NAPHTHENIC ACIDS CATEGORY ANALYSIS AND HAZARD CHARACTERIZATION
SPECTROMETRIC IDENTIFICATION OF NAPHTHENIC ACIDS
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SPECTROMETRIC IDENTIFICATION OF NAPHTHENIC ACIDS ISOLATED FROM CRUDE OIL THESIS Presented to the Graduate Council of Texas State University-San Marcos in Partial Fulfillment of the Requirements for the Degree Master of SCIENCE by Pratap Rikka, B. Pharm. San Marcos, Texas December 2007
Transcript of SPECTROMETRIC IDENTIFICATION OF NAPHTHENIC ACIDS
SPECTROMETRIC IDENTIFICATION OF NAPHTHENIC ACIDS
ISOLATED FROM CRUDE OIL
THESIS
Presented to the Graduate Council of Texas State University-San Marcos
in Partial Fulfillment of the Requirements
for the Degree
Master of SCIENCE
by
Pratap Rikka, B. Pharm.
San Marcos, Texas December 2007
SPECTROMETRIC IDENTIFICATION OF NAPHTHENIC ACIDS
ISOLATED FROM CRUDE OIL
Committee Members Approved:
Walter Rudzinski, Chair
Chang Ji
Kevin Lewis
Approved:
J. Michael Willoughby
Dean of the Graduate College
ACKNOWLEDGEMENTS
I would like to express my greatest gratitude and heartfelt thanks to my parents,
Sumathi and Late Lokanath Rao, for their love and support all through my life. I would
like to take this occasion to thank my siblings, Sujatha and Shivaji, for all the advice and
moral support in completing my thesis.
I would like to gratefully acknowledge my thesis advisor Dr. Walter Rudzinski,
Professor of Analytical Chemistry, Texas State University-San Marcos, for giving me an
opportunity to work in his labs. It has been a great experience working with him and
learning the concepts of analytical science. He is not only a great scientist but also a great
man to be with. I thank him for all the help and support; he has given during my graduate
studies of my life.
I extend my thanks to my committee members Dr. Chang Ji, Assistant Professor
of analytical Chemistry and Dr. Kevin Lewis, Assistant Professor of Bio-Chemistry,
Texas State, for being on the committee.
Special thanks to Maheedhar Guntupalli for helping me with the NMR
experiments and the moral support while working on my thesis. Thanks to Michael for
helping me in my research.
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I sincerely thank Dr Jitendra Tate, Assistant Professor of Mechanical Engineering,
College of Engineering, Texas State, for giving me a chance to do research in his lab and
the support he has offered to continue my graduate studies.
I would also thank the Department of Chemistry and Biochemistry for giving me
the opportunity to complete my graduate studies.
Finally, I would like to thank all the fellow students, my friends, philosophers and
guides for making my thesis a great success.
This manuscript was submitted on August 17th, 2007.
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TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS............................................................................................... iii
LIST OF TABLES........................................................................................................... viii
LIST OF FIGURES ........................................................................................................... ix
CHAPTER
1. INTRODUCTION ...........................................................................................................1
1.1. Naphthenic acids...............................................................................................1
1.2. Problems associated with naphthenic acids ......................................................4
1.3. Techniques for the isolation of naphthenic acids from crude oil......................4
1.4. TAN number .....................................................................................................5
1.5. Characterization of naphthenic acids using spectroscopic techniques .............6
1.5.1. MS (Mass Spectrometry) ...................................................................6
1.5.2. NMR (Nuclear Magnetic Resonance)................................................9
1.5.3. IR (Infrared) .......................................................................................9
1.6. Extraction of naphthenic acids with ammoniated ethylene glycol and characterization using MS, NMR and IR................................................9
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2. EXPERIMENTAL..........................................................................................................11
2.0. Materials .........................................................................................................11
2.1. Isolation of naphthenic acids ..........................................................................11
2.1.1. Extraction Protocol .............................................................................12
2.1.2. TAN number .......................................................................................14
2.2. Mass Spectrometry (MS) ................................................................................15
2.2.1. ESI (-) MS Experiments.......................................................................15
2.2.2. ESI (-) MS/MS Experiments................................................................16
2.3. Nuclear Magnetic Resonance (NMR) Spectroscopy ......................................16
2.4. Infrared Spectroscopy (IR) .............................................................................17
3. RESULTS ......................................................................................................................19
3.1. Isolation of Naphthenic acids..........................................................................19
3.1.1. Extraction yield...................................................................................19
3.1.2. TAN number .......................................................................................19
3.2. Mass Spectrometry (MS) ................................................................................20
3.2.1. MS and MS/MS of standard compounds ............................................20
3.2.2. MS and MS/MS of Merichem.............................................................22
3.2.3. MS and MS/MS of naphthenic acids extracted from 5% Merichem in Tufflo...........................................................27 3.2.4. MS and MS/MS of naphthenic acids extracted from crude oil...................................................................................32 3.2.5. MS and MS/MS of naphthenic acids extracted
from crude oil: samples run in 20% dichloromethane / 80% acetonitrile ...............................................................................37
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3.3. Nuclear Magnetic Resonance (NMR) Spectroscopy ......................................40
3.3.1. NMR of Merichem..............................................................................40
3.3.2. NMR of 5% Merichem in Tufflo after extraction...............................42
3.3.3. NMR of Tufflo supernatant after contact with ethylene glycol ............................................................................... 43 3.3.4. NMR of ethylene glycol after removal of ammonia...........................44
3.3.5. NMR of naphthenic acids extracted from crude oil............................44
3.3.6. NMR of crude oil supernatant after contact with ethylene glycol .................................................................................45 3.3.7. NMR of ethylene glycol after contact with crude oil after removal of ammonia................................................................46
3.4. Infrared Spectroscopy (IR) .............................................................................47
3.4.1. IR of polyethylene and petroleum ether..............................................47
3.4.2. IR of Merichem...................................................................................47
3.4.3. IR of 5% Merichem in Tufflo extraction ............................................48
3.4.4. IR of naphthenic acids extracted from crude oil .................................49
4. CONCLUSIONS............................................................................................................50
REFERENCES ..............................................................................................................52
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LIST OF TABLES
Table Page
1. ESI(-) ion mode MS/MS analysis of naphthenic acids standards....................................7
2. MS/MS results for naphthenic acids extracted from crude oil run in acetonitrile ........................................................................................................33 3. MS/MS results for naphthenic acids extracted from crude oil in 20% dichloromethane / 80% acetonitrile. The fragment ions are arranged in decreasing order of intensity..............................................................38
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LIST OF FIGURES
Figure Page
1-1: Cyclopentane and Cyclohexane carboxylic acids ........................................................2
1-2: Aromatic naphthenic acids ...........................................................................................2
1-3: Z in relation to ring structure........................................................................................3
1-4: MS/MS of 1,2,3,4- Tetrahydro-2-naphthoic acid showing a loss of water, carbon monoxide and carbon dioxide ................................8 1-5: MS/MS of 5β-cholanic acid showing a loss of water, carbon monoxide and carbon dioxide..................................................8 2-1: Overview of the extraction protocol...........................................................................14
3-1: MS/MS of Benzoic acid (121Da) in acetonitrile using ESI negative ion mode with mass range 50-125 m/z...............................................20 3-2: MS/MS of Butylbenzoate (178m/z) using ESI in negative ion mode with mass range 50-182 m/z in acetonitrile...............................21 3-3: MS/MS of 1-decanesulfonic acid using ESI in negative ion mode in the mass range 60-225 m/z in methanol ..............................................22 3-4: Merichem in ESI negative ion mode in the mass range 180-425 m/z in acetonitrile ................................................................23 3-5: Merichem in ESI negative ion mode in the mass range 256-350 m/z in acetonitrile ...................................................................23
