Macromolecules by total reflection X-ray fluorescence: marking techniques

Spectrochimica Acta Part B 58 (2003) 2169–2175

Macromolecules by total reflection X-ray fluorescence: marking techniques夞 a,b ´ S. Boeykensa,*, C. Vazquez , N. Tempranoa a

´ ´ ´ Universidad de Buenos Aires, Paseo Colon ´ 850, Laboratorio de Quımica de Sistemas Heterogeneos. Facultad de Ingenierıa, Buenos Aires 1063, Argentina b ´ ´ Nacional de Energıa ´ Atomica, ´ Unidad de Actividad Quımica, Comision Av. Gral. Paz 1499, Buenos Aires 1650, Argentina Received 10 January 2003; accepted 1 July 2003

Abstract This paper proposes an optimized method to mark polysaccharide macromolecules with heavy atoms in order to make possible their detection by total reflection X-ray fluorescence. A chemical reaction was employed to produce the substitution of OH groups of the polymer macromolecules by iodine atoms. Temperature, time, concentration of reactants and pH of the labeling chemical reaction were the variables and the relative sensitivities of the introduced atoms in the TXRF determinations were the optimization parameters for the TXRF determinations. Control of the physical properties of the polymer and the labeled product were made in order to prevent chemical alterations. The quantification of the labeled macromolecules was made by a previous careful calibration. 䊚 2003 Elsevier B.V. All rights reserved. Keywords: Marking; Labeling; TXRF analysis; Macromolecules; Polysaccharides

1. Introduction The applicability of the total reflection X-ray fluorescence (TXRF) analysis for polymer systems have been proposed in previous works w1,2x. TXRF is a variation of energy-dispersive X-ray fluorescence with a different optical arrangement and is used for chemical micro, ultramicro and trace analyses because of its high detection power and 夞 This paper was presented at the 9th Symposium on Total Reflection X-Ray Fluorescence Analysis and Related Methods, held in Madeira, Portugal, September 2002, and is published in the Special Issue of Spectrochimica Acta Part B, dedicated to that conference. *Corresponding author. Fax: q54-11-43431852. E-mail address: [email protected] (S. Boeykens).

sensitivity. Matrix effects cannot be built up within the thin layer of the sample used. In conventional spectrometers, only elements of atomic number higher than Si are determined w3x, for this reason, polymer analysis mainly composed by carbon, hydrogen and oxygen cannot be detected. The polysaccharides have an important use in enhanced oil recovery (EOR), food and pharmaceutical industries, based on their ability to alter viscosity, density and solubility of the water solutions w4x. The solutions of these macromolecules are non-Newtonian fluids. This means that the viscosity of the solutions is not constant but depends on the shear rate in the fluid showing non-linear flow curves. The relation used to

0584-8547/03/$ - see front matter 䊚 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.sab.2003.07.005

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Fig. 1. Two steps chemical iodation reaction for labeling scleroglucan macromolecules.

characterize this behavior is expressed by: hskg˙ yn

(1)

where, h is the viscosity, g˙ is the shear rate, k is a measure of the consistence of the fluid and n is a measurement for the degree of non-Newtonian behavior w5x, both are constants for a particular fluid. The aim of this work was to label the scleroglucan, a neutral b glucan polysaccharide with an average molecular weight of 4=106 Da, with iodine atoms by a fast and simple chemical reaction. The introduction of iodine atoms into the polysaccharide makes possible the TXRF detection of this macromolecule. The working variables as the concentration of reactants, pH, temperature and time were optimized for the best sensitivity in the TXRF detection. 2. Experimental 2.1. Reagents Scleroglucan (Actigum CS11) was provided by Sanofi Bio Industries (France). The polymeric systems were prepared by shaking a weighted mass of solid in ultrapure water (18 mV cm resistivity) and after purified by dialysis using a A.H. Thomas Co., No 4465-A2 membrane. These processes were made following a protocol especially tested and

described in a previous work w6x. p-toluenesulfonyl chloride (tosyl chloride) was provided by Aldrich (T3595.5), sodium hydroxide and hydrochloric acid by Merck and sodium iodide u.s.p. (granular) by Mallinckrodt. Nitric acid was prepared by subboiling distillation of reagent grade feed stocks. Standard solutions of S and I for TXRF calibration were Tritisol Merck. The glass vessels used in this work, were pre-cleaned by continuous steaming with gently diluted nitric acid prepared as before. Surface of the reflector was cleaned with dilute nitric acid, rinsed with high-purity water and wiped with a non-bleached paper towel. 2.2. Labeling the macromolecule The chemical reaction of iodation for labeling the scleroglucan is performed in two steps. The first step consists on adding, with continuous stirring, a saturated solution of p-tosyl or p-toluensulfonyl chloride, w(C6H6)–CH3–SO2 xq Cly, to a scleroglucan solution. The electronegativity of O atoms causes the introduction of p-tosyl group into the scleroglucan molecule, through the S atom. In this way, the scleroglucane–p-tosyl complex is chemically activated and, under these conditions, the p-tosyl which is a very good leaving base, is displaced by the introduction of in situ Iy ions in a second step. Fig. 1 shows the chemical process. The proposed chemical reaction to label the polymer is fast and simple and it has an easily controlling performance as relevant characteristics.

