Jiugao Yu,
Dongzhi Yang
School of Science, Tianjin University, Tianjin, China
Synthesis and Application of Starch 3,5-Dinitrobenzoate
A novel starch ester was prepared by the reaction of starch and 3,5-dinitrobenzoyl chloride (DNBZCI). The product (starch 3,5-dinitrobenzoate, DNBZ-ST) was characterized by means of elemental
analysis, Fourier Transform Infrared (FTIR) and 13C NMR spectroscopy. The influences of molar
ratio of DNBZ-CI to anhydroglucose (AGU), reaction temperature and time, and amount of
pyridine on the degree of substitution (DS) were studied. The esterification reaction is essentially
complete after 2 h. Increase in the molar ratio of DNBZ-CI to AGU leads to an increase in DS when
the former varies from 2:1 to 5:1. DS increases with the reaction temperature when the latter is
varied from 70 to 100 °C. DS first increases and then decreases with amount of pyridine, the highest
DS was obtained when l/(pyridine)/m(starch) was 53 mL/g. The thermal stability of DNBZ-ST
increases when the DS increases, and the degradation starts at 353 °C for the sample of DS 2.14.
Creatinine is a toxin accumulated in the blood of chronic renal failure (CRF) patients and a special
adsorbent for creatinine is not reported in literature. DNBZ-ST displayed specific adsorption ability
for creatinine, the adsorption equilibrium is reached after 4 h. The adsorption capacity increases
with the DS of adsorbent and creatinine concentration. When the creatinine concentration is higher
than 300 mg/L, concentration has no apparent effect on the adsorption capacity. As the temperature
of the solution is varied from 19 to 49 °C, adsorption capacity first decreases and then increases,
being lowest at 37 °C. The adsorption capacity first increases and then decreases as the pH value
increases, and is highest at pH 8.The highest adsorption capacity obtained was 25 mg of creatinine
per gram of adsorbent at 37 °C, pH 7 and a creatinine concentration of 100 mg/L. The study on the
FTIR and UV-VIS spectra suggested that some chemical reaction took place between the DNBZ-ST
and creatinine in buffer solution.
Keywords: Starch 3,5-dinitrobenzoate; Adsorbent; Creatinine
1 Introduction
Although starch has been used for centuries, it continues to be attractive as a raw material for its
abundant supply, low cost reproduction, biodegradability, biocompatibility and ease of chemical
modification. Starch molecules possess a multitude of functional groups, i.e. three reactive hydroxyl
groups per repeating unit, which are accessible to the typical conversions of primary and secondary
alcoholic groups. However, most of the starch derivatives, which are available commercially, have
a low degree of substitution (DS = 0.01-0.20). Examples are starch acetate, starch succinate,
hydroxyalkyl starch obtained by reaction of starch and ethylene or propylene oxide, and
carboxymethyl starch obtained by reaction of starch and sodium monochloroacetate. Many other
types of starch derivatives have been prepared, including products with high DS (>0.5), but most of
these compounds had not
Correspondence: Dongzhi Yang, Department of Chemistry, School of Science, Tianjin University,
Tianjin 300072, China. Phone: +86 22 2740 0612, e-mail: ydz7654@sohu.com.
been commercialized, as was shown by Wurzburg [1]. Chemical modification continues to provide
a dominant route towards starch utilization in polymer-based materials.
In this paper, our interest was focused on starch 3,5-dinitrobenzoate, which was formed by reaction
of starch and 3,5-dinitrobenzoyl chloride. This derivative was employed as a new adsorbent used
for removal of creatinine in buffer solution.
Creatinine (2-imino-1-methylimidazolidin-4-one) is a byproduct of nitrogen metabolism related to
muscular mass, it is excreted in human urine in the range of 1.2 to 1.8 g/day for a normal kidney
function.
