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ANALYTICAL APPLICATIONS OF GC-MES IN THE ULTRAVIOLET-VISIBLE AND VUV REGIONS OF THE SPECTRUM (VACUUM-ULTRAVIOLET, GAS, CHROMATOGRAPHY, MICROWAVE, EMISSION)

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8229959
Pierson,Duane Aron
ANALYTICAL APPLICATIONS OF GC-MES IN THE UV-VISIBLE AND VUV
REGIONS OF THE SPECTRUM
PH.D. 1982
The University o f Iowa
University
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ANALYTICAL APPLICATIONS OF GC-MES IN THE UV-VISIBLE
AND VUV REGIONS OF THE SPECTRUM
by
Duane Aron Pierson
A thesis submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy in Chemistry
in the Graduate College of
The University of Iowa
July, 1982
Thesis supervisor:
Professor Clyde W. Frank
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Graduate College
The University of Iowa
Iowa City, Iowa
CERTIFICATE OF APPROVAL
PH.D. THESIS
This is to certify that the Ph.D. thesis of
Duane Aron Pierson
has been approved by the Examining Committee
for the thesis requirement for the Doctor of
Philosophy degree in Chemistry at the July,
1982 graduation.
Thesis committee:_
Thesis supervisor
M
JLts
Member
__
I.,
Member
Memb
Member
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ACKNOWLEDGEMENTS
This dissertation is the culmination of many long years
of hard work.
I wish to thank my parents, my family and my
friends for their support and encouragement.
I would
especially like to thank my wife Dee, for her love and
support when I needed it most.
As my advisor, Dr. Clyde Erank provided the assistance
and the opportunity to grow as an analytical chemist.
The
type of training I have received will be of great value in
making the transition from academics to industry.
Finally, I would like to thank Dr. H. Bruce Friedrich
for assisting me in preparing this document by computer.
ii
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TABLE OF CONTENTS
Page
LIST OF T A B L E S ............ . . .. ................ . . . . vi
LIST OF F I G U R E S ..........
vii
LIST OF ABBRE V I A T I O N S .................
. . . . . . . . . ix
PART A. ANALYTICAL APPLICATIONS OF GC-MES IN THE UVVISIBLE ..................... . . . . . . . . .
1
CHAPTER
I.. INTRODUCTION
2
...................
2
Background . . . . . . . .
Microwave Induced Plasmas
...................
3
System Components ........
3
Discharge Properties
.....................
8
Sample I n t r o d u c t i o n ..........
. . . . . . 12
Gas Chromatography-Microwave Emission
Spectroscopy .
..........
16
Method Development
................. . . 17
Applications
.............................. 25
S u m m a r y ..............
28
Element Selective Detectors for GC . . . .
28
Conclusions
........ .. . . . . . . . . . 3 1
Purpose of S t u d y .........................
31
Background
......................... 31
Lactose In t o l e r a n c e ............... .. . . . 33
Hydrogen Breath Test
........ .. . . . . 35
Previous Application of Breath Tests
. .. 3 7
Determination of Hydrogen in Breath . . . . 38
II. EXPERIMENTAL .
.......................
Introduction . . . .
............... . . . .
Gas C h r o m a t o g r a p h
.
Control Panel . . . . . . . . . . . . . . .
Injection P o r t ....................... . 4 7
Exit Port
ii.i
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42
42
43
43
50
Page
Column . . . . . . . . .
........
. . . .
Detection System . . . . . . . . .
...........
Plasma Capillary . . .
..............
..........................
Cavity Mount
Plasma Ignition ............... . . . . . .
Light Pipe
. . . . . .
.. . . . . . . . .
................
Spectrometer Modifications
Wavelength Readout
........................
Photomultiplier Tube Mount
................
Optical Adjustment
. . . . . . .
.........
III.
RESULTS AND DISCUSSION . . .
.........
. . . . .
50
53
53
53
53
56
56
61
64
65
72
Plasma Characteristics .....................
72
Plasma Background Spectrum
. . . .. . . . 72
Spectroscopic Temperature . . . . . . . . . 7 6
Detection of Hydrogen Gas
.. . . . . . . . . 7 7
S e p a r a t i o n ......................
Detector Optimization .
.. . . . . . . . . 8 1
Wavelength Calibration .
................ 83
Calibration of R e s p o n s e ........ .. . . . . 88
Evaluation of Hydrogen Detection . . .. . . . 9 3
Sensitivity and Detection Limit . . . . . . 93
Selectivity . . . . . . . .
.............. 97
A c c u r a c y ............
98
Linear R a n g e ..............
99
Reproducibility
. . . . . . . .
100
Hydrogen Breath T e s t ............... . . . . 105
Procedure
. 105
Breath Collection .........................
105
Collection Apparatus
................
107
Sample Storage
....................108
Breath Test A p p l i c a t i o n s ........... ' . . . . 110
Lactose Studies . . . . . . . . . . . . .
110
Ambient Hydrogen Levels . •. . r' . . . . . . 110
Rebreathing vs. Hyperventilation
. . . . 112
Lactose Malabsorption in MS Patients
. . 115
Lactulose Study
...............
120
Method Validation . . . .
................. 121
C o n c l u s i o n s ......................
iv
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80
126
Page
PART B.
ANALYTICAL APPLICATIONS OF GC-MES BELOW 2000 A 129
CHAPTER
IV. INTRODUCTION ..............
. . . . . . . . . .
130
Recent Analytical Applications . . . . . . .
Purpose of S t u d y ............
. . . . . . .
131
133
V................ EXPERIMENTAL
...
134
Purging the Optical System
................. 134
D e t e c t i o n ................................. 135
VI.
RESULTS AND DISCUSSION
..
137
Oxygen Removal . . . ............
Detection of Iodine Emission .......... . . .
Wavelength Calibration .................
Method Development
...........
Detection of Sulfur Emission . . . . . . . .
Wavelength Calibration .
...............
Method Development
........
Conclusions
.
..............................
137
138
138
139
152
152
154
163
APPENDIX A.
SPECTROMETER ALIGNMENT PROCEDURE . . . .
165
APPENDIX B.
GC-MES ALIGNMENT PROCEDURE . . ........
167
APPENDIX C.
BENZENE - SPECTRUM 1000 - 2500 A . . . .
168
APPENDIX D.
NITROETHANE - SPECTRUM 1000 - 2500 A
..
170
APPENDIX E.
MALATHION - SPECTRUM 1000 - 1500 A
. ..
172
APPENDIX F.
BROMOBUTANE - SPECTRUM 1000 - 2500 A
..
174
APPENDIX G.
IODOBUTANE - SPECTRUM 1000 - 2500 A
..
176
APPENDIX H.
ANALYTICAL APPLICATIONS OF GC-MES
. ..
178
LIST OF REFERENCES
. . . . . . . . . . . . . . . . .
v
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189
LIST OF TABLES
Table
1.
Page
Experimental Apparatus
. . ........
46
.................
73
2.
Plasma Background Spectrum
3.
Operating Conditions for H2 .
4.
Comparison Between Analytical Linesfor Hydrogen
5.
Storage Stability for Breath Samples
6.
Storage Stability vs. Temperature
7.
Ambient Hydrogen L e v e l s ..........
113
8.
Comparison between Rebreathing and
Hyperventilation . . ...................
114
9.
...
. . . . . . .
. . . . .............. 82
. .
..
. 95
. . 109
.
Hyperventilation Collection Method
. .
Ill
116
10.
Residual Lung Volume
. . . ....................... 118
11.
Reproducibility of Hydroden Determinations
12.
Validation of Hydrogen Method . . . .-
13.
Observed Iodine Transitions ... ...................
142
14.
Separation of Organoiodides .
...........
149
15.
Observed Sulfur Transitions .....................
153
16.
Sulfur Response vs. Compound Type . . . .
17.
Selectivity for Sulfur vs. Slit W i d t h ..........
.. .
.
.
. . 119
..
..
...127
. . 161
vi
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163
LIST OF FIGURES
Figure
Page
. . ..............
45
1.
GC-MES Experimental Set-up
2.
Injection Port Design . . . . . . .
3.
Exit Port D e s i g n .........................
52
4.
Microwave Cavity Mount
55
5.
Adjustable Light Pipe Mount .....................
6.
Light Pipe Support
7.
Wavelength Readout Device
8.
PMT - Exit Slit Mount . . . . . . . . . . . . . . .
67
9.
Slit Width C a l i b r a t i o n ..........
69
10.
Spectrometer Mount
71
11.
Argon Plasma S p e c t r a .................
12.
Detector Optimization -
(4861 A ) ................. 85
13.
Detector Optimization - ^
(6720 A)
14.
Hydrogen Calibration Peaks
. . . . . . . . . . . .
90
15.
Hydrogen Calibration Curve
.................
92
16.
Hydrogen Linear Range . . . . . . . . . . . . . .
102
17.
Reproducibility of Standard Injections
104
18.
Breath Sample Chromatogram
19.
Lactulose study - HBT
20.
Plasma Doping Apparatus .
21.
Detector Optimization - Iodine (1830 A) .........
.............. . 4 9
.......................
.58
...............
.............. .
. . . .63
. . . . . .
79
87
........
...........
..............
60
123
...
. .
...............
vii
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125
141
144
22.
Detector Optimization - Iodine (1844 A) . . . . . .
23.
Separation of O r g an o i o d i d e s .....................
24.
Detector Optimization - Sulfur (1807 A) . . . .
25.
Sulfur Compounds
148
151
.
156
............................. 159
viii
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LIST OF ABBREVIATIONS
A
Angstroms
ARL
Applied Research Laboratories
b
y-intercept
C
Centigrade
cal
calorie
cm
centimeter
CMP
capacitivly coupled plasma
dc
direct current
EC
electron capture
EGA
evolved gas analysis
eV
electron volt
FID
flame ionization detector
fg
femtogram (1x10 "15gram)
FPD
flame photometric detector
g
gram
GC
gas chromatograph
h
Plank's constant
HBT
hydrogen breath test
HID
helium ionization detector
ICP
inductively coupled plasma
ix
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i.d.
inside diameter
I.P-.
ionization potential
K
Kelvin
kcal
kilocalorie (lxl03calorie)
kg
kilogram (lxl03gram)
LTE
local thermodynamic equilibrium
LTT
lactose tolerance test
X
wavelength
m
slope
MDL
minimum detectable limit
MES
microwave emission spectroscopy
MIP
microwave induced plasma
MHz
megahertz
mL
milliliter (1x10_3liter)
mm
millimeter (1x10-3meter)
MS
multiple sclerosis
yg
microgram (1x10 "'gram)
yL
microliter (1x10 "'liter)
ng
nanogram (1x10 "9gram)
NPT
National
V
frequency
o.d.
outside diameter
Pipe Thread
Ohms
pg
picogram (1x10 _12gram)
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PMT
photomultiplier tube
ppm
parts per million
ppt
parts per thousand
psig
pounds per square inch (gauge pressure)
QA
quality assurance
r
correlation coefficient
RIR
radiative ionization - recombination model
RDS
relative standard deviation
s
second
S.D.
standard deviation
TC
thermal conductivity
UV
ultraviolet
VUV
vacuum ultraviolet
V
voltage
v/v
volume/volume
W
watts
w/v
weight/volume
xi
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PART A
ANALYTICAL APPLICATIONS OF GC-MES IN THE
UV-VISIBLE
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2
CHAPTER I
INTRODUCTION
Background
A plasma state is established when sufficient energy is
transferred to a gas so that ions and electrons dominate the
behavior of the system (1).
Plasmas produced by the
interaction of electromagnetic fields with inert gases such
as argon and helium have been developed as spectral
excitation sources since the mid-1950's (2).
The inductively coupled plasma (ICP) has received an
extaordinary amount of attention for the past ten years.
The ICP is powered by inductive coupling with the magnetic
field of a radio frequency supply.
Excellent reviews on the
capabilities and instrumentation associated with the ICP
have been written by Fassel and Kniseley (3/4), Greenfield
et al. (5) and Sytz (6).
Plasmas produced by the interaction of microwave
electrical fields with gases have also shown considerable
potential as spectral excitation sources.
exist.
Two general types
In the capacitively coupled microwave plasma (CMP),
microwaves are conducted through a coaxial waveguide to the
tip of a conductive electrode.
A flamelike plasma is formed
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3
when an inert gas is forced through the center of the
electrode.
The second type of microwave discharges are
electrodeless devices known as microwave induced plasmas
(MIP).
Energy is coupled to a flowing inert gas contained
in a nonconductive quartz tube via an external cavity or
antenna.
Excellent reviews dealing with the MIP have been
presented by Greenfield et al. (7), Skogerboe and Coleman
(2), and more recently by Zander and Hieftje (8).
A
complete list of references can also be obtained in
Analytical Chemistry Reviews on Emission Spectrometry
(9,10,11).
The following overview will deal exclusively
with microwave induced plasmas.
Microwave Induced Plasmas
System Components
Microwave Power Supplies
The commercially available power units operating at
2450 MHz are commonly in use.
The power supplies are
normally variable between 0 and 125 watts.
Availability of
units that operate at other scientific frequencies with the
power, stability and operational requirements for
spectroscopic applications is still limited (8).
West (12)
compared a 2450 MHz MIP with an excitation system operated
at 30 MHz and reported certain advantages for both.
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4
Microwave Cavities
A standing electromagnetic wave is established within
the resonant cavity.
For microwave frequencies, the size of
the cavity necessary to form this standing wave is on the
order of centimeters.
The purpose of the cavity is to
transfer power from the microwave source to the inert gas
flowing through the plasma capillary.
The efficiency of
this power transfer depends on the impedance match between
the cavity (including the plasma) and the coaxial cable
leading to the magnetron.
been described (13,14).
Impedance matching devices have
Maximum power is transferred to the
plasma when the amount reflected back to the magnetron is
tuned to a minimum.
The electromagnetic fields and currents produced within
the cavity decrease with penetration into the cavity wall.
Energy can be lost as thermal heating of the cavity when
penetration is high.
The choice of construction material
for the cavity usually involves a compromise.
The metals
which best inhibit penetration are good conductors such as
silver, copper, gold and aluminum.
Unfortunately, these
metals are either difficult to machine or corrode, making
them less efficient.
Brass is often used and in some cases
is coated with silver or gold (8).
One of the original cavity designs was the tapered
rectangular (TEq ^3 ) type which was introduced by Broida
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5
(15,16).
The plasma capillary tube is positioned in a slot
at the short-circuited end, in the electric field direction.
Since there is no physical contact between the cavity and
plasma capillary, the cavity can be moved without disturbing
the discharge.
McCormack et al. (17) found the tapered
cavity to be more sensitive than the 3/4-wave Evenson
coaxial cavity, but that it could not accept as much sample.
Two types of foreshortened 3/4-wave coaxial cavities
have been described (18).
As in the case of the tapered
cavity, the discharge is viewed perpendicular to the plasma
capillary.
However, when the plasma is operated at
atmospheric pressure, axial viewing is also possible
(19,20).
Moye (21) reported difficulties such as high noise
and low discharge tube life using the Evenson type 3/4-wave
cavity and favored the tapered design.
The foreshortened 1/4-wave coaxial cavity is a
modification which, unlike the 3/4-wave, allows adjustment
of the cavity without breaking vacuum lines to the discharge
tube.
Another modification is the foreshortened 1/4-wave
radial cavity (18).
This cavity operates well at reduced
pressures and is normally viewed axially.
The most significant cavity design improvement was
reported by Beenakker (22,23).
This cavity was designed for
efficient operation in either argon or helium at atmospheric
pressure.
The cavity operates in the
mode with the
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6
plasma capillary positioned axially.
When helium is used at
atmospheric pressure, the plasma is self igniting and is
highly stable (24).
The plasma emission is viewed.axially.
Mulligan et al. (25) compared the performance of the
Beenakker cavity to several others in the simultaneous
determination of As, Ge, Sb and Sn.
The Beenakker TM q ^q
cavity represents the state of the art in microwave cavity
design and is now commercially available (26).
Modifications of this cavity to improve coupling efficiency
and to permit direct introduction of aqueous samples is a
very active area of research (27,28,29).
Plasma Capillary
The plasma is contained by a nonconductive tube through
which the support gas and analyte species flow.
Quartz is
the material of choice because of its high dielectric
character and transparency in the UV-visible region of the
spectrum.
The inner diameter of the tube is an important
parameter which affects plasma stability and emission
intensity (17,21,30,31).
The method employed in viewing emission from the plasma
varies with the type of resonant cavity.
As mentioned
previously, the plasma may be viewed axially down the length
of the plasma or transversely through the capillary wall.
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7
Commercial Instrumentation
Rapid development and proven utility of the microwave
induced plasma have led to its commercial availability.
An
inventory of the various systems available was recently
presented by Broekaert (26).
Manufacturers and a
description of the components are given.
Radiation Exposure
The hazards of stray microwave radiation are not
completely understood and remain a controversial topic.
This is reflected in the large discrepancy between the
safety standards set in the U.S.
U.S.S.R.
(0.01 mW/cm2).
(10 mW/cm2) vs. the
Stanley et al. (32) measured stray
radiation levels for four cavity designs.
Microwave power
density dropped to 10 mW/cm2 within 18 cm of the tapered
cavity.
Stray levels were this high 22 cm from an Evenson
1/4-wave cavity, and 50 cm from a Raytheon type "C" cavity.
The Broida 3/4-wave cavity exhibited a maximum power density
of 1 mW/cm2 at the tuning adjustment.
f
Power densities were found to fall off rapidly with
distance from the cavities.
When metal objects were placed
close to the cavities, power levels at points of
reinforcement were an order of magnitude larger.
These
authors constructed a shield from 16 guage sheet metal with
2.38 mm holes.
Radiation leakage was reduced to less than
0.05 mW/cm2.
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8
Van Dalen et al. (33) measured stray radiation levels
for an actual laboratory situation employing a 1/4-wave
Evenson type cavity.
When reflected powers were adjusted to
<1%, a maximum of 2 mW/cm2 was measured.
This stray
radiation level was reduced to 0.1 mW/cm2 by enclosing the
system in an aluminum box.
