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Determination of carbonyl compounds in the air by lc/ms

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Title:
Determination of carbonyl compounds in the air by lc/ms
Creator:
Fan, Shuang
Publication Date:
Language:
English
Physical Description:
xiv, 111 leaves : ; 28 cm

Thesis/Dissertation Information

Degree:
Master's ( Master of science)
Degree Grantor:
University of Colorado Denver
Degree Divisions:
Department of Chemistry, CU Denver
Degree Disciplines:
Chemistry

Subjects

Subjects / Keywords:
Carbonyl compounds -- Measurement ( lcsh )
Air -- Analysis ( lcsh )
Liquid chromatography ( lcsh )
Mass spectrometry ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Bibliography:
Includes bibliographical references (leaves 110-111).
Thesis:
Department of Chemistry
Statement of Responsibility:
by Shuang Fan.

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|University of Colorado Denver
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Auraria Library
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
516013090 ( OCLC )
ocn516013090
Classification:
LD1193.L46 2009m F36 ( lcc )

Full Text
DETERMINATION OF CARBONYL COMPOUNDS
IN THE AIR BY LC/MS
by
Shuang Fan
B.S., Dalian University of Technique, 2006
A thesis submitted to the
University of Colorado Denver
In partial fulfillment
Of the requirements for the degree of
Master of Science
Chemistry
2009


The thesis for the Master of Science
degree by
Shuang Fan
Has been approved
By
Larry Anderson
Mark Anderson
Date
Xiaotai Wang


Fan, Shuang (M.S., Chemistry)
Determination of Carbonyl Compounds in the Air by LC/MS
Thesis directed by Professor Larry Anderson
ABSTRACT
Mass spectrometry with the atmospheric pressure chemical ionization in the negative ion mode
APCI(-) combined with high-performance liquid chromatography (HPLC) was the most useful
detection method for identification of 2.4-dinitrophenylhedrazone derivatives of carbonyl
compounds in the air. Two different instrumentations were used in this research, one was
HPLC-UV and another was HPLC-MS. Both HPLC-UV and HPLC-MS have a limit of
detection at the level of 100 u g/L by visual test. The precision of HPLC and MS is less than 8%,
2%, respectively. A 15 carbonyl mixture standard was used to make the calibration curves for
air samples. The isomers of C3 (acetone, and propionaldehyde), C4 (crotonaldehyde,
methacrolein, methyl ethyl ketone, and butylaldehyde), and C5 (isovaleraldehyde, and
valeraldehyde) carbonyl derivatives were identified by fragment ions produced by MS2. Air
samples were taken from downtown Denver. Formaldehyde, acetaldehyde, acetone,
propionaldehyde, crotonaldehyde, and butyraldehyde were presented in the air.


This abstract accurately represents the content of the candidates thesis. I recommend its
publication.
Larry Anderson


DEDICATION
I dedicate the thesis to my mom and dad.


ACKNOWLEDGMENT
My thanks go to my advisor, Larry Anderson, for his help and patience with me during the set up
of the instruments, and also for his contribution and support with me during the research. I
would also like to thank Jeff Cahill for his advice and support. I also wish to thank all the
members of my committee for their valuable participation and insights.


TABLE OF CONTENTS
Figures................................................................................ix
Tables................................................................................xii
Chapter
1. Introduction........................................................................1
1.1 Purpose of the Study...............................................................1
1.2 Carbonyl Compounds.................................................................2
1.3 Health Effects of Carbonyl Compounds...............................................5
1.4 DNPH Coated Cartridge Technique....................................................5
2. Experimental........................................................................7
2.1 Chemicals..........................................................................7
2.2 Standards and Samples..............................................................7
2.2.1 Standards........................................................................7
2.2.2 Samples.........................................................................11
2.3 Instrument Analysis................................................................13
3. Results and Discussion.............................................................15
3.1 Purity and Calibration Curves of Standards........................................15
3.2 Analysis of Samples by HPLC......................................................35.
vii


3.3 Parameter Optimization and Precision
43
3.4 Atmospheric Pressure Chemical Ionization.......................................45
3.4.1 C3 Carbonyl Derivatives.......................................................47
3.4.2 C4 Carbonyl Derivatives......................................................56
3.4.3 C5 Carbonyl derivatives......................................................77
3.5 Analysis of Samples by HPLC-MS.................................................86
3.6 Limit of Detection.............................................................96
3.6.1 HPLC-MS.......................................................................97
3.6.2 HPLC-UV.....................................................................102
4 Conclusions......................................................................107
Appendix
A-Calculations.......................................................................109
References...........................................................................110
Vlll


LIST OF FIGURES
Figure
2.1 LpDNPH S10 CARTRIDGE......................................12
3.1 H NMR SPECTRUM OF THE C4 CARBONYL DERIVATIVES............17
3.2 CHROMATOGRAM OF THE STANDARD..............................32
3.3 CALIBRATION CURVES OF THE STANDARD........................36
3.4 CHROMATOGRAMS OF THE BLANK................................39
3.5 CHROMATOGRAMS OF THE SAMPLES..............................41
3.6 STRUCTUES SCHEME OF CARBONYL-DNPH DERIVATIVES BY APCI (-).45
3.7 FRAGMENTATION SEQUENCES OF DNPH...........................46
3.8 MS SPECTRA FOR C3 CARBONYL-DNPHs..........................48
3.9 MS2 SPECTRA FOR C3 CARBONYL-DNPHs.........................48
3.10 FRAGMENTATION SEQUENCES FOR ACETON-2,4-DNPH.............51
3.11 FRAGMENTATION SEQUENCES FOR PROPIONALDEHYDE-2.4-DNPH....52
3.12 TOTAL ION CURRENT CHROMATOGRAM FOR C3 CARBONYL-DNPHs
MIXTURE..................................................54
3.13 MS2 SPECTRA FOR C3 CARBONYL-DNPHs MIXTURE...............55
3.14 MS SPECTRO FOR C4 CARBONYL-DNPHs........................58
3.15 MS2 SPECTRA FOR C4 CARBONYL-DNPHs.......................59
3.16 FRAGMENTATION SEQUENCES FOR METHYL ETHYL KETONE-2,4-DNPH.63
ix


3.17 FRAGMENTATION SEQUENCES FOR BUTYRALDEHYDE-2,4-DNPH
64
3.18 FRAGMENTATION SEQUENCES FOR METHACEOLEJN-2,4-DNPH.........65
3.19 FRAGMENTATION SEQEUNCES FOR CROTONALDEHYDE-2,4-DNPH.......66
3.20 TOTAL ION CURRENT CHROMATOGRAM FOR MEK/BUTYRALDEHYDE-DNPHs
MIXTURE.................................................68
3.21 MS2 SPECTRA FOR MEK/BUTYRALDEHYDE-DNPHs................69
3.22 TOTAL ION CURRENT CHROMATOGRAM FOR
METHACROLEIN/CROTONALDEHYDE-DNPHs MIXTURE...............71
3.23 MS2 SPECTRA FOR METHACROLEIN/CROTONALDEHYDE-DNPHs MIXTURE.72
3.24 TOTAL ION CURRENT CHROMATOGRAM FOR 4 C4 CARBONYL DERIVATIVES...75
3.25 MS2 SPECTRA OF THE 4 C4 CARBONYL MIXTURE...............76
3.26 MS SPECTRA FOR C5 CARBONYL-DNPHs.......................78
3.27 MS2 SPECTRA FOR C5 CARBONYL-DNPHs......................79
3.28 FRAGMENTATION SEQUENCES FOR ISOVALERALDEHYDE-2,4-DNPH..81
3.29 FRAGMENTATION SEQUENCES FOR VALERALDEHYDE-2,4-DNPH.....82
3.30 TOTAL ION CURRENT CHROMATOGRAM FOR C5 CARBONYL-DNPHs
MIXTURE.................................................83
3.31 MS2 SPECTRA FOR C5 CARBONYL-DNPHs MIXTURE..............84
3.32 TOTAL ION CURRENT CHROMATOGRAM FOR THE 15 MIXTURE STANDARD... .86
3.33 TOTAL ION CURRENT CHROMATOGRAM OF SAMPLES..............92
3.34 MS SPECTRA OF PEAKS IN THE SAMPLE BLANK................93
3.35 TOTAL ION CURRENT CHROMATOGRAPHY FOR ACETONE OF DIFFERENT
CONCENTRATIONS..........................................99


3.36 MS SPECTRA OF THE DILUTED ACETONE.....................101
3.37 CHROMOTAGRAM OF DILUTED ACETONE.......................105
xi


LIST OF TABLES
Table
1.1 The List of Common Carbonyls........................................................3
1.1 (Cont.) The List of Common Carbonyls................................................4
2.1 C3 Carbonyl Derivatives..............................................................8
2.2 C4 Carbonyl Derivatives..............................................................8
2.3 C5 Carbonyl Derivatives..............................................................9
2.4 C7 Carbonyl Derivatives..............................................................9
2.5 Concentrations of Carbonyl Derivatives in the Standard..............................11
2.6 Volume of Air in the Cartridges.....................................................12
2 7 Gradient Program of LC Separation....................................................13
3.1 Variation of the Peak Areas of the Standard.........................................18
3.1 (Cont.) Variation of the Peak Areas of the Standard.................................19
3.1 (Cont.) Variation of the Peak Areas of the Standard.................................20
3.1 (Cont.) Variation of the Peak Areas of the Standard.................................21
3.1 (Cont.) Variation of the Peak Areas of the Standard.................................22
3.1 (Cont.) Variation of the Peak Areas of the Standard.................................23
3.1 (Cont.) Variation of the Peak Areas of the Standard.................................24
3.1 (Cont.) Variation of the Peak Areas of the Standard.................................25
xii


