Anal. Chem. 1983,55,2067-2070
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Characterization of Chemically Modified Carbonaceous Electrode Materials by Diffuse Reflectance Fourier Transform Infrared Spectrometry Robert M. Ian~niello,~ Henry J. Wieck,2 a n d Alexander M. Yacynych*
Department
of
Chemistry, Rutgers, The State University of New Jersey, New Bruaswick, New Jersey 08903
The Infrared spectra of surface specles on modifled carbonaceous materials have been obtained by use of diffuse reflectance Fourler transform infrared spectrometry (DRIFT). Comparlsons between graphite, graphitic oxide, and adlvated carbon Indicate that surface oxides can be observed, provlded that the material has hHgh surface area and relatlveiy high surface oxide concentration. The covalent attachment of histldine to actlvated carbon via carbodlimide modification is confirmed by the appearance of the cyclic amide band (1726 cm-’) In the infrared spectrum. Various characteristics of the spectrum indlcate that covalent attachment takes place at an Imidazole ring nitrogen In the histidine molecule. Eland intensities are compared to those of standard spectra and Undicate a surface IOadlng of 20.9%. While covalent attachment of cyanuric chloride is suggested by the appearance of a C-0-C stretchlng band (1398 cm-‘), there is significant evidence for the presence of adsorbed cyanurlc chloride, cyanurlc acid, and various chlorinated triazine derivatives.
The characterization of chemically modified surfaces is of current interest in the areas of chemically modified electrodes, immobilized enzymes, and stationary phases in high-performance liquid chromatography. Kuwana et al. have discussed prospects in tlhe analysis of chemically modified electrodes in a recent review ( I ) . In most cases, the amount of active agent which is ultimately bonded to the support material is dependent upon the identity and surface concentration of a covalently lbound linking agent. Despite the use of elegant methods for surface analysis (e.g., ESCA, Auger electron spectroscopy), details concerning the identity of functional groups are often lacking. Without this knowledge, it is difficult to ascertain the efficiency of the immobilization process. For example, decomposition of a linking site during reaction or storage often cannot be detected. Structural information can be obtained for modified surfaces by using secondary ion mass spectrometry (SIMS). However, this method is both destructive and necessitates the use of a high vacuum environment. Clearly, the need exists for a nondestructive technique capable of yielding structural information without introducing the sample into a “hostile” environment. Infrared spectrometry has been used for over 40 years in studying surface species. The wealth of standard spectra and functional group frequency assignments coupled with the availability of commercially built Fourier transform infrared spectrometers has made the study of adsorbed molecules much more convenient. In addition, the renaissance of reflectance and photoacoustic infriued methods (2-6) now allows for the detection of small quantities of surface species in the presence of “dark” substrates. Of particular interest is the use of diffuse Present address: GAF Corp., Wa ne, NJ 07470. Present address: Department of ?hemistry and Physics, Kean College of New Jersey, Union, NJ 07083.