Quimby et al.
(34) suggested
shielding may be necessary when argon is used as a support
gas.
Discharge Properties
Unequivocal mechanisms for plasma formation and analyte
excitation cannot be found in the literature (8).
The
following discussion is a summary of the processes taking
place in microwave induced plasmas that have received
general acceptance.
The plasma gas, usually argon or helium, flows through
a quartz tube which confines the discharge.
This tube is
positioned along the axis parallel to the electric field
inside of the resonance cavity.
When a seed electron is
introduced by a Tesla coil, it oscillates in the field.
As
the electron accelerates, it collides with plasma gas atoms
as shown in the following equations:
Ar + e --- * Ar(+) + e + e
(1)
Ar( + ) + e —
(2)
* Arm + hvcont±nuum
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The ionization of an argon atom produces two electrons
characterized by different velocities and temperatures
(35,36,23).
A high-energy, low-density group is responsible
for subsequent ionization of neutral argon atoms via
equation 1.
The second group, which is characterized by
low-energy and high-density recombines with an ionized argon
atom to form a metastable species.
A stable plasma results
from the equilibium between these two processes and is
p ressure dependent.
When atmospheric pressures are
employed, higher electric fields or lower frequencies are
required.
The excitation mechanism in a MIP is believed to
involve the metastable species.
levels at 11.49 and 11.66 eV.
Argon has two metastable
Helium has two levels of
higher energy at 19.73 and 20.53 eV.
Electrons promoted to
a metastable state are forbidden by selection rules to
release energy by emitting radiation.
Instead, they loose
their energy through collisions with other atoms.
Because
only radiationless processes are allowed and the lifetime of
a metastable state is relatively long, they are believed to
be responsible for spectral excitation (37,23,38).
The
following mechanism is consistent with the proposed
radiative ionization-recombination (RIR) model:
Ar
m
+ M
M( + ) + e —
Ar + M(+) + e
» M*
M* -- > M + hv
characteristic
(3)
(4)
(5)
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10
Equation 3 represents Penning ionization of the analyte
species, M, whose ionization potential must be less than the
excitation potential of the metastable state.
Excess energy
is transferred to the electron in the form of kinetic
energy.
Radiative recombination of the analyte ion with a
low-energy electron produces an excited state which
spontaneously decays with emission of characteristic
radiation.
The RIR model is the most commonly accepted mechanism
for plasma excitation.
Recently, Brassem et al. (39)
re-evaluated the model for a low pressure MIP.
The RIR
model was favorable to a purely thermal excitation model but
was still off by a factor of two.
Direct excitation of
analyte atoms by metastable species or high energy electrons
is possible but definitely not the dominant mechanism
(37,39,23).
Bush and Vickers (37) characterized excitation
conditions for the MIP by measuring spectroscopic
temperature, electron temperature, electron concentration,
relative argon metastable state concentration and argon
emission intensity.
They found that the parameters which
controlled excitation were the concentration and energy of
the two different electron groups and the concentration of
the metastable argon atoms.
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11
These authors state that it is unnecessary to define
microscopic processes which constitute excitation in a
plasma at atmospheric pressure.
This is due to the fact
that local thermodynamic equilibrium (LTE) is often attained
by plasmas at atmospheric pressure where particle densities
are high (40).
Under these conditions, the population of
energy levels follows the Boltzmann distribution, and the
Saha equation describes the yield of ionization products
(37).
The state of the plasma' is defined by its temperature
and density.
Bush and Vickers (37) measured spectroscopic excitation
temperature in the MIP by observing relative radiances of
the support gas spectral lines.
Use of this method required
that wavelengths, energy levels and transition probabilities
were known quantities.
A plot of log (IX/gA) vs. log E for
the spectral transitions yields a straight line with a slope
of -1/kT where:
I = line intensity
g = statistical weight-of the upper level
A = transition probability
X = wavelength of the observed line
E = energy of the upper level
k = Boltzmann constant
T = excitation temperature (Kelvin)
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12
The spectroscopic temperature of an argon MIP at
atmospheric pressure has been reported to be approximately
5000 K at 100 watts of applied power (41,42).
For a helium
plasma at atmospheric pressure and 100 W, a temperature of
7250 K was observed (24).
This increase in temperature is
due to the higher energy of helium metastable state.
Sample Introduction
The early applications of the MIP to spectrochemical
analysis reflected the fact that only small amounts of
sample could be introduced, preferably already in the gas
phase.
Hence, the MIP was initially used as a detector for
gases, gas chromatographic effluents or volatilized
compounds.
The discussion of analytical applications of the MIP
will be limited to those which employ introduction of the
sample as a vapor.
The introduction of aqueous samples has
taken on a whole new direction since the advent of the
Beenakker cavity (22).
Greenfield presents an excellent
review of the literature with respect to pneumatic
nebulization, ultrasonic nebulization and sealed tube
excitation of aqueous samples (7).
A review by Zander and
Hieftje (8) contains a comprehensive listing of elements
determined by solution analysis in microwave induced
plasmas.
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13
Gases
The simplest examples of sample introduction involve
direct injection of gases.
The first successful use of a
MIP in spectrochemical analysis was reported by Broida (16)
who determined isotopic concentrations in nitrogen and
nitric oxide.
Taylor et al. (43) determined trace
impurities in the argon plasma support gas.
Serravallo and
Risby (44) found that the presence of air severely limited
the application of direct injection techniques for the
determination of gaseous air pollutants, since oxygen and
nitrogen quenched the emission from atomic species.
et al. (45) determined CC^, NO and SO2
Dagnall
in air by GC-MES.
Heated Filament Vaporization
Runnels and Gibson (46,47) evaporated the analyte
species onto a platinum filament.
After insertion into the
plasma gas stream, the filament was electrically heated to
vaporize the sample.
Aldous et al. (48) used a platinum or
tungsten loop to vaporize samples into a plasma supported at
the mouth of the quartz capillary.
Kawaguchi et al. (30,49)
used a tantalum filament and found that the addition of KC1
enhanced spectral emission for several elements as well as
suppressing interference effects.
A variety of similar applications employing filament
vaporization have been reported (50,51,52).
The main
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14
consideration in interfacing these types of sample
introduction systems is that the analyte species must not be
allowed to plate out on the walls, of the system before
entering the plasma (8).
Carbon cup (53,54), carbon rod
(55) and platinum boat (56) devices have also been used.
Watling (57) amalgamated trace amounts of mercury in
seawater onto silver wool after reduction by tin (II)
chloride.
Mercury was vaporized by heating the silver wool
in the argon stream of a microwave plasma.
Generation of Gaseous Species
The most common approach for many elements is to form
the volatile hydride.
Lichte and Skogerboe (58) designed an
arsine generator which was directly attached to an argon
MIP.
Barret and Copeland (59) compared various hydride
generation techniques with MIP excitation with respect to
sensitivity and precision.
Mulligan et al. (25) compared
four different microwave cavity designs for the simultaneous
determination of As, Ge, Sb and Sn as volatile hydrides.
Other methods of chemical modification of the analyte
to produce a volatile species include reduction of mercury
compounds (60) or conversion to volatile chlorides (61).
In
the latter method, plasma gas containing HC1 was passed over
the sample after preheating to 850°C.
Conversion and
determination of Bi, Cd, Ge, Mo, Pb, Sn, Tl and Zn was
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15
accomplished.
Runnels and Gibson (46) introduced Cu, Co,
Cr, Fe and Mn as acetylacetonate chelates.
These were
vaporized from a platinum filament as previously described.
Tanabe et al. (62) determined ultratrace levels of
ammonium, nitrite and nitrate nitrogens using a gas
generation technique coupled with MIP detection.
Quimby et
al. (34) trapped volatile trihalomethanes on a porous
polymer adsorbent and thermally desorbed the analyte into
the plasma.
A novel method of introducing solid samples is by
direct laser vaporization (63).
Picogram levels of Al, Cr,
Cu, Fe, Mn, Mo, Ni, Pb, Ti and Zn were determined in metals
and required no sample preparation (i.e. dissolution).
A
similar technique was applied to the analysis of aluminum
and zinc samples (64).
Layman and Hieftje (20) employed a
computer controlled, microarc sample atomization system for
trace elemental analysis.
Bauer and Natusch (14) have utilized MIP emission
detection in evolved gas analysis (EGA).
heated in the support gas stream.
The sample is
Compound identification
is based on the coincident observation of its metal and
nonmetal components at its temperature of vaporization.
Coincidence exists for pure halide, sulfide and sulfate
salts of Cd, Hg, Pb and Zn.
In a subsequent article (65),
carbonate compounds were determined in coal fly ash.
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Carbon
16
dioxide was monitored as the evolved gas.
One obstacle to
the method was the possibility of chemical reactions
altering compound identity before vaporization.
Gas Chromatography-Microwave
Emission Spectroscopy
The most common-application of the MIP has been as a
detector for gas chromatography (GC-MES).
The first
practical application of this type was reported in 1965 by
McCormack, Tong and Cooke (17).
The spectra emitted by
organic molecules showed that fragmentation had taken place
to produce atomic and diatomic species.
This emission could
be used in the sensitive and selective determination of
compounds containing C, Cl, F, I, P and S.
Bache and Lisk
simultaneously published their work on the determination of
organophosphorous pesticides by GC-MES (66).
The popularity of GC-MES is evidenced by the volume of
work that has been done since its introduction.
The basic
requirements for this method are as follows:
1.
The analyte must be present in the gaseous state
following chromatographic separation.
2.
The plasma must be able to handle the amount of
effluent coming from the gas chromatographic column.
3.
The ionization potential of the desired analyte
transition must be smaller than the excitation
potential of the metastable state.
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Taking these limitations into consideration, the detector
can be made element selective simply by varying the
wavelength of observation.
Method Development
Wavelength Calibration
A variety of methods have been used to optimize the
wavelength of observation.
Early investigators set the
monochromator to the desired setting and optimized response
by injecting standards at small wavelength intervals.
McCormack et al.
(17) passed argon support gas over volatile
compounds of interest and recorded the resulting spectra.
A
similar method was used by van Dalen et al. (33) who
controlled diffusion from capillaries by regulating
temperature in relation to solute volatility.
Estes, Uden
and Barnes were able to optimize wavelength settings for Si,
C and H by observing background emission resulting from the
quartz capillary tubing and low level hydrocarbon impurities
in the carrier gas (67).
Tanabe et al. (68) recently
published wavelengths, transition assignments and relative
intensities for nonmetal species observed in an atmospheric
pressure helium MIP.
Sensitivity
The first GC-MES systems employed tapered cavities with
argon as a support gas at atmospheric pressure (17,66,69).
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18
The first attempt to improve sensitivity was reported by
Bache and Lisk who. used reduced pressure plasmas in argon
(70) and helium (71,72).
Sensitivity and linear range were
improved at low pressures by minimizing collisional
deactivation of the analyte species.
Plasma temperature,
electron concentration and resulting emission intensities at
low pressures were very dependent on flow rate (36).
The
use of helium further improved results due to the higher
energy of the helium metastable state.
Microwave plasmas in
helium could only be sustained at reduced pressures between
5 and 30 Torr when tapered cavities were used.
Moye
achieved optimum sensitivity for organophosphorous
pesticides in a helium plasma containing 15% argon (21).
Slit width and He/Ar ratio were found to have a great effect
on the signal to noise ratio.
Detection limits for certain elements may be limited by
background emission due to impurities in the plasma gas.
Runnels and Gibson reported background emission from
molecular species such as C 2 , CH, N2 , NH, OH and CN (46).
Subsequent investigators have further cataloged the species
commonly found in MIP background spectra (24,67).
Reamer et
al. (73) used a wavelength modulation technique for
background correction.
To decrease background emission, purification of the
plasma gas has also been investigated.
Parkinson (74) used
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19
a glass U-tube packed with activated carbon and cooled with
liquid nitrogen.
This system effectively removed
C>2 / Ar and CC> 2 from He at flow rates of 3 L/min.
^z
Runnels
and Gibson purified argon by passing it over Mg at 550°C
(46).
Brenner (75) used molecular sieve 5A cooled in a dry
ice/acetone bath for the ultrapurification of helium used in
a microwave emission detector.
Estes et al. (67) found that
scrubbing helium with liquid nitrogen cooled molecular sieve
5A prevented emission from NH and OH but not ^
or N 2 (+).
Experimental parameters such as microwave power and
carrier gas flow rate also have a significant effect on
sensitivity.
An increase in power can affect emission
intensity by influencing excitation conditions such as
metastable concentration.
Increased analyte emission is
often accompanied by larger background levels, however.
The
flow rate influences excitation conditions and determines
analyte residence time in the detector.
Emission intensity
varies along the length of the plasma when viewed in a
transverse configuration (76,77) and spacially in the crosssection of a plasma viewed axially (67).
Selectivity
When characteristic emission of the analyte species is
observed, a great degree of selectivity is achieved.
can be an important advantage when chromatographic
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This
20
separation is incomplete or chromatograms are very
complicated, as is often the case when using capillary
columns.
The MIP can also be used as a general detector for
organics by observing carbon emission.
I
The MIP has been used in conjunction with a general
t
detector in gas chromatographic applications.
McLean et al.
(78) split the gas chromatographic effluent between MIP and
flame ionization detectors (FID) as did other investigators
(79,76).
Tanabe et al. (80) combined thermal conductivity
(TC) detection with an atmospheric pressure, helium GC-MES
system.
Dual strip chart recordings were obtained which
aided in compound identification.
The selectivity ratio is a term which compares analyte
emission intensity at its characteristic wavelength to
background emission at that same wavelength.
Since the
background emission is usually due to either atomic or
molecular carbon emission (C, C 2 , CH, CN, CO, etc.), the
selectivity ratio is calculated with respect to carbon.
Early investigators calculated the ratio in terms of the
volume of solvent required to give an equivalent signal
(17,66).
Subsequent articles defined the selectivity ratio
in terms of response per gram-atom of the element vs. carbon
(79,34).
Dagnall et al.
(81) based the selectivity ratio on the
molar concentration of carbon vs. analyte species resulting
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21
in equivalent peak areas.
This method of calculation seems
more appropriate since emission is directly proportional to
the specific number of atoms present.
Selectivity ratios
reported recently in the literature seem to be following
this trend in calculation although there is still some
inconsistency (23,67,80).
Selectivity is mainly a function of spectral
interference from carbon band emission.
This being the
case, the selectivity ratio can be increased by decreasing
the amount of band emission.
The use'of helium as the
plasma support gas leads to better fragmentation efficiency
(71).
Better selectivity can be achieved in a helium plasma
because atomic lines can be observed with a narrow bandpass,
and molecular background emission due to impurities is
reduced (23).
Estes et al. (67) used a quartz refractor
plate background corrector to improve selectivity ratios for
elements whose emission lines occur in high carbon
background regions.
Braun et al. (82) observed transitions
in the vacuum ultraviolet region and found molecular
background emission to be less of a problem.
Selectivity is
strongly dependent on the resolution of the monochromator
employed and slit width.
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22
Response vs. Structure
McCormack et al. (17), as well as Bache and Lisk (66),
found that response for nonmetals was dependent on.the
structural environment in the parent compound.
This
required individual calibration for each compound of
interest.
In subsequent studies with organic mercury
(83,84,85) and arsenic compounds (86), no such dependence
was found.
They attributed this phenomenon to the relative
bond strengths between the heteroatom and other atoms in the
compound.
Nonmetal atoms, unlike metals, form strong bonds
with atoms such as carbon and oxygen.
Dagnall et al. (87) reported that response for sulfur
compounds in an argon plasma was dependent on the structural
environment in the parent compound.
In a subsequent article
(88), the detector was improved by using an alternate cavity
with helium as the support gas and inserting a platinum wire
catalyst into the plasma tube.
The platinum partially
vaporized and aided in fragmentation.
These same
investigators eventually returned to an argon plasma at
atomospheric pressure because of simplicity (31).
With this
system, they described the feasibility of determining
interelement ratios between Br, Cl, I, P, S and C (81).
These ratios were valid for I, P and S compounds since
atomic emission was observed.
Results were not good for Br
and Cl since band emission was monitored and response was
flow rate and concentration dependent.
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23
McLean and coworkers used a low pressure helium MIP for
determining empirical formulas of compounds (78).
The gas
chromatographic effluent was split between a MIP and a flame
ionization detector for non-specific detection.
Elemental
ratios were determined for C, H, D, F, Cl, Br, I, S, P, N
and 0.
A GC-MES instrument based on McLean's work was
manufactured commercially by Applied Research Laboratories.
The MPD-850 bulletin claims, as do authors (78,89), that
sensitivity in the low pressure helium MIP for a particular
element is independent of the type of compound introduced
and proportional to the number of atoms present.
Closer
inspection of the published data reveals that this claim is
valid only in homologous series with exclusion of the lowest
members (33).
Better results have been obtained in
atmospheric pressure helium plasmas using the Beenakker
cavity design (23).
Reproducibility
Two major problems have plagued GC-MES since its
introduction:
i) the inability of the low power plasma to
accept large amounts of material; and ii) the loss of
transparency of the quartz plasma tube when viewing in the
transverse configuration.
Even though the MIP has a high
excitation temperature, it does not have sufficient energy
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24
to vaporize or atomize materials' introduced in large
quantities.
To reduce the effect of the solvent peak on
both of these factors, many investigators have simply
extinguished the plasma during this time (17).
This has
been effective when the plasma has time enough to stabilize
before the elution of analyte species.
A second approach to minimize the solvent effect has
been to vent the solvent away from the detector (87).
Quimby et al. (79,34,90) used a high temperature valve
system which allowed the effluent to be directed either to a
vent or to the plasma.
Estes et al. (91,67) employed a
chemically deactivated, low volume, valveless fluidic logic
gas switching interface to vent large quantities of solvent.
Inertness was demonstrated by determining chemically active
and thermally labile trialkyllead chlorides.
An atmospheric
pressure helium plasma was used which permitted venting.
The plasma could, therefore be viewed axially, circumventing
any reproducibility problems due to the loss in transparency
of the quartz capillary.