3-6: MS/MS of 245.1 m/z for Merichem in ESI negative ion mode within the mass range 100-248 m/z in acetonitrile ..................................24 3-7: MS/MS of 287.1 m/z for Merichem in ESI negative ion mode with the mass range 100-290 m/z in acetonitrile .....................................25 3-8: MS/MS of 301.1 m/z for Merichem in ESI negative ion mode with mass range 100-305 m/z. in acetonitrile ..........................................26 3-9: MS/MS of 313.2 m/z for Merichem in ESI negative ion mode with mass range 100-317 m/z. in acetonitrile ..........................................26 3-10: 5% Merichem in Tufflo Extract in ESI negative ion mode in the mass range 200-400 m/z run in acetonitrile..................................27 3-11: MS/MS of 245.2 m/z from the Merichem extract in ESI negative ion mode within a mass range of 65-248 m/z in acetonitrile ......................................................................................28 3-12: MS/MS of 287.3 m/z from the Merichem extract in ESI negative ion mode within a mass range of 75-290 m/z in acetonitrile ...........29 3-13: MS/MS of 301.2 m/z from Merichem extract in ESI negative ion mode within the mass range 80-305 m/z. in acetonitrile............30
3-14: MS/MS of 313.2 m/z from Merichem extract in ESI negative ion mode within the mass range 85-315 m/z in acetonitrile.............30
3-15: MS of naphthenic acids extracted from crude oil run in acetonitrile, negative ion mode within the mass range 100 m/z to 440m/z...........32 3-16: MS/MS of 241.2 m/z of naphthenic acid extract from crude oil with a mass range 100-245 m/z.....................................................34 3-17: MS/MS of 187.2 m/z of naphthenic acid extract from crude oil with a mass range 50-190 m/z........................................................34 3-18: MS/MS of 335.3 m/z of naphthenic acid extract from crude oil within a mass range 100-340 m/z ..................................................35 3-19: MS/MS of 347.2 m/z of naphthenic acid extract from crude oil within a mass range 100-350 m/z ..................................................35 3-20: MS/MS of sulfur compound extracted from crude oil in acetonitrile in negative ion mode with a mass range 60 m/z to 220m/z ................36
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3-21: MS of naphthenic acids extracted from crude oil in 20% dichloromethane / 80% acetonitrile with 0.5% ammonia run in negative ion mode..............................................................................................37
3-22: MS in positive ion mode of naphthenic acids extracted from crude oil in 20% dichloromethane / 80% acetonitrile with a mass range of 180 m/z to 800m/z ..........................................................................40 3-23: 1H NMR of Merichem..............................................................................................41
3-24: 13C NMR of Merichem.............................................................................................42
3-25: 1H NMR of 5% Merichem in Tufflo after extraction ..............................................42
3-26: 1H NMR of Tufflo supernatant after contact with ethylene glycol...............................................................................................43
3-27: 1H NMR of ethylene glycol layer after extraction of Merichem and boil off of ammonia...................................................................44 3-28: 1H NMR of naphthenic acids extracted from crude oil ............................................44
3-29: 1H NMR of Crude oil fraction after extraction.........................................................45
3-30: 1H NMR of ethylene glycol after contact with crude oil after removal of ammonia.......................................................................46
3-31: Background IR of polyethylene and petroleum ether...............................................47
3-32: IR of Merichem dissolved in petroleum ether on a Polyethylene film...................................................................................................48
3-33: IR of 5% Merichem in Tufflo after extraction in petroleum ether on a polyethylene film .................................................................48 3-34: IR of Naphthenic acid extract from crude oil in petroleum ether on a polyethylene film ................................................................49
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CHAPTER 1
INTRODUCTION
1.1 Naphthenic acids
Napthenic acids, originally identified as carboxylic acids1 in crude oils with single
or multiple saturated ring structures, is the phrase loosely used to include all acidic
components in crude oils, even including aromatic carboxylic acids.2 Petroleum products
have a complex composition3-10, most of the compounds cannot be easily characterized
due to their compositional complexity, and they also have a variable range of
concentration based on their geographical distribution.11
Refined naphthenic acids are transparent, oily liquids. They are obtained by distilling the
petroleum fractions.12 Naphthenic acids are mainly cyclopentane derivatives with a
smaller fraction based on the cyclohexane ring (Figure 1-1). In fact, naphthenic acids are
a mixture of different compounds which may be polycyclic and may have unsaturated
bonds, aromatic rings, and hydroxyl groups. The physical and chemical properties depend
on the nature and source of the processed petroleum. As an example, Romanian
naphthenic acids have a very low aromatic and phenolic compound content, usually less
than 60 ppm.
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2
FIGURE 1-1: Cyclopentane and Cyclohexane carboxylic acids.
Empirical formula and structure of napthenic acids are complex mixtures of alkyl-
substituted acyclic and cycloaliphatic carboxylic acids. The empirical formula for the
acids may be described by Cn H2n+Z O2 13-17, where the Z value1 is the “hydrogen
deficiency” and is a negative, even integer, and more than one isomer will typically exist
for a given Z homologue.
FIGURE 1-2: Aromatic naphthenic acids.
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The complex composition is polydisperse with varying molecular weights and
structure differences.11, 18-22 Examples of the types of molecular structures that are
thought to comprise naphthenic acids are shown in Figure1-2 and Figure1-3. However
individual acids are difficult to be isolated from the mixture.
FIGURE 1-3: Z in relation to ring structure.
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1.2 Problems associated with naphthenic acids
Napthenic acids in crude oils are of concern due to their corrosivity to refinery
units.23-25 It is highly desirable to determine the ring-type distribution and the carbon
number distribution of each ring type because the corrosivity of napthenic acids is
dependent on their size and structure. The characterization of napthenic acids is also of
interest in geochemical studies, particularly migration and biodegradation26-28, and to
refinery wastewater treatment for environmental compliance.
1.3 Techniques for the isolation of naphthenic acids from crude oil
There are various chromatographic and solvent extraction methods used to isolate
napthenic acids from crude oils. One of the methods of obtaining the carboxylic acid
fraction from crude oil includes simple base extraction or adsorption onto a KOH
impregnated silica gel.29 A non-aqueous ion exchange method30-31 has also been
developed to separate liquid fossil fuels into acid, base, and neutral concentrates. The
acid concentrate obtained in this way is then separated by HPLC into sub-fractions using
tetralkylammonium hydroxide modified silica gel.32
There are difficulties in extracting napthenic acids. For example, aqueous caustic
extraction cannot be used with acids greater than C20 because they form emulsions.
Isolation efficiency is also difficult to assess because of the formation of salts in solution
and the co-extraction of weak acids like phenol and carbazoles.
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Solid phase extraction29 (SPE) is a fairly recent approach for the efficient
extraction of napthenic acids. SPE is attractive because of stationary phase uniformity,
low solvent consumption, and the potential to exploit specific interactions in order to
separate polar hydrocarbons from non-polar hydrocarbons. The sample is absorbed on a
quaternary amine ion exchange column, and then washed with hexane to remove the
hydrocarbons. The aromatics and polar species are eluted with a solvent, or a mixture of
solvents of higher eluotropic strength, e.g. toluene, dichloromethane, or acetone. The
absorbed acids are then removed by washing with diluted acids (e.g. formic or acetic
acid) and then reconstituted in an appropriate solvent.
1.4 TAN number
Crude oil typically contains napthenic acids in quantities of up to 4 wt %. One of
the important markers used to gauge the acidity of oils is the “total acid number” (TAN),
which is defined as the mass of potassium hydroxide (in milligrams) required to
neutralize one gram of crude oil. Crude oils are considered acidic if their TAN exceeds
0.5 mg KOH/g by non-aqueous titration33. The TAN of oil has frequently been used to
quantify the presence of napthenic acids, because the carboxylic acid components of oils
are believed to be largely responsible for oil acidity. However, more recent research has
begun to highlight deficiencies in relying upon this method for such a direct correlation
and the total acid number is no longer considered to be such a reliable indicator. It is
therefore essential that a more reliable method should be found for isolating and
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characterizing the naphthenic acids in crude oil in order for corrosion to be better
understood.
1.5 Characterization of naphthenic acids using spectroscopic techniques
Fossil fuels and complex hydrocarbon mixtures have long been investigated by mass
spectrometry, and various separation methods have been evaluated for general use with
complex hydrocarbon mixtures. One of the greatest difficulties posed when
characterizing the naphthenic acids found in crude oils is that it is not possible to extract
as many naphthenic acid species as separate components for analysis, and the resulting
mass spectra are, therefore, very complex.