reflectance FT-IR (DRIFT) in the analysis of powdered materials. Highly absorbing materials such as coal (3, 7) or pyrolyzed resins (8), which contain surfaces species, can be studied by this technique in a simple and nondestructive manner. This paper describes the study of chemically modified carbonaceous materials by using diffuse reflectance FT-IR. Histidine and cyanuric chloride were covalently attached to surface-modified activated carbon (AC) supports. Evidence for the covalent attachment of histidine is shown by the appearance of an intense amide band (1726 cm-l) in the infrared spectrum. While evidence is given for the covalent attachment of cyanuric chloride, the resulting spectrum indicates the presence of a multitude of different triazine species. EXPERIMENTAL SECTION Spectrometer and Attachments. All spectra were obtained with an IBM IR/97 Model 2A Fourier transform infrared spectrometer system (IBM Instruments, Danbury, CT). A globar light source and wide range, liquid nitrogen cooled, mercury cadmium telluride (MCT) photoconductive detector (Infrared Associates, New Brunswick, NJ) were used. The instrument is internally calibrated by a He-Ne laser so that the frequency scale is accurate t o 0.05 cm-l. The diffuse reflectance accessory employed was a Harrick DRA, “Praying Mantis” Model (Harrick Scientific Corp., Ossining, NY). Spectra were recorded by signal averaging 1000 interferograms at 4 cm-l resolution. Base lines and peak heights were established by using the software supplied by the manufacturer of the spectrometer (version ATS68X4). This version of the operating system did not allow for the calculation of integrated peak intensities, therefore peak heights (of well shalped peaks) were used to construct calibration curves. Unless otherwise noted, all single beam spectra were ratioed against spectroscopic grade KBr (International Crystal Laboratories, Elizabeth, NJ), which was dried at 150 “C for 24 h and ground in a “Wig-L-Bug” for 30 s before use. Ratioed spectra were base line corrected and converted to Kubelka-Munk plots for concentration studies. Materials. Granular activated carbon (Type CPG; 12 >: 40 mesh) was supplied by the Calgon Corp. A BET (nitrogen) surface area determination was performed on the activated carbon by Quantichrome Corp. (Syosset,NY) and yielded a surface area of 954 m2 g-l. Powdered graphite (Acheson no. 38) and lithium aluminum hydride (95+%) were obtained from Fisher Scientific metlhylCo. N-Cyclohexyl-~~-(2-morpholinoethyl)carbodiirnide p-toluenesulfonate (”puriss” grade) and cyanuric chloi4de (“purum” grade) were obtained from Tridom Chemical Co. (Hauppauge, NY). L.Histidine hydrochloride monohydrate (Sigma grade) was obtained from Sigma Chemical Co. (St. Louis, NIO). Benzene was extracted with concentrated sulfuric acid and distilled from 4-A molecular sieves before use. Sodium dihydrclgen phosphate (0.2 M MaH2P04,pH 4.75) and sodium potassium phosphate buffer (0.05 M, pH 7.0) were prepared in a routine manner. All aqueous solutions were prepared from distilled/ deionized water. All other chemicals were of reagent grade. Procedures. Graphite oxide was prepared from graphite powder by the KMn04-H2S04method of Hummers and Offeiman (9) without use of sodium nitrate. Activated carbon was prepared and oxygen plasma treated in the manner described previously (10). Cyanuric chloride was covalently attached to chemically reduced activated carbon according t o previously described
0003-2700/83/0355-2067$01.50/00 1983 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 13, NOVEMBER 1983 1.5
+
PI
I
rz 3
I
01 KM
------" 4000
Flgure 1.
I
3000 2000 WAVENUMBERS (CM-')
/
1000
/ 3000 zobo wavanum bers (c rn-'
Kubelka-Munk spectra of graphite (A) and graphitic oxlde
(B) in KBr.
procedures (10). Histidine was covalently attached to the activated carbon by the carbodiimide procedure of Janolino and Swaisgood (11)with minor modification. A 1-g sample of oxygen plasma treated activated carbon was reacted with 0.1 M carbodiimide in 0.2 M NaH2P04(pH 4.75) at 25 "C for 30 min. The mixture was filtered and washed with 250 mL of 0.05 M phosphate buffer (pH 7.00) at 0 "C. The material was then reacted with 0.1 M histidine in pH 7.0 phosphate buffer at 4 "C for 16 h. The solution was adjusted to the proper pH by addition of 2 M NaOH. After reaction, the material was washed three times each with cold phosphate buffer, 1 M NaCl, and distilled water. The immobilized preparation was then dried at 50 "C at 100 mtorr for 8 h and stored in a vacuum desiccator before use. The control preparation was subjected to the identical steps in the procedure with the exception of carbodiimide activation. Carbonaceous samples were sampled for DRIFT analysis by intimately mixing the material with KBr (10% w/w), followed by "Wig-L-Bug" treatment for 30 s. Samples used for concentration studies consisted of the appropriate concentration of pure material (ca. 0.5 to 10% in KBr) subjected to the identical treatment. Powdered samples were transferred to an aluminum sample cup (8 mm diameter, 2 mm deep) without compression and leveled with a spatula. The spectrometer was purged with dry nitrogen for 1-2 h before collection of interferograms.
