Journal of Chromatographic Science 2014;52:390– 394
doi:10.1093/chromsci/bmt047 Advance Access publication May 16, 2013
Article
New Determination Method for Sulfonation Degree of Phthalic Anhydride by RP-HPLC
Lijun Zhu, Lechun Song, Bin Liu, Yulu Zhou, Yuzhi Xiang and Daohong Xia*
State Key Laboratory of Heavy Oil Processing, College of Chemical Engineering, China University of Petroleum, Qingdao 266580,
China
*Author to whom correspondence should be addressed. Email: [email protected]
Received 23 September 2012; revised 05 March 2013
A novel method was developed to monitor the reaction process and
evaluate the sulfonation level in the sulfonation of phthalic anhydride
by reversed-phase high-performance liquid chromatography (RPHPLC). The product peak was identified in chromatograms through
product analysis and by comparing its retention time with that of
standard compounds. By comparing the hydrolysis and alcoholysis
methods, optimized pretreatment of the sample was found for RPHPLC. Based on the determined percentages of phthalic anhydride
and sulfonated phthalic anhydride in the mixture, the degree of sulfonation was calculated. When the sulfonation degree of phthalic anhydride was in the range of 2.8 –71%, the recovery of 97 –104% was
achieved, and the procedure was rapid and accurate.
In this paper, SPA was synthesized and its structure was identified by infrared spectroscopy (IR) and nuclear magnetic resonance (NMR). Based on the study of hydrolysis, alcoholysis
reactions and the mechanism of the RP column, a new method
was established for determining the PA and SPA contents and
degree of sulfonation; the method can be used to monitor the reaction process and study the sulfonation reaction in detail and to
lay the foundation for the sulfonation of phthalocyanine. The hydrolysis and alcoholysis reactions of PA and SPA are shown in
Figure 1.
Experimental
Introduction
The solubility of cobalt phthalocyanine can be greatly improved
in water or caustic solutions by sulfonation, which is better than
when phthalocyanine is substituted by groups such as hydroxyl,
carboxyl or halogen (1, 2). Furthermore, this sulfonated cobalt
phthalocyanine can also produce higher catalytic activity when
it serves as a catalyst in light oil sweetening (3). In general, the
synthesis of sulfonated cobalt phthalocyanine is achieved by two
steps, which include first synthesizing the cobalt phthalocyanine
and then sulfonating it with fuming sulfuric acid. Compared
with this two-step procedure, direct one-step solid-phase synthesis of sulfonated cobalt phthalocyanine from sulfonated
phthalic anhydride (SPA) has undoubtedly proved to be a promising route. The sulfonation of phthalic anhydride (PA) is not
only more economical than the sulfonation of cobalt phthalocyanine, but can also avoid the loss of cobalt phthalocyanine
during the sulfonation process. Furthermore, using SPA as a starting material to synthesize sulfonated cobalt phthalocyanine, the
number of sulfonic groups on the phthalocyanine ring and the
performance of the phthalocyanine metal can be controlled
during the reaction. Therefore, it is important to study the sulfonation of PA and establish a fast and accurate analysis method
for the sulfonation degree of the product mixture.
Reversed-phase high-performance liquid chromatography
(RP-HPLC), which has an excellent separation effect on large
polar compounds, can be used for qualitative and quantitative
analysis because it is accurate, sensitive and time-saving. It is
widely used in organic and drug analysis fields (4– 6). However,
the situation is different for the analysis of the contents of PA
and SPA, because PA and SPA are not stable enough and undergo
hydrolysis or alcoholysis easily in a polar mobile phase, so the
sample cannot be directly injected into the chromatograph.
Equipment and reagents
IR was conducted on a Nicolet 6700 FTIR (Thermo Scientific,
Waltham, MA). The NMR spectra were collected on a Varian
Mercury UX300 spectrometer in deuterated dimethyl sulfoxide
(DMSO-d6) with trimethylsilyl (TMS) as reference.
PA [analytical reagent (AR) grade], anhydrous ethanol (AR),
95% ethanol (AR), ethanol (HPLC), oleum (50%), methanol
(HPLC), ammonium dihydrogen phosphate (AR) and ultrapure
water were also used.