3.1 (Cont.) Variation of the Peak Areas of the Standard
.26
3.1 (Cont.) Variation of the Peak Areas of the Standard...........................27
3.1 (Cont.) Variation of the Peak Areas of the Standard...........................28
3.1 (Cont.) Variation of the Peak Areas of the Standard...........................29
3.1 (Cont.) Variation of the Peak Areas of the Standard...........................30
3.1 (Cont.) Variation of the Peak Areas of the Standard...........................31
3.2 The Retention Times and Linear Equations of the Standard......................33
3.3 The R2 Value of the Component in the Standard..................................35
3.4 Concentration of Carbonyls Derivatives in the Samples.........................41
3.4 (Cont.) Concentration of Carbonyls Derivatives in the Samples..................42
3.5 Atmospheric Concentrations of the Carbonyls....................................42
3.6 APCI (-) Parameters...........................................................43
3.7 Precision for Acetone in Total Ion Current....................................44
3.8 Major Fragments for C3 Carbonyl-DNPHs.........................................49
3.8 (Cont) Major Fragments for C3 Carbonyl-DNPHs..................................50
3.9 Major Fragments for C3 Carbonyl-DNPHs Mixture.................................56
3.10 Major Fragments for C4 Carbonyl-DNPHs........................................60
3.10 (Cont.) Major Fragments for C4 Carbonyl-DNPHs.................................61
3.10 (Cont.) Major Fragments for C4 Carbonyl-DNPHs.................................62
xiii


3.11 Major Fragments for MEK/Butyraldehyde-DNPHs Mixture.........................70
3.12 Major Fragments for Methacrolein/Crotonaldehyde-DNPHs Mixture...............73
3.12 (Cont) Major Fragments for Methacrolein/Crotonaldehyde-DNPHs Mixture.... ...74
3.13 Major Fragments forC5 Carbonyl-DNPHs........................................79
3.13 (Cont) Major Fragments for C5 Carbonyl-DNPHs................................80
3.14 Major Fragments for C5 Carbonyl-DNPHs Mixture...............................85
3.15 Retention Times and Major Fragments of the 15 Mixture Standard..............87
3.16 Linear Regression Equation for Some Carbonyl Derivatives in the Standard....94
3.17 The Concentration of Carbonyl Derivatives in the Samples.....................95
3.18 The Concentration of Carbonyls in the Samples...............................96
3.19 Standard Derivation of 0.1 mg/L Acetone by HPLC-MS.........................102
3.20 Standard Derivation of 0.1 mg/L Acetone by HPLC-UV.........................106
XIV


1. Introduction
1.1 Purpose of the Study
Carbonyl compounds, commonly aldehydes and ketones, have received scientific attention these
years. Measurement of carbonyls in the environment is of great interest because of their
ubiquitous presence because they play an important role in the atmospheric chemistry1'2, and also
because some lower molecular-weight carbonyls (formaldehyde, acetaldehyde and acrolein) are
known as toxic air contaminants.3'4 In the atmosphere, carbonyl compounds are significant
photochemical oxidation products of almost all organic compounds, and are precursors of free
radicals, ozone, and peroxyacyl nitrates. Based upon the roles carbonyl compounds played in
the air, it is important to have an analysis method which is both selective and sensitive in
determining the quantity and identity of these carbonyl compounds.
During the past three decades, different analysis methods of carbonyls in ambient air have been
attempted using a variety of techniques which try to overcome the polar reactivity and
semivolatile characteristic of carbonyls. Derivatization (a technique used in chemistry which
transforms a chemical compound into a product of similar chemical structure) is one of these
methods. The 2,4-dinitrophenylhydrazine (DNPH) characterization method has been established
as the most widely used procedure for the determination of carbonyls in air samples in recent
years. Air monitoring programs in the United States as well as in Europe apply the DNPH
method for the determination of C1-C5 carbonyls. The carbonyls react with acidified DNPH
1


during sampling, forming the corresponding hydrazones. These hydrazones are then determined
by high-performance liquid chromatograph (HPLC) with ultraviolet (UV) detection. However,
conventional HPLC/UV detection has some disadvantage: First, for all unsymmetrical DNPH
derivatives, two isomers may exist, syn and anti. These isomers may have very similar
chromatographic properties and complicate the HPLC separation.
X\
N=C 1 an<3 ^ NC f
R1
2
R1 and R2 are the carbonyl substituents (R1 = H for aldehydes) and X is the
(2,4-dinitrophenyl)amino substituent-NHC6H3(N02)2- Second, the detection limit of HPLC/UV
method isnt low enough to measure some trace carbonyls in the air samples. So the HPLC-UV
detection method can only perform well with lower molecular weight carbonyls which have a
higher concentration in the air. Carbonyl compounds containing four or more carbon atoms are
difficult to identify using the UV detection because of the more complex structures and lower
concentration in the air. In this way, instead of UV detection, mass spectrometry (MS) detection
with HPLC separation is investigated as a means of determining high molecular weight carbonyls.
1.2 Carbonyl Compounds
Carbonyl compounds are identified as either aldehydes or ketones. They are reactive volatile
substances. The functional group on aldehydes and ketones is a carbonyl group which is
composed of a carbon atom attached to an oxygen atom with a double bond. In aldehydes,
2


the carbonyl group is on one "end" of a carbon chain, while in ketones, it is in the "middle'
of a carbon chain.
carbonyl group aldehyde ketone
Table 1.1 shows the structure, elution order, and the molecular weight of several carbonyl
compounds existed in the air.
Table 1.1 The List of Common Carbonyls
Component Structure Carbon Number Molecular weight (amu)
Formaldehyde =0 1 30
Acetaldehyde 2 44
Acrolein 3 56
Acetone V 3 58
Propionaldehyde 3 58
3


Table 1.1 (Cont.) The List of Common Carbonyls
Component Structure Carbon Number Molecular weight (amu)
Crotonaldehyde 4 70
Methacrolein 4 70
Methyl Ethyl Ketone 0 vA 4 72
Butyraldehyde 4 72
Benzaldehyde O' 7 106
Isovalderaldehyde 5 86
Valeraldehyde 5 86
O-Tolualdehyde M-Tolualdehyde oja 8 120
P-Tolualdehyde O '' 8 120
Hexaldehyde 6 100
2,5-Dime thylbenzaldehyde oa 9 134
4


1.3 Health Effects of Carbonyl Compounds
Several carbonyls have been classified as toxic and hazardous (by the Environmental Protection
Agency EPA), such as formaldehyde, acetaldehyde, and acrolein. For instance, formaldehyde
contributes to eye, nose and throat irritation: the photooxidation products of acetaldehyde have
mutagenic (or carcinogenic) activity,4 and nasal irritation appears to be the most sensitive
respiratory effect of acrolein.5 Acrolein is not stable in the air. It can be rapidly removed to
generate hydroxyl radicals and is expected to degrade in water, soil, and air very quickly.
Carbonyl compounds in the air are either directly emitted (primary sources) or are produced in the
atmosphere (secondary sources) by chemical transformations. They can be produced from
incomplete combustion of biomass or fossil fuels and also can be obtained through the
atmospheric photooxidation of hydrocarbons.
1.4 DNPH Coated Cartridge Technique
Currently, there are several techniques used to measure gas phase carbonyl compounds. One of
the most widely used techniques is the 2,4-dinitrophenylhydrazine (DNPH)-coated cartridge
technique. Using this method, reactive carbonyl compounds in the air are pumped into the
cartridge, converted to stable hydrazones, which can be easily separated and analyzed by different
instruments. The most commonly used adsorbent for cartridges are silica gel and Cl8 silica gel.
Silica gel is a polymer of silicon and oxygen with surface hydroxyl groups. Cl 8 silica gel has a
similar structure except an octadecane hydrocarbon group attached to the surfaces, Cl8 groups.
5


The C18 group is chemically bonded to the silica support through siloxane groups (i.e., Si-O-R).6
The derivatation reaction used in the experiment is shown below:
Unlike Cl8 silica gel cartridges, it is reported that silica gel cartridges shows a negative
interference for formaldehyde and acetaldehyde in the presence of ozone (03). In order to have
an accurate concentration measurement of carbonyls in the air, the DNPH ozone scrubber is used
to prevent ozone into the cartridge. The common ozone scrubber is a closed polypropylene tube
containing high purity potassium iodide or NO titration, which is highly effective in trapping
ozone.
6


2. Experimental
2.1 Chemicals
All solvents employed were HPLC grade. The acetonitrile (ACN) was purchased from J.T.
Baker. Standard solutions containing 15 kinds of carbonyl-DNPHs ( DNPH derivatives of
formaldehyde, acetaldehyde, acetone, acrolein, benzaldehyde, butyraldehyde, crotonaldehyde,
hexaldehyde, isovaleraldehyde, m-tolualdehyde, o-tolualdehyde, p-tolualdehyde, propionaldehyde,
valeraldehyde, 2,5-dimethylbenzaldehyde) were purchased from Supelco (USA). Some other
C3, C4 and C5 carbonyl derivatives were synthesized in the lab by previous students: acetone,
propionaldehyde, methacrolein, crotonaldehyde, methyl ethyl ketone, butyraldehyde,
isovaleraldehye, valeraldehyde, m-tolualdehyde, o-tolualdehyde and p-tolualdehyde. The purity
of these derivatives was tested by HPLC and NMR (H).
2.2 Standards and Samples
2.2.1 Standards
The DNPH derivatives were made by previous students in the lab. Acetone, and
propionaldehyde (C3 carbonyls), methacrolein, crotonaldehyde, methyl ethyl ketone, and
butyraldehyde (C4 carbonyls), isovaleraldehye, and valeraldehyde (C5 carbonyls),
m-tolualdehyde, o-tolualdehyde, and p-tolualdehyde (C7 carbonyls) were used in this experiment.
Table 2.1 -2.4 contains the structure and molecular weight of these compounds.
7


Table 2.1 C3 Carbonyl Derivatives
Molecular
Components Structure Weight (amu)
Acetone-2,4-DNPH 2Nv^yN02 238
Propionaldehyde-2,4-DNPH '^'N02 238
Table 2.2 C4 Carbonyl Derivatives
Components
Methacrolein-2,4-DNPH
Crotonaldehyde-2,4-DNPH
Methyl Ethyl Ketone-2,4-DNPH
Butyraldehyde-2,4-DNPH
Structure
Molecular
Weight (arau)
250
250
252
252
8