The loss in transparency is due to the build-up of
carbonaceous and metallic oxide deposits on the interior
walls of the discharge tube and devitrification of the
quartz at high temperatures.
McLean, Stanton and Penketh
doped a helium plasma with traces of oxygen or nitrogen gas
(78).
This scavenger gas was found to reduce carbon
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25
deposits.
Serravallo and Risby also studied the effects of
doping gases in GC-MES (92,93).
Beenakker prevented carbon
deposition by adding 0.1% 02 or N2 which led to the
formation of volatile carbon oxides or nitrides (23).
This
technique also increased the linear range by one order of
magnitude without affecting the background or sensitivity.
Schwartz reported an important dependence on oxygen
scavenger concentration in the determination of hydrogen
(94).
Optimum detection was achieved with 0.4 to 1.5%
oxygen.
In a subsequent paper, an anomaly in hydrogen
response was reported due to a charge transfer reaction with
the scavenger gas (95).
To avoid viewing the plasma through the discharge tube
wall, Dagnall et al. (31) attempted to develop an "open"
plasma detector at the tip of the plasma capillary.
Unfortunately, the high flow rates required to sustain a
stable plasma were not compatible with gas chromatographic
requirements.
More recently, Gebhart (77) viewed emission
in the transverse configuration through a hole drilled into
the side of the quartz capillary.
Improved long-term
reproducibility was reported.
Applications
One method that has found widespread application is the
formation of volatile chelates followed by separation and
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26
detection by GC-MES.
Dagnall et al. (96) determined
volatile metal chelates of Al, Cr, Cu, Ga, Fe, Sc and V.
Talmi measured trace levels of selenium in environmental
samples after conversion to the thermally stable and
volatile piaselenol complex (97).
As little as 40 pg Se
could be detected after extraction of the complex into
toluene and concentration.
Talmi also reported the
determination of arsenic and antimony after conversion to
volatile complexes (98).
Other applications of metal
chelates have been reported for Al, Be (99), and chromium
(99,92,100,93).
Other methods of chemical modification prior to
analysis include reduction of alkylarsenic acids to arsines
using NaBH^ (86).
Quimby et al. (90) used capillary column
GC-MES to determine aqueous chlorination products of humic
substances in water supplies.
Non-volatile compounds were
derivatized with diazomethane before determination.
Since
the carrier gas for the capillary column was only 4 mL/min,
a make-up gas of 50 mL/min. was required.
Novel applications of GC-MES have included the use of
reflected microwave power as a general detector (101) and
the determination of isotopic concentrations (102,38).
One
of the most comprehensive applications of GC-MES was
recently published by Estes, Uden and Barnes (67).
A fused
silica gas chromatographic capillary column was interfaced
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27
to an atmosphic pressure helium MIP.
Calibration curves,
selectivity ratios and detection limits were established for
V, Nb, Cr, Mo, W, Mn, Ee, Ru, Os, Co, Ni, Hg, B, Al., C, Si,
Ge, Sn, Pb, P, As, S, Se, F, Cl, Br, I, H and D!
No attempt will be made to describe all of the
published applications utilizing GC-MES.
The interested
reader is directed to the review articles previously
mentioned.
However, an overview of the elements determined,
observation wavelength, cavity type, support gas and
pressure, detection limits and selectivity ratio is
presented in Appendix H.
Commercial Instrumentation
GC-MES instumentation is available commercially.
Hobbs
et al. (103) reviewed the use of the ARL MPD-850 for
environmental pollution studies.
The instrument was capable
of selectively monitoring up to 12 elements simultaneously
over a wide linear range.
Computer programs for selectivity
and data handling have also been described (104).
Hobbs
used a pyrolysis furnace in conjunction with the MPD-850 to
analyze river water extracts for compounds containing C, H,
Cl and S (105).
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28
Summary
Element Selective Detectors for GC
The development of element selective detectors has
received much attention.
Selective detectors simplify
chromatograms so that less efficient separation is needed,
which often leads to reduced analysis time.
When highly
efficient capillary columns are used, selective detectors
simplify the resulting chromatogram.
Element selective
detectors often provide qualitative information not
available with general detectors.
Electron Capture Detector
The electron capture (EC) detector is sensitive
primarily to halogenated compounds (F<Cl<Br<I).
Mulligan et
al. (106) found the EC detector to be more sensitive than
microwave emission spectroscopy (MES) for the determination
of polybrominated biphenyls.
however.
The MIP was more selective,
Other disadvantages of EC detection include
susceptibility to contamination, small linear range (102 10*) and unpredictability of response (107).
Coulometric and Conductometric Detectors
It is usually necessary to oxidize or reduce organic
compounds eluting from the gas chromatograph to simple,
inorganic gases before detection.
For the Ag(+) detector
cell, sulfur compounds are reduced with hydrogen to H^S.
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29
Sulfur compounds are oxidized to SC^/SO^ with oxygen at
750"C before entering the I(-) cell.
Absolute response to
ng levels with selectivities as high as 106" are possible
(107).
Since the oxidation/reduction furnaces add dead
volume to the detector, Fredericks et al. (108) suggested
that these electrochemical detectors were best suited for
packed columns and relatively simple mixtures.
A new
conductivity detector design for S, Cl and Ng was developed
by Hall (109).
Sub-nanogram sensitivity, a linear range of
10s and selectivities as high as 10s were reported.
Flame Detectors
Gutsche and Herrmann (110) obtained characteristic
emission for iodine, bromine and chlorine compounds eluting
from a gas chromatograph.
The burner contained indium which
produced band spectra with halides.
Nowak et al. (Ill)
developed a flame detector based on sodium emission from a
sodium sulfate-coated wire in the presence of halogens.
The
addition of sodium compounds to a flame ionization detector
(FID) to enhance response for phosphorous and halogen
compounds was first described by Karmen et al. (112).
The most popular flame detector is the flame
photometric detector (FPD) used primarily in the sulfur or
phosphorous mode (113).
When the 3939 A S£ band is
observed, high sensitivity can be attained, but response is
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30
proportional to the square of sulfur concentration.
-Several
authors have reported that response is dependent on the
environment of the sulfur atom in the molecule (114-116).
Beroza and Bowmann reported sulfur to hydrocarbon
selectivities of 10“ (117).
However, when sulfur compounds
were not completely separated from the hydrocarbon,
quenching of the chemiluminescent emission was possible
(118).
Golovnya et al. (119) suggested that the FPD should
not be used for quantitative gas-liquid chromatography below
0.1 mg/mL because of signal drifting.
Plasma Detectors
Ellebracht et al. have recently reported the use of a
dc discharge plasma as a gas chromatographic detector for
sulfur compounds in the vacuum ultraviolet region (120-122).
This high temperature, high energy plasma minimized
quenching effects and could handle as much as lOOyL of
solvent without being extinguished.
The relative standard
deviation for replicate injections of 259 ppm carbon
disulfide in hexane was 4.5%.
The minimum detectable level
obtained for sulfur at 1807 A with this VUV-PAES system
ranged from 100 to 300 pg S/second.
The linear dynamic
range covered three orders of magnitude with a selectivity
ratio of 1000 for a variety of organic solvents.
A similar
dc discharge for detection of organics and halogens in the
UV-visible was reported by Braman and Dynako (123).
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31
Windsor and Denton determined halogens, C, Fe, H, Pb,
Si and Sn by GC-ICP (124).
Detection limits (low ng range),
linear dynamic range (>103) and selectivities were
considered favorable to both the FPD and MIP.
Unfortunately, intensities for B, F and S were low while N
and O lines were completely absent.
Conclusions
The microwave induced plasma has made an important
contribution to selective gas chromatographic detection.
The "state of the art" GC-MES system is capable of
determining most of the elements in the periodic table with
picogram sensitivity in many cases.
The detector exhibits a
weak, and relatively- simple, background spectrum.
It is
inexpensive to operate with low power requirements and low
gas consumption.
The major problem which still requires attention is the
sample load capacity of the MIP.
The combination of
separate evaporator/atomizer units with the MIP is
promising.
In GC-MES, the trend seems to be moving towards
the use of capillary columns.
Purpose of Study
Background
A study was proposed by three faculty members at the
University of Iowa, College of Nursing, to investigate
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32
possible correlations between multiple sclerosis (MS) and a
condition known as lactose malabsorption (125).
Recent
findings indicated a connection between the two and
warranted further investigation:
1.
In the summer of 1976, a group of 45 MS patients were
questioned about their dairy product consumption.
Approximately 25% of this group indicated that they
no longer drank milk because jof the discomfort that
resulted.
2.
The Eastern Iowa MS Clinic at the University of Iowa
Hospital reported that approximately 50% of their
patients refused milk.
3.
This aversion to milk and milk products was commonly
spoken of during conversations with MS patients at
regional MS Society meetings.
Fatigue has been described as one of the initial
symptoms of MS.
This fatigue is the primary factor which
limits a patient's activity (126).
McAlpine et al. (127)
observed that factors such as infections, trauma, fatigue,
physical exertion, emotional disturbance and preventative
inoculations all appear to be capable of influencing the
onset and course of MS.
Lactose malabsorption is one possible explanation for
this fatigue.
Fatigue resulting from food allergies may be
due to the excess energy required to support a
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33
hypersensitive response (125).
Foods which are most
commonly associated with allergic reactions include sugar,
milk, chocolate, wheat, corn, citrus, yeast, spices and eggs
(128).
Lactose Intolerance
Enzymic digestion in the oral cavity, stomach and small
intestine removes most dietary constituents (129).
However,
fibrous constituents (130) and small molecular weight
carbohydrates such as stachyose in beans, raffinose in
cottonseed meal, and the artificial sugar lactulose are not
attacked by mammalian enzymes (131).
Instead, they are
degraded by the microbial community of the large intestine.
Fermentation products in the large intestine include
volatile fatty acids, methane, hydrogen and carbon dioxide.
Lactose, 0-(S-D-galactopyranosyl-(l-*-4)-0-Dglucopyranose, is a carbohydrate found in milk but otherwise
does not occur in nature (132).
Individuals who are unable
to completely digest lactose are said to be lactose- or
"milk"-intolerant.
People have been known to suffer from
this condition for centuries; however, its cause was not
identified until just recently (133).
The enzyme lactase, a p-galactosidase, hydrolyzes
lactose into its monosaccharide components of glucose and
galactose.
These can then be absorbed into the small
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34
intestine.
Individuals with decreased lactase activity have
been found to absorb as little as 25 to 58% of the lactose
they ingest (134).
If milk products are consumed,
non-absorbed lactose is transported down the digestive
tract.
As the lactose reaches the colon, it is fermented by
intestinal bacteria into lactic acid and hydrogen gas
(135,136).
The resulting symptoms are watery and acid
diarrhea, flatulence, cramps and abdominal bloating (137).
The definitive test for lactose malabsorption requires
a biopsy of the small intestine and determination of lactase
activity (138).
One common indirect diagnostic method is
the lactose tolerance test (LTT).
Blood glucose levels are
monitored following an oral dosage of lactose.
Lactose
intolerant subjects show very small increases in glucose
because of decreased absorption.
Unfortunately, this test
is influenced by factors such as gastric emptying and
intermediary glucose metabolism,
leading to possible errors
(139).
In 1968, Levitt et al. reported that breath hydrogen
excretion was a useful indicator of carbohydrate
malabsorption (140).
Diagnosis was based on the principle
that nonabsorbed lactose passes through to the colon where
hydrogen gas is formed.
Hydrogen production in normal
subjects was found to average 0.24 mL/min. in the fasting
state, increasing to 1.6 mL/min., after intestinal
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35
instillation of lactose.
Approximately 14% of the hydrogen
produced in the colon is absorbed into the bloodstream and
excreted via the lungs (136).
Bacterial degradation of
nutrients is the only source of hydrogen in the body (138).
Hence, breath measurements are directly proportional to the
amount of lactose not absorbed in the small intestine (134).
Hydrogen Breath Test
Normal subjects exhale little or no hydrogen after
fasting overnight.
When a dose of lactose is administered
orally, only those subjects with lactase deficiencies show
an increase in breath hydrogen levels over time.
Hydrogen
excretion begins to increase an hour after the dose, and
normally reaches a maximum at approximately 1.5 to 2 hours
(141,142).
Measurement of the relative increase in hydrogen
concentration over baseline levels is the basis for
diagnosis.
The use of breath hydrogen excretion as an
indicator of carbohydrate malabsorption requires that: i) no
appreciable hydrogen is produced from carbohydrates in the
small intestine; and ii) delivery of carbohydrate to the
colon results in a detectable increase in breath hydrogen
excretion (139).
An increase of 20 ppm over baseline
hydrogen levels has been suggested as a positive indicator
for lactose malabsorption when 50 g of lactose is
administered (143).
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36
The hydrogen breath test (HBT) has been described as
the most suitable test for the screening of lactase
deficiency (138).
Bond and Levitt (134) found that the
quantity of lactose absorbed, as determined by breath
hydrogen excretion, correlates very closely to the quantity
of unabsorbed lactose aspirated directly from the 'ileum.
The HBT was also found to agree with the results of standard
lactose tolerance tests (144).
The advantage of the HBT
over tolerance tests is that it reflects the quantity of
sugar not absorbed, so much smaller doses can be given.
It
is sufficiently sensitive to detect malabsorption pf as
little as 5 to 10 grams of carbohydrate (139).
Tolerance
tests require doses of 50 to 100 grams of carbohydrate.
HBT Errors
Possible errors associated with the HBT were summarized
by Solomons (144).
In some cases, elevated baseline
hydrogen levels were encountered.
This is often the case
after a period of sleep due to hypoventilation and decreased
passage of flatus.
Since interpretation of the HBT is based
on peak hydrogen concentrations relative to baseline levels,
initially elevated levels may invalidate the test.
The
simplest solution to this problem is to delay the test diet
until baseline hydrogen levels equilibrate.
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37
Delayed gastric emptying or retarded intestinal transit
can also obscure HBT results.
This problem has been
encountered in lactose tolerance tests.
The carbohydrate
could be delivered to the small intestine by direct
instillation, but this defeats the non-invasive nature of
the test.
Alternatively, the time of observation could be
extended in order to detect delayed hydrogen excretion.
The production of hydrogen in the colon depends on the
availability of bacterial flora and their ability to produce
hydrogen.
Colonic flora not capable of producing hydrogen
from carbohydrate has been reported but is believed to be
rare (139).
Lack of knowledge about the bacteria
responsible for hydrogen formation makes it difficult to
predict what factors influence this mechanism.
The use of
broad spectrum antibiotics has been shown to inhibit
hydrogen production by colonic bacteria (144).
One way to
determine a subject's hydrogen production capability is to
administer a non-absorbable carbohydrate such as lactulose.
»
Previous Application of Breath Tests
The technique of measuring exhaled breath constituents
with respect to ingested food has been used in a variety of
studies.
Calloway and co-workers evaluated the suitability
of certain diets for space (145) and studied the effects of
antibiotics (146).
Breath analysis has also been used in
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38
the study' of intestinal absorption, gross parasitism, liver
function and effects of gastro-intestinal bacteria on
endogenous and exogenous materials (147).
Determination of Hydrogen in Breath
Chromatography
The determination of trace amounts of hydrogen gas has
been reported previously in the literature.
These methods
normally employ gas-solid chromatographic separation on
molecular sieve columns.
Separation of hydrogen in breath
analysis studies have almost exclusively employed either 5A
or 13X molecular sieve columns.
Other adsorbent phases such
as PorapakfSQ, activated alumina and activated silica have
also been used successfully.
Separation of hydrogen from
other permanent gases is complete at ambient or sub-ambient
temperatures.
Hydrogen conveniently elutes from the column
before other breath constituents.
Detectors
The first quantitative determinations of respiratory
gases employed gas chromatography with thermal conductivity
(TC) detection.
Carle (148) developed a microbead
thermistor detector for sensitive determination of microbial
respiratory gases including hydrogen.
The use of this
detector resulted in a negative peak anomaly for hydrogen at
certain concentrations.
Levitt and coworkers (136,139,149)
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39
used TC detection in developing the hydrogen breath t6st for
lactose intolerance.
They reported a detection limit of 10
ppm using 2 mL breath samples.
In order to detect hydrogen
levels down to a few parts per million (ppm) using TC,
Solomons (142) increased the sample loop size to 16 mL.
More recently, Niu et al. (150) used TC to detect as little
as 2 ppm hydrogen in breath for a 1 mL sample injection.
Gearhart et al. (141) employed a helium ionization
detector (HID) to determine lactose malabsorption by breath
analysis with gas chromatography.
The lactose test diet was
reduced to 0.25 g per kg body weight because detection
limits for hydrogen were improved to sub-ppm levels.
However, breath levels below 10 ppm required extensive
purification of the helium carrier gas.
scrubbed with Ba( 0 H ) 2
Breath samples were
and anhydrous CaSO^ before injection.
There have been numerous reports about problems
associated with the helium ionization detector.
Parkinson
suggested that these detectors were useful over a very
limited concentration range due to anomalous response for
hydrogen (151).
When extremely pure helium carrier gas was
used, a negative peak was observed for traces of hydrogen.
As the hydrogen level was increased, the response became
progressively less negative and eventually positive!
Payne-Bose and coworkers (152) compared the HID to thermal
conductivity detection.
They found the HID to be variable
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40
in performance and difficult to maintain.
Similar comments
were obtained through personal communication with
representatives from Varian Instrument Company who formerly
marketed the HID.
The use of argon ionization detectors has also been
reported.
Gawlowski et al. (153) found that at flow rates
of 25 to 75 mL/min., the detector is mass-flow sensitive.
At higher flow rates, sensitivity decreased sharply.
The
detection limit for hydrogen'was 100 pg/second with a linear
dynamic range of 100.
The operational mechanism for the
argon ionization detector was discussed in a subsequent
article by the same authors (154).
The development of electrochemical detectors for
hydrogen and other permanent gases has also received some
attention.
Bergman et al. (155) used a metallized-membrane
electrode to measure hydrogen production of bacteria in the
human gut.
This detector was used in a simple portable gas
chromatograph.