Sulfonation of phthalic anhydride
The PA (10.01 g, 67.6 mmol) was added to a round bottom flask
(RBF) with three necks and heated at 1408C. A condenser was
installed and the end of the condenser was connected with an
absorption bottle. The flask of fuming sulfuric acid was heated so
that the sulfur trioxide was released, which went into the flask
though the glass tube and contacted the melted PA. The sulfonation reaction was conducted at 1408C for 2.5 h. Approximately
7.1 g (88.7 mmol) of sulfur trioxide (SO3) was used. After reaction, the mixture was purified by vacuum distillation. After the
SO3 and PA were removed, the pure SPA was obtained and its
purity was confirmed by thin-layer chromatography (TLC) and
HPLC, which met the criteria of the analysis.
The IR spectra of SPA were 3,426 cm21 (a stretching vibration
peak of O–H appeared from the sulfonic group), 1,200 cm21
(a strong peak family showed S ¼ O symmetric and asymmetric
stretching vibration peaks mixed with the C–O –C stretching vibration) and 580 cm21 (C –S stretching vibration peak).
The three peaks confirmed the presence of the benzenesulfonic acid group.
1
H-NMR (DMSO) showed the following: d: 12.90 (s), 7.872
(d, J ¼ 1.507 Hz, 1H), 7.798 (dd, J ¼ 8.289 Hz, J ¼ 1.507 Hz, 1H),
7.661 (d, J ¼ 8.289 Hz, 1H), 2.5 (s).
# The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
RP-HPLC conditions
The RP-HPLC system consisted of a Waters 2695 separations
module, a Waters 2996 photodiode array detector and a C18
column (Venus-C18-5-100, 200 4.6 mm). The 42:58 (v/v)
mixture of methanol– water (with 0.15 mol/L of ammonium
dihydrogen phosphate in water) was used as the mobile phase in
the chromatography experiments. The chromatographic test
was conducted under the flow rate of 0.6 mL/min, the detection
wavelength of 240 nm, the column temperature of 308C and the
injection volume of 20 mL.
Sample pretreatment for RP-HPLC
PA and SPA are not stable and decompose easily in the mobile
phase, so anhydride and its analogs needed to be converted into
stable compounds for analysis. Another advantage is that it can
be converted into proper polar substances for RP-HPLC analysis
(9, 10). Two methods are commonly used: hydrolysis and
alcoholysis.
Hydrolysis
Fifty milligrams of PA or sulfonation mixture was added to a
50 mL RBF and 15 mL of water was added. The mixture was
heated and stirred for 30 min at 908C. After this, 10 mL of ultrapure water was used for flushing the inner wall of the condenser.
The solution was combined, transferred into a 50 mL flask and
completed to final volume with water.
Figure 1. Reactions of sulfonation, hydrolysis and alcoholysis of PA and SPA.
According to the 1H-NMR spectra, the single peak at approximately 13 ppm belongs to the hydroxyl group, indicating the existence of benzene sulfonic acid groups. The peaks at 2.5 ppm
are from the solvent (DMSO-d6) (7). The coupling constants
of o-phenyl hydrogen are 7 –10 Hz and the coupling constants of
m-phenyl hydrogen are 1 –3 Hz. The coupling constants
of P-phenyl hydrogen are generally less than 1 Hz (8). The single
peak at 12.90 ppm belongs to hydrogen from the sulfonic group.
The double peaks at 7.789 ppm belong to Hb, some belong to Ha
(J ¼ 8.289) and a long distance couple belong to Hc (J ¼
1.507 Hz). The Ha, Hb and Hc were difined as the hydrogen on
the benzene ring of SPA, shown in Figure 1. Therefore, the
double peaks at 7.661 ppm belong to Ha and the double peaks at
7.872 ppm belong to Hc. Hc is located in the lowest field
because of the electron withdrawing effect from the sulfonic
acid and carbonyl groups; Ha is located in the highest field,
because of the single effect from the carbonyl group, which is in
accordance with the analysis of coupling constants. According
to the analysis, the compound is four-substituted SPA.