Creatinine is accumulated in the blood of CRF patients and may promote the progression of CRF
[2-4]. In the clinical therapy, creatinine is removed mainly by he-modialysis (HD). Despite the
success in this treatment, there are some physical and economical disadvantages in these HD
procedures, as was shown by Babb [5]. Creatinine was reported to appear in gastrointestinal tracts,
and earlier studies suggested that oral adsorbents could improve uremia symptoms and delay the
initiation of HD. In order to support HD, the use of oral adsorbents such as activated charcoal or
ion-exchange resins is typical, as was shown by Koide [6], Liu [7] and Takashi [8]. However, the
adsorbents have some disadvantages, e.g. poor selectivity, low adsorption capacity, poor
biocompatibility, etc. Up to now, an ideal adsorbent of good selectivity and high adsorption
capacity for creatinine has not been found.
The reaction of picric acid (2,4,6-trinitrophenol) with creatinine, which is the basis of the method
for the clinical determination of creatinine concentration, has been thought to yield red complex
tautomers of creatinine-picric acid (1:1 and 2:1) [9-11]. 3,5-Dinitrobenzoic acid (DNBA) and 3,5dinitrobenzoyl chloride were more specific reagents for creatinine than picric acid. During their
reaction with creatinine a purple complex is formed [12]. The mechanism of the reaction of DNBA
or DNBZ-CI with creatinine is similar to that of picric acid, but up to now, the exact structure of the
purple complex has not been determined.
In this paper the preparation of a novel starch derivative DNBZ-ST with a wide range of DS is
reported, using pyri-dine as catalyst. The products are characterized by elemental analysis, FTIR
and 13C NMR spectroscopy. Furthermore, the thermal stability of samples was investigated. The
DNBZ-ST samples' adsorption properties for creatinine were examined. Finally, the adsorption
mechanism was investigated fundamentally.
2 Materials and Methods
2.1 Chemicals
Corn starch (25% amylose) was provided by Langfang Chemical Co. (Langfang, China) and used
after drying at 100 °C for 24 h under vacuum, having a moisture content less than 1% (w/w).
Reagent grade DNBZ-CI was obtained from Shanghai Chemical Co. (Shanghai, China), and used
after purification by recrystallization from carbon tetrachloride. Others reagent grade chemicals,
purchased from Tianjin Chemical Co. (Tianjin, China) were used without further purification.
2.2 Synthesis of starch 3,5-dinitrobenzoate
The synthesis of DNBZ-ST was carried out in two steps. First, activation of starch was performed
by mixing starch
and pyridine at 115 °C and stirring for 1 h. Second, the reaction mixture was cooled down to the
desired temperature, DNBZ-CI was added and the mixture was stirred at this temperature for a
definite time. The synthesized DNBZ-ST samples were isolated by precipitation in 500 ml_ of
absolute ethanol. Washing was performed as follows: the samples were washed repeatedly by
stirring in 500 mL 80% (v/v) ethanol at 50 °C for 1 h, then filtered. When any color impurities and
by-products were eliminated, the washing was finished. After filtration, the starch ester was dried at
50 °C overnight under vacuum.
2.3 Characterization of DNBZ-ST
The FTIR of samples were performed on KBr pellets containing 3 mg of sample in 300 mg of KBr.
The spectra were obtained on a Perkin-Elmer 2000 Fourier Transform infrared spectrometer
(Perkin-Elmer, Norwalk, CT). Elemental analysis was carried on a Vario Elemental Analyzer
(supplier, place?) after drying the samples at 100 °C for 24 h. Standard 13C NMR spectra of samples
were measured in solid-state on a Varian Unity-Plus 400 spectrometer (400 MHz) (Varian, Palo
Alto, CA). The UV-VIS spectra of samples were measured on a Hewlett Packard 8453
Spectrophotometer (Hewlett Packard, Palo Alto, CA).
2.4 Determination of the thermal stability
Thermogravimetric (TG) measurements were performed on a WCT-1 TG Analyzer (Beijing Optical
Instruments Factory, Beijing, China). Each sample was heated up to 700 °C with a heating rate of
15 °C/min in nitrogen gas. Before thermal analysis, the samples were dried in a vacuum oven at 80
°C for 24 h.