The system was capable of determining less
than 10 ppm hydrogen with a precision of ±0.1 ppm.
Guglya
developed a platinum-coated lithium niobate plate as an
electrochemical sensor for as little as 0.2 ppm hydrogen
(156).
Taylor and coworkers (43) determined trace impurities
in argon carrier gas using a microwave induced plasma (MIP).
By observing atomic emission from the plasma, element
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41
specific detection of the impurities, including hydrogen,
was obtained.
The detection limit for hydrogen at 4861 A
was 1.8 ppm by weight.
This study was performed on a
constant flow of sample (the plasma gas) which improved
sensitivity.
Lefebure used a 4 MHz discharge in helium at
atmospheric pressure for the determination of ppm levels of
H, N, 0 and CH^ (157).
When both N and H were present in
the plasma, a "line" at 3360 A characteristic of the NH
radical was observed.
Atomic hydrogen emission in the
Balmer Series was found to decrease in intensity with an
increase in hydrogen concentration!
Recently, Schwarz
characterized the emission from atomic hydrogen in a MIP
(95).
Helium was used as the plasma gas at reduced
pressure.
An anomaly in hydrogen response was observed in
the presence of either oxygen or argon.
This was believed
to be due to a charge transfer reaction between O 2
and H(+)
which competed with excitation by metastable helium atoms.
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42
CHAPTER II
EXPERIMENTAL
Introduction
The first objective of this work was to assemble a
GC-MES system from individual components developed
previously in our laboratory.
A Nester Faust, Model 750,
gas chromatograph was completely modified.
The vacuum
ultraviolet spectrograph built by Dreher (158) was converted
into a scanning spectrometer.
The plasma observation work
reported by Gebhart (77) was the basis for the optical
interface between the gas chromatograph and the
spectrometer.
The wavelength readout system was a
modification of the digital integration circuit developed by
Cox (159).
A block diagram of the experimental set-up is given in
Figure 1.
A quartz capillary tube extended from the end of
the chromatographic column.
A microwave cavity, or antenna,
was positioned so that microwave energy was focused on a
small portion of the quartz capillary.
The microwave power
could be adjusted between zero and 120 watts at a fixed
frequency of 2450 MHz.
After initiating the plasma with a
Tesla coil, it was self-sustained inside the quartz
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43
capillary when argon was used as the carrier gas.
This
plasma served as an excitation source for species eluted
from the GC.
As an analyte species passed through the plasma,
excitation occurred with subsequent emission of radiation.
The wavelength of the emitted radiation was specific for the
species being excited.
Emission was observed through a one
millimeter hole drilled into the side of the quartz
capillary.
Light passing through this observation port
propagated through a light pipe, based on the principle of
total internal reflection, to the entrance slit of the
spectrometer.
The emission signal was detected by a
photomultiplier tube and associated electronics.
A summary
of the instrumental components is presented in Table 1.
Gas Chromatograph
Control Panel
A control panel was set up for all GC functions and
mounted on an instrument rack.
Controls for the oven,
injection and exit port heaters, oven blower fan, and oven
vent were included.
Iron-constantan thermocouples were used
to monitor column, injection, and exit temperatures.
Additional thermostat adjustments were located on the rear
of the GC.
The zero control was used to set the low
temperature limit to ambient conditions.
The damping
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44
Figure 1
GC-MES Experimental Set-up
1)
2)
3)
4)
5)
6)
7)
8)
9)
10)
11)
12)
13)
14)
Gas chromatograph
Argon carrier gas
Drying column
Rotameter
Microwave power supply
Microwave cavity
Plasma capillary
Light pipe
Monochromator
Photomultiplier tube
HV power supply
Electrometer
Electronic filter
Recorder
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
m
Os
Figure
CN
00
CN
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46
Table
1
Experimental Apparatus
Excitation
Microwave generator
Raytheon, Model P6M-10X1
Microwave cavity ......
Tapered
Monochromator
Mount ..................
Czerny-Turner modification
Focal length ........... 1.075 m
Gratings .... .
7500 A blaze, 1180 grooves/mm
4000 A blaze, 1180 grooves/mm
Signal measurement
HV power supply .......
EMI Gencom, Model 3000R
Photomultiplier tube ... Hamamatsu R212
Electrometer ..... ....
Keithly, Model 610CR
Electronic filter .....
Spectrum, Model 921
Recorder ......... ....
Esterline Angus, Model MS401B
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47
control was set at its maximum clockwise position to
minimize temperature fluctuations.
The calibration control
could be adjusted so that the potentiometer dial setting
closely approximated actual temperature.
Injection Port
The injection port was designed to prevent the sample
from coming in contact with any metallic surfaces.
The body
was fabricated out of brass with a quartz insert as shown in
Figure 2.
Carrier gas entered the injection port through a
1/8" Swagelock® to 1/8" NPT connection and flowed through to
the column via the 1 mm hole in the side of the quartz
insert.
A constriction in the quartz insert minimized
sample migration back into the dead volume adjacent to the
septum.
One end of the quartz insert was sealed with a septum
(Supelco 2-0404) arrangement consisting of a 1/4" Swagelock®
to 1/4" NPT connection.
Soldered to the septum connection
was a heat sink consisting of a circular metal disk and coil
of 1/4" o.d. copper tubing.
In high temperature GC
applications/ water could be flushed through the tubing to
protect the septum from deterioration.
The opposite end of
the injection port was sealed by modifying a 6 mm Swagelock®
union.
A union connection was required that would hold the
quartz insert tightly against the septum in addition to
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 2
Injection Port Design
(drawn to scale)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
49
o
u
u
u
G
<U
>
o
o
u
fl
G
CN
Figure
o
-U
rH
3
W
•H
H
G
u
o
0000
OOOO
4J
O.
<D
c/i
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
50
providing a gas tight connection to the column.
To
accomplish this, a hole was drilled in the block concentric
with the quartz insert (0.499" diameter x 13 mm).
.A 1/4"
Swagelock® nut was turned down until its outside diameter
was approximately 0.001" larger than the hole into which it
fit.
After the nut was cooled in liquid nitrogen, it was
rapidly forced into the hole before warming.
This resulted
in a rigid, leak-free bond which required neither adhesives
nor solder which could fail at high temperatures.
A 6 mm
Swagelock® union sealed with 6 mm graphite ferrules (Supelco
2-2493) completed the flow system to the column.
Exit Port
The exit port provided the connection between the GC
column and the plasma capillary as shown in Figure 3.
Graphite ferrules were used in all connections to quartz
tubing and provided a leak-free seal at one quarter turn
past finger tight.
Under these conditions, the plasma
capillary could still be removed with careful twisting.
This removal was convenient for cleaning, optical
adjustment, or replacement purposes since disassembly of the
exit port was not required.
Column
Quartz tubing ( 6 m m o . d .
chromatographic column.
x4mmi.d.)
was used for the
Variable lengths could be used
depending on separation requirements.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 3
Exit Port Design
(drawn to scale)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
52
Figure 3
Plasma Capillary
I Observation Hole
\
\
\
V
\
I Asbestos
Fiberglass
GC Insulation
GC Column
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53
Detection System
Plasma Capillary
The specifications for the plasma capillary tube were
optimized in previous work done by Gebhart (77).
Most
important in these considerations were the 1 mm i.d. and. the
1 mm observation port.
with a 6 mm o.d.
The capillary was 25 cm in length
The observation port was placed as close
to the exit port as physically possible to minimize cooling
and possible condensation of the analyte species.
The
resulting distance between the column exit and the
observation port was 11.5 cm.
Cavity Mount
A tapered microwave cavity was mounted in a device
which allowed vertical adjustment along the length of the
plasma capillary.
The cavity mount was attached to the GC
as illustrated in Figure 4.
By raising and lowering the
cavity, the portion of the plasma observed could be varied.
The observation height was defined as the distance between
the observation port and the center of the cavity.
Plasma Ignition
The plasma was initiated with a Tesla coil mounted at
the rear of the GC.
A push-button switch on the instrument
rack initiated the Tesla coil which provided seed electrons
to the plasma along a wire between the coil and the plasma
capillary.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 4
Microwave Cavity Mount
(drawn to scale)
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55
Figure 4
v
Microwave
Cavity
JZJ
HF
Asbestos
Fiberglass
GC Insulation
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
56
Light Pipe
Optimal dimensions for the light pipe were determined
previously in this laboratory (160).
Light pipes were
purchased from Wilmad Glass Co. with dimensions of 50
14 mm o.d. x 5 mm i.d.
These pipes were
cm x
specified to be
optically flat to 1/3 of a wavelength at 3000 A.
The light pipe was supported at the
mount illustrated in Figure 5.
plasma end by the
Adjustment of the set screws
provided for alignment with the plasma observation port.
The opposite end of the light pipe was held in position by
an iris mechanism centered in relation to the entrance slit.
An illustration of the iris mechanism and its mount to the
spectrometer is given in Figure 6.
A 1/4" Swagelock® union
was soldered to this mount which provided a means of
flushing the light pipe with an inert gas.
Spectrometer Modifications
The original spectrograph was not designed for scanning
purposes.
The conversion of the instrument to a scanning
spectrometer was accomplished by modifying the grating
rotation and scan mechanism from a Perkin Elmer 303 Atomic
Absorption Spectrometer.
The grating rotation unit was
fastened to the optical shelf inside the spectrometer by
three pairs of opposing set screws.
an adjustment for grating tilt.
These screws provided
The rotation shaft extended
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 5
Adjustable Light Pipe Mount
(drawn to scale)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
58
Figure 5
Top View
Asbestos
Fiberglass
O
Gas
Chromatograph
O
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 6
Light Pipe Support
(drawn to scale)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
60
CD
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>
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a
o
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CO
View
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vo
□
□
o
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Xt <D
(7> 0 .
■H ‘H
J 0.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
61
through the bottom wall of the spectrometer and was
supported by a bearing assembly attached to an exterior
shelf.
This design provided true rotation with very little
friction.
The sine bar was attached to the rotation shaft below
the exterior shelf.
The gear assembly which moved the sine
bar and a motorized scan mechanism were mounted to the
bottom of the shelf.
A 30 rpm motor provided forward and
reverse wavelength scanning at approximately 1100 A/minute.
A second 1/2 rpm motor could only be used to scan in the
forward direction at approximately 15.6 A/minute..
Microswitches were positioned on either side of the sine bar
to automatically shut off the drive mechanism at the scan
limits.
When either of these switches shut off the drive,
the scan direction had to be be reversed and started
manually.
Wavelength could also be scanned manually by
depressing the pin which disengaged the 1/2 rpm motor.
Wavelength Readout
The wavelength readout system was based on a voltage to
frequency conversion circuit previously developed in our
laboratory for peak integration (159).
A block diagram for
the readout device is illustrated in Figure 7.
The
objective was to convert a signal representing grating angle
to a digital signal which could be calibrated to display in
Angstrom units of wavelength.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 7
Wavelength Readout Device
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
63
Figure 7
•GND
f out
Ve
R>'
Rx $<■
wyw
AD537
V/F CONVERTER
in
r\
— VNAAAA
+ 20 V
MP 1029A FREQUENCY METER
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
64
A 25 turn, 0-100 k£2 potentiometer was mechanically
attached to the grating rotation gear mechanism.
Rotation
of the grating resulted in a linear change in resistance
(R^).
When the proper voltage (V^) was applied between the
stationary terminals of the potentiometer, the voltage drop
to the slider terminal was linearly proportional to the
grating angle.
After this point the readout system was very
similar to the integration system previously developed.
An
Analog Devices AD537 voltage to frequency converter
integrated circuit chip was employed.
Adjustment of
controlled the slope of the curve
relating readout to actual wavelength.
Before attaching the
potentiometer to the drive mechanism, R^ had to be adjusted
to approximate the correct wavelength.
Fine adjustment
(±40 A) was made with R^' , a 0-500 k£2 potentiometer.
R^1
could be set to give the correct readout for a standard
wavelength in the region of interest.
Calibration was then
accurate to within a few Angstroms over a 500-1000 A range.
Photomultiplier Tube Mount
The instrument was originally built as a spectrograph
with photographic recording and no exit slit.
A mount was
designed and fabricated from brass to hold a slit mechanism
and either side mount or end-on PMT housings.
A Hilger slit
mechanism was placed into the brass mount illustrated in
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65
Figure 8.
The slit adjustment screw was lengthened so that
it extended through the top of the mount.
calibration is illustrated in Figure 9.
Slit width
A side mount PMT
housing could be attached directly to the slit mechanism.
Holes were included for mounting an end-on PMT housing.
Optical Adjustment
The gas chromatograph was supported by an adjustable
mount.
Half-inch bolts were secured at the corners of a
base slightly larger than the bottom of the gas
chromatograph.
These adjustment bolts extended through a
rectangular arrangement of one inch angle iron attached to
the bottom of the GC.
A pair of half-inch nuts on either
side of the angle iron support provided independent vertical
adjustment at the four corners.
This arrangement permitted
the entire GC to be moved up, down, or even tilted to align
the plasma observation port with the optical path.
Another modification of the spectrometer included
adjustable supports to aid in alignment with the plasma.
*
Each end of the spectrometer was supported on an adjustable,
vibration-absorbing mount as illustrated in Figure 10.
The
adjustment screws were positioned in a triangle, two for
side-to-side tilt and a third for vertical displacement.
Alternating layers of cork and rubber padding were used in
the base to minimize vibrations.
A summary of the alignment
procedures are presented in Appendices A and B.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 8
PMT - Exit Slit Mount
(drawn to scale)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 8
Slit Width Indicator
(# turns open)
Mounting Holes for
End-on PMT (3)
Slit Adjustment
Extension
J
Purge/Vacuum Port
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 9
Slit Width Calibration
Key:
O Entrance slit
• Exit slit
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 9
Slit Width, mm
.50
.25
2.0
Number of Turns Open
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 10
Spectrometer Mount
(scale 10mm = 1 inch)
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71
Figure 10
Monochromator
I
Adjustment
1 Screws
r
1
Cork
^ Rubber
-,L \ V k..k. \ >>■
\ N-.k. k '
-1
Wood
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72
CHAPTER III
RESULTS AND DISCUSSION
Plasma Characteristics
Plasma Background Spectrum
The plasma background spectrum for argon carrier gas
and argon with 5% helium is presented in Table 2.
The
second positive system of nitrogen was the most prevalent
band system in the background spectra.
The presence of
significant amounts of nitrogen in argon plasmas has been
reported (67).
This nitrogen system is composed of close,
triple-headed bands which are degraded towards shorter
wavelengths.
Several bands were also observed due to N£(+).
These bands are single-headed and degraded towards shorter
wavelengths.
A final band system centered at 3360 A originates from
the NH radical.
Bands are degraded in either direction with
the Q-branches forming a strong central maximum.
Under low
dispersion, the Q-maxima of the 0,0 and 1,1 bands are often
mistaken for an atomic doublet (161).
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73
Table
2
Plasma Background Spectrum
(3000 - 5000 A)
X, A
Assignment*
3063.6
OH
3090.4
°3
(0,0)
3097.5
3106.1
3136.0
°3
I
Argon
IAr+5%He
^'theor
19
-
10
69
5
8
34
-
4
24
-
5.
N2
(2,1)
10
11
8
N2
(1/0)
19
47
9
3360.0
NH
(0,0)
60
29
9
3370.0
NH
(1,1)
90
>160
3159.3
3371.3
3469.0
3500.5
3536.7
3548.9
3563.9
3576.9
3582.1
3606.5
3641.7
N2
10
10
(0,0)
(3,4)
-
1
0
(2,3)
-
3
4
N2
(1,2)
6
19
8
V
V
(3,2)
5
4
3
(2,1)
5
6
4
N2
(0,1)
42
127
10
N2+
(1,0)
-
6
4
3
4
1000
3
3
3
^2
N2
Ar
N2
(4,6)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 2 (cont'd.).
3649.8
Ar
3671.9
3710.5
3755.4
3370.4
3834.7
3857.9
3884.3
3894.6
3914.8
3943.0
3947.5
3949.0
3998.4
-
<1
800
N2
(3/5)
-
1
6
N2
(2/4)
1
4
8
• N2
d/3)
4
12
10
-
1
400
14
' 38
10
4
3
800
Ar
3804.9
-
N2
Ar
(0/2)
N2
(4,7)
-
<1
5
V
d/1)
2
3
3
N2
(3/6)
-
1
7
N2 +
(0,0)
2
12
6
N2
(2,5)
-
1
8
Ar
[ 9
L
Ar
1000
» '
9
N2
Ar
d/4)
(0,3)
4158.6
N2
Ar
4164.2
2000
2
4
9
21
25
1200
4
7
8
>58
120
1200
Ar
19
13
1000
4181.9
Ar
18
21
1000
4190.7
Ar
47
48
4044.4
4059.4
4191.0
Ar
600
1200
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75
Table 2 (cont'd.).
4198.3
Ar
4200.7
Ar
4251.2
50 .
126
1200
>56
126
1200
Ar
7
8
800-
4259.4
Ar
40
43
1200
4266.3
Ar
28
29
1200
4272.2
Ar
42
42
1200
4300.1
Ar
33
34
1200
4333.6
Ar
34
34
4335.3
Ar
14
34
4343.6
N,
(0,4)
'
800
-
12
1000
1
12
4
4345.2
Ar
4510.7
Ar
14
15
1000
4596.1
Ar
5
4
1000
4628.4
Ar
4
3
1000
4702.3
Ar
5
5
1200
J
.
1000
* Assignments in brackets were unresolved.
** Molecular intensity values were obtained from reference 161.
Atomic intensity values were obtained from reference 162.
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76
Spectroscopic Temperature
Spectroscopic temperatures were based on the relative
intensities of argon lines at 4259 A, 4345 A and 4511 A.
These lines were reported to be free of self-absorption by
Adcock and Plumtree (163).
This method utilizes the fact
that a plot of log (IX/gA) vs. E should be a straight line
that is inversely proportional to temperature.