The sulfonation of PA cannot be completely conducted: it is
limited by the sulfonation equilibrium. Therefore, the mixture of
PA and SPA with a certain sulfonation degree was obtained,
which was called a sulfonation mixture in this paper. This sulfonation mixture can be used to directly synthesize sulfonated
cobalt phthalocyanine with different amounts of sulfonic acid
substituents. This procedure is very economical, because there
is no need to separate the SPA from the mixture, and saves the
usage of raw materials. Therefore, it is needed to analyze the
contents of PA and SPA in the product mixture.
Alcoholysis
Fifty milligrams of PA, SPA or sulfonation mixture was added to a
50 mL RBF and 15 mL of ethanol was added. The mixture was
heated to a certain temperature and stirred for a certain time.
After this, 10 mL of ethanol was used to flush the inner wall of
the condenser. The solution was combined, transferred to a
50 mL flask and completed to final volume with water.
For the alcoholysis reaction, the reaction temperature and
time were the primary factors on the reaction, which was
studied systematically. Six samples of PA (50.0 mg) were accurately weighed and placed into a 50 mL RBF, 15 mL of ethanol
were added and the mixture was heated at 50, 60, 70, 80, 90 and
1008C and stirred for 30 min. The samples were analyzed by
HPLC. The same procedures were used for six samples of SPA
(50.0 mg).
Five samples of PA (50.0 mg) were accurately weighed and
placed into a 50 mL RBF, 15 mL of ethanol were added and the
mixture was heated at 1008C and stirred separately for 15, 30,
45, 60 and 90 min. The samples were analyzed by HPLC.
The same procedures were used for five samples of SPA
(50.0 mg).
Determination of the contents of PA and SPA
A standard solution of PA and SPA was prepared and the concentration in the solution was determined by external standard
curve method. Therefore, the sulfonation degree of the product
could be calculated from the content of PA and SPA.
For the standard solutions of PA, 25.2 mg of PA was weighed
accurately, placed into a 50 mL RBF and 15 mL of ethanol was
added. The mixture was heated at 1008C for 45 min, transferred
New Determination Method for Sulfonation Degree of Phthalic Anhydride by RP-HPLC 391
into a 50 mL flask and completed to final volume with water.
A solution of 504.0 mg/L was prepared. Two, 4, 6, 8, 10 and
12 mL were pipetted into six 50 mL volumetric flasks and completed to final volume with water. Therefore, 20.16, 40.32, 60.48,
80.64, 100.8 and 120.96 mg/L PA solutions were obtained and
analyzed by HPLC.
For the standard solutions of SPA, 25.2 mg of SPA was used for
the standard solutions. According to the procedures for PA, solutions of 20.16, 40.32, 60.48, 80.64, 100.8 and 120.96 mg/L SPA
were obtained and analyzed by HPLC.
By associating the peak area and concentration, the standard
curve was obtained, by which the concentration of PA in the
mixture could be calculated from the HPLC peak area.
Determination of the sulfonation degree of PA
After the sulfonation reaction, 50 mg of the PA/SPA mixture or
sulfonation mixture was used for alcoholysis. The solution was
analyzed by HPLC, so the concentrations of PA and SPA were calculated by the external standard method. The degree of sulfonation was calculated from the concentrations of PA and SPA by
using the relation of Eq. (1). Y1 and Y2 represent the contents of
PA and SPA, respectively, in the mixture and S% is the sulfonation
degree.
S% ¼ Y2 / (Y1 þ Y2) 100% .
(1)
To study the robustness of the determination, one chromatographic condition was changed in a certain range while the
other conditions remained unchanged. The solution was determined at the sulfonation degree of 23.9% by HPLC.
Results
RP-HPLC analysis of the hydrolysis products or alcoholysis
products of PA and sulfonation mixture
The contents of the hydrolysis of PA and sulfonation mixture
were analyzed by RP-HPLC and the results are listed in Figures 2
and 3. The contents of alcoholysis of PA and the sulfonation
mixture were analyzed by RP-HPLC and the results are listed in
Figures 4A and 4B.