RESULTS AND DISCUSSION The theory governing adsorption, scattering, and emission of radiation in a medium has been stated by a number of authors (12-15). In addition, various workers have systematically determined the proper sampling techniques needed for obtaining high-quality diffuse reflectance infrared spectra of powdered materials (7, 8, 16). In this study, previously reported optimized sampling conditions (16) were used for obtaining reproducible Kubelka-Munk spectra. Diffuse reflectance infrared spectrometry can be particularly useful for identifying surface species bound to high surface area supports. In the case of carbonaceous materials, surface oxides can be observed by DRIFT provided the material is of high surface area and relatively high oxygen content. This is demonstrated by the Kubelka-Munk spectra of graphite and graphitic oxide shown in Figure 1. The spectrum of graphite is almost featureless, except for the broad band a t ca. 800 cm-l due to aromatic C-H out of plane bending. This is in direct contrast to the spectrum of graphitic oxide which not only contains bands due to C-OH, C-0, and C=O vibrations (1070, 1400, and 1750 cm-l, respectively), but also S04H- and water modes (1600 and 3450 cm-l, respectively). The latter features are due to adsorbed species that result from the method of preparation and the hydrophilicity of graphitic oxide. While these materials are "extremes'! in terms of oxygen content and surface area (9, 17), it is evident that DRIFT should be applicable for surface analysis of carbonaceous materials which possess high surface area and significant surface concentrations of bound species. Histidine Attachment. Histidine was chosen for covalent attachment on carbodiimide activated AC for a number of practical reasons. First, the material is water soluble and
o b i
1
Figure 2. Kubelka-Munk spectrum of L-histidine hydrochloride in KBr (5.2% w/w). OA9'lo
20001.4%
1000 5.4'10
2000-
10 0
WAVENUMBERS (CM-')
Kubelka-Munk spectra of histkline at various concentrations in KBr in the range 1000 to 2000 cm-l. Slopes and correlation coefficientsof the 1641, 1609, 1501, and 1337 cm-' peaks are 0.105 and 0.997, 0.1 18 and 0.998, 0.107 and 0.988, and 0.095 and 0.972, respectively. Figure 3.
available in high purity. Second, the total charge on the molecule can be adjusted by variation of the solution pH. This can allow one to direct the covalent attachment to a specific site on the molecule. Third, the characteristic imidazole ring breathing frequencies of histidine occur in a region where the support material is devoid of fundamental vibrations. Thus, the imidazole frequencies can be used as a quantitative marker for histidine loading on AC. Finally, histidine can serve as a simplified model for the attachment of enzymes to insoluble supports. The Kubelka-Munk (K-M) spectrum of L-histidine hydrochloride (solid state) is shown in Figure 2. The zwitterionic character of the material is confirmed by the NH3+ stretching (3105-3017 cm-l), overtone (2037 cm-l), and bending (1641 cm-l) bands and the free carboxylate band (1609 cm-l). The presence of the imidazole ring is confirmed by the appearance of bands due to indole-type N-H stretching (3414 cm-l), =NH stretching (2700-2400 cm-l), imidazole ring breathing modes (1501 cm-' and 1337 cm-l), and -CH deformations in the imidazole ring (868 cm-' and 825 cm-l). The high intensity of the band at 3414 cm-l is due to the fact that both ring nitrogen atoms are protonated in the solid-state material. Peak heights of the bands at 1641, 1609, 1501, and 1337 cm-' were measured for solid-state histidine samples in the concentration range 0.5-10% (in KBr) and used to construct calibration curves. This is shown in Figure 3. In each case, satisfactory linear correlation coefficients are obtained (see legend, Figure 3), indicating that the diffuse reflectance method can yield useful calibration plots up to ca. 1 K-M unit. While theory (13) predicts degradation of linearity for K-M values in excess of 0.6, previous experimental work (7) has shown otherwise (i.e., linear response up to 1 K-M unit). The Kubelka-Munk spectrum of activated carbon that has been treated with carbodiimide and histidine is given in Figure 4. Direct evidence of covalent attachment is shown by the presence of a new band at 1726 cm-l. In addition, the strong
ANALYTICAL CHEMISTRY, VOL. 55, NO. 13, NOVEMBER 1983
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M x
'
wavenumbers (cn? )
wavenumbers (cm-1)
Figure 4. Kubelka-Munk spectrum of plasma treated activated carbon which contains covalently attached histidine (9.50% wlw in KBr).
