Resolution
Resolution may be limited by the size of the grating as

well. The grating resolution, R, is given by:
R / = nN
n= Diffraction order
N= Number of grooves of the grating that are

illuminated
Grating resolution imposes a kind of effective

bandwidth limitation just like the EFF defined
above
Please note these three things:
1. Both grating resolution and effective bandwith

define a minimum resolvable for the
monochromator.
2. A given monochromator will be limited by the
worse of the two parameters.
3. This is usually EFF
Page 130 of 316

6.11 Grating Resolution Exercise:
How large must a 1000 groove/mm

grating be to give a resolution of 1nm at
500nm? (Assume that the
monochromator is functioning in first
order.)
R= / = nN
N= /n = 500 nm /1*1nm = 500 grooves
500 grooves / 1000 grooves/mm = 0.5 mm!
Page 131 of 316

If a Czerny-Turner monochromator has the following specifications:
Holographically-ruled diffraction grating with 1582 grooves per mm.
250 mm focal length
Grating position such that the diffracted angle is 45 degrees
Operation in first order
A slit width of 0.5 mm
a. What is the reciprocal linear dispersion effective bandwidth of this

monochromator (use appropriate units)?
b. What is the effective bandwidth of this monochromator (use

appropriate units)?
A Czerny-Turner monochromator is set to 300.00 nm and the slits are set so that
the effective bandwidth is 1.0 nm. If a broadband UV light source is directed into
the monochromator, what wavelengths of light will exit?
What type of light source might be appropriate for this experiment?
If a Czerny-Turner monochromator has the following specifications:

a. Holographically-ruled diffraction grating with 940 grooves per mm.
b. 250 mm focal length
c. Grating position such that the refracted angle is 45 degrees
d. Operation in first order
What slit width is required to give this monochromator an effective bandwidth of

0.5 nm?
How big must the grating be in order to achieve this resolution (R).
Page 132 of 316

6.12 High Resolution and Echelle Monochromators

Atomic spectroscopy demands high resolution
because atomic absorption / emission lines are:
1. narrow 2. numerous
So we want effective bandwidth EFF = small

wD-1 = w d cos(r) / nF to be:
We also want resolution (R = / = nN) large

to be:
EFF R
w should be small -
d should be small small
F should be large large
n should be large large
But there are limitations to the above:

If w is too small 1 no light 2 exceeds
w the ability to focus light
d Limitations in the fabrication of gratings
F How big of a spectrometer is tolerable?
What about n?
n is the key
Page 133 of 316

A monochromator that operates in very high order is

called:
An Echelle monochromator
The grooves are machined to reflect light at high

incident angles i and r are nearly identical and are
called
An echelle monochromator outperforms a Czerny-

Turner monochromator in high-resolution applications:
Czerny-Turner Echelle
F/m 0.5 0.5
Groove density 1200 / mm 79 / mm
i,r or 10 63
R (300nm) 60,000 700,000
-1
D (nm / mm) 1.6 0.15
Page 134 of 316

But Echelle monochromators have a strange

handicap. At a given angle , many wavelengths may
simultaneously be striking the detector!
n = 2dsin( ) n1 = n2 2
1
63x300 = 2dsin( ) 2 = n1 1/n2
but
62x??? = 2dsin( ) also!
n / nm

61 309.83
62 304.84
63 300
64 295.31
65 290.77

This problem is called: Order overlap
Is this a problem for Czerny-Turner monochromators?

If n1=1 and 1=300nm then for n2=2, 2 = 150 nm
If n1=1 and 1=800nm then for n2=2, 2 = 400 nm
Czerny-Turner monochromators handle this problem
with:
Echelle Monochromators handle the order-sorting
problem with cross dispersion usually a silica SiO2
prism. UV-absorption is a problem with glass
Page 135 of 316

Page 136 of 316

This means that the Echelle monochromator has a 2-

dimensional focal plane!
What kind of detector is tailor-made for an Echelle

mnochromator
The CCD array detector!
Page 137 of 316

7 Photometric Issues in Atomic Spectroscopy

Skoog Chapters Covered: 9B, 9C
Question: How can one quantify low concentrations of

atoms that have extremely narrow band absorption
spectra? How can one do this when the atoms are
contained in a glowing flame that contains many
broadband emitting species.
Answer: Interrogate the sample of atoms with an

equally narrow band and very bright source of
radiation.
Where do you find such a source?

Hollow Cathode Lamp
Cathode is made of analyte metal e.g. Fe or Zn

Ne+ ions in plasma accelerate into cathode, sputter
metals atoms, excite them and emission collected out
lamp end.
Page 138 of 316

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Three photometric problems:

1. EFF >> ATOMIC so stray light is a major issue. The maximum
absorbance that one could realistically expect with a 0.25 nm EFF is
roughly 0.01 using a broadband source, and this is going to be noisy so:
a. Use hollow cathode lamp with emission identically matched to
absorption. This makes maximum absorbance much higher, and
less noisy. For the remaining source and flame stray light, one can:
b. Modulate the source to help to distinguish it from flame and other
radiation that leaks into detector around atomic line.
2. Flame and molecules in flame present a broadband absorption
interference. These are direct interferants, so they must be measured in
some way that is not sensitive to the analyte atoms:
a. Use broadband (D2) source that is approximately blind to analyte
to approximate the broadband flame absorbance.
b. Use Zeeman splitting to polarization select between resonant
absorption and off-peak background absorption.
c. Use Smith-Hiefje splitting to approximate the same effect.
Major Learning Objectives:

#1 Understand the exponential relationship between transmitted light power
and concentration.
#2 Understand how stray light can affect computed values of absorption.
#3 Understand how narrow atomic bands, that are narrower than the
attainable effective bandwidth, yield a difficult stray light problem.
#4 Understand how the hollow cathode lamp addresses the problem of

atomic absorption lines that are narrower than the effective bandwidth of
the monochromator by matching the spectum of the source (hollow
cathode lamp) to that of the absorber (free atoms in flame).
#5 Understand how the absorbance of broadband D2 radiation actually

approximates the blank absorbance of the flame even when there is
analyte present in the flame.
#6 Understand how Zeeman splitting samples the flame background but not
the analyte absorbance.
Page 151 of 316

8 Practical aspects of atomic spectroscopy:

Sample introduction and plasma chemistry.
Skoog chapters covered: 8C, 9A
Flame or Plasma
Monochromator
Atomic Emission Atomic PMT

from Absorption
P
Hollow and / or
Cathode
Lamp Atomic P0
Emission in
Light source Flame/Plasma
for absorption
measurement Molecular gas Absorbance:
only. P
Dry solids A Log b[C ]
PO
Flame or
Plasma
Aerosol Emission:
Nebulizer / [C] k ( P PO )
Spray Chamber
Analytes in
Solution
In AAS and AES solutions of analytes are a. aerosolized, b. measured as

they race past the detector as a very dilute gas and in a flame or plasma.
This a. very strongly dilutes the analyte and b. limits the time it spends in
front of the detector. ---- Why is this tradeoff worthwhile?
Intense and narrow emission and absorption bands make

atomic spectroscopy sensitive and selective compared to
most comparable solution methods.
Page 152 of 316

8.1 Nebulization (sample introduction):
Sample introduction in AAS and AES is usually

through a nebulizer.
The nebulizer makes a very fine mist of the analyte

solution.
The finest droplets in this mist are then selected by

the spray chamber.
These droplets are then swept into the flame or

plasma.
This sample introduction process is necessary but:
a. inefficient
b. problematic
c. hard to do reproducibly
Page 153 of 316

Nebulizer types:
1. concentric
2. cross flow
3. fritted disk pneumatic
4. Babbington
5. ultrasonic
Concentric:
Page 154 of 316

Nebulizers, cont:
Cross flow:
Babbington:
Fritted disk:
Page 155 of 316

Ultrasonic:
Page 156 of 316

8.2 Atomization
Atoms and
ions diluted
Atoms
Optimal AES
region
Optimal AAS
region
Molecules
Particles
Droplets
Entire process happens in: milliseconds
So large droplets are: bad
Page 157 of 316

8.3 Flame Chemistry and Matrix Effects
Our understanding of what happens is based on the

idea of:
Local Thermodyamic Equilibrium
(LTE)
This means that we theorize and analyze the process

occurring in the flame/plasma as though they were
equilibrium processes.
Some flame / plasma properties (Table 9-1):

Fuel Oxidant Temp. C Burn velocity
(cm/s)
Natural Air 1800 40
Gas
Natural O2 2800 400
Gas
Acetylene Air 2400 230
Acetylene O2 3100 2000
Acetylene N2O 2700 300
Page 158 of 316

8.4 Flame as sample holder:
1. For best sensitivity:
Interrogate with hollow cathode

radiation through atom rich region.
Changes in observation height:

2. Change sensitivity so dont move
observation region during experiment.
Page 159 of 316
8.5 Optimal observation height:
Optimize:
Observation region
Flame stoichiometry
1. Increased absorbance corresponds to:

More gas phase atoms
2. Absorbance normally starts low because:
Molecules have not been broken down

into atoms yet
3. Absorbance normally ends low because:
Gas in flame expands and atoms are

diluted.
4. Cr profile decreases because:
Cr atoms form stable oxides and this

increases with height.
5. Ag profile increases because:
Ag atoms do not form stable oxides
Page 160 of 316

8.6 Flame Chemistry and Interferences:
1. Chemical equilibria can occur in flames.

Common oxyanions can react with atoms:
- -
H2PO4 + Fe FePO4 + 2H+ + 3e
-2 -
SO4 + Fe FeSO4 + e
2. Electrons can be a reagent.