Table 2.3 C5 Carbonyl Derivatives
Molecular
Components Structure Weight (arau)
Isovaleraldehyde-2,4-DNPH OzN^J-yNOz 266
Valeraldehyde-2,4-DNPH 2NY^rN2 H 266
Table 2.4 C7 Carbonyl Derivatives
Components Structure Molecular Weight (amu)
o-Tolualdehyde-2,4-DNPH NOz 300
02N^;S\.N02
m-Tolualdehyde-2,4-DNPH 300
H
p-Tolualdehyde-2,4-DNPH
N-NH N02
rs

300
9


lmg of acetone, m-tolualdehyde, o-tolualdehyde, and p-tolualdehyde derivatives were weighed
and transferred to separate 10 mL volumetric flasks. HPLC grade acetonitrile is used as the
solvent and added to the calibration mark in the flask. The lOOug/mL acetone-2,4-DNPH
standard served as the solutions needed to optimize certain parameters for the mass spectrometer.
The C7 carbonyl derivatives were used to identify the retention times of these isomers for they
were difficult to separate by HPLC. Four different standard mixtures were used in the
experiment. The first three, mixture standards of C3, C4 and C5 derivatives were used for
gauging elution positions and confirm mass spectra of isomers. The last one, containing 15
carbonyl derivatives, was used to make calibration curve in order to determine the quantity of
carbonyls in the air sample. The purity of each compound is 99%. The different points for the
calibration curve were obtained as below: 20pL, 50pL, 200|iL, 300pL, and lOOOpL of mixture
standards were transferred into separate 10 mL volumetric flask, then added ACN to the
calibration mark. Concentrations of different components in the standard were written in Table
2.5.
10


Table 2.5 Concentrations of Carbonyl Derivatives in the Standard
cone. Diluted Concentration ( u g/mL)
component
Formaldehyde-2,4-DNPH
Acetaldehyde-2,4-DNPH
Acrolein-2,4-DNPH
Acetone-2,4-DNPH
Propionaldehyde-2,4-DNPH
Crotonaldehyde-2,4-DNPH
Butyraldehyde-2,4-DNPH
Benzaldehyde-2,4-DNPH
Isovalderaldehyde-2,4-DNPH
Valeraldehyde-2,4-DNPH
o-Tolualdehyde-2,4-DNPH
m-Tolualdehyde-2,4-DNPH
p-Tolualdehyde-2,4-DNPH
Hexaldehyde-2,4-DNPH
2,5-Dimethylbenzaldehyde-2,4-Dl
(ug/mL) 20 uL 50 UL
105.03 0.21 0.525
76.35 0.153 0.382
63.15 0.126 0.316
61.5 0.123 0.308
61.5 0.123 0.308
53.55 0.107 0.268
52.22 0.104 0.261
40.35 0.081 0.202
46.36 0.093 0.232
46.35 0.093 0.232
37.52 0.075 0.188
37.51 0.075 0.188
37.51 0.075 0.188
42 0.084 0.21
35.13 0.07 0.176
200 uL 300 uL 1000 u L
2.101 3.151 10.503
1.527 2.291 7.635
1.263 1.895 6.315
1.23 1.845 6.15
1.23 1.845 6.15
1.071 1.607 5.355
1.044 1.567 5.222
0.807 1.211 4.035
0.927 1.391 4.636
0.927 1.391 4.635
0.75 1.126 3.752
0.75 1.125 3.751
0.75 1.125 3.751
0.84 1.26 4.2
0.703 1.054 3.513
2.2.2 Samples
The atmospheric samples were taken from downtown Denver. Samples were collected by
drawing the air with a sampling pump through the LpDNPH S10 cartridge (Figure 2.1). In this
process, carbonyls were converted to the hydrazone derivatives inside the cartridge. The
sampling rate was 284mL/min. Four samples were collected. The sample duration was 1 day, 2
11


days, 3 days, and 4 days, respectively (Table 2.6). The sampled cartridges and a blank cartridge
were eluted with 5mL of ACN into a 5mL volumetric flask.
Table 2.6 Volume of Air in the Cartridges
Air Volume (L)
1 Day 2 Days 3 Days 4 Days
408.96 817.92 1226.88 1635.84
Seriogicai
Grade ____
Polypropylene
Tube
Reservoir____
(holds 2mL)
Pdyefilene
Frits
'Vacuum Pump
Tubing
Male Liter Fitting
(Cat. Mo. 21010 )
Cartridge Adapter
----(Cat Mo. 21018-U )
Air Flow
-2,4-DNPH-CoaSed
SSca
Luer Tip
Figure 2.1 LpDNPH S10 CARTRIDGE
12


2.3 Instrument Analysis
The analysis instrumentation consisted of a Finnigan Surveyor LC System equipped with
Surveyor MS Pump, Surveyor Autosampler and Surveyor PDA Detector and a Finnigan LCQ
mass spectrometer with electrospray (ESI) and atmospheric pressure chemical ionization (APCI)
and multiple MS/MS capacity. An Agilent ZORBAX SB-C18 column (250-mm lengthx4.6-mm
i.d.) filled with 5-pm solid core particles was used for separation. A guard column is employed
in case to prevent possible contamination. Deionized water and acetonitrile were the eluants for
this system. Separations were carried out at about 25 C and optimal gradient program was
shown below in Table 2.7.
Table 2.7 Gradient Program of LC Separation
Time c(CH3CN)(%) c(H20)(%) Flow Rate Injection
(min) (mL/min) Volume (pL)
0 60 40
20 70 30
23 100 0 1 15
27 100 0
28 60 40
33 60 40
13


The eluent system was ACN-H20, starting with a gradient that contained 60% ACN then changed
to 70% ACN over 20 minutes. The gradient was then increased to 100% ACN within 3 minutes
(from t=20 min to t=23 min) and kept that ratio for 4 minutes. After that, the ratio of ACN in the
gradient was sharply reduced from 100% to 60% in 1 minute, and ran this ratio for the last 5
minutes of the gradient. The detector was operated at wavelength of 360 nm. APC1 negative
mode was used in the experiment because ESI mode was too powerful to obtain for the molecular
ions of molecules in the sample. It fragmented the molecules to very tiny pieces which cannot
be used to analyze the structures of the molecules. The optimal MS parameter was carried with
acetone-2,4-DNPH solution (~100ug/ml) mentioned in the earlier section. Mass spectrometry
parameters which were adjusted by the computer include vaporizer temperature (550C), sheath
gas flow rate (35arb), discharge current (5 P A), capillary temperature (150"C), capillary voltage
(27 V), and tube lens offset (-10V).
14


3. Results and Discussion
3.1 Purity and Calibration Curves of Standards
The carbonyl derivatives were made by previous students and had been stored in a freezer for a
long time. So it is necessary to prove the purity of the standards by analysis of the
chromatograms and H NMR spectrum of the four C4 hydrazones is shown in Figure 3.1
The 15 carbonyl mixture standard was injected 3 times a day and repeatedly for three days. The
variation of the peak areas of the mixture standard was calculated and listed in Table 3.1. Figure
3.2 is a typical chromatogram of mixture standard. The standard was made up of 15 carbonyl
derivatives while there were only 14 peaks on the chromatogram. The reason was that
m-tolualdehyde and p-tolualdehyde derivatives were overlapped. According to the data in Table
3.1, peak areas of some carbonyl derivatives had a relative standard deviation (RSD) higher than
5%. Incomplete separation and overlapping of two peaks is the possible reason. Furthermore,
the shift of retention time is another possible reason for the increasing of the RSD value. The
pump used to control the ratio of the eluents has a limited precision because of the unstable
pressure and flow rate within the pump. The ratio of the eluents would determine the
repeatability of the retention time.
15


METHYL ETHYL KETONE-2,4-DNPH
Sample_1
Solvent: Chloroform-d
Temp. 25.0 C
Varian Inova 400 MHz
h4 NO,
3^
2
NO,
J' J J /
l4 1l C0O3
i..
5 J M/) 7 TMS
Lj I \j>
' '"I 1 ! I 1 !.1. 1 I " ...T-T-T-T").
11 10 9 8 7 6 5 4
0.90 0.76 0.88 0.60
0,90
i.'"> t >->.'.* i .>.!-r>
3 2 1 ppm
0.19 0 3so.17
2.00 2.86 2.90
CROTONALDEHYDE-2,4-DNPH
Sam pie2
Solvent: Chloroform-d
J
11
V Hr*
0.17
1.02
10
1.T-T-l-r-r-,-;-!.i1 .....1.1-T
9 8 7 6
0.98 1.13 0.78 2.00
1.14 1.01 0.38
T ' I
5 4
TT_r 1 I .r-T-r |-rrrrp
321 ppm
3.14
0.59 0.49
16


METHACROLEIN-2.4-DNPH
Sample_3
Solvent; Chloroform-d
Temp. 25.0 C
Varian Inova 400 MHz
no2
l4
j~ j y i I coci3
-It 5
M.O
7
j L.A..
rMS
T T } I r ?<>( 1 T"TT-| t , , > | I
11 10 9 8 7
1,08 0.94 1.031.021.16
1.03
6 5 4
1 02
0.99
5 , ,
321 ppm
3.00 0.33
0.12 103
BUTYRALDEHYDE-2,4-DNPH
Sample_4
Solvent: Chloroform-d
Temp. 25.0 C
Varian Inova 400 MHz
7 V H4 *t*
5
NQj
j j . . -
j* 1 2i3 5 COCij
6 | 7
HjO
11 10 9 8 7 6
'i1 V "TT1 V w
0,98 0.78 0.91 0.98
0.93 0.52
-TT^-r-r-rr--T-T-T-r-t--t^^
5 4 3 2 1 ppm
2.00 0.38
1.98 2.94
Figure 3.1 'H NMR SPECTRUM OF THE C4 CARBONYL DERIVATIVES
17