Spectroscopic temperatures were determined for a
variety of carrier gas compositions:
Argon
4300 K
Argon
+ 5% Helium
4500 K
Argon
+ 9% Helium
6800 K
Argon
+ 12% Helium
6100 K
These results were obtained at an observation height of 12
mm below the center of the microwave cavity at a total
carrier gas flow rate of 120 mL/min.
The increase in
temperature with helium content was expected due to the
higher excitation potential of the helium metastable state
(19.8 eV).
A decrease in temperature was observed for helium
concentrations greater than approximately 10%.
This
probably represents a limit for temperature enhancement with
helium at 60 watts of applied power.
When higher power
levels were applied to the 12% He plasma, the plasma
capillary began to glow due to the extreme heat.
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77
Detection of Hydrogen Gas
In order to determine the feasibility of observing
hydrogen emission in an argon microwave plasma, the
background emission spectrum was recorded.
The argon
carrier gas was then doped with a trace amount of hydrogen
gas.
Figure 11 is a comparison between background argon
plasma emission with and without traces of hydrogen.
The main features of the plasma spectrum were atomic
argon emission lines in the 4000 A to 4500 A region, atomic
hydrogen emission at 4861 A and 6563 A, and second order
molecular NH emission at 6720 A.
The atomic hydrogen
emission was from the Balmer series and has been used
previously for spectrochemical analysis (43,94).
The NH
band emission at 3360 A has not been used previously for the
analytical determination of hydrogen.
This molecular
emission has been used for the determination of NH^ in
argon, however (43).
This emission was investigated in the
interest of improving sensitivity for hydrogen.
Weak NH emission was observed in the background argon
spectrum as well.
By observing the plasma through the
capillary wall, emission due to hydrogen and nitrogen
impurities in the carrier gas were selectively observed.
Since argon purity was specified to be 99.998%, NH emission
was negligible.
The majority of the background NH emission
observed at the observation port was assumed to be due to
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 11
Argon Plasma Spectra
a) Argon carrier gas
b) Argon + trace hydrogen gas
Grating
Slits
Ar flow
Observation ht.
Power
4000 A
0.5 mm
120 mL/min.
-12 mm
70 watts
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79
Figure 11
a)
4000
5000
6000
7000
o
CM
4000
5000
6000
7000
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80
atmospheric hydrogen and nitrogen.
This contribution led to
a small, positive background interference which was found to
be very constant.
It was believed that hydrogen passing
through the chromatographic column must combine with
atmospheric nitrogen at the observation port.
This was
confirmed by injecting trace quantities of hydrogen gas and
comparing emission through the wall and through the
observation port.
A much larger chromatographic peak
corresponding to hydrogen was observed through the port.
On
this basis, observation of t h e N H band was investigated for
the analytical determination of hydrogen gas.
Separation
Even though the MES system is element specific,
baseline emission is affected by large amounts of any
species passing through the plasma.
plasma is actually extinguished.
In extreme cases, the
Hydrogen, therefore, had
to be separated from the rest of the constituents of the
breath sample.
Narrow peaks and a minimum separation time
were desired.
Molecular sieves have been used extensively in the
separation of permanent gases.
Types 5A and 13X have both
been used since separation is not actually based on a
sieving mechanism but on electrostatic interactions (164).
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81
A molecular sieve 13X column will separate, in order,
hydrogen, oxygen, nitrogen and methane, with carbon dioxide
being completely adsorbed (165).
Hydrogen was very
efficiently separated from the rest of the gases.
A 60/80
mesh size was chosen since flow rates required for
maintenance of the plasma were.not possible with smaller
sized particles.
This problem was assumed to be due to
fines which were difficult to remove.
Separation was accomplished at ambient temperatures.
Flow rate was optimized with respect to peak height, shape
and time of analysis.
separation parameters.
Table 3 is a summary of the optimized
Under these conditions, the
retention times for hydrogen, oxygen and nitrogen were 24,
42 and 72 seconds, respectively.
Detector Optimization
To maximize sensitivity, microwave power as well as
observation height required optimization.
These two
parameters were important because of their relation to
excitation temperature.
Since plasma length increases with
power, the optimum height of observation also changes.
In
order to maximize both power and observation height, these
parameters were investigated simultaneously.
Detector
response to a standard injection of hydrogen was observed at
various positions along the length of the plasma for
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82
Table
3
Operating Conditions for
GC column dimensions ..... 6 ft.
x 6 mm o.d., 4 mm i.d.
GC column packing
mesh molecular sieve 13X
....
60/80
Carrier gas ..... ........ Argon
Column temperature ......
.
Plasma c a p i l l a r y
(99.998%), 120 mL/min.
Ambient, 20 - 25°C
26 cm x 1/4 in. o.d., 1 mm i.d.
Plasma observation hole .. 1 mm diameter, 12 cm from column
Microwave power output ... 60W forward, <1W reflected power
Observation height ......
-12 mm from cavity center
Light pipe dimensions .... 50 cm x 14 mm o.d., 5 mm i.d.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
different power settings.
Results for atomic hydrogen
emission at 4861 A and second order NH emission at 6720 A
are illustrated in Figures 12 and 13, respectively.
Atomic
emission appears to increase with applied microwave power
with maximum emission at 80 watts and a distance of 12 mm
from the center of the cavity.
A large amount of energy is
required to excite atomic hydrogen emission.
The profiles
for NH emission are similar except at high power levels,
where emission falls off drastically toward the center of
the cavity.
This may be due to the fact that conditions are
too energetic for the formation of the NH radical.
Wavelength Calibration
Wavelength calibration for the determination of metal
species is very straightforward due to the availability of
hollow cathode lamps.
Obtaining wavelength standards for
nonmetals or gases is a different problem.
As mentioned
previously, hydrogen emission lines could be obtained by
doping the argon carrier gas with hydrogen.
Unfortunately,
this approach resulted in contamination of the
chromatographic column.
After wavelength calibration, it
took two to three hours for the hydrogen signal to return to
a normal baseline.
An alternate approach was devised that
by-passed the chromatographic column completely.
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Figure 12
Detector Optimization -
(4861 A)
Grating
4000 A blaze
Slits
0.2 mm
Ar flow
120 mL/min.
Injections 0.2 mL x 16 ppt
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85
Figure 12
30
25
15
20W
Peak
Height,
mm
20
40 W
60 W
80 W
10
5
50
Distance from Cavity Center, mm
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 13
Detector Optimization. - H 2 (6720 A)
Grating
Slits
.
Ar flow
Injections
7500 A blaze
0.2 mm
120 mL/min.
0.2 mL x 98 ppm H 2
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87
Figure 13
30
20
20 W
60 W
80 W
Peak
Height, mm
25
IQ-
20
30
40
Distance from Cavity Center, mm
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50
88
An argon/trace hydrogen mixture was added through the
top of the plasma capillary tube.
The total flow rate
through the top closely matched the column flow rate.
This
method was possible only because of the observation port in
the plasma capillary.
Once the optimum wavelength was
found, the connection to the top of the plasma was removed
and analysis could begin immediately.
Calibration of Response
A calibration gas mixture was purchased from Matheson
(H77-252).
This standard was specified to be 98 ± 2 ppm
hydrogen gas in nitrogen.
The disposable cylinder was
equipped with a syringe adapter (H77-901) so that samples
could be transferred directly to the injection port.
To calibrate the detector, the 98 ppm hydrogen
calibration gas was injected in amounts varying from 0 to
0.2 mL.
Sharp, reproducible peaks were obtained, as shown
in Figure 14.
Breath concentrations based on the
calibration curve ranged from 0 to 10 ppm hydrogen for a 2
mL injection.
A representative calibration curve for
hydrogen at 3360 A is illustrated in Figure 15.
Peak height was used as a measure of response.
The use
of variable injection volumes and peak height measurement
was considered valid because of minimal peak broadening.
This assumption was tested by preparing a range of dilutions
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 14
Hydrogen Calibration Peaks
Wavelength
Concentration
Slits
Ar flow
Observation ht.
Power
6720 A
98 ppm H,
0.2 mm
120 mL/min.
-12 mm
60 watts
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
14
Figure
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 15
Hydrogen Calibration Curve
Wavelength
6720 A
Correlation, r .9997
Slope, m
2.8
Intercept, b
-2.2
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92
Figure 15
50
40“
-c
O)
*«
30-
20-
10-
0
2
6
4
M
,
8
ppm by volume
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10
93
of the 98 ppm hydrogen standard.
The corresponding
calibration using variable concentrations was found to be
identical to using variable injection volumes.
The only
difference seemed to be the larger error associated with
preparing dilutions.
Evaluation of Hydrogen Detection
The primary requirement for the detection of hydrogen
eluting from the chromatographic column was adequate
sensitivity.
Since breath hydrogen concentrations range
from 1 to 200 ppm, sensitivity and linearity over this range
were essential.
Selective detection for hydrogen was
actually unnecessary because it was separated
chromatographically.
Increased selectivity for hydrogen
over other breath components would simplify the
chromatogram, however, and ease separation requirements.
Since changes in hydrogen excretion were measured
relative to baseline levels in the hydrogen breath test,
precision was more important than absolute accuracy.
Finally, a rapid detection method was desired for fast
sample turn-around.
Sensitivity and Detection Limit
One of the main purposes of developing the GC-MES
method for the determination of hydrogen was to improve on
the sensitivity of existing methods.
The parameters that
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94
had the greatest effect on sensitivity were wavelength
setting accuracy and observation height in the plasma.
Other parameters which improved sensitivity to a smaller
extent included decreasing carrier gas flow rate, increasing
microwave power, and opening the slits.
Slit width was
limited by the amount of background plasma emission.
One of the major reasons for observing NH emission was
to improve sensitivity.
Sensitivity for this molecular
emission was found to be approximately one order of
magnitude better than atomic emission at 4861 A.
Table 4 is
a comparison between analytical results for hydrogen in
breath at 4861 A vs. 6720 A.
Note that the slope (m) of the
calibration curve is much greater for the molecular NH
emission.
Results for the two wavelengths are identical
within experimental error.
atomic hydrogen is 13.6 eV.
The ionization potential for
This is greater than the argon
metastable levels at 11.49 and 11.66 eV.
Due to the fact
that atomic hydrogen emission was observed in the argon
plasma at all, a mechanism other than the one involving
ionization by argon metastables must be possible.
However,
the high ionization potential could explain why atomic
hydrogen emission was not as intense as the NH emission.
Increasing the slit widths to improve sensitivity was much
more beneficial when observing NH.
Because of the inherent
band structure associated with molecular emission, slits
could be opened up to increase the bandpass.
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95
Table
4
Comparison Between
Analytical Lines for
Hydrogen
Calibration data:
4861 A
6720 A
.9924
.9969
slope, m
0.5
2.9
intercept, b
1.5
-0.6
correlation, r
Breath sample concentration (ppm)
Sample 1
5 ± 4
7.3 ± .5
2
10 ± 4
11.0 ± .5
3
14 ± 4
13.8 ± .5
4
18 ± 4
17.5 ± .5
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96
Another method of increasing detection was to increase
the volume of sample injected.
The major limitation to the
injection volume size was peak broadening.
This would have
been a problem since quantitation was based on peak height.
Small amounts of helium were added to the argon carrier
gas in another attempt to increase sensitivity.
For argon
containing approximately 10% helium, large, positive
interferences were observed when air, nitrogen or argon
samples were injected.
In these cases, the plasma length
increased at the hydrogen retention time.
The signal,
therefore, was probably due to an increase in background as
the plasma expanded and contracted.
The reason for this
behavior specifically at the hydrogen retention time could
not be explained.
Since this phenomenon was observed only
when helium was added, pure argon carrier gas was used
thereafter.
Detector sensitivity was specified by determining the
minimum detectable level (MDL).
This is the "level" of
sample in the detector at the peak maximum, when the signal
to noise ratio is 2 (166).
For the determination of
hydrogen at 3360 A, the MDL was found to be 15 pg
hydrogen/second.
This specification can be used for
comparison to other GC detectors because it is independent
of column parameters and sample size.
The absolute
detection limit for hydrogen was found to be 0.1 ng.
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97
Hydrogen concentrations in this text are given as-ppm
hydrogen by volume in air (v/v).
These concentrations would
be approximately one order of magnitude lower if presented
as the weight of hydrogen per volume of air (w/v).
Selectivity
Observation of characteristic emission results in
excellent selectivity.
’When molecular NH emission was
monitored, however, a positive response was observed for
nitrogen as well as for hydrogen.
This "spectral
interference" was due to the presence of an intense
molecular nitrogen bandhead at 3371 A.
Better selectivity
was possible when observing second order NH emission at 6720
A due to increased dispersion.
The response to nitrogen did not result in a positive
interference for hydrogen determination because of the
chromatographic separation.
Hydrogen response was observed
and recorded before* the nitrogen peak eluted from the
column.
The only consequences of this response to nitrogen
were increased analysis time and slightly elevated
background emission.
Since breath samples are mainly
composed of nitrogen, a very large peak was observed.
It
took approximately five minutes for the signal to return to
a normal baseline.
At this time another sample could be
injected.
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98
Greater selectivity was observed at the 4861 A atomic
hydrogen line.
t
This was due to the absence of nitrogen band
emission in this region.
The nitrogen did, however,
extinguish the plasma for a short period of time.
After
approximately two to three minutes, the plasma was
re-ignited and the next sample could be run.
Hence, by
observing atomic hydrogen, analysis time could be cut in
half at the expense of sensitivity.
Accuracy
An analytical accuracy of 10% was desired for this
method.
Absolute measurements such as ambient hydrogen
levels or baseline human breath levels could then be
compared with literature values.
To monitor the accuracy of
the hydrogen determinations, a quality assurance (QA)
standard was run periodically as an unknown.
The QA sample was prepared in a 2 liter volumetric
flask.
A rubber stopper was fit with a septum and length of
Tygon® tubing which could be clamped.
The total volume of
the system with the stopper and clamp in place was
calculated to be 2.06 liters.
The flask was evacuated and
flushed with nitrogen three times.
Atmospheric pressure was
approximated by opening the clamp and slowly releasing
nitrogen until leakage stopped.
Injecting 0.1 mL of
hydrogen gas into the flask resulted in a calculated
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99
concentration of 49 ± 1 ppm hydrogen.
Error propagation
revealed that uncertainty in this calculation was almost
entirely due to the inaccuracy of the hydrogen injection
volume.
The QA sample was treated as an unknown and was
analyzed along with each sample set.
Using injections of
the 98 ppm standard for calibration, an average
concentration of 50 ppm was determined for the QA sample.
This represented a relative deviation of 2.5% from the
calculated value.
Linear Range
To determine the linear range of detector response,
hydrogen standards of various concentrations were prepared.
To achieve a complete range of absolute levels, injection
volumes were also varied.
Since the range of hydrogen
levels extended over four orders of magnitude, electrometer
and recorder settings had to be adjusted to keep the
response on scale.
Correction factors were required to
account for nonlinearity in these settings.
The linearity
of hydrogen determination at the second order NH emission
band is illustrated in Figure 16.
The linear range extended
over approximately two orders of magnitude.
Using 2 mL
injection volumes, this range represented breath hydrogen
concentrations from 0.5 to 50 ppm.
The linear range for
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100
atomic hydrogen emission at 4861 A ranged from 10 to 1000
ppm.
Reproducibility
The precision of the analytical determination of
hydrogen was actually more important than accuracy.
By
definition, the hydrogen breath test monitors the increase
in concentration above baseline, a relative measurement.
A
reproducibility of 10% was desired in these studies.
The short term precision was determined by injecting
replicate amounts of the 98 ppm calibration standard.
chromatograms are illustrated in Figure 17.
These
Plasma
instability and electronic noise contributed to the 3%
relative standard deviation (RSD) of the analytical method.
Results were not expected to be as precise for larger sample
injections and smaller concentrations.
Long term or day-to-day reproducibility was determined
by comparing results for the quality assurance standard.
The average concentration was found to be 50 ± 3 ppm
hydrogen, which represented a relative error of 6%.
Short-term reproducibility for the quality assurance samples
averaged 1.5% RSD.
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101
Figure 16
Hydrogen Linear Range
Wavelength
Slits
Ar flow
Observation ht.
Power
6720 A
0.2 nun
120 mL/min.
-12 mm
60 watts
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102
r -rr
vo
Log
» CN
H 2 Concentration,
nl
■ cn
CN
uiui 'esuodssy 601
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103
Figure 17
Reproducibility of Standard Injections
Wavelength
Slits
Ar flow
Observation ht.
Power
6720 A
0.2 mm
120 mL/min.
-12 mm
60 watts
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
ZF
A
7
CN
Figure
17
Nf
CO
a*
CN
NT
CO
CO
O.
y
O'
J
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105
Hydrogen Breath Test
Procedure
Subjects were required to fast overnight before the
test.
Breath samples were taken initially to determine
baseline hydrogen levels.
Lactose was then administered as
a suspension in 100 mL of water.
0.6 g lactose per kg body weight.
The dose was measured at
Duplicate breath samples
were taken at half-hour intervals for 3 to 4 hours.
Samples
were analyzed the same day.
Breath Collection
The method used in collecting a breath sample depends
on the sensitivity of the analytical method.
Collection
methods were also evaluated with respect to reproducibility
and degree of difficulty for the subject.
Previous
investigators used a "rebreathing" technique which
concentrated hydrogen levels in the breath sample (136).
This method involves breathing the same volume of air for a
period of 2 to 4 minutes.
All of the hydrogen excreted
during this time is concentrated in a volume of air equal to
the sum of the subject's lung volume and the volume of the
collection system.
Rebreathing for 2 minutes resulted in consistent breath
hydrogen levels but required an extreme effort on the part
of the subject.
It was decided that many multiple sclerosis
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106
(MS) patients would not be able to undergo this type of
testing because of their condition.
As detection limits for
the determination of hydrogen were improved, concentration
of the breath hydrogen was unnecessary.
In a second collection method, the subject was
instructed'to breathe deeply four times to clear any
residual hydrogen from the lungs.'
After the fourth breath
was used to flush out the collection bag, the sample was
obtained.
Using this hyperventilation technique, a group of
MS patients was tested and showed very little response to
the hydrogen breath test (HBT).