Ionization can be suppressed by addition of KCl:
-
KCl K+ + Cl + e
-
Fe+ + e Fe
3. Oxides can be suppressed by:
a. Higher Temp: Fe + Ox FeOx

b. Competition: Ti + Ox TiOx
a. Ti absorbs O from flame
c. richer flame: more C2H2, less O2
4. Hydroxides can be suppressed by:
Higher Temp: CaOH Ca + OH
Page 161 of 316

8.7 Matrix adjustments in atomic spectroscopy:
Releasing agents:
Compete with analyte for interferant:
Adding Sr+2 to react with PO4 and release
Ca+2 and Mg+2 for analysis.
Protecting agents:
Form stable but volatile complex with
analyte e.g. EDTA
Radiation buffering (when sensitivity is not an issue):

Add interferant to samples and standards
Standard Additions:
Match matrix of sample and standards
Page 162 of 316

Chem 155 Unit 9 Atomic Emission Page 163 of 316
9 Atomic Emission Spectroscopy
Responsibilities in the Chapter:

Introduction and all of 10A
Overview
Nomenclature
Plasma Types
Pros and Cons
Inductively Coupled Plasma

Torch Design
Operating Principles
Plasma Properties
Plasma Optimization
Direct Current Plasma

Design
Properties
Spectrometers
Bandwidth Considerations
Multi-channel Designs
Quantitation and Accuracy
Comparisons with Atomic Absorption
Page 163 of 316

9.1 AAS / AES Review:
M+ - ions
-e- +e-
Emission
M*h [M] = k(P-Po)
h
Absorption
HCL Atomic gas [M] = -klog(P/Po)
M
Molecular
gas
Solid
particles
Nebulize
Liquds Solids Gases
Page 164 of 316

9.2 Types of AES:
9.2.1 PLASMA
Plasma is ionized gas typically Ar, Ar+, e-

Ionization is maintained in one of three ways:
A DC electrical discharge called Direct Current

Plasma, DCP or Plasma Jet
An induction coil operating at 27 or 41 MHz
Inductively Coupled Plasma , ICP
A Microwave Cavity called Microwave Induced
Plasma or MIP
9.2.2 FLAME
Flame Emission Spectrometry or FES

Flame is chemical plasma typically
comprising CO2, H2O, other Molecules, e- e-
Flame types include: Air - C2H2 , N2O - C2H2 ,
O2 C2H2
FES is limited to easily excited species such as
Na, Li, K
9.2.3 ARC / SPARK

Arc = Continuous Discharge
Spark = Pulsed Discharge
These two are like DCP, but are done in air, on
solids (e.g. pressed powders) or metal
surfaces
Page 165 of 316

9.3 Inert-Gas Plasma Properties (ICP,DCP)
Ar mostly, also Ne and He (MIP)

Chemically Inert
9.4 Predominant Species are Ar, Ar+, and electrons
Very Hot
5,000 to 10,000 K vs. 2,000 to 3,000 K for flames
Superior Atomizers
B2O3, PO4, WOx, VOx, ZrOx and other
REFRACTORY compounds can be analyzed
Refractory compounds are extremely thermally
stable, and therefore hard to atomize.
Nonmetals Can Be Analyzed
Cl, Br, I, S for example: Try making a hollow
cathode lamp out of a non-metal!
Plasma Emission Flame Atomic Absorption
Simultaneous Detection One or Two at a Time
Optimization Simpler Easier to Use
Excellent Dynamic Range Higher Precision
Wide Range of Elements Metals Only
Costly to Use / Buy Significantly Cheaper
Page 166 of 316

9.5 Inductively Coupled Plasma AES: ICP-AES
Induction and Eddy Currents:
Switch on electromagnet & eddy currents (circular)

flow in conductor around changing magnetic field
lines:
Page 167 of 316

9.6 ICP Torches
Intensely luminescent, 5000K

plasma tauroidal core.
Water-cooled induction
coils with 27 or 41 MHz
large AC currents
flowing through them.
Quartz envelope
Tangential Ar
flow pushes
Sample plasma away
aerosol from quartz
housing
Page 168 of 316

9.7 Atomization in Ar-ICP
Excitation =
Optimal
observation
region.
Desolvation /
Atomization
Properties:
Inert
Hot
Electron rich
Inert = lowAtomization
Analyte oxide formation, chemical interference low
/ Ionization:
Hot = efficient atomization
Electron rich = low ionization
Excellent atomizer
Page 169 of 316

9.8 Direct Current Plasma AES: DCP-AES
Ar flow:
Argon Plasma Jet:

Argon:
Relative to ICP:
Page 170 of 316

9.9 Advantages of Emission Methods

One of the main advantages of emission methods is:
Simultaneous multielement determinations
With the appropriate spectrometer design, it is

possible to measure many elements at once using
emission. This means:
Simultaneous light detection at different
wavelengths i.e. different points along the focal
plane.
With conventional spectrometers, this means many

photomultiplier tubes (PMTs) placed along the focal
plane of the Czerny-Turner, or similar
monochromator. But, PMTs are neither small, nor
cheap. So, PMT-based multi-channel spectrometers
are both BIG, and EXPENSIVE.
With the echelle design, there is a 2-dimensional

focal plane that happens to be extremely well suited
to the lithographically designed semiconductor
charge-transfer array detector such as the CCD and
CID.
Page 171 of 316

P0
Page 172 of 316

9.10 Accuracy and Precision in AES
The Good News:
Plasmas of Inert Gas are Excellent Atomizers so:
Atomization is High
Chemical Interference is Low
Oxidation is Low
The Bad News:
AES relies on the existence of excited states, but

excited state populations:
Are small relative to ground states

Depend strongly on temperature
E
Excited Excited
N g kT
Recall: e
No go
The Solution: (when very high accuracy or precision
is needed)
Internal Standards
Page 173 of 316

A perfect internal standard will have:

Identical Activation Energy ( E)
EA
g kT
SA QA NA e
g0
EIS
g kT
SIS QIS NIS e
g0
Exercise: Derive an equation for the ratio of the

analyte to IS signals as a function of temperature:
a
e a b
e
Recall: b
e
EA E IS
The
answer:
SA QANA kT
e
S IS Q IS N IS
So:
Page 174 of 316

A good internal standard can compensate for drift

i.e. changes that appear between measurements
due to:
Sample introduction rate
Instrument Gain
Atomization Efficiency
Temperature
Do you need one?
Do the experiment
Check your precision and accuracy and see!
Page 175 of 316

Two examples of precision / accuracy assessments of

ICP-AES:
Page 176 of 316

Page 177 of 316

10 Ultraviolet-Visible and Near Infrared Absorption

Responsibilities in the Chapter 13
Measurement of Transmittance / Absorbance (13A)
Beers Law (13B)
Mixtures (13B-1)
Limitations (13B-2)
Effect of Instrumental Noise on Spectrophotometric
Analysis (13C)
Instrumentation (13D, 13D-1, 13D-2)
10.1 Overview
Ultraviolet 190-400 nm
Visible 400-750 nm
Near Infrared 750-2500 nm
We group these wavelengths together for two
reasons.
Analytes:
This wavelength range covers many atomic

and molecular electronic absorptions.
Optics:
Pure SiO2, called fused silica or quartz is

transparent in this range. Even air
absorbs strongly at <180 nm.
Page 178 of 316

10.2 The Blank

There are two problems with calculating the
absorbance of a liquid sample in a container.
These are that losses of light other than
abosrobance by analyte molecules.
b. Absorption by solvent or non-analyte solute
in the sample.
a. Reflections at each interface (Fresnel eqn).

Scattering particles in the sample.
Reflection losses are about 7% for glass, air and

water. So even without analyte, the transmitted
power for the sample, P, is less than the incident
power.
How do we compensate for this loss in intensity in the
sample power P that is not due to the analyte?
Measure Po with a blank in place that has nearly
the same reflection and scattering losses as the
cuvette so the only difference in P and Po is
due to absorbance.
Page 179 of 316

10.3 Theory of light absorbance:

Absorbance theory is based on the assumption that
molecules have a cross section through which no
light can pass.
Each slice of the sample removes a fixed fraction of

the light entering it so an exponential relationship
between light power and distance results:
P(x) = Po exp(-aNx)
a = the cross section of the absorber in cm2
N = number of absorbers per cm3 (# concentration)
x = distance
Page 180 of 316

10.4 Cross section a and molar extinction :
The cross-section a can be related to the molar

extinction coefficient, , in Beers law.
Use this to answer the question: can the absorption

cross section of a molecule be bigger than the
molecule itself?
Beers law: A = bC = -Log(P/Po)
M max 105 M-1 cm-1
a is about 1 nm
theoretical cross section is about
the same size as the molecule!
Page 181 of 316

10.5 Limitations to Beers Law:

1. Fundamental Deviations:
1.1. Electrostatic or other interactions between
the absorbing ions or molecules can change the
energy levels and symmetries of molecules, and
therefore change ( ). These appear at high
(>0.01 M) absorber concentration.
1.2. Refractive Index depends on refractive
index (n) so, in cases where comparisons
between absorbance in solutions of greatly
differing n must be made, then you can re-write
Beers Law as: A = n/(n2+2)2 b c.
2. Apparent Chemical Deviations: Concentration

dependent equilibria may occur such as
HA H+ + A-. If for HA is different from for A-
then cal curve may be nonlinear!
3. Instrumental Deviations: Several instrumental

factors are known to cause non-Beers law
behavior:
3.1. The monochromator effective bandwidth is
large compared to the spectral bandwidths
analyzed.
3.2. Stray light is getting into the detector.
3.3. The light levels are too low for the detector to
accurately represent.
Page 182 of 316

10.5.1 Instrumental Limitations to Beers Law:
10.5.1.1 The Stray Radiation Problem:

Light reaching the detector that a. does not come from the
source, b. does not go through the sample. or c. is not the
intended wavelength contributes a background power,
PSTRAY that, in addition to making the absorbance deviate
from Beers law.
Does stray radiation make a positive or a negative

deviation from Beers law (A = bC)
Stray radiation can come from the source!

In a conventional Czerny-Turner monochromator operating
in first order, if the monochromator setting is 700nm, 350nm
radiation diffracted in second order will also fall on the exit
slit! Order-sorting filters that are moved into the light beam
according to the wavelength normally remove this but if a
filter fails to remove all of the 350nm light then there will be a
PSTRAY component.
Pi Pi P stray
P stray 0.05 A true log A app log
i Po i P o P stray
Pi A true A app
i i 4
0
1 0
1 3
0.1 0.845
2
0.01 1.243
3 A true 2
110 -3 1.314 i
4
110 -4 1.321
1
0
0 1 2 3 4
C
i
Page 183 of 316

10.5.1.2 Instrumental Noise:

Consider the signals P and Po you actually
measure these, and they always contain noise.
Po P Po Po
A= A= A=
Case 1. Case 2.
Hard to distinguish P and Po Hard to distinguish P and zero
For example:
For example: P=0.010.01
P=0.990.01 Po=1.000.01
Possible : Possible: Possible :
P = 0.00
0.99
P/Po 1.00
A = -Log(P/Po)
1.01
A=-Log(P/Po)
Infinite!
NEGATIVE
Page 184 of 316

10.5.2 Apparent Deviations due to Equilibria:

For many molecules, especially weak acids, only one of the
conjugate acid / base pair may absorb at a given
wavelength.
This is how acid-base indicators work!
Suppose only the conjugate base of a weak acid is colored.