2.5 Analysis for creatinine adsorbed by DNBZ-ST
Creatinine (10 mg) was dissolved in 100 mL Na2HPO4 -NaH2PO4 buffer (pH 7.0). Dry adsorbent
(DNBZ-ST) was added to the creatinine buffer solution and the mixure shaken at 37 °C for 4 h.
Creatinine concentration was measured according to the Jaffe procedure [13]. The adsorption
capacity of DNBZ-ST was calculated from the decrease of creatinine concentration in the solution.
3 Results
3.1 Preparation of DNBZ-ST
The degree of substitution (DS) for starch is defined as the moles of substituted hydroxyl groups per
mole of D-glucopyranosyl structural units. Since each repeating unit contains three hydroxyl
groups, the theoretical maximum of DS is three. Tab. 1 shows the C, H and N content of the
Tab.1. Dependence of DS on the esterification reaction condition.a
synthesized DNBZ-ST determined by elemental analysis. The DS was calculated from the N
content as follows:
The optimization of the process of esterification was performed by varying the reaction parameters
such as the molar ratio of DNBZ-CI to AGU, reaction temperature, duration of reaction and amount
of pyridine. Each parameter was varied while other parameters were kept constant, as shown in
different sets of experiment in Figs. 1-4.
3.1.1 Influence of the molar ratio DNBZ-CI to AGU
To evaluate the accessible range of the DS, the molar ratio of DNBZ-CI to AGU was varied from
2:1 to 5:1 (see Fig. 1). The DS increases with increasing amount of DNBZ-CI per AGU, but it
increases only slightly when the molar ratio was varied from 3:1 to 4:1. This is probably because of
such factors as follows: the esterification reactivity of three groups in AGU is different, as the molar
ratio reached 3, most of the hydroxyl groups with higher reactivity had been substituted. As the
molar ratio was increased from 4:1 to 5:1, the DS again increased this is
probably because of the esterification of hydroxyl groups with lower reactivity.
3.1.2 Influence of reaction temperature
The DS of starch increases with raising of reaction temperature in the investigated range of reaction
temper-
Fig. 1. Dependence of DS on n(DNBZ-CI)/n(AGU). Other reaction conditions: starch 3 g, reaction
temperature 90 °C, reaction time 2 h, pyridine 80 mL, stirring rate 80 rpm.
atures, the largest DS was obtained at 100 °C (see Fig. 2), but the reaction mixture became a
viscous mass and the precipitating and washing were difficult at this temperature. In order to obtain the products with higher DS and homogeneous structure, the esterification
reaction in Fig. 1, Fig. 3 and Fig. 4 was carried out at 90 °C.
Fig. 2. Dependence of DS on the reaction temperature. Other reaction conditions: starch 3 g,
n(DNBZ-CI)/n(AGU) 4:1, reaction time 2 h, pyridine 80 mL, stirring rate 120 rpm.
Fig. 3. Dependence of DS on the reaction time. Other reaction conditions: starch 3 g, n(DNBZCI)/n(AGU) 4:1, reaction temperature 90 °C, pyridine 80 mL, stirring rate 80 rpm.
3.1.3 Influence of reaction time
The esterification reaction was terminated after 1, 2, 3 and 4 h. The DS values of the obtained
products are given in Fig. 3. It can be seen that the DS increases with reaction time and reaches an
almost constant value after 2h.
3.1.4 Influence of pyridine
The results obtained by investigating the influence of the amount of pyridine on the DS of starch are
shown in Fig. 4. The amount of reactants during the experiment was constant, but the reactant
concentration decreased by increasing the amount of catalyst, i.e. pyridine. In Tab. 1, the highest
DS was obtained when 53 mL pyridine was used per gram of starch. This is probably due to the fact
that on the one hand the catalytic effect of pyridine increases with concentration, on the other hand
the pyridine dilutes the reactants.