1.5.1 MS (Mass Spectrometry)
MS is well suited for characterization of acidic components at the molecular level.14,
17, 22, 11, 34-37 Due to their polarity, acids are commonly converted to their corresponding
esters for detailed GC/MS38 analysis. However, direct acid analysis is more desirable
because it can provide rapid analysis without the concern of losing material through
derivatization. Several MS ionization methods have previously been developed for such
purposes, including fluoride-ion chemical ionization13, negative-ion fast atom
bombardment (FAB)14, chemical ionization (CI), and liquid secondary-ion mass
spectroscopy (LSI-MS). We have evaluated alternative soft ionization techniques,
including atmospheric pressure chemical ionization (APCI)17, electrospray ionization
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(ESI)36,39, and nanospray ionization (nanoESI)40, which can yield molecular or pseudo-
molecular ions for the determination of the molecular distribution of napthenic acids.
When ESI coupled with a MS/MS experiment is extremely valuable in the
determination of the molecular weight distribution as well as confirmation of the identity
of napthenic acid samples. Carboxylic acids generate neutral losses of 44, 28 and 18
Dalton that can be respectively attributed to M-CO2, M-CO, and M-H2O.34 Table 134
shows an MS/MS analysis of naphthenic acids in negative ion mode. Figure1-4 and
Figure1-5 show the representative spectra that illustrate the fragmentation that can be
observed in MS/MS spectra.
TABLE - 1: ESI (-) ion mode MS/MS analysis of naphthenic acids standards. Table
taken from reference 34.
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FIGURE 1-4: MS/MS of 1,2,3,4- Tetrahydro-2-naphthoic acid showing a loss of water,
carbon monoxide and carbon dioxide. Figure taken from reference 34.
FIGURE 1-5: MS/MS of 5β-cholanic acid showing a loss of water, carbon monoxide
and carbon dioxide. Figure taken from reference 34.
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1.5.2 NMR (Nuclear Magnetic Resonance)
Nuclear magnetic resonance spectroscopy11, 42, 43 can give information about the
functional groups present in a sample. In 1H NMR, the naphthenic acids present in crude
oil will exhibit a peak that can be attributed to carboxylic acid hydrogen as well as peaks
that can be attributed to hydrogens in aromatic and aliphatic hydrocarbons. 13C NMR can
also be used to identify carbonyl, aromatic and primary, secondary and tertiary carbons
associated with aliphatic hydrocarbons.41
1.5.3 IR (Infrared)
Infrared spectroscopy31 can also give information about the functional groups present
in a sample. Naphthenic acids from crude oil should show a broad OH and a sharp C=O
stretch associated with a carboxylic acid group, C-O-H out of plane and O-H out of plane
bending.
1.6 Extraction of naphthenic acids with ammoniated ethylene glycol and
characterization using MS, NMR and IR
Recently ammoniated ethylene glycol has been reported as an efficient extraction
medium for carboxylic acids44. Though the reported protocol seemed to extract
naphthenic acids, no verification was included that naphthenic acids and only naphthenic
acids were extracted. A TAN number was reported but under the conditions of titration,
10
acids may have been produced from esters formed during the extraction protocol. In this
project the extraction efficiency of the procedure will be evaluated. A series of analytical
techniques including mass spectrometry, nuclear magnetic resonance, and infrared
spectroscopy will also be used to characterize the naphthenic acids extracted from
ammoniated ethylene glycol.
CHAPTER 2
EXPERIMENTAL
2.0 Materials
Petroleum crude oil was obtained from Chevron Texaco, Houston. Merichem
standard, and Tufflo were donated by Exxon-Mobil research and engineering (Baton
Rouge, LA). Benzoic acid, butyl benzoate, 1-decane sulfonic acid, acetone, deutrated
chloroform, deutrated acetone, deutrated dichloromethane were obtained from Sigma-
Aldrich (St. Louis, MO) and used without further purification. Methanol, ethanol,
dichloromethane, acetonitrile, acetic acid, and ammonium hydroxide were all
chromatographic grade and obtained from EM Science (Gibbstown, NJ).
Phenolphthalein, ethylene glycol, and petroleum ether were obtained from J T Baker
(Phillipsburg, NJ), Matheson Coleman & Bell (Norwood, Ohio), and Mallinckrodt (St.
Louis, MO) respectively.
2.1 Isolation of naphthenic acids
Evidence from earlier studies on crude oil indicates the presence of up to 5%
naphthenic acids in crude oil samples from different parts of the world. A naphthenic acid
11
12
standard mix and a naphthenic acid extract from a crude oil have been prepared in order
to evaluate the extract efficiency of ammoniated ethylene glycol. In addition, ESI/MS and
tandem mass spectrometry, (MS/MS) as well as NMR and IR (when applicable), have
been used to identify the classes of chemical compounds.
2.1.1 Extraction Protocol
Tufflo is a crude oil simulates (generously donated by Exxon-Mobil) that contains
aliphatic and aromatic hydrocarbons in a ratio similar to that found in crude oils.
Merichem is a standard mixture of naphthenic acids. A control mixture that mimics the
properties of petroleum crude oil was prepared by adding Merichem to Tufflo. 100
milligrams of Merichem was added to 1.9 grams of Tufflo, followed by sonication for 10-
15 minutes to attain a uniform 5% Merichem in Tufflo solution. Tufflo is a clear viscous
liquid and Merichem is a dark yellow viscous liquid; upon sonication, the whole mixture
turns into a uniform light yellow liquid. Anhydrous ammonia was bubbled at a constant
flow rate through 9-10 ml of ethylene glycol in a three-necked, round bottom flask
maintained at 0 °C in an ice bath. It usually takes about 1.5 - 2 hours for the solution to
be saturated.
Two grams of 5% Merichem in Tufflo or 2 g of petroleum crude oil was added to
9-10 ml of ammoniated ethylene glycol and then stirred for 1 hour at 0 °C. The whole
system was transferred into a single neck round bottomed flask capped with a rubber
stopper, then heated at 60-65 °C for an hour. The solution was then transferred into a
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separating funnel and, upon cooling in about 20-25 minutes, two layers formed: an oil
phase (top layer) and an ethylene glycol phase (bottom layer). (Note: If the ammonium
naphthenate salts in the ethylene glycol phase are left too long in the separation funnel,
they may go back into the oil phase). The top layer was then discarded, and the ethylene
glycol layer, which contains ammoniated naphthenate salts, was then transferred into a
round bottom flask and heated at 130-135 0C using an oil bath for 1.5 – 2 hours or until
no ammonia gas evolves; the ethylene glycol layer turns to a yellow color on heating.
The heat removes the ammonia, which has a low boiling point, from the ammoniated
naphthenate salt, and the naphthenic acids that have high boiling points remain
undecomposed. The ethylene glycol layer was transferred to a separatory funnel while
hot and left to cool to room temperature.
Five-ten ml of petroleum ether was added to the funnel and all the naphthenic
acids are extracted from ethylene glycol. The suspension was left to settle for 15-20
minutes. The bottom layer (ethylene glycol) was discarded. The petroleum ether layer
containing the naphthenic acids was then washed with 3-5 ml of water which removes the
residual ethylene glycol from the petroleum ether. The naphthenic acids are obtained by
evaporation of the solvent at 40 0C for 5-8 minutes. The end result was a dry extract of
naphthenic acids. Figure 2-1 illustrates the entire sequence of steps.
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Ethylene glycol
Bubble with ammonia gas for 1.5-2 hours Ethylene glycol saturated
with ammonia
Add crude oil
Naphthenic acids in crude oil react with ammonia in ethylene glycol solution to form ammoniated naphthenic acid
The process is carried out in an ice bath
Top layer is non-polar contains crude oil remnants
Bottom layer is polar with naphthenicacids in ethylene glycol
Heated at 1300 C for 2 hours
Bottom layer is ethylene glycol which has more species
Top layer (supernatant) contains insoluble naphthenic acids
Naphthenic acids are extracted into petroleum ether followed by a water wash to remove ethylene glycol
FIGURE 2-1: Overview of the extraction protocol.
2.1.2 TAN number
Naphthenic acids were extracted from either 2 g of petroleum crude oil or the 5%
Merichem in Tufflo mixture using the ammoniated ethylene glycol extraction procedure.
The naphthenic acids from the crude oil or the Merichem extract were titrated with 0.01
N KOH in ethanol solution using phenolphthalein indicator. The TAN number is
15
reported as the mass of potassium hydroxide (in milligrams) required to neutralize one
gram of the 5% Merichem in Tufflo mixture or petroleum crude oil.