In a third breathing method, the lungs were never
cleared out by hyperventilation.
The subject exhaled
normally, after which the residual lung volume was collected
with a maximum exhalation effort.
This breath collection
technique has been described in the literature as an
end-expiratory sample(152).
Hydrogen concentrations were
expected to be higher in the residual lung volume.
The final breath collection mebhod was similar to the
interval sampling techniques described in the literature
(144).
A normal exhalation was collected without
rebreathing, hyperventilating or clearing the residual lung
volume.
The obvious advantage of this method was that it
required no special effort on the part of the subject.
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107
Collection Apparatus
Three liter breath collection bags were constructed of
Mylar®-coated foil.
Samples were collected by breathing
through a short length of Tygon® tubing attached to the bag.
These bags were designed and supplied by Dr. D. H. Calloway,
Department of Nutrition, University of California at
Berkeley.
Samples containing trace amounts of hydrogen were
shown to be stable in these bags for 47 days (142).
During the HBT, approximately twenty samples were
collected from each subject.
For convenience in transport
and storage, samples were transferred to 50 mL syringes.
These syringes were lubricated with ethylene glycol,
rendering them air-tight.
The sample was drawn into the
syringe through a septum arrangement after the Tygon® tubing
had been clamped off.
Between sample collections, the bags were flushed twice
with dry nitrogen to remove residual hydrogen and moisture.
The efficiency of this flushing procedure was determined for
a bag which originally contained 80 ppm hydrogen.
The
second nitrogen flush was stored in the collection bag for
10 minutes.
The resulting hydrogen concentration was below
the 0.5 ppm detection limit.
Because of the large moisture content of human breath,
its effect on hydrogen concetration was studied.
A small
filter containing absorbent paper was used in this study.
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108
The filter (DuPont P101) was originally used to protect
personal air sampling pumps from dust and moisture.
After
the absorbent paper was saturated with moisture, the 98 ppm
calibration standard was drawn through the filter and
injected on the column several times.
Response was compared
to samples taken directly from the calibration gas cylinder.
Response for the "dry" samples was found to be 43 ± 1
mm while "moist" samples averaged 43 ± 3 mm.
equivalent within experimental error.
Results were
The greater standard
deviation observed for the "moist" samples was possibly due
to the effect of moisture on the chromatographic column.
Sample Storage
Sample storage in the 50 mL gas-tight syringes was
found to be both convenient and stable.
When the syringes
were stored in a rack, tip down, a positive pressure was
placed on the sample.
out of the syringe.
Any sample leakage would therefore be
The ethylene glycol lubricant did not
interfere with either the storage or determination step.
The stability of breath samples stored in the syringes over
a period of 18 days is presented in Table 5.
When breath sampling was performed during the hot
summer months, samples were transported under high
temperature conditions.
The effect of high temperature on
sample storage stability was investigated.
Dilute hydrogen
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109
Table
5
Storage Stability for Breath Samples
Storage
Hydrogen concentration (ppm)
Breath sample 1
(days)
Breath sample 2
0.3
4.7 ± 0.8
5.0 ± 0.8
1.0
5.9 ± 0.8
5.6 ± 0.6
1.4
5.1 ± 0
5.0 ± 0.9
6.0
5.8 ± 0
5.8 ± 0
11.0
6.0 ± 0.5
5.6 + 0.7
14.0
5.7 ± 0.5
5.5 ± 0.5
18.0
4.3 ± 0.3
5.1 ± 0
Average 5.4 ± 0.7 ppm
5.4 ± 0.3 ppm
RSD
6%
13%
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110
samples were prepared and distributed between two sets of
syringes.
One set was placed in an oven at 42°C.
The
remaining set, initially identical in concentration, was
stored at a room temperature of approximately 27 °C.
The
results of this study for a one week storage period are
presented in Table 6.
No significant differences were found
between the two sets of samples as long as they were allowed
to cool to room temperature before injection.
Breath Test Applications
Lactose Studies
.The hydrogen breath test (HBT) was used to diagnose
lactose malabsorption in a group of subjects with multiple
sclerosis (MS).
This portion of the study was initiated and
set up by three faculty members from the University of Iowa,
College of Nursing.
Toni Tripp Reimer, Laura Hart and
Mildred Freel authored the initial proposal to the Johnson
County MS Society (125).
These nurses took responsibility
for background research, recruiting and scheduling subjects,
as well as collecting the samples.
Breath collection and
analytical methodologies were developed as part of this
doctoral research.
Ambient Hydrogen Levels
Each time a new set of tests were run, air was
collected from the room to determine background hydrogen
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Ill
Table
6
Storage Stability vs. Temperature
Time
27°C
42 °C
Sample 2
27°C
42 °C
Sample 3
27°C
42 °C
0
1.1
1.1
2.6
2.6
5. 9
5.9
6
1 .6
1.5
2.4
2.8
6.5
6.6
20
1.3
1.3
to
•
o
(hours)
Sample 1
2.8
6.1
6.0
168
1.7
1.1
2.8
2.1
5.4
5.7
AVE ± SD
1.2 ± 0.3
2.5 ± 0.3
6.0 ± 0.4
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112
levels.
The results for room hydrogen concentrations are
presented in Table 7.
to be 1.8 ± .5 ppm.
The average concentration was found
These hydrogen levels varied less than
0.4 ppm during a single test period.
Outdoor air was found to contain less hydrogen.
average concentration of 0.8 ppm was measured.
An
This value
compares favorably with values reported in the literature
for atmospheric hydrogen concentration (167,168).
The
difference between indoor and outdoor hydrogen levels could
have been due to experimental error.
Rebreathing vs. Hyperventilation
Table 8 is a comparison of HBT results (ppm hydrogen)
between rebreathing and hyperventilation breath collection
methods.
Subject 1, the normal absorber, was actually
classified as a malabsorber when rebreathing was used.
Since breath hydrogen was concentrated by this method, the
20 ppm increase criterion for positive diagnosis was
invalid.
Hyperventilation resulted in breath hydrogen
*
levels proportional to rebreathing, but much lower.
Notice
that peak hydrogen excretion occurred between 1.5 and 2
hours for subject 1.
Again, levels obtained with rebreathing were much
higher.
There were two differences between the HBT's for
subjects 1 and 2.
The malabsorber excreted larger amounts
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113
Table
7
Ambient Hydrogen Levels
Date
Room Hydrogen Concentration (ppm)
6/24
2.5
3.8
1.8
1.5
1.8
6/25
1.7
1.6
1.-9
1.8
1.8
2.3
6/26
2.3
1.7
1.3
1.3
1.5
1.7
6/27
1.7
2.9
1.8
1.5
1.5
6/30
1.5
1.5
1.8
1.8
7/7
2.2
2.0
2.3
2.7
10/2
1.0
1.2
1.9
1.2
10/21
1.4
1.3
1.4
1.4
10/22
1.6
1.7
1.5
1.4
11/20
1.2
1.2
2.3
1.1
2.3
2.5
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114
Table
8
Comparison between
Rebreathing and
Hyperventi1ati on
Hyperventilation
Subject 1*
Subject 2**
47
26
2
7
0.5
139
20
10
5
1.0
170
-
15
4
1.5
171
43
16
13
to
•
o
(hours)
Rebreathing
170
130
18
29
to
U1
Time
102
125
10
39
3.0
59
290
7
67
B1
Subject 1
Subject 2
* Subject 1 was a normal absorber
♦♦Subject 2 was known to have lactose malabsorption and MS
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115
of hydrogen, as expected, and complained of some discomfort.
The unexpected observation involved the peak time of
hydrogen excretion.
The breath hydrogen concentration for
subject 2 was still increasing after 3 hours instead of
reaching a maximum at 1.5 to 2 hours.
Lactose Malabsorption in MS Patients
It was decided that the hyperventilation technique
could be used to distinguish between lactose absorbers and
nonabsorbers.
tested.
An initial group of ten MS patients was
HBT results are presented in Table 9.
subjects were shown to be lactose malabsorbers.
None of the
It was also
significant that none of the subjects complained of
discomfort during the testing period.
Residual Lung Volume
At this time the residual lung volume collection
technique was developed to concentrate the breath hydrogen
somewhat.
Results from Table 10 show that this technique is
also capable of distinguishing between lactose absorbers and
nonabsorbers.
However, there were still no conclusive
results for MS subjects.
The hyperventilation and residual
lung volume techniques could be compared since subject 2 in
Tables 8 and 9 was the same person.
Breath hydrogen
concentration in the residual lung volume was significantly
higher than for hyperventilation.
It is also important to
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116
Table
9
Hyperventilation Collection Method
Subjects*
4
5
1
2
3
7
8
9
10
B1
4
3
5
4
2
2
5
10
6
2
B2
4
4
5
3
2
3
4
8
4
2
0.5
3
6
'5
4
2
2
4
6
4
2
1.0
2
5
4
5
2
2
5
6
4
2
1.5
3
4
4
5
2
2
6
5
3
3
2.0
4
5
4
6
2
2
6
4
3
3
2.5
7
4
4
6
1
2
5
3
5
3
12
4
4
6
2
2
3
3
5
2
3.5
-
-
4
5
2
-..
-
4
-
3
5
5
2
CO
6
o
Time
(hours)
4.0
* All subjects were MS patients
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3
117
note that the hydrogen excretion did not follow exactly the
same pattern for subject 2 in these two separate tests.
In
both cases, however, hydrogen levels were still increasing
at 3 hours.
Reproducibility of Breath Sampling
Breath sample reproducibility was monitored for the
tests using the hyperventilation collection method.
The
relative standard deviation (RSD) between duplicate
determinations of the same sample averaged 7.8%.
These
results are compared to errors associated with the
analytical method in Table 11.
The increased error associated with the breath samples
was due mainly to the increased injection volume required
for low concentrations.
It was much more difficult to
consistently inject a volume as large as 2 mL.
Since peak
height was used for quantitation, slight peak broadening
might have contributed to the loss in precision.
Finally,
the moisture present in breath samples also could have led
to some instability, with respect to column performance.
Relative standard deviation between different breath
samples collected within minutes of each other was 8.4%.
This additional loss of precision compared to the duplicate
determinations was minimal.
This seemed to indicate that
the hyperventilation technique consistently collected
representative samples.
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118
Table
10
Residual Lung Volume
Time
(hours)
Bl
2
Subjects*
3
4
5
6
16
3
2
4
2
2
-
2
4
-
-
-
0.5
11
4
2
6
3
2
1.0
3
21
2
6
2
2
1.5
5
22
3
6
2
2
to
•
o
B2
1
5
22
2
4
2
2
2.5
.4
28
2
4
2
2
3.0
3
37
2
3
2
2
♦Subject 1 was a normal absorber
Subject 2 was known to have lactose malabsorption and MS
Subjects 3-6 were MS patients
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119
Table
11
Reproducibility of
Hydroden Determinations
Standard hydrogen mixture (98 ppm)
Injection volume
0.2 mL
Number of determinations
10
Relative standard deviation (RSD)
2.8%
Breath samples (average 4 ppm)
Injection volume
2.0 mL
Number of samples
240
RSD (same sample)*
7.8%
RSD (duplicate samples)**
8.4%
* duplicate determinations of the same sample
** deviation between duplicate breath samples
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120
Lactulose Study
Lactulose, 4-0-g-D-galactopyranosyl-D-fructofuranose,
is an isomerization product of lactose.
Humans do.not have
an enzyme system capable of hydrolyzing lactulose into its
monosaccharide components (131).
Since it is very poorly
absorbed, some investigators have used it as a standard to
which partial absorption of other sugars is compared
(134,149).
In this investigation, lactulose was
administered to a normal subject.
Results of the HBT
simulated the effect that lactose would have on a milk
intolerant subject(130).
The object of this study was to
test the performance of two breath collection procedures as
well as the analytical methodology.
Lactulose was administered orally to a normal absorber.
Cephulac® is a commercial laxative containing 10 g lactulose
per 15 mL.
The dose was 0.3 g lactulose per kg body weight.
After a period of approximately one hour, the laxative
produced belching and flatulence in the subject.
Abdominal
cramps were'absent, however.
An example of a "typical” breath chromatogram is
illustrated in Figure 18.
The chromatogram is typical in
the sense that retention times are shown, with oxygen
extinguishing the plasma, and nitrogen response eventually
returning to the baseline after re-ignition.
The
concentration of hydrogen in this chromatogram was very
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121
high.
Since such'a small injection volume could be used,
the nitrogen peak returns to the baseline fairly quickly.
For a 2 mL breath injection, the nitrogen peak would have
been much larger.
Figure 19 illustrates the HBT results for the lactulose
study.
Note that baseline hydrogen levels were-
approximately 3 ppm.
Maximum hydrogen excretion occurred
1.5 to 2 hours after the lactulose was administered and then
fell off gradually.
Hydrogen concentrations were larger in the residual
lung volume.
If the results of the HBT were interpreted in
terms of a relative increase in hydrogen concentration,
these two breath sampling procedures could give very
different results.
Another important difference found between the two
breath collection techniques was the reproducibility.
Collection of the residual lung volume seemed to be
difficult for the subject to reproduce.
Any change in the
exhalation effort would result in a different breath
hydrogen concentration.
In contrast, collecting a
reproducible normal interval breath sample was very easy.
Method Validation
To insure that results were consistent with those
obtained by a universally accepted method, a collaborative
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122
Figure 18
Breath Sample Chromatogram
Wavelength
Slits
Ar flow
Observation ht.
Power
6720 A
0.2 mm
120 mL/min.
-12 mm
60 watts
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Figure 18
CO
vo
3 minutes
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124
Figure 19
Lactulose Study - HBT
(dose = 0 . 3 g/kg body weight)
Wavelength
Slits
Ar flow
Observation ht.
Power
Key
6720 A
0.2 nun
120 mL/min.
-12 mm
60 watts
• Residual lung volume collection
O Normal interval collection
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125
Figure 19
70
60
<D
E
“I> 50
x
_Q
E
40
CL
CL
M
20
10-
2
0
3
Hours
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4
126
study was set up.
A hydrogen breath test was run and
duplicate samples were divided into separate sets.
As shown
in the section on reproducibility, very little error was
associated with the breath collection procedure.
Because of
this collection consistency, hydrogen concentrations should
have been nearly identical in the two sets of samples.
One set was analyzed by GC-MES using the same
conditions as previously described.
The second set was sent
to Mr. Kabir Younoszai at the University of Iowa Hospital,
Department of Pediatrics, for analysis by GC with thermal
conductivity detection.
compared in Table 12.
Results for the two methods are
Note that measured concentrations
were identical for the two methods when experimental error
is considered.
The average relative error between the two
methods was only 2%.
Conclusions
The MES system proved to be a sensitive method for the
determination of sub-ppm levels of hydrogen in breath.
»
Improved detection limits were obtained by observing
molecular emission from the NH radical.
The linear range
was limited to about two orders of magnitude but was well
suited for breath hydrogen concentrations ranging from 1 to
100 ppm.
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Table
12
Validation of Hydrogen Method
GC - TC
GC - MES
Sample [H2 1,ppm
Sample [h 2 ],ppm
Mean ± S.D. Rel. Error
1A
10
IB
9 ± 1
10 ± 1
7%
2A
7
2B
7 ± 1
7 ± 1
0%
3A
14
3B
14 ± 1
14 ± 0
0%
4A
14
4B
14 ± 1
14 ± 0
0%
5A
11
5B
11 ± 1
11 ± 0
0%
6A
17
6B
17 ± 1
17 + 0
0%
7A
25
7B
26 ± 1
26 ± 1
3%
8A
17
8B
18 ± 1
18 ± 1
4%
AVERAGE 2%
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128
The determination method was applied to hydrogen breath
tests for lactose malabsorption.
This study failed to show
any correlation between lactose malabsorption and multiple
sclerosis as proposed.
The most significant finding was
that a portion of the multiple sclerosis patients showed a
delay in peak hydrogen excretion.
This could explain the
negative test results since breath hydrogen was not
monitored for more than four hours.
Further investigations on the digestive transit time of
multiple sclerosis patients is called for.
Hydrogen levels
should be monitored until peak excretion is is observed.
Even though the hydrogen breath test is simple and
non-invasive, longer studies would probably require
in-patient clinical studies.
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PART B
ANALYTICAL APPLICATIONS OF
GC-MES BELOW 2000 A
Preliminary Investigations
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130
CHAPTER IV
INTRODUCTION
The second part of this dissertation is devoted to
spectrochemical analysis in the vacuum ultraviolet (VUV)
region of the spectrum.
This region was not used
successfully for quantitative analysis until 1955 (169).
Relatively few investigations have been conducted below 2000
A, primarily due to the difficulties encountered in
observing emission in the VUV region (170).
The main reason for limited use of the VUV region has
been light absorption by atmospheric oxygen below 2000 A.
To circumvent this problem, oxygen is removed from the
optical path of. the spectrometer by evacuation or purging
with a nonabsorbing gas.
The reduced transmission of quartz
below 2000 A requires the use of alternate optical materials
such as magnesium flouride.
Finally, t h e 'availability of
detectors for VUV radiation is much more limited than in the
UV-visible region.
Several factors suggest that utilization of the VUV for
spectrochemical analysis would be very advantageous.
The
resonance lines of numerous elements (20%) are found in the
VUV region of the spectrum.
Greater sensitivity is often
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131
possible when observing these transitions as opposed to
lines occurring at longer wavelengths (171).
Determination
of elements such as Br, Cl, N, 0 and S in the UV-visible
region is actually very limited.
The absence of molecular
emission from atmospheric gases and carbon species in the
VUV greatly reduces background emission from excitation
sources.
Recent Analytical Applications.
Analytical investigations have been concerned mainly
with the development and characterization of new excitation
sources.
Ellenbracht et al. (120) observed sulfur resonance
lines from a dc arc for aqueous solution analysis.
The 0.5
ppm detection limit did not vary for different sulfur
species.
An argon purge system was used to reduce light
absorption by atmospheric oxygen.,
In a subsequent paper,
these authors used a dc discharge plasma as a sulfur
specific detector for gas chromatography (122).