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The reaction was managed avoiding the polymer degradation and taking into account that the macromolecules were labeled without altering their physical properties; this means the substitution of only a few OH groups per macromolecule, and reaching the lower detection limit for the polymer by TXRF. In order to obtain the maximum sensitivity in scleroglucan TXRF detection, the simplex method was used to rapidly optimize the three factors or variables of the reaction temperature (T), time (t) and pH w7x. All the experiments were carried out in an excess of p-tosyl reactive. The model algorithm was: Rsb0qb1pHqb2Tqb3t

(2)

where, in this case, the response (R) was the intensities ratio of sulphur (in the first step of the reaction) or iodine (in the second step) to cobalt, and: b1s

≠R ≠ pH

(3)

b2s

≠R ≠T

(4)

≠R ≠t

(5)

b3s

2.3. X-Ray analysis X-Ray analysis was carried out by using a total reflection system composed of an X-ray spectrometer, an X-ray tube excitation system, a total reflection module and spectrum acquisition and quantitation software w8x. The X-ray spectrometer consisted of an 80 mm2 Si(Li) detector with 166 eV of full width half maximun for 5.9 keV, a 0.008-mm thick Be window, an Ortec 672 fast spectroscopy amplifier and an analoge digital converter (ADC) Nucleus PCA2. Excitation conditions were 40 kV and 30 mA in all cases. The TXRF designed at the Atominstitut der ¨ ¨ Ostereichischen Universitaten was employed, fitted

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with a cut-off-filtered radiation from a fine focus diffraction molybdenum anode X-ray tube w9x. For spectral data analysis, the AXIL program was used. The QXAS software package from the International Atomic Energy Agency, was employed for data quantitation. TXRF technique was used for testing the development of the chemical reaction and then, it was adjusted for the analysis of scleroglucan labeled solutions. As the matrix effects are negligible, a set of calibration aqueous standard solutions containing S and I covering a range between 5 and 50 mg ly1 was made. Co was chosen as internal standard due to its non-interfering properties. The system sensitivity required 0.5 mg ly1 Co content. The fluorescent intensity of the Ka or La lines was measured for S and I, respectively. The acquisition time for each spectrum was 200 s. For measurements, an aliquot of 10 ml of standard solution or sample was pipetted onto a quartz glass sample carrier and allowed to dry to a thin film under an infrared lamp. Four replicates of each sample were analyzed. Intensity ratio between element i and internal standard (Co) was obtained by: Ii si Ci s ICo sCo CCo

(6)

where, Ii is the line intensity (counts sy1) of the element i in the sample; Ci is its concentration (mg mly1); ICo is the line intensity of the internal standard (Co) in the sample; CCo is its concentration; si is the system sensitivity (counts ml mgy1 sy1) for the element i; sCo is the system sensitivity for the internal standard (Co). Relative quantities (Ri) (mg mly1) of the element i for this system are defined by: RisSriCi

(7)

si , is the non-dimensional relative sCo sensitivity of the system for the element i. Fig. 2 shows the calibration curves for S and I in the aqueous matrix. The slope of these curves, where: Sris

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Fig. 2. Calibration curves for I and S in scleroglucan solutions using Co as internal standard. Non-dimensional sensitivity (S) and correlation coefficients (r 2) are shown beside the curves.

the relative sensitivity (Sri) and the regression coefficient (r 2) are also shown. After the optimization of the chemical reaction, in order to know the relationship between the sulphur (in the first step of the reaction) or the iodine (in the second step) intensity lines and the

scleroglucan content in the marked solutions, two new calibration curves in labeled scleroglucan solutions were made. Fig. 3 shows the relative quantity of sulphur and iodine vs. the scleroglucan content in the solution. It is interesting to note that the lost in sensitivity are due to the fact that the

Fig. 3. Calibration curves for marked scleroglucan solutions. The sensitivities (S) for this method and the correlation coefficients (r 2) are shown beside the curves.