2.2 Mass Spectrometry (MS)
MS and MS/MS experiments were performed using a Thermo Finnigan Mass
Spectrometer controlled by Tuneplus software (Excalibur), with the ESI (Electrospray
Ionization) source in negative ion mode. The concentration of the sample solutions was
1-1.5 mg of naphthenic acid extract in 2 ml of solvent; which was acetonitrile, methanol
or dichloromethane. The experiments were performed without 0.5% of ammonia in
solution except for naphthenic acids extracted from crude oil that are run in 20% / 80%
dichloromethane / acetonitrile.
2.2.1 ESI (-) MS Experiments
The sample flow rate was 4 µL – 6 µL/min; the mass scan range was between 180
to 450 m/z; mass spectra were based on 3 microscans with a 50 msec ion injection time;
automatic gain control was enabled. The sheath gas was maintained at 60 psi, the
temperature of the API stack at 220 ° C, the capillary voltage at 7 V, and the spray
voltage at 3.15 kV.
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2.2.2 ESI (-) MS/MS Experiments
For all ions, the collision activated dissociation (CAD) energy was set in such a
way that the MS/MS scan of the precursor and fragment ions could both be detected.
CAD energy was between 20-40 %. The optimum peak isolation width was 1 Da,
(whereas a wider width (more than1 Da) resulted in fragmentation of ions of species with
m/z near that of the precursor ion.) Activation Q was 0.25 and activation time is 30 msec.
MS and MS/MS experiments were performed on a series of standards: benzoic
acid, butyl benzoate, decanesulfonic acid and, a Merichem naphthenic acid standard mix.
MS and MS/MS experiments were also performed on the extract of 5% Merichem
in Tufflo and the naphthenic acids extracted from crude oil.
2.3 Nuclear Magnetic Resonance (NMR) Spectroscopy
All the NMR experiments were performed on a Varian 400 MHz NMR
spectrometer equipped with a Sun workstation. 13C NMR spectral data were collected at a
frequency of 100 MHz.
Merichem: 100 mg of Merichem was dissolved in 2ml of deuterated chloroform.
1H and 13C NMR spectra were collected.
17
Fractions of 5% Merichem in Tufflo: 1H NMR experiments were performed for
5% Merichem in Tufflo after extraction, Tufflo supernatant after contact with ethylene
glycol, and the ethylene glycol layer after removal of ammonia. 50 mg of each fraction
was dissolved in a deuterated solvent.
Fractions of crude oil: 1H NMR experiments were performed on naphthenic acids
extracted from crude oil, crude oil supernatant after contact with ethylene glycol, and
ethylene glycol after contact with crude oil after removal of ammonia. 55 mg of each
fraction was dissolved in a deuterated solvent.
All the fractions were dissolved in 2ml of deuterated chloroform except for the
ethylene glycol fraction which was dissolved in deuterated acetone.
2.4 Infrared Spectroscopy (IR)
Fourier transform infrared (FTIR) experiments were performed using Perkin-
Elmer FTIR (model 1600) instrument controlled by Spectrum One software. All spectra
were obtained in the absorbance mode with a scan range of 4000-450 cm-1 and a
resolution of 4 cm-1 for 10 scans Samples were prepared by dissolving 5-6 mg of
Merichem standard mix, 5% Merichem in Tufflo extract, or naphthenic acid extract from
petroleum crude oil in 2 mL of petroleum ether. A drop of each was placed on a
polyethylene film. The petroleum ether was allowed to evaporate. Background spectra
were collected for polyethylene and polyethylene and petroleum ether mix.
18
Petroleum ether was not used to dissolve ethylene glycol, water and crude oil
samples. These samples were in the liquid state when applied to the polyethylene film.
CHAPTER 3
RESULTS
3.1 Isolation of Naphthenic acids
3.1.1 Extraction yield
The extraction yield for the 5% Merichem was determined to be 80%.
By subtracting weight of final extract from initial weight of 5% merichem in
tufflo used and dividing with the initial weight of 5% merichem. There was a possibility
that the extract may have impurities from crude oil and also from the chemicals used in
the extraction process.
3.1.2 TAN number
7.56 mg of KOH in 0.01 N ethanol was required to neutralize 8 mg of Merichem
naphthenic acid standard mix (TAN = 0.945).
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20
3.2 Mass Spectrometry (MS)
3.2.1 MS and MS/MS of standard compounds
The MS and MS/MS study was performed on a standard carboxylic acid, an ester
and a sulfonic acid to understand the characteristic fragmentation patterns of these
compounds. Benzoic acid: The MS/MS experiment on a carboxylic acid (benzoic acid)
was run to observe the fragmentation pattern. There is a mass loss of 44Da due to carbon
dioxide loss shown by benzoate, leaving a fragment ion peak at 77 Da (benzene ring) 34
(Figure 3-1).
FIGURE 3-1: MS/MS of benzoic acid (122Da) in acetonitrile using ESI negative ion
mode with mass range 50-125 m/z.
Ester compound: Heating an acid at high temperature (120-180 °C) in presence of
a base/ water can favor the formation an ester. Our extraction procedure favors the
formation of esters from acids in the crude oil. MS/MS experiments were performed on
21
butylbenzoate45 with 179 m/z in acetonitrile in negative ion mode and showed a peak at
79.9m/z (Figure 3-2).
60 70 80 90 100 110 120 130 140 150 160 170 180m /z
0
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79.9
179.1
89.4 112.980.8 142.1117.270.7 123.6102.9 137.765.253.2 150.5110.564.3 83.7 172.5169.8162.395.178.7 128.7
[M-H]-
FIGURE 3-2: MS/MS of butylbenzoate (178m/z) using ESI in negative ion mode with
mass range 50-182 m/z in acetonitrile.
Decanesulfonic acid: (Figure 3-3) MS/MS experiment was performed on a
standard sulfur compound (1-decanesulfonic acid) in order to observe the fragmentation
pattern of the precursor ion. In negative ion mode, the precursor of 1-decanesulfonic acid
of molecular weight 222 (sodium salt peak with molecular mass of 244), showed a peak
at 221m/z in methanol, and the MS/MS experiment showed a peak at 79.9m/z, which
corresponds to SO3.
22
70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220m /z
0
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79.9
221.1
80.9 184.5151.6206.364.9 188.978.6 155.0148.9 164.0
SO3 = [M-H-C10H22]-
[M-H]-
FIGURE 3-3: MS/MS of 1-decanesulfonic acid using ESI in negative ion mode in the
mass range 60-225 m/z in methanol.
3.2.2 MS and MS/MS of Merichem
A number of peaks in the range of 200 to 400 m/z were found for the ESI
negative ion mode mass spectrum of Merichem extract. The distribution appears very
Gaussian with a maximum at around 300 and a spacing of 14 Da (due to (–CH2) between
most of the large peaks. MS spectrums are shown in Figures 3-4 and 3-5.
23
-m arch3m erichem inACN #233-417 RT: 2.77-4.93 AV: 185 NL: 1.79E4T: - p Full m s [ 100.00-500.00]
200 220 240 260 280 300 320 340 360 380 400 420m /z
0
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299.1285.1
301.1
287.1
315.1313.1271.1
273.1297.2
283.1
329.1311.1
319.2269.1
259.2 343.2
347.1255.1 361.1
371.1245.1241.1 373.1
389.2235.2 399.1225.2 403.2 425.1
221.1209.2195.1
FIGURE 3-4: Merichem in ESI negative ion mode in the mass range 180-425 m/z in
acetonitrile.
-march3mericheminACN #177-375 RT: 2.10-4.43 AV: 199 NL: 1.70E4T: - p Full ms [ 100.00-500.00]
260 270 280 290 300 310 320 330 340 350m/z
5
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285.1 299.1301.1
287.1
315.2
271.1313.1
273.1283.1 297.2
329.1311.1327.1
303.1325.2319.1307.2269.1
341.1289.2259.1 293.2 333.2 343.2
279.1 335.2321.2345.1
267.2 275.2265.1 349.1
351.1261.1
FIGURE 3-5: Merichem in ESI negative ion mode in the mass range 256-350 m/z in
acetonitrile.