Table 3.1 Variation of the Peak Areas of the Standard
Component Formaldehyde-2,4-DNPH
Conc(pg/mL) 0.210 0.525 2.101 3.151 10.503
1 16.660 32.471 130.622 187.935 561.675
2 15.536 33.400 133.022 176.005 571.993
3 15.203 33.407 141.783 174.597 609.666
Dayl Mean 15.800 33.093 135.142 179.512 581.111
STD 0.763 0.538 5.875 7.328 25.261
RSD 4.831 1.626 4.347 4.082 4.347
1 14.464 32.297 125.397 181.462 539.208
2 14.390 32.223 129.570 170.755 557.153
3 14.114 31.991 129.183 169.049 555.486
Day2 MEAN 14.323 32.171 128.050 173.755 550.616
STD 0.184 0.159 2.306 6.728 9.914
RSD 1.287 0.496 1.801 3.872 1.801
1 14.354 32.223 138.054 187.165 593.633
2 14.514 32.245 139.196 174.749 598.545
3 14.676 33.471 140.809 173.181 605.480
Day3 MEAN 14.515 32.646 139.353 178.365 599.219
STD 0.161 0.714 1.384 7.661 5.952
RSD 1.111 2.188 0.993 4.295 0.993
Total Mean 14.879 32.637 134.182 177.211 576.982
STD 0.803 0.461 5.712 3.047 24.563
RSD 5.397 1.413 4.257 1.719 4.257
18


Table 3.1 (Cont.) Variation of the Peak Areas of the Standard
Component Acetaldehyde-2,4-DNPH
Conc(pg/mL) 0.153 0.382 1.527 2.291 7.635
1 12.815 29.320 104.849 148.401 450.852
2 11.883 29.735 102.274 139.715 439.779
3 11.744 29.802 108.977 138.091 468.601
Dayl MEAN 12.148 29.619 105.367 142.069 453.077
STD 0.583 0.261 3.381 5.544 14.539
RSD 4.797 0.882 3.209 3.902 3.209
1 11.811 27.587 100.655 149.763 432.818
2 11.751 27.524 106.033 138.891 455.942
3 11.735 27.482 105.716 137.228 454.578
Day2 MEAN 11.766 27.531 104.135 141.961 447.779
STD 0.040 0.053 3.017 6.808 12.975
RSD 0.339 0.192 2.898 4.796 2.898
1 11.701 27.524 114.071 149.252 490.506
2 12.101 27.541 108.528 140.404 466.670
3 12.502 29.320 115.230 138.747 495.490
Day3 MEAN 12.101 28.128 112.610 142.801 484.222
STD 0.401 1.032 3.582 5.648 15.404
RSD 3.312 3.669 3.181 3.955 3.181
Total Mean 12.005 28.426 107.370 142.277 461.693
STD 0.208 1.075 4.579 0.457 19.690
RSD 1.736 3.783 4.265 0.321 4.265
19


Table 3.1 (Cont.) Variation of the Peak Areas of the Standard
Component Acrolein-2,4-DNPH
Conc(pg/mL) 0.126 0.316 1.263 1.895 6.315
1 12.566 29.654 109.454 148.815 470.652
2 11.905 30.943 100.769 135.226 433.308
3 11.938 30.769 110.395 133.661 474.700
Dayl MEAN 12.136 30.455 106.873 139.234 459.553
STD 0.373 0.699 5.307 8.335 22.819
RSD 3.069 2.296 4.965 5.986 4.965
1 11.240 26.758 105.076 145.677 451.826
2 11.183 26.696 99.869 138.599 429.436
3 11.092 26.686 99.570 133.226 428.152
Day2 MEAN 11.172 26.713 101.505 139.168 436.471
STD 0.075 0.039 3.096 6.245 13.313
RSD 0.667 0.145 3.050 4.487 3.050
1 11.130 26.696 111.784 144.378 480.672
2 11.589 26.713 110.079 137.994 473.340
3 12.049 27.654 108.531 134.498 466.685
Day3 MEAN 11.589 27.021 110.132 138.957 473.566
STD 0.460 0.548 1.627 5.010 6.996
RSD 3.965 2.029 1.477 3.605 1.477
Total Mean 11.632 28.063 106.170 139.119 456.530
STD 0.484 2.077 4.356 0.145 18.731
RSD 4.158 7.402 4.103 0.104 4.103
20


Table 3.1 (Cont.) Variation of the Peak Areas of the Standard
Component Acetone-2,4-DNPH
Conc(ug/ml) 0.123 0.308 1.230 1.845 6.150
1 8.316 18.216 65.900 99.375 283.370
2 7.796 18.483 68.569 97.348 294.848
3 7.830 18.621 70.337 96.576 302.448
Dayl MEAN 7.981 18.440 68.269 97.766 293.555
STD 0.291 0.206 2.234 1.446 9.605
RSD 3.643 1.115 3.272 1.479 3.272
1 8.347 19.221 73.264 100.601 315.035
2 8.305 19.177 75.006 96.590 322.528
3 8.740 19.756 74.782 92.688 321.563
Day2 MEAN 8.464 19.385 74.351 96.626 319.709
STD 0.240 0.322 0.948 3.956 4.076
RSD 2.837 1.662 1.275 4.094 1.275
1 8.237 19.177 80.670 100.778 346.881
2 8.047 19.186 77.317 97.735 332.462
3 7.858 18.216 81.513 95.380 350.504
Day3 MEAN 8.047 18.860 79.833 97.964 343.282
STD 0.189 0.557 2.220 2.706 9.544
RSD 2.354 2.955 2.780 2.763 2.780
Total Mean 8.164 18.895 74.151 97.452 318.849
STD 0.262 0.473 5.785 0.722 24.875
RSD 3.208 2.505 7.801 0.741 7.801
21


Table 3.1 (Cont.) Variation of the Peak Areas of the Standard
Component Propionaldehyde-2,4-DNPH
Conc(pg/mL) 0.123 0.308 1.230 1.845 6.150
1 9.740 22.284 80.336 114.569 345.447
2 9.612 22.784 78.533 107.430 337.693
3 9.279 22.772 83.660 106.561 359.738
Dayl MEAN 9.543 22.613 80.843 109.520 347.626
STD 0.238 0.285 2.601 4.394 11.183
RSD 2.496 1.262 3.217 4.012 3.217
1 9.169 20.817 77.123 110.403 331.629
2 9.122 20.770 81.984 105.852 352.532
3 8.928 21.016 81.739 101.607 351.478
Day2 MEAN 9.073 20.868 80.282 105.954 345.213
STD 0.128 0.131 2.739 4.399 11.776
RSD 1.409 0.627 3.411 4.152 3.411
1 9.059 20.770 88.172 113.375 379.138
2 8.944 20.780 83.269 107.484 358.055
3 8.829 21.284 89.096 104.906 383.111
Day3 MEAN 8.944 20.945 86.845 108.588 373.434
STD 0.115 0.294 3.132 4.341 13.467
RSD 1.283 1.402 3.606 3.998 3.606
Total Mean 9.187 21.475 82.657 108.021 355.424
STD 0.316 0.986 3.638 1.850 15.644
RSD 3.435 4.592 4.401 1.712 4.401
22


Table 3.1 (Cont.) Variation of the Peak Areas of the Standard
Component Crotonaldehyde-2,4-DNPH
Conc(pg/mL) 0.107 0.268 1.071 1.607 5.355
1 8.032 19.876 71.656 102.628 308.119
2 7.824 20.124 69.426 97.248 298.530
3 7.557 20.155 73.884 95.435 317.701
Dayl MEAN 7.804 20.052 71.655 98.437 308.117
STD 0.238 0.153 2.229 3.741 9.585
RSD 3.047 0.764 3.111 3.800 3.111
1 7.933 18.656 68.789 100.161 295.794
2 7.893 18.613 73.915 96.994 317.837
3 7.966 19.046 73.694 92.837 316.886
Day2 MEAN 7.931 18.772 72.133 96.664 310.172
STD 0.037 0.239 2.898 3.673 12.461
RSD 0.465 1.271 4.017 3.800 4.017
1 7.823 18.613 78.515 102.195 337.614
2 8.171 18.622 73.805 97.888 317.361
3 8.521 19.876 80.327 94.880 345.406
Day3 MEAN 8.172 19.037 77.549 98.321 333.460
STD 0.349 0.726 3.367 3.677 14.477
RSD 4.273 3.815 4.341 3.740 4.341
Total Mean 7.969 19.287 73.779 97.807 317.250
STD 0.187 0.675 3.273 0.992 14.076
RSD 2.343 3.502 4.437 1.014 4.437
23


Table 3.1 (Cont.) Variation of the Peak Areas of the Standard
Component Butyraldehyde-2,4-DNPH
Conc(pg/mL) 0.104 0.261 1.044 1.567 5.222
1 5.567 13.844 49.796 71.554 214.121
2 5.451 14.154 49.621 68.390 213.370
3 5.331 14.218 51.918 67.540 223.248
Dayl MEAN 5.450 14.072 50.445 69.161 216.913
STD 0.118 0.200 1.279 2.115 5.499
RSD 2.167 1.421 2.535 3.058 2.535
1 5.600 13.155 47.804 73.211 205.556
2 5.562 13.125 51.599 68.731 221.877
3 5.681 12.857 51.445 65.220 221.214
Day2 MEAN 5.614 13.045 50.283 69.054 216.216
STD 0.061 0.164 2.148 4.005 9.237
RSD 1.080 1.256 4.272 5.800 4.272
1 5.490 13.125 54.061 72.954 232.460
2 5.744 13.128 51.958 69.102 223.420
3 Day3 MEAN 5.999 13.844 56.075 66.905 241.123
5.744 13.366 54.031 69.653 232.334
STD 0.254 0.415 2.059 3.062 8.852
RSD 4.426 3.103 3.810 4.396 3.810
Total Mean 5.603 13.494 51.586 69.290 221.821
STD 0.148 0.525 2.119 0.320 9.112
RSD 2.637 3.893 4.108 0.461 4.108
24


