Characterization of chemically modified carbonaceous electrode materials by x-ray fluorescence and scanning electron microscopy

Analytica Chimica Acta, 167 (1985) 353-360 Elsevier Science Publishers B.V., Amsterdam -Printed

in The Netherlands

CHARACTERIZATION OF CHEMICALLY MODIFIED CARBONACEOUS ELECTRODE MATERIALS BY X-RAY FLUORESCENCE AND SCANNING ELECTRON MICROSCOPY

HENRY J. WIECK*, ROBERT Department of Chemistry, NJ 08903 (U.S.A.)

F. ANTRIMb and ALEXANDER

Rutgers,

the State

University

M. YACYNYCH*

of New Jersey, New Brunswick,

VICTOR A. GREENHUT Department of Cemmics, NJ 08903 (U.S.A.)

Rutgers,

the State University

of New Jersey, New Brunswick,

(Received 30th May 1984)

SUMMARY An attachment scheme which utilizes cyanuric chloride as a linking agent in the preparation of chemically modified electrodes was investigated by using simultaneous scanning electron microscopy and x-ray fluorescence. Certain contaminants were discovered on the surface of the material following various steps in the reaction scheme used for attachment. The iron storage protein, ferritin, was attached to the surface of spectroscopicgrade graphite rod. Attempts were made to establish the surface distribution of the ferritin and to correlate distribution with surface morphology.

Interesting results are currently being reported by workers in the field of chemically modified electrodes (c.m.e.). Several chemically modified carbonaceous electrodes which function as amperometric or potentiometric sensors [l-4] or both [5] have been described in the literature. However, there are serious problems in trying to characterize the surface of carbonaceous c.m.e.‘s. Development of these electrodes has created a need to determine the functionality of the surface. Only by understanding the surface can the interaction of solution species with the sensing surface be understood. Characterization of these surfaces has been attempted in various ways ranging from wet chemical techniques (i.e., titrations and loading studies) to more exotic spectroscopic methods (i.e., e.s.c.a. and diffuse-reflectance Fouriertransform infrared spectrometry) [6-111. To date, no single method has emerged to resolve this complex problem. It may be true that several methods must be applied to describe these materials sufficiently.

*Present address: Department of Chemistry and Physics, Kean College of New Jersey Union, NJ 07083, U.S.A. bPresent address: Allied Corporation, Morristown, NJ 07960, U.S.A. 0003-2670/85/$03.30

0 1985 Elsevier Science Publishers B.V.

354

Scanning electron microscopy (s.e.m.) was chosen to obtain highly resolved secondary electron images of the surfaces of spectroscopic-grade graphite rods. The addition of a solid-state detector to the s.e.m. provides useful information on the elements present at or near the surface of the c.m.e., via energy-dispersive spectroscopy. The combination of these two techniques allows the acquisition of morphological and elemental information simultaneously . A typical s.e.m. experiment involves the interaction of a highly focused beam of electrons with the sample. The locally “excited” sample then emits various types of signals. Depending on the detectors used, a variety of data can be gathered. The various sample components which contribute signals are depicted in Fig. 1. Typically the crystals used as detectors in energydispersive x-ray fluorescence (x.r.f.) are isolated behind a beryllium window, rendering the detector insensitive to signals produced by elements of atomic number less than that of neon. Fortunately, x-rays produced by the carbon substrate (electrode material) are undetected. Because chemical modification takes place at the surface, the s.e.m./x.r.f. combined method becomes a pseudo-surface technique, which yields morphological and elemental data regarding the surface. Previous results indicated that carbonaceous surfaces can be dramatically altered during the course of a chemical modification scheme [9]. These changes can be morphological or of a chemical nature. This paper describes the use of s.e.m. and energy-dispersive x.r.f. (microarea and bulk) to follow the course of a chemical modification scheme which utilized cyanuric chloride as a linking agent. An attempt was also made to take advantage of the information available by concurrent s.e.m. and x.r.f.

--II-

50 A electron

beam

l-*-4J Fig. 1. Electron penetration

excitation

teardrop.