This
high-temperature, high-energy plasma minimized solvent
quenching effects and was capable of handling large
injections without extinction.
A detection limit of 300
pg/second and a selectivity ratio of 103 were reported.
Dreher and Frank employed an inductively-coupled
radio-frequency emission source for VUV spectroscopy (172).
Kirkbright et al. (173,174) have investigated ICP excitation
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132
of S # P, I, As, Se and Hg at wavelengths down to 1700 A.
Detection limits for aqueous solutions were in the ppm
range.
Heine et al.
(170) reported preliminary work on the
use of the VUV region for GC-ICP.
This system was capable
of sensitive detection of oxygen and nitrogen emission,
unlike previous investigations in the UV-visible region
(124).
The only VUV observation involving GC-MES was reported
by Braun et al. (82).
Their goal was to obtain total atomic
content of organic compounds by measuring resonance and
non-resonance atomic transitions.
Since essentially
complete fragmentation could be realized in the reduced
pressure helium plasma, response was linear with atom
content in the molecules.
With this is mind, the authors
proposed that response calibration curves for a particular
element could be obtained for a stable and easily stored
compound and would be valid for any compound containing that
element.
Braun also reported that the choice of emission lines
could affect the linearity of the signal.
Emission from
lines terminating in the ground state could be strongly
reversed due to self-absorption by ground state atoms.
This
problem, however, could have been due to the fact that the
discharge was 10 mm in diameter and was viewed in an end-on
configuration.
Resonance lines for oxygen (1303 A) and
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1 33
hydrogen (1216 A) could not be observed because of the
background emission due to impurities in the carrier gas.
Purpose of Study
The purpose of the following investigation was to
capitalize on the sensitivity and selectivity possible by
observing the VUV region of the spectrum.
In combination
with the GC-MES system previously described in this
dissertation,
increased.
sensitivity and selectivity could be further
Previous investigators have not attempted to
optimize MIP detection in this region.
In this study, detection has been optimized for both
iodine and sulfur emission from organic compounds.
Resonance transitions for these two lines have been
investigated.
In addition to this preliminary work,
qualitative results for bromine, carbon, iodine, nitrogen,
phosphorus and sulfur were obtained.
These results are
presented as emission spectra in Appendices C - G.
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134
CHAPTER V
EXPERIMENTAL
The vacuum spectrometer described in this work had not
been used for approximately eight years.
Attempts to
produce a hard vacuum were unsuccessful.
Since the leaks
could not be identified, an alternate approach to removing
oxygen from the system was investigated.
Purging the Optical System
Connections were made to the spectrometer to purge the
optical system with an inert gas.
The port at the top of
the spectrometer, originally used for the cold-cathode
vacuum gauge, served as an inlet for the purge gas.
The
diffusion pump was removed and replaced with an aluminum
plate.
A 1/4" Swagelock® connection attached to this plate
was used as an outlet for constant flow purging.
Gas flow
both in and out of the spectrometer were controlled with
rotameters.
In order to remove oxygen from the photomultiplier tube
(PMT) mount, the exit window was removed from the
spectrometer.
Vacuum tubing was clamped to the port
extending from the bottom of the PMT mount.
The
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135
spectrometer was evacated by attaching this tubing to a
mechanical vacuum pump.
When not in use, the vacuum tubing
was plugged with a rubber stopper.
This stopper served as a
safety release when gauge pressures exceeded approximately
ten pounds per square inch (psig).
Spectrometer purge gas
pressure was measured by. inserting a gas chromatographic
inlet pressure gauge directly into the vacuum tubing.
Oxygen also had to be removed from the light pipe
optical path.
Tubing was attached to the mount positioning
the light ..pipe at the entrance slit.
Gas flow through the
light pipe was controlled with a rotameter.
Detection
An EMI Gencom (G-26E315) solar blind PMT and its
side-on housing (B-215FV) were attached to the exit slit
mount.
The maximum response of the Csl photocathode at 1300
A falls off sharply at 1100 A.
Toward longer wavelengths,
response decreases by one order of magnitude at 1800 A and
two orders at 2000 A.
High voltages of 2 to 3 kV were required for operation.
Under certain vacuum conditions, an arc discharge between
the photocathode and the nearest conductor outside the tube
was possible.
This is not a problem when the tube is used
under hard vacuum conditions as designed.
The breakdown
voltage where arcing occurs depends on Paschen's Law.
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There
136
is a range in the pressure-distance curve where breakdown
voltage is at a minimum.
This minimum was found to
correspond to pressures of approximately 0.01 mm to 1000 mm
of Hg outside the tube.
When attempting to operate the PMT under mechanical
pump vacuum, violent arcing was observed.
In the purging
studies approximating atmospheric pressure, small amplitude
"spikes" were recorded.'
Since these small discharges
occurred up to ten times a minute, the use of a sensitive
v
electrometer setting was not possible.
The arcing problem was solved by pressurizing the
spectrometer with the purge gas.
At pressures greater than
five psig, discharging was not observed.
A flow rate of
approximately 100 mL/min He into the spectrometer was
necessary to maintain this pressure..
The pressure did not
build up due to leaks in the system.
It.was possible to reduce dark current noise by cooling
the PMT housing.
A cold-finger type arrangement was set up
by wrapping copper foil around the housing.
The end of the
copper foil extended down into a Dewar of liquid nitrogen.
By insulating the copper foil, the temperature of the PMT
housing could be reduced to approximately 0°C.
A
substantial decrease in noise was observed at an
electrometer setting of 1x10 "9 Amperes.
Cooling was
unnecessary for attenuations of 1x10 "• amperes and above.
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137
CHAPTER VI
RESULTS AND DISCUSSION
Oxygen Removal
Two gases were studied with respect to purging
efficiency.
Nitrogen was thought to be transparent down to
approximately 1500 A.
The spectrometer was evacuated and
then filled with nitrogen.
When monitoring the 1849 A line
from a mercury source, response did increase while purging
with nitrogen.
The spectrometer was pressurized with
nitrogen up to a few psig and response at 1849 A decreased.
This was probably due to oxygen impurities in the nitrogen.
Argon background emission was scanned between 0 and
2000 A.
The only emission observed was a weak band
extending from approximately 1850 to 1900 A.
Response was
not observed at wavelengths below 1850 A.
Better results were obtained when purging the optical
system with helium gas.
The spectrometer was first
evacuated, then pressurized with helium.
The process of
releasing the helium and subsequent pressurization was
repeated several times.
Additional evacuation would allow
room air to leak into the system.
Pressurization of the
spectrometer with helium did not seem to reduce transmission
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138
of the mercury 1849 A line.
This was probably due to the
low oxygen content of the helium, specified to be less than
1 ppm.
The argon background emission spectrum was observed
below 2000 A.
The band emission observed previously between
1850 and 1900 A was absent.
Atomic emission lines were
observed at 1657 A, 1740 A and 1931 A.
The 1657 and 1931 A
lines were due to atomic carbon emission, probably from
carbon deposits on the inner wall of the plasma capillary.
Carbon emission could also have been due to slight bleeding
of the organic stationary phase.
The plasma emission line at 1740 A was due to several
atomic nitrogen lines between 1740 and 1745 A.
Apparently,
the plasma was able to fragment molecular nitrogen
impurities in the argon carrier gas.
This was not expected
due to the high bond dissociation energy for nitrogen (226
kcal/mole).
A positive response was observed for small
injections of atmospheric nitrogen at this wavelength.
Detection of Iodine Emission
Wavelength Calibration
In order to calibrate the wavelength setting for atomic
iodine emission, 1-iodobutane was added to argon gas flowing
through the top of the plasma capillary.
The apparatus used
for this purpose is illustrated in Figure 20.
This method
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139
for doping the plasma was useful for volatile compounds.
The amount diffusing into the plasma could be controlled by
varying the argon flow or the temperature of the sample
tube.
Screw top test tubes were used to store a variety of
sample compounds.
These could be attached for wavelength
calibration and then easily removed.
The most prominent iodine emission line in the VUV
region was the atomic resonance line at 1830 A.
Many other
intense iodine lines were observed in the region between
1600 A and 2100 A.
Wavelengths and relative intensities of
the observed iodine transitions are listed in Table 13.
Method Development
Detector Optimization
Detector response at 1830 A for 2 yL injections of 500
ppm (w/v) 1-iodopentane was observed at various positions
along the length of the plasma.
Results for this
observation height optimization at various power settings is
illustrated in Figure 21.
Response for this ground state
transition fell off drastically toward the center of the
cavity at 80 watts.
This was probably due to the increased
population of upper level excited states not involved in
this transition.
The most interesting result when optimizing height was
the appearance of spikes in the response curves when
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140
Figure 20
Plasma Doping Apparatus
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•141
Figure 20
.Argon
Sample
Tube
Heating
Tape
Plasma Observation
Port
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142
Table
13
Observed Iodine Transitions
X, A
Levels, 1/cm
I
*
theoretical
1observed
1640.8
7603
-
68550
2500
1642.1
0
-
60896
2000
1702.1
7603
-
66355
15000
7
1782.8
0
-
56093
12000
>100
1799.1
7603
-
63187
5000
'>100
1830.4
0
-
54633
75000
>100
1844.4
7603
-
61820
15000
91
1876.4
7603
-
60896
2000
38
2062.4
7603
56093
M
L
J
>100
* Intensity values were obtained from reference 175.
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143
Figure 21
Detector Optimization - Iodine (1830 A)
Grating
Slits
Ar flow
Injections
1900 A blaze
0.2 nun
100 mL/min.
2yL x 500 ppm
1-iodopentane
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144
Figure 21
20n
60 w
40 w
15-
E
E
20 W
05
’a>
X
10-
o
4)
Q_
80 W
5-
i
10
20
30
40
Distance from C avity Center, mm
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“ 1
50
145
observing the bottom tip of the plasma.
This effect was
very similar to the increased hydrogen response when helium
was added to argon carrier gas.
As a foreign species eluted
from the column, the plasma contracted in length.
When
observing the lower portion of the plasma, it contacted to a
point where the tip was even with the observation port.
In
this case, the plasma formed an "L" shape with the tip
extending out of the observation port.
Characteristic
emission increased somewhat, but the majority of the
emission was due to increased background.
To support this theory, response was measured at a
wavelength setting of 1820 A.
The only response for
1-iodopentane was observed at the bottom tip of the plasma.
In this region, response was observed as the plasma
contracted.
The background effect was much greater when
helium was flowing through the light- pipe.
The plasma plume
normally extending from the observation port was minimized
by large helium flow rates.
As a result, when the plasma
contracted to form the "L" shape into the port, the
observation volume increased by a factor of 3.
Observation
\.
of this increased emission at the lower end of the plasma
was not analytically useful.
Power and height optimization results for iodine
emission at 1844 A are illustrated in Figure 22.
Emission
intensity increased towards the center of the plasma.
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146
Response also increased directly with applied power.
Comparison of the power and height curves for iodine
emission at 1830 A and 1844 A illustrates why these
parameters must be optimized for each specific wavelength.
The upper level, 1844 A iodine transition requires a higher
excitation energy than the ground state transition.
Separation of Organoiodine Compounds
The chromatographic column dimensions were the same as
those used in the hydrogen studies(6' x 6 mm o.d.).
The
column was packed with 5% OV-1 methyl silicone on 80/100
mesh acid washed Chromosorb W.
Table 14 is a summary of the
optimized conditions used for the separation and detection
of organoiodides.
Figure 23 is a chromatogram showing the
separation of iodomethane, 1-iodobutane, 1-iodopentane and
l-iodo-3-methylbutane.
The mixture was prepared by sampling
equal volumes of the vapor above the pure compounds.
final injection volume was 0.2 mL.
The
Retention times were
directly proportional to boiling point.
Sensitivity and Detection Limit
Sensitivity for iodine at 1830 A was determined by
preparing a dilute standard of 1-iodopentane in petroleum
ether.
This standard contained 1 ng C ^ H ^ I per yL solvent
or 1 ppm (w/v).
The analytical conditions were identical to
those previously specified.
The minimum detectable limit
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147
Figure 22
Detector Optimization - Iodine (1844 A)
Grating
Slits
Ar flow
Injections
1900 A blaze
0.2 mm
100 mL/min.
2 yl x 500 ppm
1-iodopentane
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148
Figure 22
8n
80 w
60 W
Peak Height
mm
6-1
5-1
4H
20 W
3H
21
o
10
20
T "
T "
30
40
Distance from Cavity Center, mm
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— I
50
149
Table
14
Separation of Organoiodides
GC Conditions
Column dimensions
6' x 6mm o.d.
Column packing
5% OV-1 on 80/100
Chromosorb W (AW)
Argon flow rate
100 ml/min.
Column temperature
130°C
Inlet temperature
165°C
Detector temperature
195°C
Detector Conditions
Observation height
-14 mm
Applied power
60 watts
Slit width
0.2 mm
Wavelength
1830 A
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150
Figure 23
Separation of- .Organo iodides
Key: a)
b)
c)
d)
Iodomethane
1-Iodobutane
1-Iodopentane
l-Iodo-3-methylbutane
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151
Response
Figure 23
Minutes
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1 52
was found to be 170 pg I/second for 1-iodopentane.
It was
not determined whether response varied between different
organoiodides.
The absolute detection limit for
1-iodopentane was 2 ng I .
Selectivity
Selectivity for iodine emission was measured by
comparing detector response for 1-iodopentane vs. carbon
tetrachloride.
Selectivity is defined as the ratio of the
molar amounts of iodine and carbon required to give an
equivalent response.
This selectivity ratio was found to be
7000 at 1830 A.
Detection of Sulfur Emission
Wavelength Calibration
The same wavelength calibration method was used as
described previously for organic iodine compounds.
Carbon
disulfide was doped into the plasma to produce
characteristic sulfur emission.
Because of the exteme
volatility of carbon disulfide, the argon flow through the
top of the plasma capillary was actually turned off.
Diffusion of CSj into the plasma was sufficient at room
temperature.
Wavelengths and relative intensities of the
observed sulfur transitions are listed in Table 15.
The
most intense emission was found for the resonance line at
1807 A.
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153
Table
15
Observed Sulfur Transitions
X, A
Levels, 1/cm
I
*
theoretical
1666.7
9239 - 69239
1 25
52
1782.3
22181 - 78290
12
10
1807.3
0 - 55331
25
>100
1820.4
397 - 55331
25
>100
1826.3
574 - 55331
25
>100
1900.3
0 - 52624
20
25
1914.7
397 - 52624
15
10
1observed
* Intensity values were obtained from reference 175.
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154
Method Development
Detector Optimization
The results obtained for hydrogen and iodine studies
revealed that the most useful power range was from 50 to 70
watts.
In studying detector response for sulfur, an applied
power of 70 watts was chosen.
Plasma observation height
profiles were compared between argon and argon/helium
carrier gas systems.
Results for this study at 1807 A are
illustrated in Figure 24.
The most obvious result of adding helium to the argon
carrier gas was the shortened plasma length.
content was approximately 10 to 14%.
The helium
At 70 W and an
observation height of -14 mm, response was twice as high
when helium was added to the carrier gas.
This increased
response was again assumed to be due to background emission
when the plasma contracted.
When observing emission at a
point near the lower tip, the plasma shrank just enough so
that it extended into the observation port.
Another interesting observation was the fact that the
background emission profile, measured just before injection,
closely paralleled the response to sulfur.
There was a
large increase in background corresponding to maximum sulfur
response when helium was added.
This being the case, one
could simply set the observation height for maximum
background emission to- approximate the region of greatest
response for sulfur.
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155
Figure 24
Detector Optimization - Sulfur (1807 A)
Grating
Slits
Ar flow
Injections
Key:
1900 A blaze
0.2 mm
100 mL/min.
2 y L x 50 ppm
Methyl parathion
Argon plasma background
▲ Argon plasma, sulfur emission
O Argon + 10% He background
• Argon + 10% He, sulfur emission
A
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156
Figure 24
60
Height,
40
Peak
mm
50
30-
10 -
o -o -o '
20
Distance
from
40
C avity Center, mm
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50
157
Because of the relatively small region of maximum
response, reproducibility was a problem.
Any small changes
in the plasma such as flow rate or helium content led to a
large change in response.
The large error bars for the data
points between 10 and 16 mm from the cavity center
illustrate this point.
This effect probably could have been
minimized by reducing the helium content in the plasma.
The relatively "flat" response profile observed in the
argon plasma led to more consistent results.
Note that
background emission again closely paralleled the sulfur
response profile.
As a compromise between sensitivity and
reproducibility, an observation height of 14 mm below the
cavity center was chosen for further studies using argon as
the carrier gas.
Separation
Names and structural formulas for the organic sulfur
compounds used in this study are presented in Figure 25.
/
These compounds are usually referred to as organophosphorous
pesticides.
The same OV-1 column used for the iodine
compounds was used in separating these pesticides.
Column
temperature was set at 200°C.
At an argon carrier gas flow rate of 110 mL/min.,
retention times were as follows:
diazinon, 195 sec.; methyl
parathion, 270 sec.; malathion, 337 sec.; ethyl parathion,
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Figure 25
Sulfur Compounds
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Figure 25
Methyl Parathion
S
(CH30)2-P-0
N02
Ethyl Parathion
S
(C2H 50)2-P-0
N02
Diazinon
Malathion
Ethion
Sf3
s.
N
CH S OC-Hp
'OH-C.
C-O-P
2 S
CH'
'N'
s0C2H 5
CH,
§
o
(CH30)2-P-S-CH-CH2-C-OC2H5
I
C-OC-Hc
II
2 5
O
C-H-O S
S 0CoH c
2 5 P-S-CH9-S-P
2 5
C2H50/
X°C2H5
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160
348 sec.; and ethion 818 sec.
Column efficiency was
measured with respect to the total number of theoretical
plates.
A theoretical plate height of 6 mm was measured,
which corresponded to a total of 300 theoretical plates.
Sensitivity and Detection Limit
Optimum instrumental conditions were used to determine
sensitivity for methyl parathion.
Seventy watts of
microwave power were applied and observation height was set
at 14 mm below the cavity center.