Table 3.1 (Cont.) Variation of the Peak Areas of the Standard
Component Benzaldehyde-2,4-DNPH
Conc(pg/mL) 0.081 0.202 0.807 1.211 4.035
1 5.463 13.265 47.681 68.084 205.028
2 5.185 13.188 46.498 63.817 199.942
3 5.262 13.182 49.355 63.536 212.226
Dayl
MEAN 5.304 13.212 47.845 65.146 205.732
STD 0.144 0.046 1.435 2.549 6.172
RSD 2.710 0.349 3.000 3.913 3.000
1 5.479 12.354 45.774 66.735 196.827
2 5.451 12.325 48.335 63.418 207.840
3 5.412 12.600 48.190 62.245 207.218
Day2
MEAN 5.447 12.426 47.433 64.132 203.962
STD 0.034 0.151 1.439 2.329 6.186
RSD 0.617 1.214 3.033 3.631 3.033
1 5.369 12.325 52.307 67.942 224.919
2 5.366 12.328 49.280 64.120 211.904
3 5.364 13.065 52.527 63.387 225.867
Day3
MEAN 5.366 12.573 51.371 65.150 220.897
STD 0.003 0.426 1.815 2.446 7.802
RSD 0.050 3.391 3.532 3.754 3.532
Total Mean 5.372 12.737 48.883 64.809 210.197
STD 0.072 0.418 2.165 0.586 9.309
RSD 1.342 3.279 4.429 0.904 4.429
25


Table 3.1 (Cont.) Variation of the Peak Areas of the Standard
Component Isovalderaldehyde-2,4-DNPH
Conc(pg/mL) 0.093 0.232 0.927 1.391 4.636
1 4.794 11.483 41.441 59.484 178.195
2 4.631 11.734 40.892 55.831 175.836
3 4.465 11.688 43.496 55.342 187.034
Dayl MEAN 4.630 11.635 41.943 56.886 180.355
STD 0.165 0.134 1.373 2.263 5.903
RSD 3.553 1.149 3.273 3.979 3.273
1 4.721 11.092 39.783 59.683 171.067
2 4.697 11.067 42.279 56.420 181.799
3 4.621 10.854 42.152 54.119 181.256
Day2 MEAN 4.680 11.004 41.405 56.741 178.041
STD 0.053 0.130 1.406 2.796 6.046
RSD 1.122 1.186 3.396 4.927 3.396
1 4.611 11.067 45.078 60.054 193.834
2 4.721 11.068 43.201 56.569 185.766
3 4.831 11.483 45.946 55.163 197.569
Day3 MEAN 4.721 11.206 44.742 57.262 192.389
STD 0.110 0.240 1.403 2.518 6.032
RSD 2.326 2.141 3.136 4.398 3.136
Total Mean 4.677 11.282 42.697 56.963 183.595
STD 0.046 0.322 1.792 0.269 7.703
RSD 0.973 2.854 4.196 0.472 4.196
26


Table 3.1 (Cont.) Variation of the Peak Areas of the Standard
Component Valeraldehyde-2,4-DNPH
Conc(|ig/mL) 0.093 0.232 0.927 1.391 4.635
1 4.510 11.330 41.439 59.407 178.186
2 4.487 11.673 40.810 55.417 175.483
3 4.417 11.682 43.112 54.825 185.383
Dayl MEAN 4.471 11.562 41.787 56.549 179.684
STD 0.048 0.201 1.190 2.492 5.117
RSD 1.084 1.738 2.848 4.407 2.848
1 4.699 11.021 39.781 58.087 171.058
2 4.675 10.996 42.288 56.170 181.838
3 4.695 11.017 42.161 53.037 181.294
Day2 MEAN 4.690 11.011 41.410 55.765 178.063
STD 0.013 0.014 1.412 2.549 6.072
RSD 0.273 0.123 3.410 4.572 3.410
1 4.589 10.996 44.319 59.211 190.572
2 4.557 10.997 43.041 56.234 185.074
3 4.526 11.330 45.956 54.357 197.610
Day3 MEAN 4.557 11.107 44.438 56.601 191.086
STD 0.032 0.193 1.461 2.448 6.284
RSD 0.693 1.734 3.288 4.324 3.288
Total Mean 4.573 11.227 42.545 56.305 182.944
STD 0.110 0.294 1.650 0.469 7.097
RSD 2.407 2.619 3.879 0.832 3.879
27


Table 3.1 (Cont.) Variation of the Peak Areas of the Standard
Component 0-Tolualdehyde-2,4-DNPH
Conc(pg/mL) 0.075 0.188 0.750 1.126 3.752
1 3.619 8.576 30.371 43.268 130.594
2 3.332 8.642 30.092 41.238 129.397
3 3.374 8.579 31.803 40.961 136.751
Dayl MEAN 3.442 8.599 30.755 41.822 132.247
STD 0.155 0.037 0.918 1.259 3.946
RSD 4.512 0.431 2.984 3.012 2.984
1 3.513 8.259 29.156 45.336 125.370
2 3.496 8.240 31.840 43.297 136.911
3 3.358 7.943 31.745 42.034 136.502
Day2 MEAN 3.455 8.148 30.913 43.556 132.928
STD 0.085 0.178 1.523 1.666 6.548
RSD 2.467 2.180 4.926 3.825 4.926
1 3.403 8.240 33.125 44.652 142.437
2 3.398 8.239 31.678 42.602 136.215
3 3.393 8.576 34.602 41.825 148.787
Day3 MEAN 3.398 8.352 33.135 43.026 142.480
STD 0.005 0.194 1.462 1.460 6.286
RSD 0.158 2.328 4.412 3.394 4.412
Total Mean 3.432 8.366 31.601 42.802 135.885
STD 0.030 0.226 1.331 0.888 5.721
RSD 0.877 2.703 4.210 2.076 4.210
28


Table 3.1 (Cont.) Variation of the Peak Areas of the Standard
Component M,P-ToluaIdehyde-2,4-DNPH
Conc(pg/mL) 0.150 0.375 1.500 2.251 7.502
1 7.537 18.781 67.206 96.472 288.986
2 7.281 19.162 66.701 91.226 286.815
3 7.317 19.163 70.347 90.952 302.493
Dayl MEAN 7.379 19.035 68.085 92.884 292.765
STD 0.139 0.220 1.975 3.111 8.494
RSD 1.879 1.158 2.901 3.349 2.901
1 8.064 18.031 64.518 99.678 277.427
2 8.024 17.990 70.516 93.509 303.217
3 8.139 17.971 70.305 91.188 302.310
Day2 MEAN 8.076 17.997 68.446 94.792 294.318
STD 0.058 0.031 3.404 4.388 14.635
RSD 0.723 0.172 4.973 4.629 4.973
1 7.544 17.990 74.425 96.834 320.026
2 8.015 17.998 76.127 93.097 327.348
3 8.486 18.781 76.632 91.790 329.518
Day3 MEAN 8.015 18.256 75.728 93.907 325.631
STD 0.471 0.454 1.157 2.618 4.974
RSD 5.878 2.488 1.527 2.788 1.527
Total Mean 7.823 18.429 70.753 93.861 304.238
STD 0.386 0.540 4.312 0.955 18.543
RSD 4.935 2.931 6.095 1.017 6.095
29


Table 3.1 (Cont.) Variation of the Peak Areas of the Standard
Component Hexaldehyde-2,4-DNPH
Conc(|ig/mL) 0.084 0.210 0.840 1.260 4.200
1 3.848 9.244 33.847 48.849 145.542
2 3.801 9.522 33.709 45.816 144.947
3 3.838 9.458 35.629 45.553 153.205
Dayl MEAN 3.829 9.408 34.395 46.739 147.898
STD 0.025 0.145 1.071 1.832 4.606
RSD 0.640 1.546 3.114 3.919 3.114
1 3.903 9.026 32.493 47.864 139.720
2 3.884 9.005 34.845 46.525 149.834
3 3.817 9.120 34.741 45.735 149.386
Day2 MEAN 3.868 9.051 34.026 46.708 146.313
STD 0.045 0.061 1.329 1.076 5.714
RSD 1.170 0.677 3.905 2.303 3.905
1 3.793 9.005 36.220 48.738 155.747
2 3.957 9.004 35.427 46.535 152.335
3 4.122 9.244 37.868 46.005 162.831
Day3 MEAN 3.957 9.085 36.505 47.093 156.971
STD 0.164 0.138 1.245 1.449 5.354
RSD 4.151 1.520 3.411 3.078 3.411
Total Mean 3.885 9.181 34.975 46.847 150.394
STD 0.066 0.197 1.337 0.214 5.750
RSD 1.695 2.148 3.824 0.456 3.824
30


Table 3.1 (Cont.) Variation of the Peak Areas of the Standard
Component 2,5-Dimethylbenzaldehyde-2,4-DNPH
Conc(pg/mL) 0.070 0.176 0.703 1.054 3.513
1 3.425 8.365 30.754 43.754 132.242
2 3.452 8.507 29.995 41.495 128.979
3 3.421 8.457 31.958 41.040 137.421
Dayl MEAN 3.433 8.443 30.902 42.096 132.880
STD 0.017 0.072 0.990 1.453 4.257
RSD 0.500 0.857 3.204 3.453 3.204
1 3.534 8.271 29.524 43.259 126.952
2 3.516 8.252 31.375 41.075 134.912
3 3.438 7.898 31.281 39.915 134.509
Day2 MEAN 3.496 8.141 30.727 41.416 132.124
STD 0.051 0.210 1.043 1.698 4.484
RSD 1.459 2.581 3.394 4.100 3.394
1 3.024 8.252 33.571 43.850 144.353
2 3.431 8.251 31.830 41.611 136.867
3 3.838 8.365 34.096 40.797 146.615
Day3 MEAN 3.431 8.289 33.166 42.086 142.612
STD 0.407 0.065 1.187 1.581 5.102
RSD 11.865 0.788 3.578 3.756 3.578
Total Mean 3.453 8.291 31.598 41.866 135.872
STD 0.037 0.151 1.360 0.390 5.849
RSD 1.076 1.822 4.305 0.931 4.305
31


Figure 3.2 CHROMATOGRAM OF THE STANDARD
Angilent 20RBAX SB-C18 column is used for reversed-phase chromatography. Because of the
non-polar stationary phase, polar compounds are eluted first while non-polar compounds are
retained. For organic compounds, chemical polarity is reduced by increasing the number of
carbons in the carbonyls. In this way, the elution order of those carbonyl derivatives is exactly
answered for the retention times mentioned below (Table 3.2). Table 3.2 also shows the linear
regression equation of the calibration curve for each component in the mixture standard.
32