355

measurements to determine the distribution of a protein bound to the surface of a spectroscopic graphite rod. This method can aid in understanding and refining the attachment process and consequently improving performance of the c.m.e. EXPERIMENTAL

Apparatus and materials An EDAX International Model 902D x-ray fluorescence spectrometer equipped with a Data General Nova-3 was used for survey (bulk) work. The 1.487- and the 8.041-keV lines of a Al/Cu standard were used to calibrate the energy scale. All survey spectra were obtained with a lo-kV accelerating voltage, 500-PA current to the rhodium target, over the range O-16 keV at 20 eV/channel. The scanning electron microscope with secondary electron images was an ETEC Autoscan in the Department of Ceramics. The s.e.m. was operated with a 20-kV accelerating voltage, a 150ym aperture, and a tilt angle of 45”. A Canberra Industries Si(Li) energy-dispersive detector and a Model 8100 multichannel analyzer were used for x-ray (micro) fluorescence (s.e.m./x.r.f.) studies on the s.e.m. A copper tape was placed on the s.e.m. planchette so that the L, (0.928 keV) and the K, (8.041 keV) lines could be used for energy calibration of the spectra. A Harrick Scientific Model PDC-3XG, plasma chamber was used at a power setting of 10 (approximately 16 W, up to 10 r.f. coupled) to subject the samples to an oxygen plasma. Oxygen was admitted to the plasma chamber via a micrometer-adjustable bellows-sealed needle valve (Whitey, SS2 2RS4). Oxygen pressure was monitored with a Sargent-Welch Model 1515 thermocouple vacuum gauge. National spectroscopic-grade electrodes (grade AGKSP, Union Carbide Corp., Chicago, IL) were obtained as rods (6.1 mm diameter, 305 mm long). Lithium aluminum hydride (95%; Aldrich Chemical Co.) and cyanuric chloride (98%; Fluka) were used. Ferritin (type 1) from horse spleen (Sigma Chemical Co,) was stored refrigerated. All other chemicals were of reagent grade. Only distilled/deionized water was used. Procedures The graphite rods were cut into small pieces (approximately 100 mm2 total geometric surface area). They were extracted with methanol in a Soxhlet apparatus for 24 h and then dried overnight at 150°C in vacuum (50 mtorr). The lithium aluminum hydride was purified by a procedure similar to that used by Davis et al. [13], except that a dry box was not used. Significant reduction of a chloride contaminant was achieved. Samples were subjected to the various steps in a cyanuric chloride attachment scheme shown in Fig. 2. The conditions and sample designations are given in Table 1.

356

I-

CHp-Na++

N-f’ N

Cl 0

“-$I

_

I

Fig. 2. Cyanuric chloride reaction scheme used. Encircled numbers (l-5) surfaces chosen for study by s.e.m.-x.r.f.

represent the

TABLE 1 Sample treatments and designations Sample

Treatment

1

Cut spectroscopic-grade rod extracted with methanol for 24 h and vacuum-dried Subjected to oxygen plasma for 10 min at a pressure of 20 Pa followed by equilibration with the gas at 260 Pa for 10 min. Reduced with a saturated solution of purified lithium aluminum hydride in ether, refluxed for 3 h, filtered, washed with cold ether, cold 1M nitric acid and cold water, soaked in 2M sodium hydroxide for 1 h, dried overnight at 150” C. Refluxed for 1 h in neat cyanuric chloride at 19O”C, washed with cold anhydrous acetone, cold water, and again with acetone, immersed in cold anhydrous acetone and stirred for 15 min, dried in vacuum oven at room temperature. Reacted with a solution containing 100 mg of ferritin in pH 7.3 phosphate buffer for 30 min, washed with phosphate buffer. Left in contact with a solution containing 100 mg of ferritin in pH 7.3 phosphate buffer for 30 min, washed with phosphate buffer; used as control sample.

2 3

4

5 Adsorbed

RESULTS AND DISCUSSION

In a previous report [ 121, the effects of various methods of oxidative pretreatment on pyrolytic graphite disks and spectroscopic-grade graphite rods were discussed. As suggested earlier, the work here utilized a radiofrequency oxygen plasma as the oxidative method. Preliminary work showed

357

a significant amount of chloride on samples subjected to reduction with lithium aluminum hydride, which had a chlorine contaminant when received from the supplier. Ensuing reductions were done with purified lithium aluminum hydride. Spectra (s.e.m./x.r.f .) for samples l-5 and the adsorbed sample (see Table 1 and Fig. 2) are presented in Fig. 3. In comparing the spectra, one can see the effect of the various reagents used on the sample during the course of chemical modification. The spectra for samples 1 and 2 are similar and without any significant peaks showing a typical background signal The spectra shown for sample 3 shows some peaks that are not present in its precursor. The large peak at 1.041 keV is due to sodium &, which is introduced into the sample while soaking in sodium hydroxide which is a step in the preparation of sample 3. The smaller peak at 2.62 keV is due to chlorine which is still present in the purified hydride but which is many times reduced in comparison with samples reacted with the unpurified hydride. Sample 4 has been reacted with the linking agent, cyanuric chloride and the chlorine peak at 2.62 keV increases because of the chlorine present in the linking agent. A surface subjected to completely effective washing ideally should not show any sodium at this point. The sodium introduced in sample 3 is considerably reduced, but is not entirely removed. The exact nature, distribution, and relative reactivity towards cyanuric chloride of the surface groups is not fully known (i.e., -CH20-Na+ vs. -@Na’); this makes speculation about the remaining sodium difficult. Work is being continued to remove totally this sodium contamination; apparently its presence at this level of concentration does not seem to hinder the operation of the c.m.e.‘s. Comparison of the chlorine peak at 2.62 keV for samples 4 and 5 and the adsorbed blank all show similar levels of chlorine. Sodium chloride is present in the buffer (0.15 M) in which the ferritin is supplied. This additional source of chloride complicates the interpretation in sample 5 and in the adsorbed blank, as the chlorine signal may be due to chlorine from the buffer. Sample1

2

Fig. 3. Spectra (s.e.m.-x.r.f.) blank.

in the 0.5-3.3

keV range for surfaces l-5

and the adsorbed

358

Up to this point, only the steps in one of several commonly used attachment schemes have been analyzed. These steps provide a surface that is reactive with lysine residues of certain proteins [14]. This technique is used to attach enzymes to a carbonaceous surface. Ferritin, an iron storage protein, was made to react with the chemically modified surface. The ferritin contains a large percentage of iron (ca. 4500 atoms of iron(II1) per molecule [ 151). The signal from this iron was monitored by energy-dispersive x.r.f. The K, (6.041 keV) and the Kp (7.057 keV) peaks of iron are clearly visible in the x.r.f. (bulk) spectrum of the dried ferritin sample shown in Fig. 4. Each of the 24 protein subunits of ferritin contains a number of chemically reactive lysine residues which can attach to the electrode surface. The ferritin was attached via a cyanuric chloride linkage (sample 5), and was also adsorbed onto a virgin surface (adsorbed). As was seen in other work [6], the chemically modified surface actually has less total protein than the adsorbed blank. Because the surface was in contact with a variety of reagents during the modification process, it is not surprising that its absorption capacity diminished. However, a discernible iron signal is observed in both cases. It should be possible to detect proteins which contain a smaller percentage of an element, provided that the element is observable in the x-ray region, if the protein is confined or aggregated to a relatively small percentage of the surface area of the electrode. Examination of the electrode surface at 200X (Fig. 5) shows many small features. An overall evaluation of the surface at 200X magnification was compared with several randomly chosen smaller areas. There was a definite difference in the concentration of iron on various regions of the electrode surface. An attempt was made to correlate the concentration of iron on the surface of the electrode with certain morphological features of that surface

2

4

ENERGY

6

CkeV I

Fig. 4. Bulk x.r.f. spectra of dried ferritin.

8

359

Fig

5 Scanning

electron

mlcrograph

(200x ) of electrode

Fig 6 Scanning electron edge or plane areas.

micrograph

(2000x

) of electrode

surface surface

showmg

predommantly

(sample 5). Features which resembled “edges” or “ridges” were seen at a magnification of 2000X , as shown in Fig. 6. To investigate this heterogeneous surface, six sites of equal area (ca. 9 pm*) were chosen and monitored for 500 s each. The net counts and their standard deviations for these six areas and the overall area of 20000 pm2 (on which the six areas resided) are presented in Table 2. The counts were corrected for the non peak and normalized to background in order to make legitimate comparisons, and standard deviations were determined. There was a factor of approximately 5.5 between the extremes in the iron results. It appears that the distribution of ferritin on the areas referred to as edges is more uniform than that on the areas designated as planes, as indicated by the ranges of the two data sets. The average edge counts (2.8 + 0.2) and the average plane counts (3.3 + 0.2) are not very far from the overall value, which represents an area some 100 times greater than the area of any one of the individual components, indicating a representative sampling, in spite of the small number of areas examined.

Conclusion The carbonaceous materials used as substrates for c.m.e.‘s are essentially invisible in x.r.f. The method provided a quick analysis for elements, and in this particular case, gave information regarding contaminants in the lithium aluminum hydride. When used in conjunction with an appropriate tag (e.g., the iron in ferritin), the combination enables the distribution of the material on the substrate surface to be visualized.

360 TABLE 2 Net counts for iron and average deviations for the overall surface and six selected smaller areas (sample 5) Area

Normalized net counts (10’)

Area

Normalized net counts (10’)

Overall Edge (1) Edge (2) Edge (3) Average edge

3.2 i 0.1 1.6 i 0.1 2.9* 0.1 3.9 f 0.1 2.8 * 0.2 Range = 2.3

Plane (4) Plane (5) Plane (6) Average plane

2.2 f 0.1 6.7 f 0.1 1.1 f 0.1 3.3 f 0.2 Range = 5.6

A.M.Y. thanks the National Science Foundation 8022237) for research support.

(grant number CHE

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