Figure 2. Chromatogram of PA after hydrolysis (Peak 1: phthalic acid).
392 Zhu et al.
Optimization of alcoholysis temperature
A study was conducted on the temperature of PA alcoholysis,
which revealed that as the temperature increases, the peak
area of phthalic acid decreases and the peak area of
2-(ethoxycarbonyl) benzoic acid gradually increased, as shown
in Figure 5. When temperature reached 908C, the phthalic
acid peak disappeared completely. At 1008C, the peak areas of
2-(ethoxycarbonyl) benzoic acid were stable. Similarly, the study
of SPA alcoholysis revealed that when the temperature was
above 908C, the peak of sulfophthalic acid did not appear, and
only peaks from SPA appeared (Peaks 4 and 5). At the same time,
the peak areas were stable at 1008C. Therefore, 1008C was
chosen as the alcoholysis temperature for PA and SPA.
Optimization of alcoholysis time
When the alcoholysis of PA was more than 30 min, the
peak of phthalic acid disappeared and the peak area of
2-(ethoxycarbonyl) benzoic acid reached maximum, as shown
in Figure 6. When the time for alcoholysis of the SPA was more
than 45 min, the alcoholysis of SPA was completed. Therefore,
45 min was selected as the best alcoholysis time. After optimization of the alcoholysis conditions, PA, SPA and sulfonation
mixture were subjected to alcoholysis at 1008C for 45min and
the chromatograms in Figures 4C, 4D and 4E were obtained.
There are no peaks of phthalate acid and 4-sulfophthalic acid,
only alcoholysis products from PA and SPA at three peaks on
the chromatogram, which indicates that the alcoholysis was
complete.
Quantitative analysis of PA and SPA
The standard curve method was used to determine the content
of PA. Using the integral area of the peaks as the horizontal axis
and the content of PA as the vertical axis, the standard curve
equation in Table I was obtained with a correlation coefficient of
0.9994. The accuracy of the concentration of PA calculated by
this formula can meet analysis needs. The same procedure was
applied to set up the equation for SPA. The difference was that
there were two peaks. Both represented the alcoholysis product
of SPA, so the sum of the two peak areas was used as the horizontal axis and the content of SPA was the vertical axis. The standard
curve equation in Table I was obtained with a correlation
Figure 3. Chromatogram of PA and sulfonation mixture after hydrolysis (Peak 2:
4-sulfophthalic acid; Peak 1: phthalic acid).
Figure 6. Effect of time on the alcoholysis reaction.
Table I
Parameters of the Analytical Performance of the Proposed Method (n ¼ 6)
Figure 4. Chromatogram of alcoholysis products: PA after incomplete alcoholysis (A);
sulfonation mixture after incomplete alcoholysis (B); PA after complete alcoholysis (C);
SPA after complete alcoholysis (D); sulfonation mixture after complete alcoholysis (E).
Peaks: 1, phthalic acid; 3, 2-(ethoxycarbonyl) benzoic acid; 4, 2-(ethoxycarbonyl)-4sulfobenzoic acid; 5, 2-(ethoxycarbonyl)-5-sulfobenzoic acid.
Compound
Slope
Y-intercept
Correlation
coefficient (r)
Linear
range
(mg/L)
LOD
(mg/L)
LOQ
(mg/L)
PA
SPA
8.00 1026
3.00 1026
2.433
3.600
0.9994
0.9989
20–120
20–120
5
9
20
20
coefficient of 0.9989. By using the peak area the contents of PA
and SPA can be calculated.
Under the optimum conditions, the characteristics of the new
method were investigated and the results are shown in Table I. It
was found that the method is linear over the range of 20 –
120 mg/L of PA and SPA, with correlation coefficients of 0.9994
and 0.9989. The limit of detection (LOD) was defined as the concentration of PA and SPA in the standard solution after dilution
producing a peak in the chromatogram with a signal-to-noise
ratio of 3; LODs were 5 and 9 mg/L. The limit of quantification
(LOQ) for the two compounds was 20 mg/L. These results indicated that the method is a simple and reliable procedure for the
rapid determination of the contents of PA and SPA in the
mixture of the product of PA sulfonation.