(think phenolphthalein!) What would a Beers law plot of an
indicator like phenolphthalein (HA) in pure water look like?
absorbing
HA H+ + A- Concentration of HA in
solution, the
1. KEQ = [H+][A-] / [HA] undissociated fraction.
2. Conservation of mass: [HA0] = [HA] + [A-]

so: [HA] = [HA0] [A-]
Number of moles of HA
- + -7
4. [A ] = [H ] (if >> 10 M) divided by volume of in
solution. Total or formal
concentration.
KEQ = [A-][A-] / [HA]

5=1+ 4
6=5+ 2 KEQ = [A-]2 / [HA0] [A-]
Page 185 of 316

KEQ = [A-]2 / [HA0] [A-]
try to solve for [A-]
[A-]2 + KEQ[A-] KEQ[HA0] = 0
2
a=1
2 b b 4ac
ax bx c 0 x
2a b = KEQ
c = KEQ[HA0]
2
- K K 4 K HAo
[A ] = x( K HAo )
2
Page 186 of 316

Let KEQ = 10-5

[HA] in solution: HA( K HAo) HAo x( K HAo)
4
4 10
4
3 10
x K HAo
4
2 10
HAo
4
1 10
0
5 4 4 4 4 4
0 5 10 1 10 1.5 10 2 10 2.5 10 3 10
HAo
Fraction ionized:
x K HAo z
HAo 0.1
z
0.01
7 6 5 4 3
1 10 1 10 1 10 1 10 1 10 0.01 0.1
HAo
z
Page 187 of 316

10.6 Monochromator Slit Convolution in UV-Vis:
A monochromator may qualitatively distort absorbance

peaks as well as quantitatively distort them if the EFF is too
large.
Page 188 of 316

So if monochromator slits are too wide (relative to

peaks):
Peaks are broadened
Non-Beers law behavior occurs
But if the monochromator slits are too narrow:
Low light power leads to noise
Low light power can lead to spurious

positive spikes positive deviations from
Beers law!
Page 189 of 316

10.7 UV-Vis Instrumentation:
Light Sources
a. D2 Lamps 190-400nm,
excellent UV sources
Electrical Discharge in D2 gas in a sealed, SiO2
bulb.
D2 D2* (dissociative molecular state)
D2* Da + Db + h
E = KE(Da) + KE(Db) + h
Wavelength
Kinetic Energy Continuum continuum
b. Tungsten(W)-Halogen 350-2500nm ,
excellent Vis-NIR (visible-near Infrared) sources
blackbody radiators (e.g. 2500K)
halogen helps to re-deposit W onto filament and
improves lifetime for very hot filaments
c. Xe- or Hg-arc lamps 200-1000nm ,

excellent UV-Vis sources
high-atomic number noble gas in electrical
discharge many states many wavelengths
that coalesce into a continuum.
Materials:
silica = SiO2 (190-2500nm)
glass = SiO2 + NaBO4, etc. (350-1500nm)
sapphire = Al2O3 (180-5500nm) $$
Page 190 of 316

10.8 Single vs. double-beam instruments:

Single-beam design: Measure blank signal (Po),
digitize and record the number, replace blank
with sample cuvette, measure P, calculate A.
Advantages of single beam designs:
Simple design, relatively inexpensive.
Disadvantages of single beam designs:
P and Po are measured at different times so a

particular noise source is prominent:
drift
i.e. slow change in:
o source intensity
o detector sensitivity
gives
absorbance error.
Page 191 of 316

Double-beam instruments continually monitor

or alternate between P and Po using a second,
blank cuvette
Advantages of double beam designs:

Accuracy:
P and Po are measured:
almost simultaneously
so source and detector drift are:
minimized!
Hence absorbances are more:
accurate.
Disadvantages of double beam designs:

Higher Cost: More complicated design
Often: Two detectors
Often: More optics
Page 192 of 316

Photodiode Array and Charge Coupled Device

(CCD) Array Multichannel Spectrometers (13D-3)
Advantages of Multichannel UV-Vis:

The spectrometers are FAST because:
no need to mechanically rotate a grating
all detectors are measuring the signal at once
The spectrometers are LESS EXPENSIVE because:
they have few moving parts and CCD
detectors are a mass produced technology.
The Wavelengths are VERY ACCURATE because:
the grating position never changes.
Disadvantage of Multichannel UV-Vis:

Single-beam design limits photometric accuracy
Page 193 of 316

Chem 155 Unit 11 194 of 316
11 UV-Visible Spectroscopy of Molecules

Skoog Ch. 14A all, 14B all, 14E-1 and 14E-3
Consider the simplest carbonyl:
H
C O Lewis Structure of
Formaldehyde
H
3 types of valence electrons are important:

n non-bonding
sigma-bonding
pi-bonding
Orbital geometries
Page 194 of 316

Chem 155 Unit 11 195 of 316
11.1 Spectral Assignments 10 *

9 *
8
.
7
1
Energy
6
5 10
4
5
Absorbance
4 n
2 3 3
0
200 250 300 350 400
2
Wavelength / nm 1
0
UV spectrum & energy levels of a fictitious
carbonyl:
If: * * n * n * are allowed,
assign peaks 1, 2 and 3 to specific transitions:
Transition Energy
From Diagram From Spectrum
Possible Energy / Peak # / nm / cm-1
Transitions arbitrary units
* 10-0 = 10 1 200 50,000
n * 10-4 = 6 2 250 40,000
* 9-1 = 8 3 330 30,000
n * 9-4 = 5
E (spectrum) E (digram) / nm Assignment
50,000 10 200 *
40,000 8 250 n *
30,000 6 330 *
n *
Page 195 of 316

Chem 155 Unit 11 196 of 316
11.2 Classification of Electronic Transitions
The following simplified classifications apply to

organic molecules:
1. *
1.1. Highest energy!
1.2. Shortest wavelength < 200 nm
1.3. O2, N2, all molecules have -bonds so: this
radiation is absorbed by air and SiO2 so
spectroscopy must be done in vacuum and is
very difficult called VUV or vacuum
ultraviolet spectroscopy
2. n *
2.1. 150-250nm most all transitions are found
in the ultraviolet
2.2. Molecules with lone (nonbonding) pairs of
electrons participate such as: O, N, Halogens
2.3. 100 to 1000 medium strength absorbers
3. n *, *
3.1. Longest wavelength transitions 200-700 nm
3.2. Strongest absorbers 100 to
-1 -1
100,000 M cm
3.3. Limited to molecules with -bonds e.g.
alkenes, alkynes, aromatics, carbonyls, azides
etc.
Page 196 of 316

Chem 155 Unit 11 197 of 316
11.3 Spectral Peak Broadening

Why are solution-phase electronic spectra hundreds
of nm wide if atomic emission and absorption peaks
are only ca. 0.005 nm wide?
Page 197 of 316

Chem 155 Unit 11 198 of 316
They are
molecules, not
atoms so
electronic
transitions,
vibrational
transitions,
rotational
transitions all
happen
simultaneously
Solution phase
interactions
perturb the energy
levels of each
molecule
differently. The
spectrum
averages over
many molecules,
creating a
continuum of
energy levels for
the transition
Page 198 of 316

Chem 155 Unit 11 199 of 316
11.3.1 Influence of Solvent Spectral Fine Structure:
11.3.2 Temperature and Spectral Fine Structure:
Page 199 of 316

Chem 155 Unit 11 200 of 316
11.3.3 Solvatochromism:
The shift in wavelength of an absorbance band with
changes in polarity (dielectric constant) of the solvent.
Conjugation: The presence of alternating single and

double bonds in organic molecules.
Page 200 of 316

Chem 155 Unit 11 201 of 316
11.4 Aromatic UV-Visible absorptions:
Three sets of * bands are prominent in the

spectrum of benzene:
Primary E2 B
(nm) 184 204 256
-1 -1
MAX (M cm ) 60,000 7,900 200
V. Strong Strong Intermed
-OH and NH2 red-shift and intensify the B-band
Based on this, does the nonbonding pair stabilize or

destabilize the orbitals? (Also, assuming that it
acts predominantly on as opposed to *)
Page 201 of 316

Chem 155 Unit 11 202 of 316
11.5 UV-Visible Bands of Aqeuous Transition Metal

Ions
Transitions are known as d-d because electronic

states correspond to changes in the population of:
Page 202 of 316

Chem 155 Unit 11 203 of 316
d-d transitions imply that d-orbitals have different

energies.
When ligands such as H2O coordinate (bond) to the

metal ion, they interact with the d-orbitals and change
their energies predictable ways depending on the
bonding geometries. They split the d-orbitals by and
amount , the ligand field strength.
Page 203 of 316

Chem 155 Unit 11 204 of 316
Ligand field splitting energy varies with the type of

ligand in the approximate following series:
I- < Br- < Cl- < F- < OH- < C2O4-2 < H2O < SCN < NH3
< Ethylenediamine < o-phenanthroline < NO2- < CN-
d-d transitions are common, 0.1 < < 1, but seldom

used for detecting or determining the concentration of
metal ions why?
Page 204 of 316

Chem 155 Unit 11 205 of 316
11.6 Charge-Transfer Complexes

Strongly Absorbing Metal Complexes:
Example:
Fe(III)3+-SCN- + h Fe(II)2+SCN0
electron transfer from SCN- to Fe(III)
5,000
Fe(II)2+(o-phenanthroline)3 + hv
Fe(III)3+(o-phenanthroline)-1(o-phenanthroline)2
electron transfer from Fe(II) to (o-phenanthroline)
12,000
Assuming that the absorption noise, A = 0.001, what

is the best detection limit for Fe+2 aquo and FeSCN+2
in the 300-800 nm range? Recall, Cm=3sb/m relate
sb to sA and m to eb!
Page 205 of 316

Chem 155 Unit 11 206 of 316
11.7 Lanthanide and Actinide Ions:

Lanthanide and Actinice ions have unusual spectral
properties.
Lots of weak visible

and infrared lines
Very narrow bands!

The 4f and 5f orbitals
are smaller than the
filled 6s and 7s orbitals
so the f orbitals are not
split or broadened
very much by solvent
ligands
Electronic
configurations differ in
energy due primarily to
orbital angular
momentum, i.e.
magnetic interactions
described by Russell-
Saunders and other
coupling schemes.
Page 206 of 316

Chem 155 Unit 11 207 of 316
11.8 Photometric Titration
Trace metals may be analyzed by by UV-Vis

absorption using a method known as photometric
titration.
For the complexation reaction:
S+TP
S = Analyte metal ion e.g. Fe3+
T = Titrant e.g. SCN-
P = Product e.g. FeSCN2+
Consider a big, stirred cuvette that you titrate with T.
To which of the above schemes would the FeSCN2+ titration above

correspond?
Why might titration be a more accurate and precise way to compute C than
using C=A/ b (i.e. from an absorbance calibration curve).
Page 207 of 316

Chem 155 Unit 11 208 of 316
11.9 Multi-component Analyses:

Consider a cell containing two analytes with overlapping absorbance
spectra:
Cons ider two absorbing s pecies A and B:
Bi
( 0.25A) i ( 0.75B) i 4
( 0.5A) i ( 0.5B) i
( 0.75A) i ( 0.25B) i
2
Ai
0
200 250 300 350 400
wi
MAX(A) = 233 nm, FWHM = 60 nm

A(233) = 5.3 A(263) = 3.2 A(293) = 0.72
MAX(B) = 293 nm, FWHM = 60 nm

B(233) = 0.72 B(263) = 3.2 B(293) = 5.3
Page 208 of 316

Chem 155 Unit 11 209 of 316
Unknown mixture of A and B
Given: 4
A(233) = 5.3 A(293) = 0.72