To identify whether the extraction of the Merichem actually yielded naphthenic
acids, we performed MS/MS experiments on the Merichem naphthenic acid mix focusing
24
on all of the more intense common peaks. The MS/MS of Merichem peaks that were
chosen for MS/MS experiments had a relative abundance of 40 and higher in the MS
spectrum. The results showed mass losses of M-1, M-18, M-28, and M-44, which
corresponds to a loss of hydrogen, water, carbon monoxide, and carbon dioxide.
-24 5 .1 m s m s m arch 3m e riche m in AC N #1 7 -37 R T: 0 .3 6 -0 .77 AV: 2 1 N L : 1 .04 E3T: - p Fu ll m s 2 24 5 .10 @ 35 .0 0 [ 10 0 .00 -2 48 .0 0 ]
11 0 12 0 13 0 14 0 15 0 16 0 17 0 18 0 19 0 2 0 0 2 1 0 2 2 0 2 3 0 2 4 0m /z
0
5
1 0
1 5
2 0
2 5
3 0
3 5
4 0
4 5
5 0
5 5
6 0
6 5
7 0
7 5
8 0
8 5
9 0
9 5
1 00
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2 27 .1
1 83 .0
2 45 .22 01 .1
2 17 .3
2 11 .2
1 98 .9 2 10 .61 89 .0 2 02 .61 65 .0 1 97 .11 63 .2 1 67 .1 1 81 .1 2 30 .11 4 3 .51 0 7 .4 2 09 .0 2 20 .515 6 .91 1 3 .0 11 9 .2 2 43 .11 38 .9
[M-H-H20]-
[M-H]-
[M-H-C0]-
[M-H-CO2]-
FIGURE 3-6: MS/MS of 245.1 m/z for Merichem in ESI negative ion mode within the
mass range 100-248 m/z in acetonitrile.
25
-287.1m s m s m arch3m erichem inAC N #17-53 R T: 0 .35 -1 .12 AV: 37 N L : 2 .88E3T: - p Fu ll m s 2 287.10@ 35.00 [ 100.00-290 .00 ]
110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290m /z
0
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269 .1
225.2243.1
253 .1287 .3
259.0237 .1223 .4209 .1205 .2187.1 244 .9 271 .9160 .9138 .1 285.3104 .6 175 .1127.3 267 .0114.8 145 .0
[M-H]-
[M-H-H20]-
[M-H-C0]-
[M-H-CO2]-
FIGURE 3-7: MS/MS of 287.1 m/z for Merichem in ESI negative ion mode with the
mass range 100-290 m/z in acetonitrile.
Figures 3-6, 3-7, 3-8, and 3-9, illustrate representative MS/MS spectra for peaks
at 245, 287, 301, and 313 (found in the MS spectrum), which confirm that these are due
to naphthenic acids. There was a mass loss of M-34 and M-62 from the parent molecule
which have not been identified. The M-1 hydrogen loss can be seen in each spectrum.
The M-18 water loss was prominent and was found in all of the spectra. The mass loss of
M-28 corresponding to carbon monoxide was small in some of the MS/MS spectra. All of
the spectra exhibited a mass loss of M-44.
26
-301.1msmsmarch3merichem inACN #25-56 RT: 0.52-1.20 AV: 32 NL: 3.27E3T: - p Full ms2 [email protected] [ 100.00-304.00]
120 140 160 180 200 220 240 260 280 300m/z
0
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45
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283.1
239.1267.1257.2
301.3
237.1223.1 273.2251.3 286.3154.9 218.9169.1 205.7189.0180.8144.9125.1107.6 121.1 140.7
[M-H]-
[M-H-H20]-
[M-H-C0]-
[M-H-CO2]-
FIGURE 3-8: MS/MS of 301.1 m/z for Merichem in ESI negative ion mode with mass
range 100-305 m/z in acetonitrile.
-313.1m s m s m arch3m erichem inACN #18-46 RT: 0.38-0.99 AV: 29 NL: 1.66E3T: - p Full m s 2 [email protected] [ 100.00-316.00]
120 140 160 180 200 220 240 260 280 300m /z
0
5
10
15
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45
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295.1
251.1
269.1
279.1 313.3
263.0 285.2249.0211.0205.2 232.9 308.1198.5132.7 160.2 213.9153.3112.9 144.8 168.8 185.5123.2
[M-H]-
[M-H-H20]-
[M-H-C0]-
[M-H-CO2]-
FIGURE 3-9: MS/MS of 313.2 m/z for Merichem in ESI negative ion mode with mass
range 100-317 m/z in acetonitrile.
27
3.2.3. MS and MS/MS of naphthenic acids extracted from 5% Merichem in
Tufflo
To identify whether the extraction of the Merichem actually yielded naphthenic acids,
we performed MS (see Figure 3-10) and MS/MS experiments on the 5% Merichem in
Tufflo extract. Merichem peaks chosen for MS/MS experiments had a relative abundance
of 40 and higher in the MS spectrum.
-msACN12feb #124-224 RT: 1.37-2.47 AV: 101 NL: 2.40E4T: - p Full ms [ 100.00-500.00]
220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390m/z
0
5
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293.2
279.4 335.3
323.3
313.3307.4 331.3251.4 287.3295.3220.3
301.2265.3317.3
275.3237.3337.4267.4221.4 351.2
223.3341.3
363.3263.5
357.3 383.3239.3235.3 371.2 397.4245.3387.3225.3 377.0211.3
FIGURE 3-10: 5% Merichem in Tufflo Extract in ESI negative ion mode in the mass
range 200-400 m/z run in acetonitrile.
A number of peaks in the range of 200 to 400 m/z were found for the ESI
negative ion mode mass spectrum of 5% Merichem in Tufflo extract. The distribution no
longer appears to be Gaussian. There was an underlying Gaussian distribution centered
28
around 300, but it was punctuated by a number of peaks with higher intensity. There was
still a spacing of 14 Da (due to (–CH2) between most of the large peaks.
The results for MS/MS experiments show mass losses of M-1, M-18, M-28 and
M-44, which correspond to the loss of hydrogen, water, carbon monoxide and carbon
dioxide. Figures 3-11, 3-12, 3-13, and 3-14, illustrate representative MS/MS spectra for
peaks at 245, 287, 301 and 313 found in the MS spectrum and confirm that these are due
to naphthenic acids. The M-28 peak has a lower intensity than the other peaks. Peaks
with a loss of M-34 and M-62 were also found upon fragmentation of some of the peaks
in the MS spectrum.
-245.2msmsACN12feb #34-63 RT: 0.71-1.34 AV: 30 NL: 3.00E2T: - p Full ms2 [email protected] [ 65.00-248.00]
70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240m/z
0
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227.2
245.2
201.2
175.2 199.3217.0159.3
78.0 229.1171.198.9 209.193.2 225.0185.5161.0 235.290.3 189.2124.973.1 101.7 135.0 157.287.2 142.4 161.5
[M-H-C0]-
[M-H-H20]-
[M-H]-
[M-H-CO2]-
FIGURE 3-11: MS/MS of 245.2 m/z from the Merichem extract in ESI negative ion
mode within a mass range of 65-248 m/z in acetonitrile.
29
Figure 3-11 is a representative MS/MS spectrum for the peak at 245 m/z. The
normal fragmentation peaks associated with a carboxylic acid are present, but there are
no peaks found at M-34 and M-62. More fragmentation was observed for the extract than
Merichem before extraction.
-287.3m sm sACN12feb #22-38 RT: 0.47-0.82 AV: 17 NL: 1.31E3T: - p Full m s2 [email protected] [ 75.00-290.00]
80 100 120 140 160 180 200 220 240 260 280m /z
0
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269.2
243.3
287.3253.3
267.2227.3 271.3241.3189.3 203.3171.3 222.7118.9 181.3101.3 154.1129.1 151.091.5
[M-H-H20]-
[M-H]-[M-H-C0]-
[M-H-CO2]-
FIGURE 3-12: MS/MS of 287.3 m/z from the Merichem extract in ESI negative ion
mode within a mass range of 75-290 m/z in acetonitrile.
Figure 3-12 illustrates a representative MS/MS spectrum for the peak at 287 m/z.
In addition to the normal fragmentation peaks associated with a carboxylic acid, there
was a peak found at M-34, but no peak was found at M-62.