Slits were set at 0.5 mm.
The minimum detectable limit (MDL) for methyl parathion was
4 pg S/second.
This respresented an absolute detection
limit of 120 pg sulfur.
Response vs. Compound Type
Sulfur emission response for the different pesticide
compounds was compared on an absolute basis.
summary of these results.
Table 16 is ..a
Response was not found to be
directly related to the number of sulfur atoms in the
pesticide compounds.
This discrepancy may have been due
either to the inability of the argon plasma to fragment
certain sulfur bonds or to differences associated with the
separation or determination of the compounds.
The
difference in response between methyl and ethyl parathion
could not be due to differences in fragmentation efficiency.
Diazinon is also similar in structure but showed greatly
reduced response.
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161
Table
16
Sulfur Response vs. Compound Type
Compound
Injection
Response
(mm2/compd.)
Response
(mmz/ng/s)
Methyl Parathion
0.1016 yg
903
7.4
Ethyl Parathion
0.0768 yg
1302
12.0
Diazinon
0.0500 yg
126
1.2
Malathion
0.0984 yg
789
4.1
Ethion
0.0904 yg
2268
6.8
Selectivity
Selectivity for the sulfur resonance line, at 1807 A was
found to be dependent on slit width.
Increasing the slit
width led to increased sensitivity for sulfur.
However,
background emission due to carbon increased to an even
greater extent.
The argon carrier gas contained
approximately 10% helium in this study.
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162
Sulfur response was measured for 2 yL injections of
methyl parathion at 50.8 ng/yL.
This represented an
absolute injection of 12.2 ng sulfur.
was used to measure carbon response.
Carbon tetrachloride
Column temperature was
reduced to 40°C so that the plasma would not be extinguished
by 0.9 yL injections of CC14 .
This represented an absolute
injection of 112 yg carbon.
A comparison of selectivity ratios at different slit
widths is presented in Table 17.
a slit width of 0.5 mm.
Selectivity was highest at
Increasing slit widths led to
greater sensitivity but lower.selectivity.
This effect was
due to the line width of the sulfur emission.
Increasing
the bandpass increased response for sulfur until it became
larger than the line width.
Background response to carbon
emission increased exponentially with slit width as shown by
the normalized response factors given in Table 17.
Sensitivity and' selectivity suffered at slit widths less
than 0.5 mm.
Selectivity was also determined with respect to
observation height.
widths of 0.5 mm.
Pure argon carrier gas was used at slit
The selectivity ratio for sulfur vs.
carbon response was greatest (2x10**) at an observation
height of -18 mm.
Selectivity did not vary greatly along
the length of the plasma.
Results of this study did show
that the selectivity ratio dropped by more than an order of
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163
T a b le
17
Selectivity for Sulfur vs. Slit Width
Slit Width
(mm)
Detector Response (cm2/mole)
Sulfur
Carbon
Selectivity Ratio
0.25
2.4x10s ( D *
4.2x10® (1)
5.7x10“
0.5
1.3x10® (6)
1.6x10“ (4)
8.1x10“
0.75
2.2x10® (9)
4.2x10“ (10)
5.2x10“
1.0
2.3x10® (0)
8.2x10“ (19)
2.8x10“
* Normalized response values are in parentheses.
magnitude when the 10% helium was removed from the argon
carrier gas.
Conclusions
Purging the spectrometer with helium proved to be an
effective alternative to evacuation.
The lowest useful
wavelength with helium purging was probably about 1600 A in
this study.
Purification of the helium to remove traces of
oxygen should lower this wavelength limit even further,
considering the optical path of the monochromator is nearly
4 meters long.
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164
Observation of VUV emission from iodine compounds did
not represent an improvement over sensitivities reported
elsewhere in the literature for longer wavelengths..
The
selectivity ratio determined for iodine in this study was
favorable, however.
Much better results were obtained for
resonance sulfur emission lines.
Sensitivity and
selectivity was improved by one order of magnitude over
previous GC-MES’investigations at UV-visible wavelengths.
Monitoring resonance transitions in quantitative work
can lead, to problems when self-absorption of the analytical
source is significant.
This effect was minimized by using
small plasma capillary diameters and transverse viewing to
provide an optically thin source.
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165
APPENDIX A
SPECTROMETER ALIGNMENT PROCEDURE
1.
The table which supported the spectrometer was
leveled.
2.
Precise leveling was accomplished by adjusting the
screws on the vibrationless mounts at each end of the
spectrometer.
3.
-
A laser was set up approximately twenty feet from the
entrance slit of the instrument.
The light beam from
this laser defined the optical path of the
instrument.
The first requirements were that: i) the
beam was level; ii) the beam entered the spectrometer
at the exact center of the slit; and iii) the beam
hit the horizontal center of the collimating mirror.
At this point, the beam was found to be 6.3 cm above
the inside shelf of the spectrometer.
4.
The collimating mirror height was adjusted so that
its vertical center was 6.3 cm above the shelf.
5.
The collimating mirror tilt was adjusted so that the
incoming beam hit the center of the grating at a
height of 6.3 cm from the shelf.
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166
6.
The grating tilt was adjusted so that various orders
of the diffracted beam could be reflected back to the
center of the collimating mirror, and back to the
laser.
7.
The "camera” mirror height was adjusted so that its
center was 6.3 cm above the shelf.
8.
The grating was rotated until one of the orders hit
the center of the "camera" mirror.
9.
The "camera" mirror tilt was adjusted so that the
laser beam was reflected towards the center of the
exit slit.
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167
APPENDIX B
GC-MES ALIGNMENT PROCEDURE
1.
The light pipe mount (Figure 5) was adjusted so that
emission from the observation hole passed through the
center of the light pipe.
Since the position of the
observation hole with respect to the GC was fixed,
this adjustment should not change.
2.
After removing the microwave cavity and plasma
capillary, the GC mount was adjusted so that the
laser beam entered the center of the light pipe.
After replacing the plasma capillary, the observation
hole should still line up with the light pipe.
There
should only be a space of approximately 2 mm between
the plasma capillary and the end of the light pipe
when the other end is touching the entrance slit.
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168
A P P E N D IX C
BENZENE -
SPECTRUM 1 0 0 0 -
Grating
Slits
Ar flow
Observation ht.
Power
2500 A
1900 A blaze
0.25
100 mL/min.
-9mm
80 watts
Assignments of Intense Transitions
X, A
Assignment
I
observed
1657.0
Carbon (I)
1742.7
Nitrogen (I)
>100
1930.9
Carbon (I)
>100
2478.6
Carbon (I)
>100
75
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
— , 1000
A
169
-
I
_
_f
t
v
A
VnA
v
A
2000
A
_
1500
A
I
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170
A P P E N D IX D
NITRO ETHANE -
SPECTRUM 1 0 0 0 -
Grating
Slits
Ar flow
Observation ht.
Power
2500 A
1900 A blaze
0.25 mm
100 mL/min.
-9 mm
70 watts
Assignments for Intense Transitions
X, A
Assignment
1657.0
Carbon (I )
1742.7
Nitrogen (I )
>100
1930.9
Carbon (I )
>100
2478.6
Carbon (I)
28
observed
95
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171
<
©
o
o
7\— /A.
<
o
o
in
H
<
o
o
o
CM
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172
APPENDIX E
MALATHION - SPECTRUM 1000 - 1500 A
Grating
Slits
Ar flow
Observation ht.
Power
1900 A
0.25 mm
100 mL/min
-9 mm
70 watts
Assignments for Intense Transitions
X, A
Assignments
1657.0
Carbon (I)
57
1666.7
Sulfur (I)
52
1742.7
Nitrogen (I)
1782.3
Sulfur (I)
10
1807.3
Sulfur (I)
>100
1820.4
Sulfur (I)
>100
1826.3
Sulfur (I)
>100
1900.3
Sulfur (I )
25
1914.7
Sulfur (I)
10
1930.9
Carbon (I )
>100
observed
>100
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173
<
o
o
m
iH
ILJ^V^ W aJUJLvV
I
I
I
o
o
(N
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
I
174
APPENDIX ]
BROMOBUTANE - SPECTRUM 1000 - 2500 A
Note:
Grating
Slits
Ar flow
Observation ht.
Power
Attenuation
1900 A blaze
0.25 mm
100 mL/min.
-9 mm
70 watts
1x10_9 (1000-1500 A)
1x1 0 '8 (1500-1500 A)
Assignments of Intense Transitions
X, A
Assignment
*observed
1574.8
1576.4
Bromine (I )
5
1633.4
Bromine (I )
15
1657.0
Carbon (I )
>100
1742.7
Nitrogen (I )
>100
1930.9
Carbon (I)
>100
2478.6
Carbon (I )
>100
1582.3
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1000
A
175
JIaAJ
2000
A
1500
A
Ju
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
A
176
APPENDIX G
IODOBUTANE - SPECTRUM 1000 - 2500 A
Grating
Slits
Ar flow
Observation ht.
Power
1900 A blaze
0.25 mm
100 mL/min.
-9 mm
80 watts
Assignments of Intense Transitions
A
Assignments
*observed
1640.8
Iodine (I)
j
1642.1
Iodine (I)
)
1657.0
Carbon (I )
.>100
1702.1
Iodine (I)
7
1742.7
Nitrogen (I)
1782.8
Iodine (I)
97
1799.1
Iodine (I)
52
1830.4
Iodine (I)
>100
1844.5
Iodine (I)
10
1930.9
Carbon (I )
91
2062.4
Iodine (I)
8
2478.6
Carbon (I )
11
31
>100
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1500
A
1000
A
177
/\w
2000
A
u
JL
JL
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178
APPENDIX H
ANALYTICAL APPLICATIONS OF GC-MES
This information is included to review the following
features of GC-MES:
1.
Elements that can be determined and the wavelength of
observation.
2.
The influence of cavity t y p e ,
gas type, gas pressure
and analytical wavelength on sensitivity and
'
selectivity.
3.
Additional references related to the analyte species
of interest.
Sensitivities are presented in terms of the mass flow rate
required to give an analyte signal equal to a certain
multiple of the noise.
This multiple is generally two but,
in some cases, multiples of one or three times the noise
have beexi chosen to define "detectability.”
No attempt has
been made to normalize sensitivity data.
As mentioned previously, there are also inconsistencies
in the method used for calculating selectivity ratio.
this data is included for approximate comparisons only,
values were left as found in the literature.
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Since
179
KEY
X, A
Plasma Gas
Cavity
Sensitivity
Selectivity
Ref.
Aluminum (see also: reference 99)
3962
Ar(atm)
1/4-wave
3962
Ar(atm)
tapered
3952
He (atm)
™ 0 io
20 pg/s
0.5 ng
5 pg/s
990
96
-
176
39°?
67
Antimony
2598
Ar(.atm)
tapered
50
pg
2000
98
pg
2x10“
86
6 pg/s
5x10“
67
Arsensic (see also: references 98,103)
2288
2288
Ar(atm)
He (atm)
tapered
™ 0io
20
Beryllium
2349
Ar(atm)
tapered
10 pg
-
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99
180
Boron (see also: reference 177)
2498
He (atm)
™ qio
4 pg/s
9259
67
Bromine (see also:' references 81,89,34,33,103)
2985
Ar(atm)
tapered
200 ng/s
10
17
4786
He(red)
tapered
20 pg/s
38
71
4705
Ar(atm)
3/4-wave
-
31
4705
He(red)
1/4-wave
90 pg/s
1300
78
4705
He(atm)
™010
5 pg/ s
220
23
4705
He (atm)
™ qio
10 pg/ s
1400
79
™010
S ’p'g/s
530
80
4705
He(atm)
159 ng
4705
He (atm)
™ 0io
67 pg/s
1060
67
4786
H e (atm)
™010
34 pg/ s
599
67
Carbon (see also: references 17,31,81,89,33,103)
2479
Ar(atm)
3/4-wave
20 pg/s
1
31
2479
He(red)
1/4-wave
80 pg/s
1
78
1931
1931
He (atm)
He(atm)
™oio
™010
400 fg/ s
9 pg/s
1
1
23
80
2479
He (atm)
™ qio
3 pg/s
1
67
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
181
Chlorine (see also: references 17,21,31,81/89,33,103)
4795
He(red)
tapered
60 pg/s
4795
He(red)
1/4-wave
60 pg/s
510
78
4795
He(atm)
™010
7 pg/s
200
23
4810
He (atm)
™010
16 pg/s
2400
79
4810
He(atm)
16 pg/s
730
34
4795
He (atm)
5 pg/s
1000
80
4795
He (atm)
43 pg/s
610
67
3930
96
™010
™010
™010
44 .
71
Chromium (see also: references 92,100)
3579
Ar(atm)
1/4-wave
3 pg/s
4254
Ar(atm)
tapered
1 ng
3579
He(red)
1/4-wave
3579
Ar(atm)
1/4-wave
2677
He(atm)
™010
15 pg/s
900 fg
7000
-
99
93
100
7 pg/s
1x10s
67
6 pg/s
2x10s
67
Cobalt
2407
He(atm)
™010
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
182
Copper
3248
3247
Ar(atm)
Ar(atm)
tapered
1/4-wave
1 ng
8 pg/s
2250
103
96
Deuterium (see also: references 102,89,103)
6561
He(red)
1/4-wave
6561
He (atm)
™010
90 pg/s
880
78
7 pg/ s
194
67
3 pg/s
10
17
60 pg/s
2300
78
Fluorine (see also: references 89,33,103)
5166
Ar(atm)
tapered
6856
He(red)
1/4-wave
6856
He (atm)
™010
8 pg/ s
3500
79
6856
He (atm)
™010
2 pg/ s
820
80
6856
He (atm)
™ 010
180 pg/s
1x10*
67
3 pg/s
1170
96
Galium
2944
Ar(atm)
1/4-wave
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
183
Germanium
2651
He(atm)
™010
^
pg/ s
8x10“
67
Hydrogen (see also: references 89,33,94,103)
4861
He(red)
1/4-wave
4861
He (atm)
™010
6563
He (atm)
30 pg/s
-
78
2 P9/s
“
23
™010
® P9/s
80
4681
He(atm) .
TMQ10
16 pg/s
-
67
6563
He (atm)
™ 0io
7 pg/s
"
67
Iodine (see also: references 69,70,21,89,31,33,103)
2062
Ar(atm)
tapered
70
fg/s
1x10“
17
5338
He(red)
tapered.
50 pg/s
38
71
2062
Ar(atm)
3/4-wave
100 pg/s
1000
81
5161
He(red)
1/4-wave
50 pg/s
400
78
5161
2062
He (atm)
He (atm)
™ 010
™ 010
3 pg/s
31 pg/s
130
1100
23
79
2062
He (atm)
™ 0io
31 pg/s
140
34
2062
H e (atm)
™ 0io
7 P^/s
530
80
2062
He (atm)
™ 0io
21 pg/s
5010
67
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
184
Iron
2599
3441
He (atm)
Ar(atm)
™ qio
1/4-wave
300 pg/s
13 pg/s
3x10s
1610
67
96
Lead (see also: references 103,91)
™ 0io
2833
4058
He (atm)
Ar(atm)
500 fg/s
6 pg
1x10s
8x10'*
79
73
3/4-wave
4058
He (atm)
™oio
2
pg/ s
2x10s
67
2833
He (atm)
™ 010
200
fg/s
2xl°S
67
™ 0io
™oio
250
fg/s
2x10s
79
2 pg/s
lxl°5
67
Manganese
2576
He (atm)
2576
He (atm)
Mercury (see also: references 178,179,84,57,85,103)
2537
Ar(atm)
tapered
100 pg
lxlO4
83
2537
He(red)
1/4-wave
50 fg
-
38
™ 0io
™ qio
1 pg/s
600 fg/ s
9x104
8x10*
79
67
2537
2537
He (atm)
He (atm)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
185
Molybdenum.
2816
He (atm)
™oiO
5 pg/s
™ 0 10
3 pg/s
6470
67
™ 010
69 pg/s
3x10“
67
2x10“ .
67
Nickel
2316
He (atm)
Niobium
2883
He (atm)
Nitrogen (see also: references 17,31,33,89,103)
7469
He(red)
*
1/4-wave
3 ng/s
-
78
Osmium
2256
He (atm)
Oxygen (see
7772
He(red)
™ qio
6 pg/s
5x10“
67
also: references 89,33,103)
1/4-wave
3 ng/s
-
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
78
186
Phosphorous (see also: references 17,21, 81,89,103)
2536
Ar(atm)
tapered
5 pg/s
100
66
2536
Ar(red)
tapered
600 fg/s
1000
70
2536
He(red)
tapered
9 pg/s
1000
71
2536
He(atm)
™010
2 pg/s
3x10“
79
2536
He(atm)
™010
3 pg/s
1x10“
67
™010
8 pg/s
1x10*
67
1/4-wave
3 pg/s
1620
96
Ruthenium
2403
He(atm)
Scandium
3614
Ar(atm)
Selenium (see also: reference 103)
2040
Ar(atm)
tapered
40 pg
1x10“
97
2040
He (atm)
™ qio
5
lxl0'‘
67
pg/s
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
1 87
Silicon (see also: reference 76)
2616
He(atm)
™ qio
29 pg/ s
2516
He(atm)
™ qio
9 pg/ s
3900.
79
1580
67
Sulfur (see also: references 17,87,47,88,89,33,103)
5454
He(red)
tapered
50 pg/s
22
71
1820
Ar(atm)
3/4-wave
40 pg/s
460
81
5454
He(red)
1/4-wave
90 pg/s
390
78
5454
He(atm)
TM
±ra010
25 pg/s
200
23
5454
He(atm)
™010
63 pg/s
250
79
5454
He(atm)
™010
39 pg/s
70
80
5454
He(atm)
™010
52 pg/s
4590
67
™oiO
2 pg/ s
4x10s
67
Tin
2840
He (atm)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
188
Tungsten
2555
He (atm)
™oio
51 pg/s
5450
67
™oio
1/4-wave
10
8
6x10“
1400
67
96
Vanadium
2688
3184
He (atm)
Ar(atm)
pg/s
pg/s
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
189
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