Table 3.2 The Retention Times and Linear Equations of the Standard
Component Elution Order Retention Time (min) Linear Regression Equation
Formaldehyde-2,4-DNPH 1 5.561 y = 54.313x4-8.0572
Acetaldehyde-2,4-DNPH 2 6.885 y= 59.654x4-7.3411
Acrolein-2,4-DNPH 3 8.557 y = 71.342x4-6.8381
Acetone-2,4-DNPH 4 8.706 y = 51.207x + 4.617
Propionaldehyde-2,4-DNPH 5 9.454 y = 57.049x + 5.1862
Crotonaldehyde-2,4-DNPH 6 11.118 y = 58.486x 4- 4.8757
Butyraldehyde-2,4-DNPH 7 12.58 y = 41.928x4-3.6094
Benzaldehyde-2,4-DNPH 8 13.842 y = 51.421x4- 3.2496
Isovalderaldehyde-2,4-DNPH 9 15.956 y = 39.077x 4- 2.9586
Valeraldehyde-2,4-DNPH 10 16.71 y= 38.959x4-2.8186
o-Tolualdehyde-2,4-DNPH 11 17.61 y = 35.734x4-2.3174
m,p-Tolualdehyde-2,4-DNPH 12 18.113 y = 40.027x 4- 4.7323
Hexaldehyde-2,4-DNPH 13 21.817 y = 35.332x 4- 2.4602
2,5-Dimethylbenzaldehyde-2,4-DNPH 14 22.403 y = 38.174x4- 2.1073
33


The calibration curve of the mixture standard consisted of the peak area and the concentration of
each component in the standard (Figure 3.3). A calibration curve is a method used to determine
the concentration of a component in a sample by comparing the response to that of a known
concentration of a standard. For this calibration curve, the x-axis was the concentration of the
standard and y-axis is the peak area of different components in the standard. There were five
standard concentrations used in the calibration curve and there was very good linear relationships
(R2 >0.999) for the average values of 3 days between the concentrations and responses for all
carbonyls identified. The linear relationship within a day was good as well, which had a R2
range between 0.9982 and 0.9993. The R2 value of each component is listed in Table 3.3.
34


Table 3.3 The R2 Value of the Component in the Standard
Component Day 1 R2 Day 2 R2 Day 3 R2 Mean R2
Formaldehyde-2,4-DNPH 0.9992 0.9992 0.9988 0.9991
Acetaldehyde-2,4-DNPH 0.9993 0.9992 0.9988 0.9992
Acrolein-2,4-DNPH 0.9991 0.9992 0.9987 0.9991
Acetone-2,4-DNPH 0.9988 0.9991 0.9982 0.9991
Propionaldehyde-2,4-DNPH 0.9993 0.9992 0.9985 0.9991
Crotonaldehyde-2,4-DNPH 0.9992 0.9992 0.9988 0.9992
Butyraldehyde-2,4-DNPH 0.9992 0.9991 0.9989 0.9992
Benzaldehyde-2,4-DNPH 0.9992 0.9992 0.9988 0.9992
Isovalderaldehyde-2,4-DNPH 0.9992 0.9992 0.9989 0.9992
Valeraldehyde-2,4-DNPH 0.9992 0.9992 0.9988 0.9992
o-Tolualdehyde-2,4-DNPH 0.9993 0.9989 0.9990 0.9992
m,p-Tolualdehyde-2,4-DNPH 0.9992 0.9992 0.9984 0.9992
Hexaldehyde-2,4-DNPH 0.9992 0.9992 0.9990 0.9992
2,5-Dimethylbenzaldehyde-2,4-DNPH 0.9992 0.9992 0.9988 0.9992
35


Formaldehyde-2,4-DNPH
Concentration (ug/ml)
Acetaldehyde-2,4-DNPH
500.0
400.0
300.0
s
< 200.0
100.0
0.0
0.0 2.0 4.0 6.0 8.0 10.0
Concentration (ug/ml)
Acrolein-2/4-DN PH
Acetone-2,4-DNPH
Concentration (ug/ml)
Concentration (ug/ml)
Propionaldehyde-2,4-DNPH Croto.uldehyde-2,4-ONPH
Concentration (ug/ml)
Concentration (ug/ml)
36


Butyraldehyde-2,4-DNPH
Benzaldehyde-2,4-DNPH
Concentration (ug/ml)
Concentration (ug/ml)
lsovaleraldehyde-2,4*0NPH
Concentration (ug/ml)
Valeraldehyde-2,4-DNPH
Concentration (ug/ml)
0-Tolualdehyde-2,4-DNPH
M,P-Tolualdehyde-2,4~DNPH
Concentration (ug/ml)
Concentration (ug/ml)
37


Hexaldehyde-2,4-DNPH
2,5-Dimethyl benzaldehyde-2,4-DN PH
Concentration (ug/ml)
Concentration (ug/ml)
Figure 3.3 CALIBRATION CURVES OF THE STANDARD
3.2 Analysis of Samples by HPLC-UV
Figure 3.4 and 3.5 show the chromatography of blank cartridge and 4 samples (1 day, 2 days, 3
days, and 4 days samples), respectively. According to the retention times and linear regression
equations for the components in the standard mentioned above, it is easy to analyze and calculate
the concentration of carbonyls in the sample. The process of the calculation is shown in
Appendix A. However, according to the data shown in Table 3.4, the increasing concentration
for each carbonyl in the air samples was not directly proportional with the increasing of collection
time period. There are some possible reasons. First of all, the concentration of carbonyls in the
air would be variable within 24 hours. Secondly, some carbonyls in the cartridge may be
decomposed through a longer collecting time. In the end, there was no ozone scrubber attached
prior to the cartridge which can reduce the negative effect of concentrations for formaldehyde and
acetone in the air. Table 3.5 is the atmospheric concentration of the carbonyls in the air sample.
38


BLANK SAMPLE
Figure 3.4 CHROMATOGRAMS OF THE BLANK
1 DAY SAMPLE
39


2 DAYS SAMPLE
3 DAYS SAMPLE
40


4 DAYS SAMPLE
Figure 3.5 CHROMATOGRAMS OF THE SAMPLES
Table 3.4 Concentration of Carbonyls Derivatives in the Samples
Components
1 Day 2 Days
Area Conc.(mg/L) Area Conc.(mg/L)
Formaldehyde-2,4-DNPH 77.223 1.273 159.157 2.782
Acetaldehyde-2,4-DNPH 220.144 3.567 657.93 10.906
Acetone-2,4-DNPH 306.64 5.898 911.11 17.703
Propionaldehyde-2,4-DNPH N/A N/A 56.237 0.895
Crotonaldehyde-2,4-DNPH N/A N/A 13.669 0.15
Butyraldehyde-2,4-DNPH N/A N/A 31.087 0.655
N/A: below detection limit
41


Table 3.4 (Cont.) Carbonyls in the Samples
Components Area 3 Days Conc.(mg/L) Area 4 Days Conc.(mg/L)
Formaldehyde-2,4-DNPH 133.269 2.30537 227.915 4.04798
Acetaldehyde-2,4-DNPH 380.712 6.25894 181.719 2.92316
Acetone-2,4-DNPH 16.641 0.235 N/A N/A
Propionaldehyde-2,4-DNPH 27.362 0.389 45.839 0.713
Crotonaldehyde-2,4-DNPH 24.177 0.33 25.699 0.356
Butyraldehyde-2,4-DNPH 34.824 0.744 39.665 0.86
N/A: below detection limit
Table 3.5 Atmospheric Concentrations of the Carbonyls
Components 1 Day Atmospheric Concentration (pg/m3) 2 Days 3 Days 4 Days
Formaldehyde 2.2 2.4 1.3 1.8
Acetaldehyde 8.6 13.1 5.0 1.8
Acetone 17.6 26.4 0.2 N/A
Propionaldehyde N/A 1.3 0.4 0.5
Crotonaldehyde N/A 0.3 0.4 0.3
Butyraldehyde N/A 1.1 0.9 0.8
N/A: below detection limit
42


3.3 Parameter Optimization and Precision
Electrospray ionization (ESI) in the positive and negative ion modes showed little or no response
to the carbonyl derivatives.
The MS parameter optimization was carried with a solution of acetone-2,4-DNPH. Three
vaporizer temperatures were chosen, 350"C, 450C, and 550C. The signal abundance of m/z
237 [M-H]' was maximized at 550C. The best signal-to-noise (S/N) ratio was obtained at a
discharge current of 5pA. Other optimized parameters are listed in Table 3.6.
Table 3.6 APCI (-) Parameters
Parameter Setting
Sheath Gas Flow Rate (arb) 35
Capillary Temperature (C) 150
Capillary Voltage (V) 27
Tube Lens Offset (V) -10
Structural information was obtained by further fragmentation using MS/MS. The parameters
were as follows: collision voltage, 20; normalize energy, 35%.
The precision was determined by measuring the reproducibility of acetone. It was evaluated in
terms of percent relative standard deviation (%RSD). 10 u L of acetone was injected into the
43


HPLC/MS 5 times a day and repeated 3 days. The mobile phase was ACN/H20 (v/v 70/30).
The flow rate was 1 mL/min. Table 3.7 lists the total ion current of acetone in different injections
at the optimized parameters. The RSD was less than 2% which indicates a good precision from
injection to injection.
Table 3.7 Precision for Acetone in Total Ion Current
Acetone Day 1 Day 2 Day3
3.44E+07 3.38E+07 3.49E+07
3.45E+07 3.47E+07 3.47E+07
Total Ion Current 3.48E+07 3.43E+07 3.51E+07
3.33E+07 3.49E+07 3.41 E+07
3.41E+07 3.43E+07 3.41 E+07
Mean 3.42E+07 3.44E+07 3.46E+07
STD 5.72E+05 4.24E+05 4.60E+05
%RSD 1.67 1.23 1.33
44


3.4 Atmospheric Pressure Chemical Ionization
A structure elucidation scheme for carbonyl-DNPH derivatives was developed by interpreting
hundred mass spectra from 11 selected compounds (Figure 3.6).7 The different kinds of DNPH
carbonyl derivatives can be identified as follows:
(M-H) of DNPH-dorivativ*
MSMS
mfr 183 179
imt-sw
*m/tiS2
biggest
j fTHOCfrCHO | not ortho>ubtiUit*d
Figure 3.6 STRUCTURES SCHEME OF CARBONYL-DNPH DERIVATIVES BY APCI (-)
In APCI (-), the peaks at m/z 120, 122,152,179, and [M-H-30] are the characteristic fragment
ions for the 2.4-dinitrophenylhydrazone. Fragmentation sequences are shown in Figure 3.7
45