B(233) = 0.72 B(293) = 5.3
Absorbance
2
A(233) = A(233)bCA + B(233)bCB

A(293) = A(293)bCA + B(293)bCB 0
200220240260280300320340360380400
Wavelength / nm
A(233) = 2.55
A(293) = 3.47
What are the concentrations of A and B?
Answer: CA = 0.4, CB = 0.6

A1 CA B1 CB A
1
A2 CA B2 CB A2
I 5.3 CA 0.72 CB 2.55
II 0.72 CA 5.3 CB 3.47
0.72
III II
5.3
0.72 0.72 0.72
III 0.72 CA 5.3 CB 3.47
5.3 5.3 5.3
III 0.098 CA 0.72 CB 0.471 Subs titute CA into I and II (to check):
I III 5.3 CA 0.72 CB 2.55 I 5.3 0.4 0.72 CB 2.55

2.55 5.3 0.4
0.098 CA 0.72 CB 0.471 CB 0.60
0.72
5.202 CA 2.079
II 0.72 0.4 5.3 CB 3.47
2.079 3.47 0.72 0.4
CA 0.40 CB 0.60
5.202 5.3
Page 209 of 316

Chem 155 Unit 11 210 of 316
Matrix approach:
A1 CA B1 CB A
1 A
1
A1 B1
A2 CA B2 CB A2 C A
A2 B2 A 2
.
A3 B3 C A
A3 CA B3 CB A
3 B 3
A4 B4 A
A4 CA B4 CB A 4
4
Ai = the extinction coefficient of s pecies A at wavelength i
Bi = the extinction coefficient of s pecies B at wavelength i
C = the concentration of s pecies A

A
C = the concentration of s pecies B

B
A = the abs orbance at wavelength i

i
Use computers to solve matrix for CA and CB, CC etc.
Overdetermine matrices to average out noise.
Page 210 of 316

Chem 155 Unit 12 211 of 316
12 Intro to Fourier Transform Infrared

Spectroscopy
Skoog Chapters Covered

Fourier Transform Optical
Measurements
16A1 Vibrational
Transitions
16A2 Heteronuclear
Diatomics - Classical
16A3 Heteronuclear
Diatomics -
Quantum
16A4 Vibrational Modes
16A5 Vibrational Coupling
16C1 Fourier Transform
Instruments
Page 211 of 316

Chem 155 Unit 12 212 of 316
12.1 Overview:
Infrared (IR) spectroscopy 1 molecular
deals mainly with: vibrations
IR radiation is low energy relative to

visible light.
IR wavelengths range (approximately) from:
Wavelength (nm) Frequency (cm-1)

Near IR 1,000 2,500 10,000 4,000
Mid IR 2,500 20,000 4,000 - 500
Far IR 20,000 200,000 500 - 50
Page 212 of 316

Chem 155 Unit 12 213 of 316
12.2 Why is IR spectroscopy important?
1. UV-Vis spectra are useful, but: all molecules:

Vibrate
So most all molecules have:
Vibrational absorption spectra
And most vibrational modes absorb:
Strongly
So IR spectroscopy is a very general and sensitive
analytical tool.
2. Vibrational spectra have a lot of information:

a. Fingerprinting / Identification
Each molecule has a complex but unique
signature.
b. Quantitation
Beers law holds for vibrational

absorption too!
c. Measurement of Interactions / Configurations
Vibrational frequencies (spectra) are

sensitive to intermolecular interactions
so one can identify phenomena such as
hydrogen bonding, trans or gauche
interactions that may control reactivity of
molecules.
Page 213 of 316

Chem 155 Unit 12 214 of 316
12.3 Some Applications of IR spectroscopy
Identification of organic molecules
Identification of functional groups
Elucidation of molecular structure
Elucidation of intermolecular interactions
Monitoring of atmospheric pollutants.
Some breathalizers use IR absorption of ethanol.
Quantitation of amide nitrogen for the determination
of protein content in food and other materials.
IR Spectroscopy applications will follow in a later

lecture section.
Page 214 of 316

Chem 155 Unit 12 215 of 316
12.4 IR Spectroscopy is Difficult!

IR beams are invisible to the eye.
IR bands are narrow, so EFF must be small.
SiO2 absorbs IR radiation.
Water and other solvents absorb IR radiation.
IR photons are low energy, so they are hard to
detect!
Why cant you use a photomultiplier tube in the IR?
Infrared radiation has a longer wavelength
than visible radiation: IR > VIS
so
IR radiation has a lower frequency: IR = c/ IR
so
IR radiation has a lower energy: EIR = h IR,

so
Photomultiplier tubes dont work because:
For a PMT to work, the photon energy must be greater
than the work function of the metal!
E
But
EIR
Page 215 of 316

Chem 155 Unit 12 216 of 316
12.5 Monochromators Are Rarely Used in IR
Because:
IR sources are somewhat weak and
IR radiation is somewhat hard to detect, and
IR radiation is hard to focus and
IR spectroscopy must be done at very low effective
bandwidth,
Grating or prism-monochromator based IR
spectrometers are usually:
slow noisy
What is done instead?
Interferometry
In interferometry all light frequencies strike the

detector at once and the information comes from
signal oscillation as a function of:
time!
Could you measure the frequency of light by

measuring the time between maxima in the
electric field?
8 1 6 14
c 3 10 m s 10 10 m 3.333 10 s
c
Page 216 of 316

Chem 155 Unit 12 217 of 316
12.6 Interferometers measure light field vs. time
Imagine you were a light-speed gremlin with a

voltmeter in hand and a light beam passed by:
1
c c
c
Signal / Volts
0 5 10 15 20 25 30
Time / femtoseconds
This can be done with an optical device called an:
Interferometer
Take this on faith for now, and lets explore what that
might look like
Page 217 of 316

Chem 155 Unit 12 218 of 316
12.7 The Michelson interferometer:
Consider that S, A, B, and D are fixed i.e.

unmoving, but Mirror 2, C, can move. In terms of the
variable distance AC, and the fixed distance AB,
When will there be a bright light at the detector as
opposed to darkness?
When 2AB - 2AC = n
Page 218 of 316

Chem 155 Unit 12 219 of 316
12.8 How is interferometry performed?
Recall light interference is how gratings work.
10 Light Electric
cos( ) 6
Fields Sum
5
cos( ) 4
cos( ) cos( )
Constructive
0
Interference
5
0 0.5 1 1.5 2 2.5 3
2
10
Destructive
cos( ) 6
5 Interference
cos( ) 4
cos( ) cos( ) 0
5
0 0.5 1 1.5 2 2.5 3
For gratings constructive

interference occurs when the path
length difference between the rays,
, is an integral multiple of the
wavelength, :
= d(sin(I)+sin(r)) = n
Constructive interference:
n = f( sin(i)+sin(r) ) for a grating
n = f( time ) for an interferometer
Page 219 of 316

Chem 155 Unit 12 220 of 316
12.9 Signal Fluctuations for a Moving Mirror

Consider the case of mirror 2 moving at constant
velocity, v (cm/s).
AC(t) = Distance from beamsplitter to mirror 2

AB = Distance from beamsplitter to mirror 1
AC(0) = AB
AC(t) = AB + vt Constant Velocity Mirror
We can define the retardation - - as the difference

in pathlength between the rays going to the fixed and
moving mirrors respectively:
(t) = 2(AC(t) - AB)

(0) = 2(AC(0) - AB) = 2(AB - AB) = 0
What is the time interval, (s) between the conditions

= 0 and = ?
Find for which ( ) = = 2(AC( ) - AB)

= ? ( hint: substitute AB + vt for AC(t) )
( )= = 2(AC( )-AB) = 2( (AB-v ) -AB) = 2v
= 2v = / 2v
Page 220 of 316

Chem 155 Unit 12 221 of 316
So at every time interval the condition = n is

true and the detector sees a bright light:
What is the frequency (s-1)of the detector signal in

terms of , and in terms of the instrumental
parameters v and ?
f (s-1) = 1/ (s) = 2v(cm/s) / (cm)
For v = 1 cm/s and = 1000 nm, what will be the

detector frequency, f?
f (s-1) = 1/ (s) = 2 1(cm/s) / 1000x10-7 (cm) =

20,000Hz
Page 221 of 316

Chem 155 Unit 12 222 of 316
12.10 Mono and polychromatic response
The interferometer makes

a low frequency oscillating
signal from a high
frequency light signal.
But we still need to see the

signal as a function of
frequency (or wavelength)
to understand it.
Source: interferogram Spectrum
monochromatic-
one frequency
only.
Two frequencies
(lines) only.
Polychromatic
many
frequencies.
Page 222 of 316

Chem 155 Unit 12 223 of 316
12.11 Interferograms are not informative:
IR and other spectra are typically presented in the

frequency domain i.e. as a function of frequency or
wavelength not as a function of time.
How can one transform:

2000
Amplitude
1000
x
0
1000
0 20 40 60 80 100
T ime
Into something informative like:
In other words, the frequency or wavelength spectrum

of the light is where the information is, how can one
get this information from time domain signals?
Page 223 of 316

Chem 155 Unit 12 224 of 316
12.12 Transforming time frequency domain

1
2
1 .5
Amp li tud e1 0
Amp li tud e 1
0 .5
1
0 0 .5 1 1 .5 0
ti me_s econ ds 0 1 2 3 4
Freq uency_ Hz
1 2
1 .5
Amp li tud e2 0 Amp li tud e 1
+ 1
0 0 .5 1 1 .5
0 .5
0
0 1 2 3 4
ti me_s econ ds Freq uency_ Hz
------------------------------------------------------------------------------------------
2
2
1 .5
Amp li tud e3 0
Amp li tud e 1
0 .5
2
0 0 .5 1 1 .5 0
0 1 2 3 4
ti me_s econ ds
Freq uency_ Hz
signals:
Time Domain Frequency Domain conversion is

done by computer using an algorithm called the:
In what domain does a conventional monochromator operate?
Discrete Fourier Transform
Frequency or Wavelength Domain A vs.
Page 224 of 316

Chem 155 Unit 12 225 of 316
12.13 The Centerburst:

For a broad spectrum of frequencies going into a
Michelson interferometer, for what value of is the
constructive interference condition, n = satisified
for all wavelengths?
n = AB-BC = is always true if:
= 0!
Page 225 of 316

Chem 155 Unit 12 226 of 316
12.14 Time vs. frequency domain signals:

It is simple to imagine deciphering the E(t) signal of a
monochromatic light beam, but, what would a
broadband light signal look like in the time domain?
Broadband spectrum: Line spectrum:
100 20
Amplitude
Amplitude
10
50
0
0 10
0 0.5 1 1.5 0 20 40
Frequency Frequency
2000
Interferogram
Amplitude
2000
0 20 40 60 80 100
T ime
10
Amplitude
10
20 25 30 35 40
T ime
Page 226 of 316
Chem 155 Unit 12 227 of 316
12.15 Advantages of Interferometry.