Determination of the degree of sulfonation
To test the accuracy of this method, four samples of PA and SPA
mixtures were prepared with sulfonation degrees of 2.8, 23.9,
56.4 and 71.2%. The sulfonation degree was determined by
following the previously described procedure. The results were
compared with those previously prepared. In Table II, when the
sulfonation degree was in the range of 2.8 –71%, the recovery
rate was between 97 and 104%. The results of the robustness test
are shown in Table III. The chromatographic conditions were
changed within a certain range and the relative error of the
sulfonation degree was less than 3%.
Figure 5. Effect of temperature on the alcoholysis reaction.
Discussion
PA and SPA decomposed easily in the mobile phase, such as in a
methanol–water solution, so hydrolysis was first taken into
New Determination Method for Sulfonation Degree of Phthalic Anhydride by RP-HPLC 393
the allowable range, indicating that this method is feasible for determination of the degree of sulfonation.
Table II
Recovery of the Degree of Sulfonation in the PA and SPA Mixtures
Number
Prepared (%)
Measured (%)
Relative error (%)
Recovery (%)
1
2
3
4
2.8
23.9
56.4
71.2
2.9
24.5
55.0
69.8
3.57
2.51
– 2.48
– 1.97
103.6
102.5
97.5
98.0
Table III
Robustness Test of the Determination of Sulfonation Degree
Number
Item
Condition
Measured
(%)
Relative error
(%)
1
Flow rate (mL/min)
2
Column temperature (8C)
3
Mobile phase composition (v/v of
methanol –water)
4
Injection volume (mL)
5
Detector wavelength (nm)
0.5
0.6
0.8
25
30
35
40:60
42:58
45:55
15
20
236
240
242
24.5
23.5
23.7
23.2
24.5
24.2
24.2
24.5
23.6
24.4
24.5
21.2
23.4
26.1
2.51
–1.67
–0.84
–2.93
2.51
1.26
1.26
2.51
–1.26
2.09
2.51
–11.3
–2.09
9.21
Conclusions
IR and NMR spectra analyses show that the 4-position SPA is
obtained after sulfonation of PA. When determining the sulfonation degree by RP-HPLC, pretreatment is necessary and alcoholysis is better than hydrolysis. According to the study on the
alcoholysis temperature and time, 1008C and 45 min are the best
conditions for complete alcoholysis. A new determination
method is established for the sulfonation degree of PA by
RP-HPLC. When the sulfonation degree is between 2.8 and 71%,
the relative error is less than 4%; the recovery of the determination is between 97 and 104%. The robustness test results are satisfactory, and the sulfonation degree can be accurately measured
in a wide range of chromatographic conditions. This is of great significance for the synthesis of sulfonated metal phthalocyanine.
Acknowledgments
This work is supported by the scholarship under State
Scholarship Fund (2009103222) and China Postdoctoral Science
Foundation (2012M521386).
References
account in sample pretreatment. The retention times of phthalic
acid and 4-sulfinophthalic acid are approximately 2 –4 min.
It was very short, almost the same as the dead time. The retention time of 4-substituted phthalic acid is shorter, because of its
stronger polarity. The retention time is so short that it will cause
peak overlap and result in inaccurate calculation of the peak
area. By comparing the spectrum of alcoholysis products of PA
and the sulfonation mixture (Figures 4A and 4B) with the spectrum of the hydrolysis product (Figures 2 and 3), it is clear that
the retention time is long enough, the peaks are distributed in
2 –9 min and there is no overlap. Therefore, alcoholysis is better
than hydrolysis for analysis. However, the hydrolysis products 4sulfophthalic acid and phthalic acid were detected, which indicates that the alcoholysis was incomplete and the peaks came
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the standard curve is not applicable at 242 and 236 nm and the
peak area of sulfonation mixture was influenced in varying
degrees. The relative error recovery and robustness test are in
394 Zhu et al.
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