[M-H]
[M-H-30]
m/z 152 m/z 122
-RCHN203
----
+H
N
m/z 120
[M-H]
m/z 179
Figure 3.7 FRAGMENTATION SEQUENCES OF DNPH
In this section, single MS and MS2 spectra of pure and mixture C3, C4, and C5 isomers were
obtained and analyzed respectively.
46


3.4.1 C3 Carbonyl Derivatives
Figure 3.8-3.9 shows the MS and MS2 spectra for C3 carbonyl derivatives (acetone,
propionaldehyde) and the major fragment ions including the relative abundance for each fragment
are shown in Table 3.8. The fragment at m/z 163 is present in the mass spectra of
propionaldehyde-2,4-DNPH in a high relative abundance, but not for acetone-2,4-DNPH. This
fragment is a significant distinction between these two isomers because a strong m/z 163 daughter
ion is typical for aldehyde-DNPHs. This ion is either absent or has a relative low abundance
<2% in mass spectra of ketone-DNPHs.7 An ion with m/z [M-H-30] (207) and m/z 152 is
present in the mass spectra of Acetone-2,4-DNPH with a high relative abundance. These two
fragment ions are characteristic of saturated ketone-DNPHs. The fragment at a m/z of 191 with
a high relative abundance (-15%) indicates that propionaldehyde-2.4-DNPH is a straight chained
carbonyl. The probable fragmentation sequences for acetone and propionaldehyde derivatives
are listed in Figure 3.10-3.11.
47


ACETONE
PROPIONALDEHYDE
Figure 3.8 MS SPECTRA FOR C3 CARBONYL-DNPHs
ACETONE PROPRIONALDEHYDE
Figure 3.9 MS2 SPECTRA FOR C3 CARBONYL-DNPHs
48


Table 3.8 Major Fragments for C3 Carbonyl-DNPHs
ACETONE
m/z Ion Relative Abundance
120.5 [M-H-117] 3.73
144.5 [M-H-93] 2.45
151.6 [M-H-86] 54.48
152.5 [M-H-85] 21.28
153.5 [M-H-84] 2.43
174.4 [M-H-63] 5.54
175.4 M-H-62] 8.73
179.4 [M-H-58] 94.75
191.3 [M-H-46] 3.15
207.2 rM-H-301 100
237.3 [M-H] 2.31
238.1 [M] 55.77
49


Table 3.8 (Cont) Major Fragments for C3 Carbonyl-DNPHs
PROPIONALDEHYDE
m/z Ion Relative Abundance
152.5 [M-H-85] 13.56
163.3 [M-H-74] 100
179.2 [M-H-58] 68.12
191.3 [M-H-46] 15.9
192.2 [M-H-45] 6.73
207.1 [M-H-30] 8.32
237.1 [M-H-l] 75.09
50


o*
-C2H3O3N3
---- NH
Figure 3.10 FRAGMENTATION SEQUENCES FOR ACETONE-2,4-DNPH
m/z 237
m/z 207
m/z 191
m/z 179
m/z 152
m/z 120
51


NO,
N02 -
-NO,
m/z 179
-C2H5N02
+H
02N
-c3h5n2o
o2n
m/z 163
m/z 152
Figure 3.11 FRAGMENTATION SEQUENCES FOR PR0PI0NALDEHYDE-2.4-DNPH
The mixture of acetone and propionaldehyde derivatives were separated by HPLC in a ACN/H20
(v/v:70/30) solvent for 10 minutes( flow rate lml/min) before going into MS detector. The
52


injection volume was 15 u L. In order to distinguishing the two components in the mixture, MS2
was used and the parent peak was at m/z of 237.4. Figure 3.12-3.13 shows the chromatography
and MS2 spectra of the C3 mixture. The major fragment ions are listed in Table 3.9. From the
most abundance fragments in this Table (those underlined), it is easy to say that the component
appeared at 6.08 minutes is acetone-2,4-DNPH. Proionaldehyde-2,4-DNPH is at 6.46 minutes.
53


uAU
Figure 3.12 TOTAL ION CURRENT CHROMATOGRAPHY FOR C3 CARBONYL-DNPHs
MIXTURE
54


RT 6.08 RT 6.46
Figure 3.13 MS2 SPECTRA FOR C3 CARBONYL-DNPHs MIXTURE
55


Table 3.9 Major Fragments for C3 Carbonyl-DNPHs Mixture
m/z Peak at 6.08 min Relative Abundance m/z Peak at 6.46 min Relative Abundance
76.4 2.77 120.4 16.4
120.5 6.84 152.6 29.99
144.6 2.54 153.6 9.87
151.5 47.08 163.6 100
152.5 32.22 164.7 9.85
175.3 7.72 165.5 2.27
179.4 78.4 179.4 70.17
191.4 7.55 191.4 27.56
207.2 100 192.4 18.98
238.1 23.25 207.3 32.84
238.1 60.01
3.4.2 C4 Carbonyl Derivatives
There were four C4 carbonyl derivatives used in the experiment, including crotonaldehyde,
methacrolein, methyl ethyl ketone (MEK), and butyraldehyde. Structures of these compounds
56


are listed in Chapter 2 (Table 2.2). The MS and MS2 spectra for these C4 carbonyl-DNPHs are
shown in Figure 3.14-3.15. The major fragments for these C4 carbonyl derivatives are shown in
Table 3.10.
Comparing the relative abundance in Table 3.10, the most abundant fragment of MEK and
butyraldehyde derivatives are at a m/z of 221 [M-H-30] and a m/z 163, respectively. This is a
significant indicator to identify the isomers.
Most of the fragments for crotonaldehyde and methacrolein derivatives are very similar except for
two things. First, only methacrolein-2,4-DNPH has a peak at the m/z of 79. Secondly, the
most abundant (100%) fragment of these two isomers is different: methacrolein at a m/z of 163:
crotonaldehyde at a m/z of 152. Both methacrolein and crotonaldehyde derivatives are
unsaturated aldehydes which have double bond at the a C. The work done by Stephan Kolliker
and Michael Oehme7 claimed that there were some typical fragments for this kind of carbonyl
derivatives, for instance, there would be a fragment at a m/z 167, [M-Fl-77] or [M-H-72].
Furthermore, there wouldnt be a fragment at the m/z of [M-Fl-30] in the spectra. These
conclusions compare favorably with the results of our spectra. Compared to
crotonaldehyde-2,4-DNPH, a m/z signal at 191 is absent methacrolein is a branched carbonyl
derivative. According to the major fragments in the spectra of MS2, probable fragmentation
sequences of C4 carbonyl derivatives are as following (Figure 3.16-3.19)
57


MEK
BUTYRALDEHYDE
METHACROLEIN CROTONALDEHYDE
Figure 3.14 MS SPECTRO FOR C4 CARBONYL-DNPHs
58


MEK
BUTYRALDEHYDE
METHACROLEIN CROTONALDEHYDE
Figure 3.15 MS2 SPECTRA FOR C4 CARBONYL-DNPHs
59


Table 3.10 Major Fragments for C4 Carbonyl-DNPHs
MEK
m/z Ion Relative Abundance
120.4 [M-H-131] 1.36
152.5 [M-H-99] 50.47
153.7 [M-H-98] 6.25
178.7 [M-H-73] 2.4
179.4 [M-H-72] 26.53
191.4 [M-H-60] 3.64
205.4 [M-H-46] 1.86
221.1 rM-H-301 100
222.1 [M-H-29] 6.52
252.1 [M] 20.35
BUTYRALDEHYDE
m/z Ion Relative Abundance
152.4 [M-H-99] 31.62
153.4 [M-H-98] 9.14
163.3 [M-H-88] 100
176.4 [M-H-75] 3.23
179.2 [M-H-72] 49.55
221.1 [M-H-30] 45.87
60


Table 3.10 (Cont.) Major Fragments for C4 Carbonyl-DNPHs
METHACROLEIN
m/z Ion Relative Abundance
79.4 [M-H-170] 3.15
122.5 [M-H-127] 14.99
152.5 [M-H-97] 30.96
163.6 [M-H-86] 100
164.6 [M-H-85] 4.48
167.5 [M-H-82] 67.88
168.8 [M-H-81] 3.25
172.6 [M-H-77] 25.05
173.6 [M-H-76] 17.27
179.4 [M-H-70] 28.31
182.5 [M-H-67] 3.5
202.4 [M-H-47] 55.27
204.2 [M-H-45] 2.14
213.4 [M-H-36] 6.37
218.3 [M-H-31] 19.58
250.1 [M] 47.82
61


Table 3.10 (Cont.) Major Fragments for C4 Carbonyl-DNPHs
CROTONALDEHYDE
m/z Ion Relative Abundance
151.4 [M-H-98] 13.58
152.5 [M-H-97] 100
153.7 [M-H-96] 4.15
163.5 [M-H-86] 32.73
167.4 [M-H-82] 15.68
172.7 [M-H-77] 47.67
173.7 [M-H-76] 35.25
179.5 [M-H-70] 41.1
192.4 [M-H-57] 39.5
202.5 [M-H-47] 39.08
203.6 [M-H-46] 6.58
204.5 [M-H-45] 13.97
214.3 [M-H-35] 5.51
218.5 [M-H-31] 9.33
232.3 [M-H-17] 32.65
249.4 [M-H] 4.76
250.1 [M] 81.3
62


m/z 251
m/z 221
m!z 205
m/z 191
m/z 179
m/z 152
m/z 120
Figure 3.16 FRAGMENTATION SEQUENCES FOR METHYL ETHYL KETONE-2,4-DNPH
63


m/z 251
m/z 221
m/z 191
m/z 179
m/z 163
m/z 152
Figure 3.17 FRAGMENTATION SEQUENCES FOR BUTYRALDEHYDE-2,4-DNPH
64


o2n
-c4h5n2o
---- o2n
-C4H5N302
o
m/z 249
m/z 218
m/z 202
m/z 179
m/z 172
m/z 163
m/z 152
m/z 122
Figure 3.18 FRAGMENTATION SEQUENCES FOR METHACEOLEIN-2,4-DNPH
65



m/z 249
m/z 232
m/z 218
m/z 202
m/z 179
-HN203
-----
m/z 172
m/z 163
m/z 152
Figure 3.19 FRAGMENTATION SEQEUNCES FOR CROTONALDEHYDE-2,4-DNPH
66