l'*
r-
AI
wavenumbers ( c m -1)
Figure 5. Kubelka-Munk spectrum of plasma treated activated carbon
subjected to histidine without prior carbodiimide treatment (10.5% w/w in KBr). band at 3414 cm-", present in the solid-state material, is not, observed. The band at 1726 cm-l is due to the amide-based carbonyl stretching mode as part of the imidazole ring system (i.e., RCONC=C). The disappearance off the band at 3414 cm-l is most likely due to two factors. The first is simply the result of an amide linkage at an indole -NH position which causes a loss of the amine hydrogen. Second, the solution pJ3 of the immobilizing solution is close to the isoelectric pH of histidine which results in deprotonation of one ring nitrogen. This evidence suggests that the covalent attachment has occurred at a ring nitrogen in the imidazole system. Further indirect proof of attachment at this site is shown by the shift of the imidazole ring breathing bands to higher frequencies, indicating a change of the related force constants due to tho presence of an attached carbonyl moiety. In addition, the presence of the WH3+ stretching modes (ca. 3100 cm-l) indicates that the zwitterionic character of the amino acid has been retained. This proves that covalent attachment to the activated carbon surface has not occurred at the amino acid portion (NH3+ or COO-) of the histidine molecule. The DRIFT spectral results ,suggest the following mode of attachment:
The Kubelka-Munk spectrum of activated carbon that has been subjected to all steps except carbodiimide modification is shown in Figure 5. It is quite difficult to observe the presence of adsorbed histidine in this spectrum. It appears that the adsorption of histidine is minimal or nonexistent on the activated carbon. Tlhis result is rather surprising considering the strong adsorption properties of activated carbon. One plausible explanation is that electrostatic interaction between the support and histidine is minimized at the solution
Flgure 6. Kubelka-Munk spectrum of cyanuric chloride in KBr (10.5%# w/w in KBr).
pH used in the experiment (net charge of histidine is 0 at pH 7.2). Another plausible explanation is that washing with 11 M NaCl reduces any residual electrostatic adsorbate-support interaction. Quantitative estimation of the loading of covalently attached histidine on AC is not straightforward in view of the fact that the imidazole bands have shifted in frequency upon immobilization. However, as the relative intensities of the 1472-cm-l and 1653-cm-l peaks in the attached histidine spectrum are similar to those of the 1337-cm-l and 1501-cm-l bands in the solid-state spectrum, these peaks heights can be used for the determination. Extrapolation of the K-M values from the calibration curves for the 1337-cm-l and 1501-cm-l peaks indicates a total amount of histidine in the sample to be 2.0 f 0.6%. As the total concentration of AC in the sample is 9.50%, the average loading of histidine on AC is calculated to be 21% for these two determinations. This corresponds to a surface loading of 1.8 (f0.5) X mol cmw2. Construction of a space filling model of the bound histidine yielded an area of 84.2 A2 per molecule. This would indicate a 9% surface area coverage based on a close packing model. However, if one allows for the fact that the bound molecule can rotate around the attaching bond, each histidine can dominate an area of 450 A2. This would correspond to 48% of a monolayer. Cyanuric Chloride Attachment. The use of cyanuric chloride (CC) as a general linking agent in the construction of chemically modified electrodes has been reported in a number of studies (18-20). Reports to date have not provided concrete evidence of the absolute identity of the bound triazine species. In view of the reactivity of CC, it is