3.2 Characterization of DNBZ-ST 3.2.1 Analysis of FTIR
The FTIR spectra of native and esterified starch are shown in Fig. 5. In the spectra of the
esterification products, compared with that of native starch, strong absorption bands at 1740 cm-1
were observed which can be ascribed to the vc=o of ester groups. Absorption bands corresponding to
an aromatic ring appear at 3100, 1600, 1079 and 719 cm-1. In addition, the characteristic band
between 3000-3400 cm-1 originates from hydroxyl band stretching. The intensity of this band
decreases in the esterified derivatives.
Fig. 2. Dependence of DS on the reaction temperature. Other reaction conditions: starch 3 g,
n(DNBZ-CI)/n(AGU) 4:1, reaction time 2 h, pyridine 80 mL, stirring rate 120 rpm.
Fig. 3. Dependence of DS on the reaction time. Other reaction conditions: starch 3 g, n(DNBZCI)/n(AGU) 4:1, reaction temperature 90 °C, pyridine 80 mL, stirring rate 80 rpm.
Fig. 4. Dependence of DS on the pyridine concentration. Other reaction conditions: starch 3 g,
n(DNBZ-CI)/n(AGU) 4:1, reaction temperature 90 °C, reaction time 2 h, stirring rate 80 rpm.
Fig. 5. FTIR spectra of starch and DNBZ-ST.
3.2.2 Analysis of 13C NMR spectra
Standard 13C NMR spectra of starch and DNBZ-ST (DS=0.54) were measured in solid state (see
Fig. 6). Assignments of the starch resonances are consistent with literature data. Signals at 99.5,
79.5 and 60.0 ppm are attributed to C-1, C-4 and C-6, respectively. The strong signal at 72.2 ppm is
associated with C-2,3,5.
The DNBZ-ST with DS 0.54 gives new signals at 126.9, 146.2, 120.2, and 162.0 ppm, because
hydroxyl groups were substituted by 3,5-dinitrobenzoyl groups, and these signals are assigned as C7,8, C-9, C-10 and C-11, respectively. Moreover, the signal of C-1'(C-1' for an O-2 substituted
AGU) appears at δ ≈ 99.5 ppm, and C-1' exhibits a upfield shift of about 4.5 ppm compared with
the corresponding carbon atom of the unsubstituted AGU. The signals of the C-2,3,5 and C-2'(C-2'
for an O-2 substituted AGU) appear at about 69.8 ppm, they can not be distinguished. The signal of
the C-6 appears at 60.5 ppm, and it refers to unmodified carbon atoms, because the shift of C-6 is
only 0.5 ppm. The signal at about 80.2 ppm corresponds more to C-4 than to C-4'(C-4' for an O-3
substituted AGU), because its shift is only 0.7 ppm compared with the unmodified AGU. However, by
the analysis above, it can be deduced that the O-2 of sample with DS 0.54 is obviously substituted.
3.3 Thermal behavior
The thermal behavior of DNBZ-ST was studied by means of thermogravimetry (TG).
Representative TG curves of the unmodified starch and DNBZ-ST show that the thermal
degradation of DNBZ-ST was initiated at a higher temperature than that of starch (302 °C). For
DNBZ-ST (DS = 1.32) the degradation starts at 326 °C and ends at 365 °C with a weight loss of
52.0%. The further heating of the sample up to 600 °C leads to carbonization of the sample and ash
formation. The stability of DNBZ-ST increases with the increase of its DS, e.g., the degradation
temperature of DNBZ-ST increases from 302 °C to 353 °C as its DS increases from 0.09 to 2.14
(see Fig. 7). This greater thermal stability of the esters is probably due to the lower amount of
unmodified starch which easily undergoes dehydration reactions between different molecules, as
was shown by Aburto [14].
3.4 Adsorption properties of DNBZ-ST 3.4.1 Influence of the DS of adsorbent
The relationship between the adsorption capacity and the DS of adsorbent was studied. The results
showed that the adsorption capacity was 10.7,15.0 and 25.0 mg of creati-nine per gram of adsorbent
as DS was in a range of 0.10, 0.54 and 2.09. The adsorption capacity of adsorbent increases with its
DS, but the increase is not linear. This trend suggested that the 3,5-dinitrobenzoyl groups play a key
role in the adsorption process, and it is most likely the functional group capable of adsorbing
creatinine.