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Table 1 Range of variables, their final selected values and optimized parameters for the first and second steps of the scleroglucan proposed marking chemical reaction

First step Second step

pH range

pH selected

Temperature range (8C)

Temperature selected (8C)

Time range (h)

Time selected (h)

Optimized parameter

7–12

12

20–80

50

2–48

24

ISyICos15

2–7

3

20–80

70

2–48

10

IIyICos20

mass of the analyte (sulphur or iodine) is only a portion of the total mass of scleroglucan. However, sulphur shows a similar sensitivity to iodine due to the major quantity of sulphur in the scleroglucan p-tosyl ester intermediate. The detection limits (DL) (mg ly1) for scleroglucan were calculated by using w10x: DLs3

CCo SriICo

yI t

i,BG

(8)

where: Ii,BG is the background intensity (counts sy1) and t the measuring time. 3. Results and discussion The chemical reaction was tested both in acidic and basic media due to the wide range of the polymer triplex stability, from pH 3 to 12 w11x. The TXRF performance study of the first step of the reaction with an excess of 150% of the ptosyl reactive was made at different pH, temperature and times by measuring the concentration of sulphur in the product after purification by dialysis. The optimal temperature, time and pH were determined by the simplex method taking into account the limits imposed by the polymer degradation and the triple helical structure destruction of the scleroglucan that could lead to some very different products. Table 1 shows the range of the variables studied, their final selected values and the optimized value of the parameter IS yICo in the TXRF analysis. In the first step, an esterification, a basic medium is indicated. The slow dissolution of the p-tosyl chloride is produced while the reaction proceeds and the liberated hydrochloric acid is neutralized by the addition of NaOH.

In the second step the conditions were also limited by the degradation of the polymer. After purification by dialysis, the study of the TXRF performance was done by measuring the iodine concentration in the product solutions, in this case, a supplementary TXRF control was done: the total elimination of sulphur. Besides, the studies on the properties of the final product were made to adjust the final conditions of the reaction, taking into account that properties of the product should not differ from properties of the reactants. Table 1 shows the range of variables studied by the simplex method and the selected conditions for better performance of labeling chemical reaction as the parameter II yICo in the TXRF analysis. In this second step, the OH group competes in advantage (hydrolysis of the ester) against the iodide for the displacement of the p-tosyl attached group and the performance of the reaction drops. The acid medium favors substitution. The lower the pH value the higher is the efficiency. Finally, quantitative TXRF analysis of scleroglucan labeled solutions showed that one p-tosyl group kept attached by every one monomer unit of the polysaccharide in the first step of the reaction. The TXRF detection limit found for these solutions was 5 mg ly1. In the second step, 60% of the introduced tosyl groups were substituted by iodide and the rest of them were hydrolyzed, in these final marked solutions the detection limit found was 2 mg ly1. Fig. 4a, b and c show the TXRF spectra coming from the scleroglucan, tosyl scleroglucan complex (S–Ka signal) and iodine scleroglucan (I–La signal), respectively. Rheological measurements were done in order to determine the change in the macromolecule behavior caused by the chemical reaction w12x.

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Fig. 4. TXRF spectra from scleroglucan (a), tosyl scleroglucan complex (b) and iodine scleroglucan macromolecules. Ka and La lines from S (b) and I atoms (c) are indicated, respectively.

The logarithmic plots of viscosity (h) and shear rate (g˙ ) measured on samples of the same concentration of scleroglucan and iodide derivatives show coincident exponent n. It was 0.72 for 0.2% macromolecule concentration. The p-tosyl ester showed an exponent slightly smaller (ns0.70). Table 2 shows the measured physical properties

for scleroglucan, p-tosyl ester and the marked macromolecule comparatively. There were no significant differences between the scleroglucan and the final marked macromolecule in all the measured properties. Rather different was the case of p-tosyl ester due to the weight and number of the p-toluensulfonic groups.

Table 2 Comparison of physical properties of 0.2% scleroglucan and derivatives solutions Rheological exponent n

Mean molecular weight Mwa

Polidispersity MwyMna

Density d (gycm3)

Water solubility (gy100 g)

DMSO solubility (gy100 g)

0.72

4=106

1.20

1

–

–

Tosyl scleroglucan

0.70

5=10

6

1.35

1

difficult

–

Iodide scleroglucan

0.72

4=106

1.23

1

–

–

Scleroglucan

a

GPC w13x.

S. Boeykens et al. / Spectrochimica Acta Part B 58 (2003) 2169–2175

Hydrolysis of these groups is very important in the second step of this method, then the final labeled polymer is much more similar to the original that the intermediate one. 4. Conclusions The iodine labeled scleroglucan showed in its molecular structure two iodine atoms for each five monomer units. It makes a difference of 5% in weight. The physical–chemical behavior was very similar to the original scleroglucan. Replacement of OH groups to obtain detectable macromolecule makes possible the experiences by TXRF analysis. Even though the p-tosyl ester can be detected by TXRF, the reaction should proceed to the second step to avoid difficulties originated by differences in the rheological behavior of these macromolecules. Acknowledgments This work was supported by grants from the following projects PICT-CONICET No 0269 and CyT No I049, Universidad de Buenos Aires (Argentina).

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