conceivable that a multitude of chlorinated and/or hydroxylated triazine species could be present on chemically modified carbonaceous supports. Due to the sensitivity of the vibrational spectra of substituted triazines to substituent effects (21),this situation can be directly addressed by DRIFT. Figure 6 illustrates the Kubelka-Munk spectrum of CC in KBr. While this spectrum is in general agreement with lit erature reports (21,22),there is a small shift of the in-plane triazine ring breathing mode (1483 cm-l) to lower frequency. This may be due to the fact that previous studies utilized the alkali disk method which may have caused some degree olf association of CC with KBr while under pressure. It is interesting to note that under the experimental conditions, little, if any, hydrolysis of CC occurs. This is suggested by the lack of bands associated with ketc-enol tautomerization of cyanuric acid (2,4,6-trihydroxytriazine)in the spectrum of CC. Figure 7 illustrates the Kubelka-Munk spectrum of actiivated carbon subjected to oxygen plasma, chemical reductive, and cyanuric chloride treatments. In view of the radical change of this spectrum compared to that of CC, it is obvious that the triazine molecule has undergone extensive modification upon immobilization. The interpretation of the
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 13, NOVEMBER 1983
R2
where R1 = alkyl or aryl group which is part of the support surface and R2 can be either C1 or OH. The presence of various adsorbed substituted triazines (C1 and/or OH) is also indicated by this spectra. These species are the hydrolysis products of unattached cyanuric chloride and are adsorbed during the course of the reaction scheme. wavenumbers (crn-l) Flgure 7. Kubelka-Munk spectrum of chemically reduced activated carbon after cyanuric chloride treatment (10% w/w In KBr).
spectrum is complicated by the fact that the substantial frequency shifts can be partly due to the environment provided by the carbonaceous material. In addition, chemical reduction of AC may introduce additional functional groups on the support which would further complicate the spectrum. Even with these problems in mind, the resulting spectrum strongly suggests the presence of etheral, hydroxy-, and chlorotriazine derivatives. It has been reported that 2,4,6-substituted triazines exhibit bands in the 800-cm-l region due to an out-of-plane ring vibration (21,23). The absolute frequency of the vibration is strongly dependent upon the types of substituents present on the triazine ring. More succinctly, the frequency of the out-of-plane vibration can be correlated with the Hammett resonance constants (uR)of the various substituents (23). In general, a shift to higher frequency is encountered for substituted triazines which contain electron-releasing groups (e.g., -0CH). Thus, the band at 800 cm-l can be due to either dichlorotriazines containing an aryl ether substituent or cyanuric acid. It is also possible that adsorbed CC is present (794 cm-l) although this is not certain due to the lack of substantial supporting evidence (e.g., 1267 cm-l band). The presence of various triazine derivatives is also supported by the appearance of several intense bands in the region 1500-1650 cm-l. Schroeder (24) has shown that the in-plane triazine ring vibration (1563 cm-l) is influenced by electrophilic substituents, i.e., electron-withdrawing groups cause a shift to lower frequency. While the 1504-cm-l band indicates the presence of cyanuric chloride (22),its shift to higher frequency (relative to the 1483-cm-l band obtained for CC in this study) more strongly indicates the presence of electron-donating groups (e.g., -OH or -OCH2R). The number of additional bands up to 1650 cm-’ strongly suggests the presence of hydroxy-, alkoxy-, and