In the IR, interferometers have advantages over
monochromators:
Advantage 1: Interferometers are fast:
Consider a grating onochromator:
If your source and detector are Multiplex
good from 200nm to 1200nm Advantage
(1000nm total) and your effective
bandwidth is 1 nm, how much light is being
measured or lost at any one time?
1 / (1000) is being measured so

1-[1/1000], or 99.9% is being thrown away!
You only get one at a time!
Advantage 2: Interferometers are high resolution.

Recall: If EFF > the width of the spectral peak:
Deviation from Peak distortion

Beers law (convolution)
Page 227 of 316

Chem 155 Unit 12 228 of 316
12.16 Resolution in Interferometry
Interferometers are easily made into high-

resolution spectrometers.
Consider two spectral lines differing in frequency by

only 2%:
How do these lines appear in the time domain?

8
7
cos( ) 6
4
cos( 1.02 ) 3
cos( ) cos( 1.02 )

2 2
0.999013 2
0 10 20 30 40 50 60 70 80 90 100
0 100
2
Page 228 of 316

Chem 155 Unit 12 229 of 316
Let us define resolution as: = 1 - 2 when 1 can

just be separated from 2.
I assert that to separate, or resolve these lines in the

frequency domain spectrum, we must scan the
retardation, , from 0 (where 1 and 2 are in phase)
to a new value, lets just call it where the two
signals are again in phase.
The frequency of oscillation of the signal in time is:
f = 2v(cm/s) / (cm) = 2 v
where is the light frequency in wavenumbers (cm-1).
The light power function will be sinusoidal:
P( ) = Pi cos(2 f t) = Pi cos(2 (2v ) t)
If we substitute v, the mirror velocity, with /2t = v we

get intensity versus retardation:
P( ) = B cos(2 )
Page 229 of 316

Chem 155 Unit 12 230 of 316
For two light rays of identical power Pi but different

frequency the total signal will be:
P( ) = Pi cos(2 1) + Pi cos(2 2)
For identical B
P( ) = P ( cos(2 1) + cos(2 2) )
What is P( ) when = 0?
B( cos(0)+cos(0) ) = 2B
So, when will P( ) be 2B again?

In other words, at what next value >0 will the two
different wavelengths 1 and 2 both give bright
signals again?
1 = some integer
2 = some integer 1
1 = 1 1
1 - 2 = 1
i.e. they have slipped one wavelength and are both

bright again
so, in this case: 1 - 2 = = 1/
Page 230 of 316

Chem 155 Unit 12 231 of 316
For a large , is small and resolution is high!
Consider an interferometer with a mirror travel

d = 0.5 cm that is operating in the mid visible at
500 nm:
= 107/500 nm = 20,000 cm-1

= 2d = 1 cm
= 1/ = 1/1cm,
so 20,001 cm-1 can be resolved from 20,000 cm-1.
107 / 20,001 cm-1 = 499.975 nm.
So, 500 nm can be resolved from 499.975 nm.
= 500 - 499.765 = 0.025nm
= 0.025nm is very difficult to achieve with a

EFF
monochromator!
Remember that is equal to 2d twice the mirror

displacement.
Page 231 of 316

Chem 155 Unit 12 232 of 316
12.17 Conclusions and Questions:
Infrared spectroscopy is important because:

1. Virtually all molecules have a unique vibrational
spectroscopic signature in the infrared.
2. Most molecules absorb strongly! So IR can be
quite sensitive for both identification and
quantitation.
3. But IR radiation is harder to manage with a
monochromator because sources are weak,
detectors are less sensitive and high resolution is
often needed.
4. So interferometry is done instead, which is both
inherently fast and high reseolution.
Interferometry converts optical frequency directly into

a frequency on the detector:
1. What frequency would a 3000 cm-1 source
produce in an interferometer operating with a
mirror velocity of 1 cm/s?
2. If the mirror travel were 1 cm, what would be the
resolution (EFF) in cm-1?
3. What will this value be in nm?
4. What focal length would be needed with a
monochromator with a 1000 nm grating spacing
and slits at 0.4 mm?
Page 232 of 316

Chem 155 Unit 12 233 of 316
12.18 Answers:
cm 1 -1
f 2v 21 3000 cm 6000s
s
1 1 1 1 1
effective bandwidth incm : EFF 0.5cm
2d 2 1 cm
7 nm 7 nm
10 10
cm cm
0.555nm
effective bandwidth in nm
: 1 1
3000 cm 3000.5cm
0.4 mm 1000 nm cos 45

w d cos ( r) w d cos ( r) 180
EFF F 0.514m
nF n EFF 1 0.55 nm
The detector frequency would be 6 kHz not a

problem.
The effective bandwidth for a 1 cm drive is 0.5 cm-1.
At 3000 cm-1 this equates to 0.55 nm.
The minimum focal length required to achieve this

bandwidth with a monochromator would be 0.5 m, a
fairly large system, especially when compared to a
1 cm interferometer drive.
Page 233 of 316

13 Vibrational Spectroscopy:
Selection Rules, Normal Modes and Group

Frequencies

16A all, 16B Sources and Transducers
16C-1, FT-Instruments
Rough overview of the optical spectroscopy ranges:

(nm) (cm-1) T (K) Designation Phenomena
100 100,000 140,000 UV - *, n- *,
n- *, - *
VIS Valence electron
excitation
1,000 10,000 14,000 Near IR low-E electronic
Hi-E vibrations
OH, C O, NH
Mid IR Group
Frequencies
10,000 1,000 1,400 Fingerprint
Inorganics,
High-Z, Low-k
Far IR
Lattice vibration,
Pure rotations
100,000 100 140 -wave
1,000,000 10 14
Page 234 of 316

13.1 Absorbance Bands Seen in the Infrared:

:
Vibrations Rotations
Solid broad rare

(phonon) (C60)
Liquid broad unresolved
Gas sharp sharp
Page 235 of 316

13.2 IR Selection Rules
How does electromagnetic radiation make a molecule

vibrate? Consider the diatomic molecule H-Cl.
E( ) sin 2
360
Electric Field Vector of Light
E( )
+ H H H
- Cl Cl Cl
In Out In Force
Page 236 of 316

Now consider the molecule N2:

E( ) sin 2
360
Electric Field Vector of Light
E( )
N N N
N N N
When will a molecular vibration absorb light?
when vibration causes change in dipole moment

dynamic dipole needed
Will interaction with electromagnetic radiation change

the FREQUENCY or the AMPLITUDE of the
vibration?
Page 237 of 316

13.3 Rotational Activity

E( ) Indicate
sin 2
360
the forces on the molecule below:
Electri c Fi el d Vector of Light
E( )
Cl H H Cl
When can a molecular rotor absorb light?

When a molecule has a permanent dipole
moment it is rotationally active.
Example:
J= J=
v= v=
Energy
Page 238 of 316
13.4 Normal Modes of Vibration:

Consider a diatomic molecule
translations
rotations
vibration!
For linear molecules, there are: 3N-5

modes of vibration.
Non-linear molecules have: 3N-6

modes of vibration.
Page 239 of 316

Modes of vibration:
For example CO2: Linear or Non-linear?
# of Modes: 3( ) - ( ) =
S = 1340 cm-1 AS = 2340 cm-1

symmetric stretch anti-symmetric stretch
S = 666 cm-1 S = cm-1

anti-symmetric stretch, IR active
symmetric stretch, IR inactive
bend modes, degenerate, IR active
For example H2O: Linear or Non-linear?

# of Modes: 3( ) - ( ) =
S(OH) = AS(OH) = S(OH) =

3562 cm-1 3756 cm-1 1596 cm-1
Page 240 of 316

13.4.1 One may see more than 3N-5/6 bands:
Overtones: v = 2, 3
Combinations: C = A + B and A - B
13.4.2 One may see fewer than 3N-5/6 bands:
not in 400-4000 cm-1 range of typical FTIR
Band may be weak
Band may be broad and overlap another band
Bands may be degenerate
Bands may be forbidden, i.e. if there may be no

change in dipole moment during the vibrational
mode.
Page 241 of 316

13.5 Group frequencies: a pleasant fiction!
Most molecules have many atoms.

That means many modes! Some very complex!
But some functional groups behave like small molecules!
Characteristic group
modes and show up
in IR spectra.
Examples of groups:
C-H
C=O
C N
NH3
OH etc.
3N-6 rule irrelevant to

this analysis
is somewhat
variable because:
environment
other bonds
other modes
influence the group
vibration.
Page 242 of 316

p. 136 of Spectrometric Identification of Organic Molecules

R.M. Silverstein, F.M. Webster, Wiley 1998
Page 243 of 316

Group frequencies can be calculated approximately:
Interatomic forces are approximated by Hookes Law:
F= ky
Since F = mA (Newtons Law)

d2y
F = mA = m = ky(t)
dt2
y(t) is proportional to its own second derivative.

This differential equation has a sinusoid solution:
1 k
y(t) = A cos(2 mt) m =
m1 m2
= reduced mass
m1 + m2
Units of are: units of mass

Page 244 of 316
Classical calculations (above) tell you the natural frequency

of a classical oscillator.
Quantuum mechanical calculations tell you the allowed

energy levels (E). From Planck and Einsteins equation
E=h we can calculate the light frequencies ( ).
E = (v + )h m
v = vibrational quantuum number = 0,1,2
v = 0,1 = the selection rule for vibrational transitions
1 k
again: m =
2
so:
h k
E= vh m = 1 =h
2
note: m is the oscillator frequency and

is the light frequency
What is the relationship between and m?
the light frequency equals the oscillator

frequency
What is the relationship between light frequency, , and light

wavenumber, (cm-1)? ( = 1/ (cm)).
=c
Page 245 of 316

13.6 Summary:
Molecules vibrate their atoms move back and forth

relative to each other.
Vibrations come in certain modes that involve the whole
molecule.
Modes wherein the molecule has a change in dipole
moment may be excited by IR radiation.
The higher the mass, the lower the mode frequency.
The stronger the bond, the higher the mode frequency.
In complex molecules with many atoms, some
individual parts of the molecule behave as though they
were independent.
The absorption bands corresponding to these group
modes are known as group frequencies and allow one
to identify functional groups such as aldehydes,
amides, olefins etc. on a given molecule.
Page 246 of 316

Chem 155 Unit 14 247 of 316
14 Infrared Spectrometry - Applications

Skoog Chapters Covered
17A1 Sample Handling in Mid-IR
17A2 Group Frequencies
17A3 Quantitation in Mid IR
17B Reflection Methods
17D Near IR applications
17E Far IR applications
The major difficulty in infrared spectroscopy is related

to the IR selection rule:
during vibration => IR active
Nearly everything absorbs IR radiation!
optics and solvents will have strong

absorption
Other difficulties relative to, e.g. visible spectroscopy:
Beam is invisible to the eye hard to align
IR Sources are often weak (blackbodies)
Detectors are not very sensitive
Page 247 of 316