30
-301m s m s AC N 12feb #20-40 RT: 0 .42 -0 .86 AV: 21 N L: 1 .22E3T: - p Fu ll m s 2 [email protected] [ 80 .00-304.00 ]
100 120 140 160 180 200 220 240 260 280 300m /z
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2 83 .2
257.1
253.2
301.3267.1
273.1239.5224.9 299.1183.796.8 165.7 221.4127.1 134.4 141.4 201.3151.083.1 110.9
[M-H]-
[M-H-H20]-
[M-H-C0]-
[M-H-CO2]-
FIGURE 3-13: MS/MS of 301.2 m/z from Merichem extract in ESI negative ion mode
within the mass range 80-305 m/z. in acetonitrile.
Figure 3-13 illustrates a representative MS/MS spectrum for the peak at 301 m/z.
In addition to the normal fragmentation peaks associated with a carboxylic acid, there are
peaks found at M-34 and M-62.
-313.3m s m sACN12feb #24-51 RT: 0.51-1.11 AV: 28 NL: 1.44E3T: - p Full m s 2 [email protected] [ 85.00-316.00]
100 120 140 160 180 200 220 240 260 280 300m /z
0
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269.3
295.2
267.2313.2279.3
251.2173.1143.9241.1 293.3149.1 225.1211.1 253.4166.9107.1 297.5199.0141.0 183.197.3 123.0
[M-H]-
[M-H-H20]-
[M-H-C0]-
[M-H-CO2]-
FIGURE 3-14: MS/MS of 313.2 m/z from Merichem extract in ESI negative ion mode
within the mass range 85-315 m/z in acetonitrile.
31
Figure 3-14 illustrates a representative MS/MS spectrum for the peak at 313 m/z.
In addition to the normal fragmentation peaks associated with a carboxylic acid, there are
peaks found at M-34 and M-62. There are also more fragmentations.
A comparison was made between samples before and after extraction of the
Merichem. The Merichem, after extraction, has a similar dispersion of peaks as
Merichem before extraction, but less intense. This could be due to the extraction process
and the loss of naphthenic acids; it also could be due to modification of some of the
naphthenic acids.
Mass spectra of Merichem before and after extraction (indicated in parentheses)
show the presence of common peaks including: 237.3 (237.3), 245.2 (245.3), 251 (251.4),
265.3 (265.3), 275.3 (275.3), 279.3 (279.4), 287.1 (287.3), 293.3 (293.2), 301.3 (301.2),
331.1 (331.3), 335.2 (335.3), and 341.3 (341.3). These provide evidence that naphthenic
acids are extracted into ammoniated ethylene glycol 44. Some of the peaks that were
found in standard Merichem, before extraction, are missing after the Merichem
extraction. It may be that some of the lower molecular weight naphthenic acids remained
in the ethylene glycol or water used in the process of extraction. Alternatively, some of
the acids may be esterified and therefore not appear in the mass spectrum.
32
3.2.4 MS and MS/MS of naphthenic acids extracted from crude oil
-m s #80 -182 R T: 0 .99 -2 .27 AV: 103 N L : 1 .24E4T: - p Fu ll m s [ 100 .00-450.00 ]
180 200 220 240 260 280 300 320 340 360 380 400m /z
0
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289 .2
271 .3
259.2
319.1301 .2
365.1 395 .2187.1241 .2 335 .3
377.3347 .2290 .3 357 .2 387 .2317 .3287 .2 329 .3 411 .4269.3 313.3257 .3 367.3214 .2 229 .1 345 .3
253.1 286 .4220 .3211 .1199 .1
FIGURE 3-15: MS of naphthenic acids extracted from crude oil run in acetonitrile,
negative ion mode within the mass range 100 m/z to 440m/z.
The MS of the naphthenic acids extracted from crude oil (Figure 3-15) does not
show the diversity present in the Merichem naphthenic acid extract. Compared with
Figure 3-10, only certain classes of naphthenic acids appear to be present in the extract
obtained from the crude oil (Chevron Texaco Cabinda oil sample). They seem to be a
homologous series (259, 289, and 319) which all lose water [M-H2O], as well as a
homologous series (241,271,301) which loses a hydride [M-2H] as well as water [M-
H2O]. Peaks chosen for MS/MS experiments have a relative abundance of 15% and
higher in the MS spectrum. All peaks are reported in table 2 with decreasing intensity.
There was a sulfur compound extracted from crude oil, so we may be extracting sulfur
compounds along with naphthenic acids. There may be some loss of naphthenic acid
from crude oil due to acids or ester derivate of acid in the extract.
33
TABLE - 2: MS/MS results for naphthenic acids extracted from crude oil run in
acetonitrile.
Precursor Fragment ions (Da)
187* 115, 143, 169, 159
214 # 150, 134, 71.7
241* 239, 223, 211,197
259 241, 257, 153, 235, 123
271* 269, 253, 235, 223, 267, 241, 225, 213, 251, 227
289 271, 153, 269
301* 253, 299, 283, 237, 235, 223, 265, 199.7
319 301
335* 317, 241, 291, 273, 306.9, 350
347* 329, 239, 241, 317, 253, 303, 285, 275
365 347, 271, 335, 229, 317, 329
377* 359, 257, 269
395* 377, 351, 359, 367, 380, 241, 271
* Shows mass loss of 44 Da that was attributed to the loss of CO2
# A sulfur compound with a mass loss of 64, which was attributed to SO2.
34
-124.1m s m s #45-80 RT: 0.94-1.67 AV: 36 NL: 2.62E3T: - p Full m s 2 [email protected] [ 100.00-245.00]
110 120 130 140 150 160 170 180 190 200 210 220 230 240m /z
0
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239.1
241.2
223.1211.3
210.7116.8 197.2136.1 221.1101.6 107.1 235.5145.8132.1 166.5 195.2155.9123.5 179.1 230.7169.1
[M-H]-
[M-H-H20]-
[M-H-C0]-
[M-H-CO2]-
FIGURE 3-16: MS/MS of 241.2 m/z of naphthenic acid extract from crude oil with a
mass range 100-245 m/z.
Figures 3-16 and 3-17, demonstrate MS/MS of the parent peak shows less
fragmentation. There was low peak signal intensity for M-CO2, M-CO, and M-H2O mass
loss. An intense peak found at 239.1 m/z and 115.1 m/z for the parent peaks 241 m/z and
187.2 m/z respectively.
-1 8 7 m s m s # 3 1 -6 0 R T: 0 .6 4 -1 .2 4 AV: 3 0 N L : 3 .6 6 E 3T : - p Fu l l m s 2 1 8 7 .1 0 @ 3 2 .0 0 [ 5 0 .0 0 -1 9 1 .0 0 ]
6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 1 9 0m /z
0
5
1 0
1 5
2 0
2 5
3 0
3 5
4 0
4 5
5 0
5 5
6 0
6 5
7 0
7 5
8 0
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9 0
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1 0 0
Rel
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1 1 5 .1
1 8 7 .2
1 4 3 .07 1 .0 1 6 9 .21 2 3 .01 1 3 .96 7 .9 1 2 5 .2 1 4 1 .9 1 5 9 .18 3 .3 1 7 0 .97 1 .8 8 6 .9 1 8 5 .91 0 1 .4 1 8 8 .7
[M-H-CO2]-
[M-H-C0]-
[M-H-H20]-
[M-H]-
FIGURE 3-17: MS/MS of 187.2 m/z of naphthenic acid extract from crude oil with a
mass range 50-190 m/z.
35
Figures 3-18 and 3-19 MS/MS of the parent peaks showing more fragmentation.
There was an intense peak signal at M-CO2, M-CO, and M-H2O mass loss. An intense
peak found at 241.3 m/z for the parent peaks 335.3 m/z. There was an intense peak loss at
239 m/z, 241 m/z, and 252.9 m/z for the parent peak 347.2 m/z.
-335.3m s m s #43-73 R T: 0.95-1 .62 AV: 31 N L: 2 .35E2T: - p Fu ll m s 2 [email protected] [ 100 .00-339.00]
120 140 160 180 200 220 240 260 280 300 320m /z
0
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317.3241.3
335.5
291.3
273.3306.9
319.8120.9305.0198.8 229.3
218.1 275.4213.0 253.4 261.7240.0193.9168.9151.1115.6 289.1189.1 207.1
279.6 330.4112.6
[M-H]-
[M-H-H20]-
[M-H-C0]-
[M-H-CO2]-
FIGURE 3-18: MS/MS of 335.3 m/z of naphthenic acid extract from crude oil within a
mass range 100-340 m/z.
-3 4 7 m s m s # 4 8 -8 6 R T: 1 .0 7 -1 .9 2 AV: 3 9 N L : 3 .1 5 E 2T: - p Fu ll m s 2 3 4 7 .2 0 @ 3 9 .0 0 [ 1 0 0 .0 0 -3 5 1 .0 0 ]
1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0m /z
0
5
1 0
1 5
2 0
2 5
3 0
3 5
4 0
4 5
5 0
5 5
6 0
6 5
7 0
7 5
8 0
8 5
9 0
9 5
1 0 0
Rel
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3 2 9 .3
2 3 9 .2
3 4 7 .52 4 1 .2
3 1 7 .1 3 4 5 .1
2 5 2 .9
2 2 6 .9
3 0 3 .02 7 5 .12 1 3 .31 2 0 .9 2 8 5 .3 3 0 1 .41 6 2 .7 1 8 1 .1 2 6 1 .92 2 5 .11 1 2 .9 3 1 3 .21 9 9 .21 2 3 .4
2 8 7 .21 9 3 .01 5 9 .6 3 3 2 .3
[M-H]-
[M-H-H20]-
[M-H-C0]-
[M-H-CO2]-
FIGURE 3-19: MS/MS of 347.2 m/z of naphthenic acid extract from crude oil within a
mass range 100-350 m/z.
36
The MS/MS spectrum in Figure 3-20 illustrates the fragmentation pattern
generated by a sulfur compound. There was sulfur associated with M-64 loss of SO2 from
the precursor ion.
-214m s m s #96-114 RT: 2.05-2.43 AV: 19 NL: 4.42E2T: - p Full m s 2 [email protected] [ 55.00-218.00]
60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210m /z
0
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Rel
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213.9
149.9
150.4
71.7 134.1
151.3
FIGURE 3-20: MS/MS of sulfur compound extracted from crude oil in acetonitrile in
negative ion mode with a mass range 60 m/z to 220m/z.
The results indicate that the extraction procedure not only extracts naphthenic
acids but also extracts other acid fractions present in crude oil, such as sulfonic acids.
There was also a possibility that during the process of extraction of napthenic acids,
napthenic acids might have been converted to their corresponding esters.
37
3.2.5 MS and MS/MS of naphthenic acids extracted from crude oil: samples run
in 20% dichloromethane / 80% acetonitrile
-1 la rge bd ich lo rod ryth en AC N de c13 #54 -87 R T: 1 .7 4 -2 .8 2 AV: 3 4 N L : 9 .3 8E5T: - p Fu ll m s [ 6 0 .00 -20 00 .0 0 ]
1 00 1 50 2 00 25 0 30 0 35 0 40 0 45 0 50 0 5 50 6 00 6 50 7 00m /z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
10 0
Rel
ativ
e A
bund
ance
2 8 9 .1
25 9 .1
3 65 .1 3 95 .1
31 9 .1
3 35 .1 4 25 .1
5 01 .247 1 .053 1 .14 41 .12 41 .12 29 .31 53 .1
57 7 .256 0 .9 60 7 .2 6 53 .2 66 6 .9 70 5 .22 26 .915 1 .0
1 99 .211 1 .4
FIGURE 3-21: MS of naphthenic acids extracted from crude oil in 20% dichloromethane
/ 80% acetonitrile with 0.5% ammonia run in negative ion mode.
Figure 3-21, using a mass range of 200 m/z to 600 m/z, there shows that are more
peaks found in 20% dichloromethane / 80% acetonitrile solvent system with 0.5%
ammonia and also showed higher signal intensity.
38
TABLE-3: MS/MS results for naphthenic acids extracted from crude oil in 20%
dichloromethane / 80% acetonitrile. The fragment ions are arranged in decreasing order
of intensity.
Precursor Fragment ions
229 227, 123, 211, 93, 199
241 239, 211, 233, 212, 135
259 241, 257, 153, 123
271 269, 253, 233, 235, 241
289 271, 153
301 253, 299, 283, 237, 265, 223, 151
319 301, 318, 302
335* 317, 241, 229, 305, 291, 199, 211, 123
347* 329, 239, 317, 253, 241, 121, 227, 213, 199, 289, 303
365 347, 271, 241, 229, 335, 259
377 359, 347, 269, 257
395 377, 271, 347, 365,289, 289, 259, 153
425 407, 377, 395, 271, 301, 289
441* 423, 335, 347, 305, 411, 317, 229, 241
454 436, 407, 419, 360, 389, 301, 299, 348, 334, 289, 319, 240
471 453, 441, 435, 365, 347, 335, 377, 423
501 483, 471, 465, 365, 377, 395, 347, 259, 271, 289
530 513, 482, 501, 377, 395, 407, 289, 271, 425, 436, 465, 259, 301, 365
* A 44 Da mass loss is attributed to carbon dioxide from carboxylic acid.
39
Table -3 reports the MS/MS results for naphthenic acids extracted from crude oil.
There was a mass loss of 18 (loss of water); other mass losses that appeared were at 30,
36, and 48. There were some extra peaks that were found in the MS when run with 20% /
80% dichloromethane/acetonitrile containing 0.5% ammonia when compared with an MS
spectrum of an extract run with acetonitrile. The extra peaks were in the range of 420m/z
- 600m/z, and found at 425, 441, 454, 471, 501, and 531. More fragmentation was
observed with 20% / 80% dichloromethane / acetonitrile with 0.5% ammonia. Both
solvent systems had common peaks, but showed different fragmentation patterns for a
particular m/z. The mass loss of M-44 is observed with an extract run in acetonitrile but a
44 mass loss was not observed with 20%/80% dichloromethane / acetonitrile with 0.5%
ammonia for the following peaks 241m/z, 271 m/z, 301 m/z, 377 m/z, and 395 m/z. It
seems that 20% / 80% dichloromethane / acetonitrile with 0.5% ammonia as a solvent
system do not favor the formation of fragments that can be ascribed to a carboxyl group.
+dichlorodrythenACNdec13 #142-180 RT: 4.56-5.76 AV: 39 NL: 1.88E5T: + p ms [ 150.00-2000.00]
200 250 300 350 400 450 500 550 600 650 700 750m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
489.3
449.0
419.1
585.1
411.0555.0
691.2642.4 751.5608.5
615.3 783.5661.3 727.5525.0705.6453.0
577.1501.1420.3335.1313.1
443.0388.7338.3306.2209.1
228.4272.5
241.1207.0182.6
FIGURE 3-22: MS in positive ion mode of naphthenic acids extracted from crude oil in
20% dichloromethane / 80% acetonitrile with a mass range of 180 m/z to 800m/z.
40
Figure 3-22 shows some peaks in the range of 400 to 500 m/z that have high
intensity in positive ion mode; they may be sulfur, nitrogen, and oxygen derivatives.45
Our extraction procedure appears to extract other compounds in addition to naphthenic
acids.
3.3 Nuclear Magnetic Resonance (NMR) Spectroscopy
Merichem is a naphthenic acid mixture; it contains aliphatic and aromatic
carboxylic acids. The NMR confirmed peaks that may be attributed to a carboxylic acid,
and aliphatic, and aromatic hydrocarbon moieties.
3.3.1 NMR of Merichem
1H NMR: Figure 3-23 shows peaks between 0.3-3 ppm. These may be attributed
to hydrogens associated with methyl and methylene carbons. The peaks between 6.8-8
ppm may be attributed to hydrogens associated with an aromatic ring, while peaks
between 11.5-12 ppm may be attributed to hydrogens associated with carboxylic acids.
The deuterated chloroform solvent shows a peak at 7.26 ppm.
41
FIGURE 3-23: 1H NMR of Merichem
C13 NMR: Figure 3-24 shows peaks between 10-60 ppm, which may be attributed
to methyl and methylene carbons, whereas peaks between 120-130 ppm may be
attributed to aromatic ring carbons. Peaks between 180-185 ppm may be attributed to a
carboxylic acid carbon. The deuterated chloroform solvent shows a peak at 77 ppm.
42
FIGURE 3-24: 13C NMR of Merichem.
3.3.2 NMR of 5% Merichem in Tufflo after extraction
FIGURE 3-25: 1H NMR of 5% Merichem in Tufflo after extraction.
The 1H NMR of 5% Merichem in Tufflo, after extraction, did not show the
expected carboxylic acid group present at 12 ppm. (See Figure 3-25) This may be due to
43
a very low concentration of naphthenic acid in the extract (5% Merichem in Tufflo) or
the fact that the carboxylic acid group was modified in the process of extraction. We did
not see the aromatic ring compounds that are known to be present, which lends further
credence to the fact that lack of signal was probably due to a concentration effect. Peaks
attributable to methyl and methylene hydrogen do appear in the range of 0.5-2 ppm, as
well as a peak at 3.7 ppm due to ethylene glycol.
3.3.3 NMR of Tufflo supernatant after contact with ethylene glycol.
The 1H NMR of Tufflo supernatant, after contact with ethylene glycol was shown
in Figure 3-26, peaks attributable to methyl and methylene hydrogens in the range of 0.5-
2 ppm, as well as a peak at 3.5 ppm due to ethylene glycol. The peak at 5.2 ppm may be
due to ester functionality, and the peak at 7.26 ppm was attributed to deuterated
chloroform.
FIGURE 3-26: 1H NMR of Tufflo supernatant after contact with ethylene glycol.
44
3.3.4 NMR of ethylene glycol layer after removal of ammonia
FIGURE 3-27: 1H NMR of ethylene glycol layer after extraction of Merichem and boil
off of ammonia.
Peaks shown in Figure 3-27, at 1.4-1-6 ppm are attributable to the hydrogen
present in methyl group, and the peak at 3.5 ppm due to hydrogens in ethylene glycol.
The deuterated acetone solvent shows a peak at 2.05 ppm.
3.3.5 NMR of naphthenic acids extracted from crude oil
FIGURE 3-28: 1H NMR of naphthenic acids extracted from crude oil.
45
Peaks in Figure 3-28, at 0.8, 1.2, and 1.6 ppm correspond to hydrogen’s in
aliphatic methyl, methylene, and methine hydrocarbons. The peak found at 2 ppm may be
due to hydrogens in an ester. The peak at 3.5 ppm was due to hydrogen’s in ethylene
glycol, and the peak at 5.2 ppm may be attributable to hydrogen’s present in a phenolic
ester. The deuterated chloroform solvent shows a peak at 7.26 ppm. The carboxylic acid
functional group was not found at 11-12 ppm.
3.3.6 NMR of crude oil supernatant after contact with ethylene glycol
FIGURE 3-29: 1H NMR of Crude oil fraction after extraction.
Figure 3-29 shows peaks at 0.8, 1.2, and 1.6 ppm corresponding to aliphatic
methyl, methylene, and methine hydrocarbons. The peak at 3.7 ppm was due to ethylene
glycol. The deuterated chloroform solvent shows a peak at 7.26 ppm.
46
3.3.7 NMR of ethylene glycol after contact with crude oil after removal of
ammonia
FIGURE 3-30: 1H NMR of ethylene glycol after contact with crude oil after removal of
ammonia.
The 1H NMR spectrum was shown in figure 3-30. The peak appears at 2 ppm was
due to deuterated acetone. The peak at 3.5 ppm was due to ethylene glycol; peaks found
in between 4.5 to 8.5 ppm may be due to phenolic derivatives.
47
3.4 Infrared Spectroscopy (IR)
3.4.1 IR of polyethylene and petroleum ether
The background IR spectrum has peaks due to the polyethylene film and residual
petroleum ether. Figure 3-31 was the background IR spectrum of polyethylene and
petroleum ether. The IR spectrum, with sample, was interpreted by discounting the peaks
shown by the polyethylene and petroleum ether background.
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.00.0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
79.9
cm-1
%T
1366.28,60.10
716.10,39.88
727.07,34.34
1462.30,9.70
2659.34,63.50
2857.14,0.192912.08,0.03
2329.67,72.372016.48,74.27
1303.18,67.60
1075.48,71.233604.39,72.36
FIGURE 3-31: Background IR of polyethylene and petroleum ether.
3.4.2 IR of Merichem.
The IR spectrum of merichem in Figure 3-32 shows a broad OH stretch found at
3300-2500 cm-1; the strong signal at 1703 cm-1 was due to a carbonyl group. The strong
signal at 1289 cm-1 was due to a C-O stretch; the signal at 1377 cm-1 was due to a C-O-H
out of plane bend; and the signal at 935 cm-1 was due to the O-H out of plane bend.
48
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.00.0
2
4
6
8
10
12
14
16
18
20
22
24
26
28.0
cm-1
%T
2664.83,12.66
1377.25,11.61
1289.46,10.65
1226.37,13.32
935.57,15.20
1703.71,2.58
2324.17,24.14
2664.83,12.66
1377.25,11.61
1289.46,10.65
1226.37,13.32
935.57,15.20
1703.71,2.58
2324.17,24.14
Broad OH O-H out of plane bend.
C-O stretch
C-O-H out of plane bend
C=0 stretch
FIGURE 3-32: IR of Merichem dissolved in petroleum ether on a polyethylene film.
3.4.3 IR of 5% Merichem in Tufflo after extraction.
In Figure 3-33, the following peaks are identified: the strong signal of the C=0
stretch at 1706 cm-1, which may be due to a carboxylic acid or a derivative containing a
C=O group, and a weak signal at 935 cm-1 due to an O-H out of plane bend. A broad OH
stretch from 3300-2500 cm-1 was not found, possibly due to the low concentration of the
extract. This issue needs further investigation.
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.00.0
2
4
6
8
10
12
14
16
18
20
22
24
26
28.3
cm-1
%T
1706.46,17.34
Weak O-H out of plane bend.
C=0 stretch
FIGURE 3-33: IR of 5% Merichem in Tufflo after extraction in petroleum ether on a
polyethylene film.
49
3.4.4 IR of naphthenic acids extracted from crude oil
In figure 3-34, a weak signal may be due to the carbonyl group identified at 1728
cm-1 and 1637 cm-1. Most of the peaks are not identified, which may be due to the low
concentration of naphthenic acids after extraction from crude oil.
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.00.3
2
4
6
8
10
12
14
16
18
20
22
24
26
2829.1
cm-1
%T
1728.40,27.771637.87,27.26
C=0 stretch
FIGURE 3-34: IR of Naphthenic acid extract from crude oil in petroleum ether on a
polyethylene film.
CHAPTER 4
CONCLUSIONS
The extraction procedure shows a high yield, approximately 80%.
The MS of standard Merichem, before extraction, was compared with the MS of
extracted Merichem. Most of the naphthenic acids were extracted but there were some
naphthenic acids that were lost or not identified after the extraction procedure. Extraction
of the petroleum crude oil and subsequent MS/MS experiments showed that not all of the
extracted compounds were naphthenic acids.
NMR experiments showed no evidence of carboxylic acids and aromatic
hydrocarbons; this might be due to the low concentrations of naphthenic acids present in
the extract. There was also a possibility of naphthenic acids being modified to some other
acid derivatives during the extraction process.
Polar and short chain naphthenic acids and other polar species might be more
soluble in ethylene glycol than petroleum ether, so there was a possibility
50
51
that the more polar species could be lost during the extraction process. IR results show
the presence of a carbonyl group in the extracts, but no –OH which would be associated
with a carboxyl group. This might be due to the formation of an ester which would be the
subject for future research.
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VITA
Pratap Rikka was born in Orissa, India, on February 5, 1978, The son of Sumathi Rikka
and Late Loknath Rao Rikka .After completing his work at S. S. V. P High school,
Andhra Pradesh, India, he entered Roland Institute of pharmaceutical Sciences, Orissa,
India in August 1998. He received a Bachelors of Pharmacy degree from Roland Institute
of pharmaceutical Sciences in August 2002. He began graduate school during the spring
of 2004 in the Department of Chemistry and Biochemistry at Texas State University-San
Marcos.
Permanent Address:
Tampara Road, Chatrapur, Ganjam-761020 Orissa, India
This thesis was typed by Pratap Rikka.