Fig. 6. 13C NMR spectra of starch and DNBZ-ST with DS = 0.54 (400 MHz).
Fig. 7. TG curves of starch and DNBZ-ST; a) starch, b) DS = 0.09, c) DS = 1.32, d) DS = 2.14.
3.4.2 Influence of adsorption time
The adsorption dynamics of DNBZ-ST were examined, as shown in Fig. 8. The results indicate that
the adsorption is complete after 4 h, i.e. DNBZ-ST is efficient in removing creatinine from the
buffer solution.
Fig. 8. Adsorption dynamics curves of DNBZ-St with DS2.09 at 37 °C, pH 7.0 and creatinine
concentration 100 mg/L.
3.4.3 Influence of creatinine concentration in the buffer solution
The data in Fig. 9 summarize the adsorption capacity at different creatinine concentrations. The
adsorption capacity of adsorbent increases with creatinine concentration in solution, but when the
creatinine concentration is higher than 300 mg/L, it has no apparent effect on the adsorption
capacity. The saturated adsorption capacity of adsorbent (DS = 0.54) is about 60 mg of creatinine
per gram of adsorbent.
Fig. 9. Adsorption isotherm of DNBZ-ST with DS 0.54 at 37 °C, pH 7.0 and adsorption time 4 h.
3.4.4 Influence of adsorption temperature
Fig. 10 showed the effect of temperature on the adsorption properties. Adsorption capacity was
measured as the temperature of the solution was varied from 19 to 49 °C. Adsorption capacity first
decreases and then increases, and is lowest at about 37 °C. It was reported that temperature affects
chemical and physical adsorption processes in opposite ways, chemical adsorption increases with
increase of temperature, while physical adsorption decreases with increase of temperature, as was
shown by Giordano [15]. So creatinine adsorption on DNBZ-ST is probably effected both by
physical and chemical adsorption.
Fig. 10. Influence of temperature on the adsorption capacity at pH 7.0, creatinine concentration 100
mg/L and adsorption time 4 h.
3.4.5 Influence of pH value of the solution
When creatinine is dissolved in water and the solution made slightly alkaline, there is immediate
exchange of the methylene protons (pKa=13.4). In fact, creatinine is a fairly strong acid, which
easily loses one of the methylene protons: the juxtaposition of the carbonyl group stabilizes the
resulting anion, as was shown by Butler [16].
The dependence of the adsorption capacity on the solution pH is presented by a curve, which goes
through a maximum around pH 8 (see Fig. 11). One reasonable possibility: there is an equilibrium
between creatinine and the creatinine anion, more creatinine anion is formed when the basicity of
the solution increases. A Meisen-heimer complex is formed by attack of the creatinine anion on the
unsubstituted position of the benzene ring of the DNBZ-ST. So, the adsorption capacity of the
DNBZ-ST increases with increase of the pH value of the solution. However, when the pH value is
higher than 8, DNBZ-ST hydrolyzes to a significant extent and the adsorption capacity decreases.
Fig. 11. Influence of pH on the adsorption capacity at 37 °C, adsorption time 4 h and creatinine
concentration 100mg/L
3.5 Specific adsorption for creatinine in the hemodialysate
Although orally administered activated charcoal and ion-exchange resins are the most popular
adsorbents used for removal of uremic toxin, the research on these adsorbents is making only slow
progress, because they could also adsorb other matters in gastrointestinal tracts except creatinine.
For this reason, the DNBZ-ST's special adsorption function for creatinine in the hemodialysate system was determined. Experimental results showed that the adsorption capacity was approximately
25 mg of creatinine per gram adsorbent. This value is just as large as that obtained in phosphate
buffer solution. It means that the DNBZ-ST did neither adsorb inorganic ions such as K+, Na+,
PO43+ or Cl-, nor organic compounds such as amino acids, proteins, urea and uric acid, etc. Thus it
can be concluded that the adsorbent containing 3,5-dinitrobenzoyl groups is a specific adsorbent for
creatinine.