Chem 155 Unit 14 248 of 316
14.1 Strategies used to make IR spectrometry work -

1. Poor sources and detectors:
use interferometer instead of monochromator
1. Optics absorb IR light:
use mirrors instead of lenses
2. Materials for transmissive optics:

use high-atomic-mass materials with weak
inter atomic bonds typical materials are:
1. CaF2 2. ZnSe 3. KBr 4. NaCl
3. Sample Handling:
use solvents transparent in selected spectral
regions
avoid solvents completely
use reflection-absorption instead of

transmission
Page 248 of 316

Chem 155 Unit 14 249 of 316
14.2 Solvents for IR spectroscopy:
14.3 Handling of neat (pure no solvent) liquids:
Sandwich your sample between IR-transparent

plates: e.g. NaCl:
Q. If your sample is 10 microns thick, and the

concentration of molecules in the neat sample is
10 M, and a typical band gives an absorbance of 0.1
a. what is ?
A= bC = A/bC = 0.1/0.001*10
= 10
b. why cant
you use a 1-cm pathlength NaCl cuvette?
A= bC = 10*1*10 = 100
T=10-100 = 0.00000000000000.01
Page 249 of 316
Chem 155 Unit 14 250 of 316
14.4 Handling of solids: pelletizing:
grind put into die
5 10 tons of pressure
a few minutes
vacuum to reduce voids
2-5 mg
14.5 Handling of Solids: mulling:
sample 2 m
Nujol =
mineral
(aliphatic ) oil
or
1. Grind sample finely Fluorolube =
fluorocarbon oil
2. Mix with mulling oil
3. Sandwich w/ KBr/NaCl
plates
Page 250 of 316

Chem 155 Unit 14 251 of 316
14.6 A general problem with pellets and mulls:
A = -log[P/Po]
How do you measure Po?
You use an open beam to approximate Po
So the spectra you obtain, are a sum of

absorbances the spectra contain:
Sample plus window plus mulling oil
absorbance plus reflection losses.
IR spectra obtained with these sampling methods are

useful for identification, not quantitation:
qualitative
The two pieces of information that are useful:
Functional group identification
Spectral fingerprinting
Page 251 of 316

Chem 155 Unit 14 252 of 316
14.7 Group Frequencies Examples

:
C-O-H versus C-Cl
Which stretching frequency is higher:
O-H or C-Cl
Why?
Reduced mass of C-Cl much
larger so (k/ )1/2 for C-Cl smaller
Page 252 of 316

Chem 155 Unit 14 253 of 316
14.8 Fingerprint Examples
Mid-IR Group Frequencies and Fingerprints

Example Branched Alkane Structural Isomers
Page 253 of 316

Chem 155 Unit 14 254 of 316
14.9 Diffuse Reflectance Methods:

A way to do IR of solids without mulling or pelletizing:
Page 254 of 316

Chem 155 Unit 14 255 of 316
14.10 Quantitation of Diffuse Reflectance Spectra:
Page 255 of 316

Chem 155 Unit 14 256 of 316
14.11 Attenuated Total Reflection Spectra:
Page 256 of 316

Chem 155 Unit 14 257 of 316
Attenuated Total Reflectance Sampling Volume and

Quantitation:
Penetration depth
Even though Snells Law says the light should totally

internally reflect, the light actually behaves as though
it were reflecting from a plane situated at:
A point just above the surface, slightly
penetrating the sample.
Page 257 of 316

Chem 155 Unit 14 258 of 316
The reflected beam interacts with the sample on the

crystal surface. It behaves as though it passes
through the sample and is reflected back by a plane
inside the sample some fraction of a wavelength
above the crystal surface (i.e. the n1-n2 interface).
Page 258 of 316

Chem 155 Unit 15 259 of 316
15 Raman Spectroscopy:
vibrational spectroscopy with
near UV, visible or near infrared

light is called:
Raman Scattering Spectroscopy
Intense monochromatic light H2O

beam usually a laser
A tiny fraction of An even tinier

the incident fraction of the
radiation scatters incident radiation
off of the molecules scatters off of the
in the solution: molecules and lose
or gain energy.
Rayleigh Raman
SCATTERED = INCIDENT SCATTERED INCIDENT
elastic scattering
inelastic scattering
Page 259 of 316

Chem 155 Unit 15 260 of 316
A Raman scattering experiment:
High resolution
monochromator /
Scattered Beam interferometer +
sensitive detector
Laser
Transmitted Beam
Incident Beam
The sample may be a Raman is not usually

concentrated solution, a neat used for dilute
liquid analyte, a transparent solutions.
solid or a surface.
If the laser is, e.g. a HeNe laser ( =632.8 nm), most of the scattered
radiation will be:
632.8 nm or 1/(632.8x10-7 cm) = 15803 cm-1
This scattered radiation is monochromatic, and is known in the spectrum

as the:
Rayleigh Line
Page 260 of 316

Chem 155 Unit 15 261 of 316
15.1 What a Raman Spectrum Looks Like
Identify in the following:
1. The Rayleigh peak.

2. The elastically scattered light peaks.
3. The inelastically scattered light peaks.
4. The Stokes peaks.
5. The anti-Stokes peaks.
6. Rationalize the relative intensity differences
between the Stokes and anti-Stokes peaks.
.
Hypothetical Raman Scattering Spectrum .
Light Power
400 425 450 475 500 525 550 575 600 625 650
Wavelength / nm
Page 261 of 316

Chem 155 Unit 15 262 of 316
15.2 Quantum View of Raman Scattering.
Energy S1
So
Vibrational coordinate
Hypothetical Raman Scattering Spectrum
Light Power
400 425 450 475 500 525 550 575 600 625 650
Wavelength / nm
Page 262 of 316

Chem 155 Unit 15 263 of 316
15.3 Classical View of Raman Scattering

Visible frequency radiation is higher frequency than IR
radiation and vibrational frequencies.
Molecular Vibration (e.g. C=O stretch, 2000 cm-1 )
r req cos 2 vib t
time
Vis ible Frequency Light (e.g. 500 nm / 20,000 cm-1 / blue-green)
E E0 cos 2 ex t
time
Page 263 of 316

Chem 155 Unit 15 264 of 316
Radiation interacts with molecules by inducing a

dipole moment.
The induced dipole m is proportional to the
polarizability, .
m E E0 cos 2
ex t
But the polarizability is itself oscillating!
d d
r re r cos 2
vib t
0 dr 0 dr A
So the induced dipole moment is oscillating in a more

complex way:
d
m E0 cos 2 ex t 0 r cos 2 vib t
dr A
The dynamic induced dipole that gives rise to Raman scattering:
Incident E-field
Oscillating
polarizability
Amplitude
modulation in
induced dipole
Page 264 of 316

Chem 155 Unit 15 265 of 316
15.4 The classical model of Raman:

The induced dipole moment has a complex
description, but how does this relate to Raman
scattering?
Recall that:
1 1
cos ( x) cos ( y) 2
cos ( x y) 2
cos ( x y)
So m becomes:
m E0 0 cos 2 ex t
1 d
E0 r cos 2 cos 2
2 dr A ex vib ex vib
Both Stokes and AntiStokes peaks are predicted by classical theory!

Clas s ical Model 'Catas trophe'
Light Power
4 4 4 4 4 4 4 4 4 4 4
1.6 10 1.68 101.76 101.84 101.92 10 2 10 2.08 102.16 102.24 102.32 10 2.4 10
Wavelength / nm
but
Page 265 of 316

Chem 155 Unit 15 266 of 316
15.5 The classical model: catastrophe!

Real intens ity dis tributions
Light Power
4 4 4 4 4 4 4 4 4 4 4
1.6 10 1.68 101.76 101.84 101.92 10 2 10 2.08 102.16 102.24 102.32 10 2.4 10
Frequency / cm-1
The classical model does not predict the Stokes /

anti-Stokes intensity ratios.
With what does an equal intensity distribution violate?
Boltzmann Distribution between v=0 and
v=1 vibrational levels.
So, the answer is: the quantum model wins, and we
accept the virtual state. Im told that time-dependent
solutions to the molecular structure allow for these
evanescent states.
The Stokes peaks are more intense than the
anti Stokes
peaks there are more molecules in the
because: ground vibrational state, that can
give rise to Stokes peaks, than in
the excited vibrational state, that
can give rise to anti-stokes peaks.
Page 266 of 316

Chem 155 Unit 15 267 of 316
15.6 Raman Activity:

mode Raman IR active?
active?
Symmetric
O stretch
C NO YES YES NO
Asymmetric
O stretch
YES NO NO YES
C
Bend
O
YES YES YES YES
C
Page 267 of 316

Chem 155 Unit 15 268 of 316
Also note that:
Purely centro-symmetric modes are only Raman

active, but IR inactive.
Most modes are both Raman and IR allowed.
Many allowed modes may be too weak to detect in

either Raman or IR or both.
Intensity distributions between Raman and IR often

differ substantially.
Page 268 of 316

Chem 155 Unit 15 269 of 316
15.7 Some general points regarding Raman:
Pro: Raman uses visible light:

Optics
Solvents
Biological samples
Imaging with Raman has better spatial resolution

because of the shorter wavelengths used.
Microscopes can be used in Raman microprobe

mode.
Silver and gold particles or rough surfaces can very

strongly enhance Raman scattering.
Different vibrational modes can be seen (e.g.

centrosymmetric)
1 k
vib
2
Very low frequency modes are accessible (e.g.

inorganics):
Depolarization ratio can distinguish between

symmetric and asymmetric stretches.
Page 269 of 316

Chem 155 Unit 15 270 of 316
Con: Raman signals are often very weak.
High-powered lasers can damage samples, so this is

not always a good solution.
Fluorescent molecules can give bad spectral

interference for Stokes lines.
Page 270 of 316

Chem 155 Unit 15 271 of 316
15.8 Resonance Raman

S1
So
Pros
1. Intensity greatly enhanced (102 106)
2. Selective for vibrations of chromophore
Cons
1. Sample degradation because of absorption.
2. Fluorescence can swamp Raman signals.
Fluorescence can be rejected with a very short pulsed laser source:
typical fluorophore - FLUOR ~ 10-9 s.

Raman - RAMAN ~ 10-14 S
Page 271 of 316

Chem 155 Unit 15 272 of 316
15.9 Raman Exercises

Given the spectrum below, compute the following:
1. The laser wavelength.

2. The vibrational frequencies of the modes in the molecule giving
rise to the following spectrum.
3. The effective bandwidth is needed in the visible wavelength
monochromator to achieve 1 cm-1 resolution with Raman given
the above laser system and chromophore.
.
Hypothetical Raman Scattering Spectrum .
Light Power
400 425 450 475 500 525 550 575 600 625 650
Wavelength / nm
7
10 1
las er frequency: 20000 cm
500
7
10 1
Stokes frequency: 17857 cm
560
7 7
10 10 1
2143 cm Effective bandwidth: Must res olve 20000 and 20001 cm-1.
500 560
7 7
7 7 10 10
10 10 1 0.025 nm high res olution needed!
2759 cm 20000 20001
500 580
Page 272 of 316

Chem 155 Unit 15 273 of 316
4. Using and Ar-ion laser at 514.5nm, at what wavelength would

you expect to see the Stokes peak for the NO2 resonance of
nitrobenzene given that it is normally observed at 1520 cm-1 in
IR absorption spectroscopy?
7 7
10 1 10
1520 17916 cm 558.2 nm
514.5 17916
5. Does the CO bond in CO2 have a dipole moment? YES
6. Does the CO2 molecule have a dipole moment? Why?