chlorotriazine derivatives with varying degrees of substitution. The band at 1720 cm-l can be attributed to carbonyl stretching characteristic of the keto form of cyanuric acid. This indicates the presence of adsorbed cyanuric acid. While this was suspected in previous studies (19,W ) ,no definite proof could be provided. Finally, concrete evidence for the covalent attachment of CC to the carbonaceous support is provided by the presence of the band at 1398 cm-l, which can be attributed to C-0-C stretching. In this case, the somewhat high value of this ether stretching frequency suggests that the carbon substituents are aromatic in nature. Thus, the various features of the spectrum of CCmodified AC suggest the presence of the following substituted triazines:
CONCLUSIONS Diffuse reflectance infrared spectrometry has been evaluated as a sampling method for the study of surface species bound to carbonaceous materials. Despite the highly absorbing nature of the carbon matrix, relatively intense Kubelka-Munk spectra are obtained. The results for histidine have indicated that this method can allow for the confirmation of covalent attachment as well as quantitization of the bound moiety. Because of the power of FT-IR, it is possible that complex molecules and/or surface interactions can be observed in future applications. Information of a structural nature is obtainable by this technique, which may allow one to critically access the efficiency of a particular modification or immobilization scheme. ACKNOWLEDGMENT We thank R. S. Lawton for the preparation of graphitic oxide. Redstry No. Graphite, 7782-42-5;graphite oxide, 1399-57-1; carbon, 7440-44-0;histidine, 71-00-1; cyanuric chloride, 108-77-0. LITERATURE CITED
(19) (20) (21) (22) (23) (24) (25)
Karwelk, D.; Miller, C.; Porter, M; Kuwana, T. I n “Industrial Applications of Surface Analysls”; Casper, L., Powell, C., Eds.; American Chemical Soclety: Washington, DC, 1970; ACS Symposium Series. Ishltani, A.; Ishida, H.; Soeda, F.; Nagasawa, Y . Anal. Chem. 1982, 54,682. Krishnan, K. Appl. Spectrosc. 1981, 35, 549. Vidrine, D. Appl. Spectrosc. IBSO, 3 4 , 314. Lowry, S.;Mead, D.; Vldrine, D. Anal. Chem. 1982, 5 4 , 546. Dowrey, A.; Marcott. C. Appl. Spectrosc. 1982, 36,414. Carroll, J.; Doyle, W. Proc. I n t . SOC. Opt. Eng. 1981, 289, 111. Fuller, M.; Griffiths, P. Appl. Spectrosc. 1980, 3 4 , 533. Hummers, W.; Offernan, R. J. A m . Chem. SOC. 1958, 80, 1339. Osborn, J.; Iannlello, R.; Wleck, H.; Decker, T.; Gordon, S.; Yacynych, A. Biotechnol. BiOeng. 1982, 2 4 , 1653. Janollno, V.; Swaisgood, H. Blotechnol. Bioeng. 1982, 2 4 , 1069. Chandrasekhar, S. “Radiative Transfer”; Dover: New York, 1960. Kller, K. J. Opt. SOC. Am. 1972, 62,882. Kubelka, P.; Munk, F. 2. Tech. Phys. 1931, 12, 593. Kubelka, P. J. Opt. SOC.Am. 1948, 38, 448. Fuller, M.; Grifflths, P. Anal. Chem. 1978, 50, 1908. MaJer, C.; Vesely, J.; Stulik, K. J. Electroanal. Chem. 1975, 45, 113. Lin, A.; Yeh, P.; Yacynych, A. M.; Kuwana, T. J. Electroanal. Chem. tQ77 .- . ., -R 4. , A. i. l. , Yacynych, A. M.; Kuwana, T. Anal. Chem. 1978. 50, 640. Iannieilo, R. M.; Yacynych, A. M. Anal. Chem. 1981, 53,2090. Padgett, W. M.; Hamner, W. F. J. Am. Chem. SOC. 1958, 80, 803. Roosens, A. Bull. SOC. Chim. Be/g. 1950, 59, 377. Heckle, W. A.; Ory,H. A.; Talbert, J. M. Spectrochlm. Acta 1961, 17, 600. Schroeder, H. J. Am. Chem. SOC. 1959, 81, 5658. Wieck, H. J.; Ianniello, R . M.; Osborn, J. A.; Yacynych, A. M. Anal. Chlm. Acta 1982, 140, 19.
RECEIVED for review March 8, 1983. Accepted July 21, 1983. A.M.Y. thanks the National Science Foundation (Grant No. CHE-8022237) for research support. R.M.I. was the recipient of a Joseph W. Richards Fellowship of the Electrochemical Society and acknowledges financial support during the course of this work.