Two C4 carbonyl derivatives were analyzed in the experiment, one is the mixture of methyl ethyl
ketone and butyraldehyde-DNPHs (MW: 252), another is the mixture of crotonaldehyde and
methacrolein (MW: 250).
The mixture of C4 carbonyl derivatives were separated by HPLC using ACN/H20 (v/v 60/40)
solvent for 20 minutes. Then the mixtures were detected by MS2 with a selected ion at the m/z
of 251.5 and 249.4, individually. Figure 3.20-3.23 are the chromatogram and mass spectra
which followed by Tables (3.11-3.12) containing the major fragments of each component. From
Figure 3.22, the chromatogram of crotonaldehyde-DNPHs and methacrolein-DNPHs mixture,
there was one more significant component that eluted just before the main carbonyl derivative
peak at the retention time of 11.41 minutes. It was suggested that the additional component was
a stereoisomer of crotonaldehyde-DNPHs because it gave a similar mass spectrum. Both of
them had the most abundant fragment at the m/z of 152 and some significant fragment ions at the
m/z of 172, and 192. The components of the mixture can be figured out by the most abundant
fragment listing in the Table (underlined fragments). The retention time of these four C4
carbonyl-DNPH is as below: crotonaldehyde-2,4-DNPH, 12.11 minutes; methacrolein-2,4-DNPH,
13.15 minutes; methyl ethyl ketone-2,4-DNPH, 14.63 minutes; butyraldehyde-2,4-DNPH, 15.19
minutes.
67


nvn

1350000-
1300000-
125G0GG-
1200000-
tBOOOO
TDGOOO-
U50OGO-
OOOOGO-
950000-
900000-
850000-
800000-
750000-
700000-
850QOO-
60GQQG-
550000-
500000-
450000-
400000-
350000-
300000-
250000-
200000-
50000-
oooco-
50000-
0" l II I 'l l
O 2
nm

T-r
8
fYi"r
13
Time (min)
TT
C

V

Figure 3.20 TOTAL ION CURRENT CHROMATOGRAM FOR
MEK/BUTYRALDEHYDE-DNPHs MIXTURE
r | i it

68


1636
lOOq
SS^
9 65t
8Q-:
7&_
7th
s ^
§ 65^
1 Sth
0 :
1 a^.
r: :
4th
35^
3Ch
25^
2(h
1th
5^
th
i l'i
100
205.5
f'fl I I I I > I I I IT-
200 250 300
RT 14.63
RT 15.19
Figure 3.21 MS2 SPECTRA FOR MEK/BUTYRALDEHYDE-DNPHs
69


Table 3.11 Major Fragments for MEK/Butyraldehyde-DNPHs Mixture
m/z Peak at 14.63min Relative Abundance m/z Peak at 15.19min Relative Abundance
152.5 53.26 120.5 4.41
153.6 3.94 152.6 41.89
178.6 3.11 153.6 23.31
179.5 36.49 163.6 100
191.5 2.75 164.7 17.52
205.5 3.21 179.5 61.03
221.2 100 191.6 4.41
222.2 7.61 205.5 14.09
252.1 18.06 221.3 47.09
251.4 1.88
252.1 84.74
70


Figure 3.22 TOTAL ION CURRENT CHROMATOGRAM FOR
METHACROLEIN/CROTONALDEHYDE-DNPHs MIXTURE
71


RT 11.41 RT 12.11
RT 13.15
Figure 3.23 MS2SPECTRA FOR METHACROLEIN/CROTONALDEHYDE-DNPHs MIXTURE
72


Table 3.12 Major Fragments for Methacrolein/Crotonaldehyde-DNPHs Mixture
m/z Peak at 12.11 min Relative Abundance m/z Peak at 13.15 min Relative Abundance
151.7 35.78 79.4 14.95
152.6 100 122.5 17.15
163.6 39.36 152.4 19.83
167.5 24.05 163.7 100
172.7 81.61 167.5 63.99
173.7 32.04 172.6 25.81
179.6 48.88 173.5 18.24
192.4 58.84 174.5 3.84
202.5 49.02 179.4 20.88
203.4 9.31 202.4 51.76
204.4 10.52 203.3 4.12
214.4 10.67 213.3 7
216.3 2.87 214.4 4.73
218.3 20.57 218.3 19.48
232.2 35.35 232.4 2.19
250.1 66.42 250.2 13.15
73


Table 3.12 (Cont) Major Fragments for Methacrolein/Crotonaldehyde-DNPHs Mixture
Peak at 11.41 min
m/z Relative Abundance
151.7 20.93
152.5 100
163.5 38.01
167.3 6.77
172.5 85.47
173.5 25.74
179.4 55.06
192.2 81.48
202.3 44.31
204.3 5.69
218.4 4.86
231 5.35
232.2 31.52
Figure 3.24 shows the total ion current chromatogram using full scan from a m/z of 50 to 500 for
the mixture of crotonaldehyde-2,4-DNPH, methacrolein-2,4-DNPH, methyl ethyl
ketone-2,4-DNPH, and butyraldehyde-2,4-DNPH. The mixture of C4 carbonyl derivatives were
separated by HPLC using ACN/H20 (v/v 60/40) solvent for 20 minutes. The F1PLC separation
74


condition produced a perfect separation by having 4 peaks. Figure 3.25 displays the MS2 spectra
of these four peaks at the m/z of 249.5, and 251.5, respectively. The peaks can easily be
identified by the significant abundant fragment ions in the spectra.
Figure 3.24 TOTAL ION CURRENT CHROMATOGRAM FOR 4 C4 CARBONYL
DERIVATIVES
75


trtz BVZ
Figure 3.25 MS2 SPECTRA OF THE 4 C4 CARBONYL MIXTURE
76


3.4.3 C5 Carbonyl derivatives
From Table 3.13, it is concluded that valeraldehyde and isovaleraldehyde have similar mass
spectra. The only difference is the fragment at m/z of 191 (appeared in
valeraldehyde-2,4-DNPH) which is the typical fragment for straight chained carbonyls. It can be
used to determine the mixture of these two C5 isomers.
The mixture of valeraldehyde and isovaleraldehyde derivatives were separated by HPLC in a
ACN/H20 (v/v: 70/30) solvent for 20 minutes( flow rate lml/min) before going into MS detector.
In order to distinguish the two components in the mixture, MS2 was used and the selected
fragment was at m/z of 265.3. According to Table 3.14, a peak appeared at 14.41 minutes that
has a fragment ions at the m/z of 191, which means it is the component of valeraldehyde-2,
4-DNPH.
77


Rataft/e Abundance
VALERALDEHYDE
ISOVALERALDEHYDE
Figure 3.26 MS SPECTRA FOR C5 CARBONYL-DNPHs
78


VALERALDEHYDE
ISOVALERALDEHYDE
Figure 3.27 MS2 SPECTRA FOR C5 CARBONYL-DNPHs
Table 3.13 Major Fragments for C5 Carbonyl-DNPHs
VALERALDEHYDE
m/z Ion Relative Abundance
152.4 [M-H-113] 22.63
163.3 [M-H-102] 100
179.3 [M-H-86] 26.46
191.3 [M-H-74] 7.08
235.1 [M-H-30] 61.61
79


Table 3.13 (Cont) Major Fragments for C5 Carbonyl-DNPHs
ISOVALERALDEHYDE
m/z Ion Relative Abundance
152.5 [M-H-113] 22.55
153.5 [M-H-112] 13.61
163.4 [M-H-102] 100
179.2 [M-H-86] 27.61
219.2 [M-H-46] 3.72
235.2 [M-H-30] 39.1
80


v
-c5h9n20 0
m/z 152
Figure 3.28 FRAGMENTATION SEQUENCES FOR ISOVALERALDEHYDE-2,4-DNPH
81


o2n
iN'N
H
o2n
NH
W
m/z 265
m/z 235
m/z 191
m/z 179
m/z 163
-c5h9n2o ^
m/z 152
Figure 3.29 FRAGMENTATION SEQUENCES FOR VALERALDEHYDE-2,4-DNPH
82


uAU
13.68 *-44 14.50
MIXTURE
83


Relative Abundance
1635
100-n
95^
9(h
65-:
eth
75^
7CH
65i
8 =
| 55^
I 5Ch
:
| 45:
* 40^
35^
3oi
25^
20^
1&i
1CH
5i
0^
1525
179.4
191.4
RT 13.6 RT 14.41
Figure 3.31 MS2 SPECTRA FOR C5 CARBONYL-DNPHs MIXTURE
2352
2661
250 300
rrfz
84


Table 3.14 Major Fragments for C5 Carbonyl-DNPHs Mixture
m/z Peak at 13.6 min Relative Abundance m/z Peak at 14.41 min Relative Abundance
101.5 3.43 152.5 35.6
152.5 17.53 153.5 21.54
153.4 17.26 163.5 100
163.4 100 164.4 7.4
164.5 3.07 179.4 32.14
179.3 26.13 191.4 26.1
205.3 3.59 205.4 11.98
218.2 2.64 219.4 3.78
235.2 52.24 220.3 3.69
235.2 62.68
266.1 2.79
85


3.5 Analysis of Samples by HPLC-MS
Figure 3.32 displays the total ion current chromatogram for 15 carbonyls mixture standard under
full scan between the m/z 50 to 500. Under a more complex condition, it is very difficult to
make out more than the [M-H] fragment. For this reason, identification of isomers is almost
impossible. Table 3.15 is a list of the components which can be detected in the mixture standard.
Under these conditions, m-tolualdehyde-2,4-DNPH and p-tolualdehyde-2,4-DNPH can not be
separated.
Figure 3.32 TOTAL ION CURRENT CHROMATOGRAM FOR THE 15 MIXTURE
STANDARD
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