No, because individual C=O bond dipoles exactly cancel.
7. Draw a series of CO2 molecules to illustrate the symmetric

stretching mode.
OCO O C O O C O
8. Is the CO2 symmetric stretch IR active? Why?

No, because at no time during the vibration does the dipole
moment change.
9. Is the CO2 symmetric stretch Raman active? Why?

Yes, because during the vibration the polarizability
changes.
Page 273 of 316

Chem 155 Unit 15 274 of 316
10. Draw a series of CO2 molecules to illustrate the
asymmetric stretching mode.
OC O O C O O CO
11. Is the asymmetric stretch IR active? Why?

Yes, because at during the vibration the dipole moment
changes.
12. Is the asymmetric stretch Raman active? Why?

No, because at during the vibration the polarizability does
not change. (increase in one CO bond polarizability is
offset by decrease in the other bond).
13. Why is N2 not a greenhouse gas?

Homonuclear diatomics have no dipole moment change
during their only vibrational mode: symmetric stretch.
Page 274 of 316

Chem 155 Unit 15 275 of 316
14. Cyclohexanone has a strong absorption peak at 5.86 m
and at this wavelength a linear relationship exists between
absorbance and concentration. 10
4
1
-1 1706 cm
a. Calculate the frequency of this mode in cm . 5.86
b. Identify the part of the molecule responsible (the group
frequency) for the absorbance at this wavelength.
This is the carbonyl or ketone symmetric stretch.
c. Suggest a solvent that would be suitable for a quantitative

analysis of cyclohexanone at this wavelength.
CCl4, CHCl3 or tetrachloroethylene
d. A solution of cyclohexanone (2.0 mg/mL) in the selected

solvent has an absorbance of 0.40 in a cell with a path
length of 0.025 mm. What is the detection limit for this
compound under these conditions, if the noise associated
with the spectrum of the solvent is 0.001 absorbance
units.
A=bC calibration slope = b
1 3 sb
A 0.40 mg 3 0.001 mg
b 0.2 Cm 0.015
C mg mL m 1 mL
2.00 mg
mL 0.2
mL
Page 275 of 316

Chem 155 Unit 15 276 of 316
15. Calculate the ratio of HCl molecules in the first vibrational
excited state at 25C relative to the ground state. The IR
absorption appears at 2885 cm-1.
E
a. The Boltzmann equation is: NN gg e kT where g0 and g1
1
0
1
0
are the degeneracies of the states 0 and 1 and for HCl35

may be considered equal, and k is Boltzmanns constant,
T is absolute
temperature k 1.38 10
23 J
T 298 K k T 4.112 10
21
J
and E is the K
energy of
1 34 10 cm 20
state 1 2885 cm 6.626 10 J s 3.00 10 5.735 10 J
s
relative to 0. cm
1 34 10
E 2885 cm 6.626 10 J s 3.00 10
s
E E
N1 kT kT 7
e e 8.784 10
N0
b. Assuming
that the bond
m1 m2
strengths are 1 k
VIB
equal, 2 m1 m2
calculate the
vibrational 1 kDCl
frequency DCl 2 DCl DCl HCl
that you since kHCl kDCl
HCl 1 kHCl HCl DCl
expect for the
2
isotope DCl35. HCl
35 1 35 2 HCl
HCl DCl 0.717
35 1 35 2 DCl
DCl 2885 0.717 DCl 2069
Page 276 of 316

Chem 155 Unit 15 277 of 316
16. Draw three simple Jablonski diagrams, e.g. for a simple
molecule and indicating just two electronic states S0 and S1
(no T state is needed).
a. On the first diagram, draw arrows corresponding to light

absorption followed by fluorescent light emission.
b. On the second, draw arrows corresponding to the Stokes
Raman peaks.
c. On the third, draw arrows corresponding to the Anti-
Stokes Raman process.
S1
Virtual state
S0
Fluorescence Raman-Stokes Raman-AntiStokes
Page 277 of 316

Chem 155 Unit 15 278 of 316
17. A peak is observed in a fluorescence spectrometer when
analyzing trace impurities in water. With the monochromator
excitation source set to 250 nm, the mysterious weak peak
appears at 274 nm, unusually close to the excitation band.
When the excitation wavelength was changed to 300 nm, the
mystery bands moved to 335 nm.
a. Calculate the Stokes shift for the mystery band in cm-1 for
the 250 nm excitation experiment.
b. Calculate the Stokes shift for the mystery band in cm-1 for
the 300 nm excitation experiment.
c. What might this mystery peak be?
7 7 7 7
10 10 10 10
3504 3483
250 274 300 335
The band moves with the excitation it is the Raman

peak for water OH stretch modes.
Page 278 of 316

Chem 155 Unit 16 279 of 316
16 Mass Spectrometry (MS) overview:

Skoog: sections 20A and B
In MS one
a. often initially performs GC or LC separation

b. gets analyte molecules into the gas phase in vacuum
c. converts analyte molecules into ions
d. that are both intact and in fragments
e. exposes the analyte ions to E and/or B fields
f. separates them based on mass / charge
g. detects them based on current (they are ions)
16.1 Example: of a GCMS instrument:
Page 279 of 316

Chem 155 Unit 16 280 of 316
16.2 Block diagram of MS instrument.
Sample
Introduction
Ionization
Source
Mass
Analyzer
Computer for Display /

storage
Pattern recognition to ID
molecule
Page 280 of 316

Chem 155 Unit 16 281 of 316
16.3 Information from ion mass

Mass spectra can distinguish
o Different isotopes of the same element.
o Small mass differences between molecules with the same
nominal molecular weight.
This makes mass spectra complex because:

There are peaks for every isotopomer!
1H35Cl, 2H35Cl, 1H37Cl, 2H37Cl
But this can be informative: Isobaric interferants
Asparagine 13CH2CONH2 (133.12)

Aspartic acid CH2CO2H (133.10)
Distinguishable only by high resolution MS
Source: Quantitative Chemical Analysis 6th Ed. By Daniel Harris
Page 281 of 316

Chem 155 Unit 16 282 of 316
16.4 Ionization Sources
16.4.1 Electron Impact (EI):
Hard ionization: extensive fragmentation
16.4.2 Chemical Ionization:

reagent gas e.g.
Derivative of EI but with 1000:1 excess of: CH4
Soft ionization: limited fragmentation
CH4 collision with electron CH4+ , CH5+, CH3+

CH4+ , CH5+, CH3+ recombination, rxn w/ CH4 C2H5+
CH5+ + MH MH2+ + CH4

Proton transfer M+1
C2H5+ + M M C2H5+
ethyl transfer M+15
Page 282 of 316

Chem 155 Unit 16 283 of 316
Example of CI vs EI MS of 1-octanol:
Electron impact
ionization
chemical
ionization (CH4)
Which is a better MS?
CI: more easily interpretable
EI: complex, but highly unique
Page 283 of 316

Chem 155 Unit 16 284 of 316
16.4.3 Matrix Assisted Laser Desorption-Ionization

(MALDI):
o Soft
o Pulsed introduction of solids
o Large molecules proteins / biomolecules
o Gives predominance of M+
sample in proteins in matrix supersonic

vacuum matrix ionizes expansion
chamber
Page 284 of 316

Chem 155 Unit 16 285 of 316
16.4.4 Electrospray Ionization (ESI):
o Soft
o Continuous introduction of solids
o Large molecules proteins / biomolecules
o Gives many different charge states: M+ , M+2 , M+3 ,
M+4 , M+5 etc.
What happens when charged droplets dry out?
Page 285 of 316

Chem 155 Unit 16 286 of 316
Which is a better ionization source for large molecules,

MALDI or ESI?
MALDI: spectra are more easily

interpretable
spectra are complex, but ESI easily
ESI:
interfaced to LC
Page 286 of 316

Chem 155 Unit 16 287 of 316
16.5 Mass Analyzers:
One important quality of mass analyzers is:
16.5.1.1 Resolution: R M/ M
Small molecule MS may demand high resolution:

For example, can one distinguish C2H4+, CH2N+, N2+, CO+
in most mass spectrometers?
Ion Nominal Mass Exact Mass

12C21H4+ 28 28.0313
12C1H214N+ 28 28.0187
14N2+ 28 28.0061
12C16O+ 28 27.9949
What resolution would be needed to distinguish these?
Smallest M = 28.0061-27.9949 = 0.012
m/ M = 28 / 0.012 = 2300
What resolution would one need to calculate the nominal

mass of immunoglobulin-G (M = 149190)?
m/ M = 149,190 / 1 = 150,000
Page 287 of 316

Chem 155 Unit 16 288 of 316
16.5.2 Magnetic Sectors:
Resolution of a single magnetic sector is ca. 2000
The properties that limit R of the ions entering it, mainly:

speed directional
distribution distribution
Page 288 of 316

Chem 155 Unit 16 289 of 316
16.5.3 Double-Focusing Electric Magnetic Sectors
Electric Sector Focuses: Speed distribution
Magnetic Sector Re-focuses:

Directional distribution
At double-focus point, all ions have narrow speed and

direction distributions, so only ions of a given M/z exit at
that point. What determines the M/z values that exit?
E-field on electric B-field on magnetic
sector sector
R attainable: 100,000
Page 289 of 316

Chem 155 Unit 16 290 of 316
16.5.4 Quadrupole Mass Filters:
Resolution up to:
10,000
< 4000 AMU

Masses limted to:
Stable trajectory is a function of: AC amplitude
DC field
M/z
A given AC amplitude + DC field
yields a stable trajectory for one:
mass filter
Like E and B sectors this is a:
One scans m/z: scanning mode (see

one at a time)
Page 290 of 316

Chem 155 Unit 16 291 of 316
16.5.5 Time of Flight (TOF):
Ion kinetic energy E = zVPULSE
But kinetic energy E = mv2
So, a drift length L gives a the flight time tF = L/v
Solve for m/Z as a function of VPULSE and distance L:
E = zVPULSE = mv2 = m(L/tF)2
2VPULSE(tF/L)2 = m/z = (2V/L) tF2
So, one measures ion m/z by measuring the ions

Arrival time at the detector: tF
Page 291 of 316

Chem 155 Unit 16 292 of 316
TOF analyzers:
1. High mass capable.

2. Fast 100 of spectra per second.
3. Efficient / sensitive all ions are collected.
4. Good Resolution up to 25000.
16.5.6 Triple Quadrupole Analyzers:
16.6 Mass Spec Questions:
1. What might you label the axes on a mass spectrum?