3.6 Adsorption mechanism
In the investigation above, the adsorption product was obtained as a purple precipitate. The FTIR
and UV-VIS spectra of this product were recorded and then compared with those of DNBZ-ST and
creatinine in order to investigate the adsorption mechanism.
Before recording the FTIR and UV-VIS spectra, the adsorption product was washed with distilled
water until creatinine could not be detected in the filtrate (by the Jaffe procedure), and then dried at
60 °C for 24 h under vacuum.
Fig. 12. FTIR spectra of a) DNBZ-ST, b) adsorption product, c) creatinine.
Fig. 13. UV-VIS spectra of a) starch, b) creatinine, c) DNBZ-ST, d) adsorption product.
The FTIR spectra of the adsorption product, creatinine and DNBZ-ST (DS = 0.54) are shown in
Fig. 12. In the adsorption product the intensity of the peak at 3100 cm-1 (vCH of the benzene ring)
slightly decreased. This is probably because creatinine interacted with the benzene ring of the
DNBZ-ST. There are new peaks at 1440, 1405 and 952 cm-1. In addition, the FTIR spectrum of the
adsorption product is similar to that of creatinine in the range of 1800-2800 cm-1 and 400-600 cm-1.
However, according to the difference of the spectra of the adsorption product and DNBZ-ST, it was
difficult to deduce the occurrence of chemical reaction and exact structure of the adsorption
product.
The UV-VIS spectra of starch, DNBZ-ST (DS = 0.54), creatinine, and the adsorption product were
recorded in 95% DMSO solution at a sample concentration of 0.4 g/L
(Fig. 13). The UV-VIS spectrum of starch did not show any adsorption in the region 200 to 800 nm
(Fig. 13 a), and that of creatinine showed two bands at 219 nm and 234 nm (Fig. 13 b). The UVVIS spectrum of DNBZ-ST had a maximum at 262 nm (Fig. 13 c), which is due to the benzene
ring, as was shown by Zhou [17]. This maximum was also found for the adsorption product, at the
same time, in the regions 340 to 460 nm and 510 to 660 nm two broad bands appeared, giving rise
to the purple color of the adsorption product.
The differences between the UV-VIS spectra of the DNBZ-ST and the adsorption product suggest
that some chemical reaction took place between DNBZ-ST and creatinine in buffer solution, but the
exact structure of the adsorption product needs further investigation.
4 Conclusions
By esterification of starch, a novel creatinine adsorbent with 3,5-dinitrobenzoyl groups was
prepared. Under optimal esterification conditions a DS of 2.14 can be obtained in the product. The
samples were characterized by means of elemental analysis, FTIR and 13C NMR spec-troscopy. The
thermal stability of products increases with the increase in DS. In vitro adsorption study DNBZ-ST
showed a good adsorption capability for creatinine. The adsorption capacity was related to the DS
of adsorbent, adsorption time, adsorption temperature, creatinine concentration and pH value of the
medium. The highest creatinine adsorption capacity was 25.0 mg of creatinine per gram of
adsorbent at 37 °C, pH 7.0 and a creatinine concentration of 100 mg/L. Studies on the factors
influencing the adsorption capacity and the FTIR and UV-VIS spectra of adsorption product
suggest that some chemical reaction took place between the DNBZ-ST and creatinine in buffer
solution. However, the detailed mechanism still needs further investigation.
In conclusion, adsorbent DNBZ-ST showed a better adsorption ability for creatinine in the buffer
solution, and is expected to remove excess creatinine in the gastrointestinal tracts of patients with
CRF. DNBZ-ST is worth to be studied more deeply in the future.
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(Received: January 25, 2002)
(1st Revision received: March 26, 2002)
(2nd Revision received: June 21, 2002)