UDC 665.613 : 547.29
BIBLID: 1450–7188 (2004) 35, 87-93
APTEFF, 35, 1-280 (2004)
Original scientific paper
STRUCTURAL AND PHYSIOLOGICAL PROPERTIES OF NATURAL
PETROLEUM ACIDS FROM MIDDLE OIL FRACTIONS
OF "KELEBIJA" OIL
Vera S. Ćirin-Novta, Ksenija N. Kuhajda, Slavko E. Kevrešan, Julijan E. Kandrač,
Ljubica M. Grbović and Petar A. Rodić
The investigated petroleum acids have been isolated from middle oil fractions (b.p.
270-380°C) of Vojvodina crude oil "Kelebija." Group-structural analysis was performed
with low resolution mass spectrometry using Field Ionization and Chemical Ionization.
The identified carboxylic acids, CnH2n+zO2, belong to the series of monocyclic, bicyclic,
tricyclic and tetracyclic carboxylic acids. Physiological activity of isolated petroleum
acids was determined using aqueous solutions of potassium salts. High auxin activity
was determined using the coleoptile test (20.5% increase in coleoptile) with petroleum
acid concentration of 10-7M, and very high biological activity of gibberellin type was
determined using the endosperm test (31.6% increase in concentration of reducing
sugars) with petroleum acid concentration of 10-7M.
KEYWORDS: Petroleum acids; low-resolution MS; auxin activity; gibberellin activity
INTRODUCTION
Natural petroleum acids are carboxylic acids of various structures, but there is a
structural analogy with hydrocarbons of the appropriate oil. The investigated "Kelebija"
crude oil of Vojvodina belongs to the group of "heavy" crude oils with high density, high
acid content and low n-parafine content (Table 1). Our previous work showed that "Kelebija" crude oil is a naphthenic oil, and that the group-structural makeup of hydrocarbons from
lower and middle oil fractions is almost exclusively of cycloalkane type with, on average,
more condensed rings and very small aliphatic chain content (1).
Dr. Vera S. Ćirin-Novta, Prof., Dr. Ksenija N. Kuhajda, Prof., Ljubica M. Grbović, M.Sc., Assist., University
of Novi Sad, Faculty of Sciences, Department of Chemistry, 21000 Novi Sad, Trg Dositeja Obradovića 3,
Serbia and Montenegro, Dr. Slavko E. Kevrešan, Prof., Dr. Julijan Kandrač, Asoc. Prof., University of Novi
Sad, Faculty of Agriculture, 21000 Novi Sad, Trg Dositeja Obradovića 8, Serbia and Montenegro, Petar A.
Rodić, dipl. chem., NIS-Oil Refinery, 21000 Novi Sad, Serbia and Montenegro
87
In our previous work, we determined the physiological activity of petroleum acids of
naphthenic type from oils of different sites in Vojvodina (2,3,4). It was determined that
low concentrations of aqueous solutions of potassium salts of petroleum acids of naphthenic type increased synthesis of CO2 in some types of yeast (5). Moreover, it was determined that K-salts of petroleum acids of naphthenic type have antistaphylococcal activity
and fungicidal activity (6), while at higher concentrations in water (as waste material)
they are toxic and endanger aquatic life (7,8). At a concentration of 0.02 mg/l, petroleum
acids block completely fertility of fish (9].
Our previous work described the methods of isolation and purification of petroleum
acids using alkaline extraction from a mixture of hydrocarbons from commercial fractions of Vojvodinian oils (10). The aim of the present work is to get a detailed groupstructural analysis of isolated acids from the middle oil fraction (b.p. 270-380°C) of
"Kelebija" crude oil by means of low resolution mass spectrometry using Chemical Ionization (CI) and Field Ionization (FI), as well as to determine the level of phytohormone
activity analogous to auxin and gibberellin activity using different concentrations of
aqueous solutions of K-naphthenates.
EXPERIMENTAL
All experiments were performed using mixtures of natural petroleum acids isolated
and purified by alkaline extraction from oil fractions (distillation interval 270-380°C) of
Vojvodinian "Kelebija" oil.
Mass spectra were recorded on a Varian MAT-311A mass spectrometer, using Field
Ionization (FI) and Chemical Ionization (CI) as ion source. The spectra recorded employing the FI source contain almost exclusively molecular ions [M]+. This is achieved
by creating a strong electronic field around the sharp metal electrodes of the small
curvature radius in the ionic source. The other electrode is placed at a distance of about
2-5 mm, so that the field gradient attains the values of 107-108 V/cm. This field causes
strong ionization of the molecules present, so that the resulting M-spectrum contains
almost exclusively [M]+ ions.
In the M-spectra recorded using CI ion source, signals of the [M+1]+ ions, almost
exclusively, are present. In this case, the sample introduced into the chamber was diluted
with a large quantity of gas-carrier (isobutane). The resulting sample concentration was
approximately 1%, and the pressure about 0.07-0.12 kPa. Under these conditions, petroleum acids corresponded almost exclusively to [M+1]+ ions.
Standard physicochemical characteristics of the oil, oil distillate and petroleum acids
have been determined using ASTM-standardized methods, elemental microanalysis performed according to Densted, and structural analysis by the standard n-d-M-analysis.
Biological tests were performed using aqueous solutions of potassium salts of investigated petroleum acids in concentrations of 10-5, 10-6 and 10-7 mol.dm-3 for coleoptile and
gibberellin tests respectively (4,11).
Gibberellin tests: Wheat seeds, after calcium hypochlorite sterilization, were cut transversely 3 mm from the distal end; the endosperm pieces were weighed in groups of 4 and
incubated for 48 h at 30°C in 14×50 mm closed vials containing 1 cm3 of test solution and
300 µg of streptomycin sulphate. Three replicates of each treatment were used. The same
88
concentration of gibberellic acid (GA3) was applied in control determination of gibberellin type activity.
Auxin tests: For auxin activity determination wheat grains were kept 4 days in thermostat at 26°C in the dark. Coleoptiles 15 mm long were cut in 5 mm segments and used
in bioassay. The same concentration of α-naphtylacetic acid was used as control.
RESULTS AND DISCUSSION
Table 1 presents physicochemical characteristics of Vojvodinian "Kelebija" crude
oil, commercial middle oil fractions (b.p. 270-380°C) of the same oil and of petroleum
acids isolated from the given fraction. Exceptionally high density, high average molecular mass of both oil fraction and petroleum acids, as well as high percentage of naphthenic type carbon and very low percentage of parafine type carbon make this crude oil
particularly different from most naphthenic oils.
Table 1. Physicochemical characteristics and structural n-d-M analysis of "Kelebija" crude oil,
middle oil distillate and isolated petroleum acids
Distillation interval (°C)
Density (kg/m3)
Refraction index
Acid number (mgKOH/g)
Viscosity
•20°C
•50°C
Structural n-d-M analysis
%C aromate
%C naphthenate
%C parafine
Elemental
%C
microanalysis % H
%N
%S
%O
Average relative
molecular mass
Average molecular formula
Crude oil
192-405
951.0
1.4800
1.89
Oil distillate
270-380
910.6
1.5001
3.08
Petroleum acids
280-340
952.9
1.5608
194.26
73.14
18.27
3.9
9.1
-
18.3
46.2
35.5
86.50
11.72
0.28
1.36
1.24
10.5
67.9
12.7
87.10
11.13
0.09
0.52
1.07
76.70
11.08
10.72
327.10
336.13
299.79
-
-
C19H34O2
Table 2 presents results of biological tests performed for three concentrations of
aqueous solutions of K-naphthenates (10-5, 10-6 and 10-7 mol/dm3). Biologically most active
are the lowest concentrations in gibberellin test, because of the 31.6% increase in reducing
sugar content as compared to the control.
89
Table 2. Physiological activity of aqueous solutions of K-naphthenates
Concentration of
K-naphthenates
(mol/dm3)
1×10-5
1×10-6
1×10-7
Auxin test
(%) of control
Gibberellin test
reducing sugars
(%) of control
119.4 ± 5
121.9 ± 2
131.6 ± 6
no activity
115.2 ± 3
120.5 ± 1
Judging by acid numbers, which are 1.89 mgKOH/g for crude oil, and 3.08 mg KOH/g
for oil distillate, "Kelebija" crude oil belongs to a group of crude oils with very high acid
content. Table 3 and 4 present results obtained by low resolution M-spectra using two different methods (CI and FI). Using this method, four Z-series of carboxylic acids (general
formula CnH2n+zO2) have been determined. Table 3 presents M-spectra taken using Chemical Ionization with isobutane as carrier gas. The spectrum contains four series of [M+1]+
ions, which represents four series of cycloalkyl carboxylic acids. The series Z= -2 represents monocyclic acid structures. Z= -4 series represents bicyclic acid structures and their
content in total acid mixture is 43.1%, which shows that bicyclic acids are predominant,
and among them especially C18H32O2 with 11.4% content. Z=-6 series, that is, tricyclic
acids make up 30.9% of total acid mixture, and Z=-8 series, i.e. tetracyclic acids, make up
4.5% of total acid mixture.
Table 4 presents low resolution M-spectra taken using FI, which represent four series of
[M]+ ions, which corresponds to the presence of four Z series of cycloalkyl-carboxylic
acids. Results from Tables 3 and 4 are similar for the same range of molecular mass, which
confirms our findings.
Table 3. Low-resolution M spectra (CI, isobutane) of the homologue series
of natural petroleum acids from middle oil fraction of "Kelebija" oil
Class of carboxylic
acid, CnH2n+zO2
Z-series
Monocyclic series
CnH2n-2O2
(Z= -2)
Bicyclic series
CnH2n-4O2
(Z= -4)
Tricyclic series
CnH2n-6O2
(Z= -6)
Tetracyclic series
CnH2n-8O2
(Z= -8)
Series of molecular peaks
[M+1]+
241(1.0)*; 255(6.3);
269(5.1); 283(4.1);
297(3.1);311(1.1);
325(0.3); 339 (0.2);
253(6.7);
267(1.0);281(9.2);
295(11.4); 309(6.5);
323(5.0); 337(3.3);
251(3.3);
265(5.8);279(6.5);
293(5.7); 307(4.1);
321(2.5);335(1.0)
305(0.2); 319(0.9);
333(2.4); 347(2.5);
361(0.8)
No. of C-atoms
in molecule
Z-series
Z-series content
in total acid
mixture (%)
14-21
21.2
15-21
43.1
15-22
28.9
19-23
6.8
* Numbers in parentheses represent abundance of molecular peak in %
90
Table 4. Low-resolution M spectra (FI) of the homologue series of natural petroleum acids
from middle oil fraction of "Kelebija" oil
Class of carbox.acid
CnH2n+zO2
Z-series
Monocyclic series
CnH2n-2O2
(Z= -2)
Bicyclic series
CnH2n-4O2
(Z= -4)
Tricyclic series
CnH2n-6O2
Series of molecular
peaks [M]+
No. of C-atoms in
acid Z-series
Content of Z-series
in total acid mixture
(%)
14-21
23.7
15-21
40.9
16-22
30.9
226(1.2)*; 240(2.1);
254(3.9); 268(4.5);
282(4.5);296(3.7);
310(3.3); 324(0.5)
238(3.2);252(4.3);
266(8.7); 280(11.3);
294(5.6); 308(4.5);
322(3.3);
250(5.6);264(4.5);
278(5.7);292(5.3);
306(5.6); 320(3.2);
334(1.0)
290(1.1); 304(2.5);
318(0.9)
(Z= -6)
Tetracyclic series
CnH2n-8O2
19-21
(Z= -8)
* Numbers in parentheses represent abundance of molecular peak in %
4.5
CONCLUSION
Isolated petroleum acids from middle oil fraction (b.p. 270-380°C) of Vojvodinian
crude oil "Kelebija" contain four classes of cycloalkylcarboxylic acids: monocyclic-, bicyclic-, tricyclic- and tetracyclic carboxylic acids. Most common are bicyclic acids (general formula CnH2n-4O2) and they belong to Z= -4 series. The best physiological activity
of investigated petroleum acids was obtained for concentration of 10-7 mol/dm3. The physiological activity of 20.5% was determined using coleoptile test, but the most pronounced activity was obtained in the gibberellin test, where the higher concentration (31.6%) of
reducing sugars can be explained by the presence of tricyclic- and tetracyclic carboxylic
acids.
ACKNOWLEDGEMENTS
This study was supported by the Ministry for Science, Technology and Development of
the Republic of Serbia (Project "Synthesis of Potential Cytotoxic Agents, Antihormones,
Enzyme Inhibitors and Growth Stimulators", No 1896)
REFERENCES
1. Ćirin-Novta, V., D. Miljković and D. Kobilarev N.: Petroleum Acids of Vojvodina
Mineral Oil "Kelebija".-Proceedings for Natural Sciences, Matica Srpska, Novi Sad,
84 (1993) 57-61.
91
2. Ćirin-Novta, V., K. Kuhajda, S. Kevrešan, J. Kandračand Lj. Radić: Biological activity
and structure of natural petroleum acids from lower oil fractions of "Velebit" oil.- Acta
Period.Technol. 33 (2002) 135-141.
3. Kevrešan, S., V. Ćirin-Novta, B. Kovacević, K. Kuhajda, J Kandrač and Lj.Radić, L.:
Efekat naftenskih kiselina na formiranje korenčića odredjenih reznica klonova sekcija
Leuce i Aigeiros.- Topola, Novi Sad, 171/172 (2003) 63-72.
4. Miljković, D., V. Ćirin-Novta and S. Repić: Biološka aktivnost naftnih kiselina.Proceedings for Natural Sciences, Matica Srpska, Novi Sad, 83 (1992) 33-39.
5. Ćirin Novta, V., K. Kuhajda, D. Miljković, S. Velimirović and S. Ćušić: Structural and
physiological properties of some natural and synthetic petroleum acids.- I. Regional
Symposium "Chemistry and the Environment", Vrnjačka Banja, 29 (1995) 215-218.
6. Rogers,V., K. Liber and M. MacKinnon: Isolation and characterization of naphthenic acids from Athabasca oil sands tailings pond water.-Chemossphere, 48 (2002) 519527.
7. Herman, D.C., P.M. Fedorak, M.D. MacKinnon and J.W. Costerton: Biodegradation
of naphthenic acids by microbial populations indigenous to oil sands tailings.- Canadian Journal of Microbiology 40 (1994) 467-477.
8. Wort., Dj. and J.G. Severson: Mechanism of plant growth stimulation by naphthenic
acid, Effect of nitrogen metabolism of Phaseolus vulgaris L.- Plant Physiol. 52 (1973)
162-165.
9. Nametkin, N.S. and G.M. Egorova: Naftenovye kisloty i produkty ikh khimicheskoi
pererabotki.- Khimiya, Moskow 43 (1982) 122-131.
10. Ćirin-Novta, V., S. Kevrešan, K. Kuhajda, J. Kandrač, Lj. Radić and P. Rodić: Optimization of petroleum acid isolation from lower oil fractions of Vojvodina "Velebit" oil.Acta Period.Technol. 34 (2003) 49-54.
11. Štajner, D., V. Ćirin-Novta and K.Bajin-Katić: Antioxidative and physiological action of naphthenic methyl esters.- Oxidation Communications 24 (2001) 515-522.
92
СТРУКТУРНЕ И ФИЗИОЛОШКЕ ОСОБИНЕ ПРИРОДНИХ
НАФТНИХ КИСЕЛИНА ИЗ СРЕДЊИХ УЉНИХ ФРАКЦИЈА
НАФТЕ "КЕЛЕБИЈА"
Вера С. Ћирин-Новта, Ксенија Н. Кухајда, Славко Е. Кеврешан,
Јулијан Е. Кандрач, Љубица М. Грбовић и Петар А. Родић
Испитиване нафтне киселине изоловане су из средње уљне фракције (т.к. 270380°C) војвођанске нафте "Келебија". Групно-структурна анализа рађена је масеном спектрометријом ниске резолуције методом јонизације поља и хемијском јонизацијом. Идентификоване карбоксилне киселине, CnH2n+ zO2, припадају серији моноцикличних-, бицикличних-, трицикличних- и тетрацикличних карбоксилних киселина.
Физиолошка активност изолованих нафтних киселина одређивана је у облику
водених раствора калијумових соли. Колеоптил тестом одређена је висока ауксинска активност (20.5% пораст колеоптила) при концентрацији 10-7M, а врло висока
физиолошка активност гиберелинског типа одређена је ендосперм тестом (31.6%
пораст концентрације редукујућих шећера) при концентрацији нафтних киселина
10-7M.
Received 19 March 2004
Accepted 6 May 2004
93