2. Consider a fragment of nominal mass 180 AMU in a
mass spectrometer. What peaks might arise from this
one fragment?
3. Consider a high resolution mass spectrum of the singly
charged fragment CHCl3+. Though varying greatly in
intensity, it will be possible to resolve 12 different
peaks for this fragment. Assign the isotopomers and
predict the masses.
4. Forensic evidence was gathered against U.S. cyclist

Floyd Landis was based on a high testosterone level
seen in a blood sample. Since testosterone is a
naturally occurring hormone, it
OH
was necessary to analyze this
further. Testosterone made
H
by biosynthesis in the human
body differs in only one way
H H
from that prepared in a
laboratory 13C/12C isotopic O
testosterone
abundances. What C19H28O2

resolution is required to Exact Mass: 288.21
Mol. Wt.: 288.42
measure this ratio? m/e: 288.21 (100.0%), 289.21 (20.6%), 290.22 (2.1%)
C, 79.12; H, 9.78; O, 11.09
Page 292 of 316

Chem 155 Unit 16 293 of 316
5. What type of mass analyzer might be useful for this?

6. Would it be necessary to perform a separation step
before analyzing Landis blood for testosterone by MS?
7. Chemists are very sloppy in the way we use the word
resolution for example, the GCMS that we use has
unit mass resolution on the quadrupole analyzer.
What does this mean?
8. In the description of C19H28O2
testosterone above, can you Exact Mass: 288.21
Mol. Wt.: 288.42
distinguish between the exact m/e: 288.21 (100.0%), 289.21 (20.6%), 290.22 (2.1%)
C, 79.12; H, 9.78; O, 11.09
mass and the molecular
weight?
9. To what do the 20% and 2% fragments correspond?
Element Iso %
12 13
Carbon C 98.90% C 1.10%
1 2
Hydrogen H 99.99% H 0.01%
16 17 18
Oxygen O 99.76 O 0.038% O 0.2%
Page 293 of 316

Chem 155 Unit 17 Chromatography Page 294 of 316
17 Chromatography Chapter 26
General and descriptive aspects of chromatographic

retention and separation: phenomenological k,
efficiency, selectivity.
Quantitative description of zone migration in partition

chromatography: migration velocity, partition
coefficient and theoretical k.
Theoretical description of efficiency: zone broadening

and the Van Deemter equation.
Solving the general elution problem: Gradient vs.

isocratic elutions.
HPLC Instrumentation: pump, injector, column,

detector, data collection.
Chromatographic modes: HPLC (reverse and normal

phase or flash), gel permeation (GPC), gel filtration,
ion exchange.
Electrophoresis.
Page 294 of 316

Chromatography: separation of chemicals in a mobile

phase by differential flow rates through stationary
phase.
Skoog Holler and Nieman Principles of Instrumental Analysis 5th ed.
Page 295 of 316

Some definitions:
Mobile Phase is the gas, liquid or supercritical fluid
that passes through separation column or slab.
Stationary Phase is the solid or immobilized liquid
into which the solutes or analytes partition, adsorb or
bind.
Band or Zone describes the region (stripe, plug) of
the separation column or slab that contains the solute
or analyte.
Retention is the word used to refer to the delay of the
solute or analyte in reaching the detector relative to
the mobile phase.
Page 296 of 316

Page 297 of 316

Page 298 of 316

Page 299 of 316

Page 300 of 316

Page 301 of 316

x
Page 302 of 316
Theoretical Plates and Plate Count:
2 2
xi i 100 25
10 0.625
1000 1000
1 1 2 2 2
S x exp x 50 12.5
2 2 2.5 0.156
2 1000 1000
x
2 2 2
1000 1000
N: 100 1600 N
100 25
2 2
1000 1000
400 6400
50 12.5
Illustration of different p late counts
0.0 3
6400 plates
0.0 25
0.0 2
signal
1600 plates
0.0 15
0.0 1 400 plates
0.0 05
100 plates
0
0 20 0 40 0 60 0 80 0 10 00 12 00 14 00 16 00 18 00 20 00
ret ent ion time or column lengt h
Page 303 of 316

Plate count depends on retention time:
Why does the plate count go up for the more retained

peaks?
x
2 2 2
400 1200
N: 1024 9216 N
12.5 12.5
2 2
800 1600
4096 16384
12.5 12.5
Illustration of different p late counts
0.0 3
0.0 25
0.0 2
signal
0.0 15
0.0 1
0.0 05
0
0 20 0 40 0 60 0 80 0 10 00 12 00 14 00 16 00 18 00 20 00
ret ent ion time or column lengt h
Page 304 of 316

The van Deemter Equation:

H = plate height
A = multipath term B
H A Cu
B = longitudinal diffusion term u
C = resistance to mass transfer
Plate
H( A B C height
u) A
B
as
C u a function of linear flow rate:
u
12
10
H ( 1 2 0.5 u)
2
0 2 4 6 8 10 12
u
Rationalize why is there an optimum flow rate:
Page 305 of 316

A 2 dp
B 2 DM
B
H A Cu
u
C = com plex function of particle
s ize, coating, diffus ion coefficient,
favored in general by large
diffus ivity and s m all particle s ize
Page 306 of 316

17.1 General Elution Problem / Gradient Elution
a.
b.
x
A mixture of molecules with strongly differing k values
is hard to separate because:
If the solvent is weak enough to separate 1 and 2:
If the solvent is strong enough to elute 5 and 6 in a

reasonable time:
Page 307 of 316

Isocratic (LC) and isothermal (GC) separations are

vulnerable to the general elution problem.
The solution to the general elution problem:
o solution gradient elution (liquid

chromatography)
x
o temperature gradient elution (gas
chromatography)
The gradient methods:

o begin with weakly eluting conditions and this
helps to separate the weakly retained species
o finish with strongly eluting conditions to
expedite the elution of strongly retained species
and to limit diffusional broadening
In LC gradient elution is a bit more difficult because:
o the columns require a fairly significant
equilibration time between runs
o the detector baseline can drift significantly if the
different solvents have different UV absorbance
at the detection wavelength if this is a problem,
then one can use
o a different wavelength
or
o a different set of solvents
Page 308 of 316

17.2 T-gradient example in GC of a complex mixture.
x
Page 309 of 316
17.3 High Performance Liquid Chromatography
x
Skoog Holler and Nieman Principles of Instrumental Analysis 5th ed.
Page 310 of 316

17.4 Types of Liquid Chromatography
Most can be performed as HPLC in high

performance instrumentation if necessary.
x
17.5 Normal Phase:
stationary phase: hydrophilic (SiO2 particles)
mobile phase: hydrophobic mobile phase
(hexane, methanol).
weak solvent: hexane
strong solvent: methanol
retained: polar compounds
eluted first: non-polar compounds
17.5.1 Reverse Phase:

stationary phase: hydrophobic (SiO2 particles
coated with hexadecane monolayer)
mobile phase: hydrophilic mobile phase (water,
methanol)
weak solvent: water
strong solvent: methanol
retained: non-polar compounds
eluted first: polar compounds
Page 311 of 316

17.5.2 Exclusion Gels
GPC
Gel permeation chromatography: porous polymer
gel particles small molecules penetrate and are
retained, large molecules elute more quickly.
x
PAGE polyacrlamide gel electrophoresis:
Aqueous gel: -CH2-CONH- used for
electrophoresis, large molecules experience more
drag and are retained.
Page 312 of 316

Ion Exchange:
Stationary phase is an ionic polymer e.g.
polystyrenesulfonic acid:
x
SO3-
*
*
n
The solid or gel phase polymer transiently binds

cations from solution. Cations that have a higher
affinity for the resin are retained longer and vice-
versa.
Page 313 of 316

17.6 HPLC System overview:
x
Injection valve
Page 314 of 316

17.7 Example of Reverse-phase HPLC stationary

phase:
x
Page 315 of 316
17.8 Ideal qualities of HPLC stationary phase:

Very fast partitioning between mobile and stationary
phase.
1. Ultimate thin film: single molecular layer.
2. Perfect defect free film with no sites for
x
strong adsorption.
Mechanically strong to withstand high pressure w/o

collapse.
Chemically inert.
Partition coefficients of solute analytes are all different

in single solvent i.e. the SP provides a measure of
selectivity without being limited to just a few chosen
analytes.
Page 316 of 316

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Chem 155 Unit 1 Page 1 of 316

1 Overview and Review

What chemicals are present in a sample?

Additionally, Analytical Chemists are asked:

What chemical forms are present?

1.1 Tools of Instrumental Analytical Chem.

Name of EM Wavelength Predominant Name of

X-Ray 0.1 to 10 nm Core x-ray absorption,

Microwave 40 m 1 rotations Rotational or

1.1.2 Chromatography Chemical Separations

Different chemicals flow through separation

Liquid Chromatography High Performance

Electrophoresis -Liquids, pump with electric

1.1.3 Mass Spectrometry

Detection method where sample is:

Very sensitive (pg) quantitation

Ion selective electrodes (ISEs):

1.1.6 Thermal Analysis

Thermogravimetric Analysis TGA

Differential Scanning Calorimetry DSC

Estimated Number of uniquely identifiable molecules by method

What are the more precise measurements that you

1.3 Vocabulary: Basic Instrumental

Analyte The chemical species that is

1.4 Vocabulary: Basic Statistics Review

Precision If repeated measurements of the same thing are

Standard A widely accepted 2

Relative Standard The standard deviation divided by the mean, and

Histogram of normally distribut ed event s

His togram of 1024 events

1.5.2 Basic Formulae

Bias or absolute systematic error = xavg

When you make real measurements of things you generally dont

1.5.3 Confidence Interval

TAKE THE AVERAGE xAVG

But, you still dont know the exact answer

SO WHAT DO YOU REALLY WANT TO SAY?

I am highly confident that the true mean lies within

How do you calculate what that interval is?

The average of the data set: x

This interval is called a confidence interval (CI). Which is better,a

How can you improve your CI?

Make more measurements (N)

Confidence Interval (continued).

The x% confidence interval for = xAVG z / N

If you have only a rough

The x% confidence interval for = xAVG ts / N

XAVG = (4.2+4.6+4.0) / 3 = 4.27

95% confidence limits for = 4.27 (4.3*0.31) / (31/2)

1.5.4 Confidence Interval (CI) in Words

In order to evaluate the precision of your camera thermometer, you

PROBLEM: The average camera reading is sometimes higher than

SOLUTION: Calculate the sample standard deviation.

QUESTION: A series of experiments, each of three measurements

Why does the sample standard deviation calculation give a different

Our criterion is this: if there is more than a 90% probability that a

We have only moments to acquire three measurements per

Passenger 1: 100.3, 101.1, 103.0.

How do we answer the question: does this persons temperature

To answer this, we must accept the following: Assuming that

For example, if we took some measurements and then computed the

10% chance 80% chance 10% chance

Use this table: Use this formula:

Complete the following table: