"New Fluorometric Methods of Analysis of Biologically Spec. f u b l . , 378, Important Compounds",in Natl. Bur. Stand. (U.S.), Accuracy in Spectrophotometry a n d Luminescence Measurements, Proceedings of a Conference held at NBS Gaithersburg, Md., March 22-24,
(5) G. G. Guilbault,
(6) (7) (8) (9)
1972. R. A Puynter, S. L. Wellons, and J. D. Winefordner, Anal. Chsm.. 46, 736 (1974). R. M: A. von Wandruszka and R. J. Hurtubise, Anal. Chem., 48, 1784 (1976). V. Pollak and A. A. Boulton, J . Chromatogr., 72, 231 (1972). G. Kortum, "Reflectance Spectroscopy",Springer-Verbg, New York, N.Y., 1969.
(IO) W. W. Wendlandt and H. G. Hecht, "Reflectance Spectroscopy", Interscience Publishers, New York, N.Y., 1966.
(11) V. Pollak, Opt. Acta, 21, 51 (1974). (12) V. Pollak, J . Chromatogr., 133, 49 (1977). (13) J. Goldman, J . Chromsogr., 78, 7 (1973). (14) J. Goldman and R R. Goodall, J . Chromatogr.. 40, 345 (1969) 115) J Goidman and R. R. Goodall. J . Chromatoor..,~32..~ 24 11968). (16) J. Goldman and R. R. Goodall; J . Chromatogr., 47, 386 (1970). (17) R. R. Goodall. J . Chromatogr., 78, 153 (1973).
~~-
RECEIVED for review July 8, 1977. Accepted October 4,1977.
Room-Temperature Phosphorescence of Compounds Adsorbed on Sodium Acetate R. M. A. von Wandruszka and R. J. Hurtubise" Department of Chemistry, University of Wyoming, Laramie, Wyoming 8207 1
The room temperature phosphorescence behavior of a number of compounds adsorbed on sodium acetate was investigated. Comparisons of molecular structures and consideration of reflectance, fluorescence, and infrared spectra allowed the postulation of certain molecular criteria for room temperature phosphorescence. The interactions of p-aminobenzoic acid wlth a sodium acetate surface were considered in detail. The adsorption was found to involve the formation of the sodium salt of p-aminobenzoic acid on the sodium acetate surface, as well as hydrogen-bonding. Only protic solvents could be used. Room temperature phosphorescence measurements of chemisorbed compounds on sodium acetate provided a novel way of surface area determination. I t was also shown that the phosphorescent compounds are adsorbed flatly on sodium acetate. Analyticai findings for several compounds are reported.
Room temperature phosphorescence of adsorbed ionic organic molecules was first reported by Roth ( I ) and later by Schulman and Walling (2, 3 ) . Paynter e t al. ( 4 ) put the phenomenon to its first analytical use. The use of a sodium acetate adsorbent in room temperature phosphorimetry was introduced by von Wandruszka and Hurtubise ( 5 ) . They employed it for the determination of p-aminobenzoic acid (PABA) in vitamin tablets. T h e phosphorescence signal of the adsorbed compound was found to be insensitive to moisture, and quantitation was achieved with a spectrodensitometer. The same approach was later applied to folic acid, p-hydroxybenzoic acid, and benzocaine (6). In this paper, the use of sodium acetate in room temperature phosphorimetry is extended to yet other compounds. Consideration is also given t o the theoretical aspects of the adsorption process. T h e phosphorescence properties of a variety of compounds adsorbed on sodium acetate were studied. Other surfaces were also investigated. Infrared, reflectance, and fluorescence spectra of adsorbed species, and surface area data for adsorbent and adsorbates were obtained. From the information gathered, generalizations could be made about the molecular requirements of the adsorbed species. A hypothesis for the adsorption process is presented. 2164
ANALYTICAL CHEMISTRY, UOL. 49, NO. 14, DECEMBER 1977
EXPERIMENTAL Apparatus. Phosphorescence measurements were made with a Perkin-Elmer MPF-2A Fluorescence Spectrophotometer and with a Schoeffel SD 3000 Spectrodensitometer as described previously ( 5 ) . Fluorescence spectra were obtained in the same fashion. Reflectance spectra were obtained with the Schoeffel spectrodensitometer in the double beam mode with a blank adsorbent sample in the reference beam. The IR spectra were obtained with a Perkin-Elmer 621 Grating Infrared Spectrophotometer. Reagents. Ethanol was purified by distillation as described by Winefordner and Tin (7). Other solvents were reagent grade and used without further purification. PABA was reagent grade and purified by recrystallization from ethanol. p-Aminohippuric acid was purified by recrystallization from water and folic acid by recrystallization from benzene. p-Aminophenol was purified by washing with ethanol, and terephthalic acid was recrystallized from ethanol. Other compounds were reagent grade and used without further purification. Procedures. The samples for the reflectance measurements were prepared by addition of ethanolic solutions of the compounds to the adsorbent and subsequent evaporation, as described previously ( 5 ) . The amount of adsorbate used for reflectance spectra was 500 ng on 10 mg adsorbent. The spectra were obtained with the spectrodensitometer and taken point-by-point at 5-nm intervals. For the determination of phosphorescence and fluorescence maxima, 200 ng adsorbate was used on 10 mg adsorbent. KBr pellets were used for the IR spectra. They were prepared with a Wilks Mini-press. For the spectra of pure compounds, 200 mg KBr was mixed with 8 mg of the compound. For the spectra of adsorbed compounds, 15 mg of the adsorbed system (sodium acetate plus compound) was used with 200 mg KBr. Of these mixtures, 60 mg was used to prepare each pellet. No more than the indicated amount of sodium acetate-adsorbed compound system could be used in a pellet because larger amounts of sodium acetate gave opaque pellets that could not be cleanly separated from the die. The amount of adsorbed compound on the sodium acetate had to be sufficient to be detected in the IR spectra. However, the systems of interest were those that showed room temperature phosphorescence, so too much adsorbate could not be used because the phosphorescence would be quenched. It was found that an amount of adsorbed compound that was 1.0%, by weight, of the sodium acetate adsorbent, satisfied both criteria. The surface area determination of anhydrous sodium acetate powder was carried out by Quantachrome Corporation, 69 Glen
Table I. Room Temperature Phosphorescence of Compounds Adsorbed on Sodium Acetate
Table 11. Phosphorescence Intensities of Compounds Adsorbed on Sodium Acetatea 77 K
Room temp.
1000
333
0.333
p-Hydroxy benzoic 1040 acid 3-Methyl-4-amino232 benzoic acid N,N-Dimethyl-4-amino- 260 benzoic acid Benzocaine 146 Terephthalic acid 238 38 Hydroquinone Folic acid 132 p-hydroxy mandelic 56 acid p -Aminohippuric 1200 acid 5-Hydroxyindole200 acetic acid 5-Hydroxytrypto210 phan
48
0.046
29
0.125
42
0.16
Compounds giving room temperature phosphorescence
Compound p-Amino benzoic
acid (PABA)
p -Amino benz oic
acid, sodium salt p-Aminohippuric acid p-Hydroxy benzoic acid Folic acid BHydroxyindoleacetic acid (5-HIAA) 5-Hydroxytryptophan (5HTP) Benzamide of PABA 3-Methyl-4-aminobenzoic acid N, N-Dimethyl-4aminobe nzoic acid rn- Amino benzoic acid Hydroquinone p-Hydroxymandelic acid
Excitation, Emission, nm nm
Compound Strength of signal
29 0
4 26
very strong
290
4 26
very strong
328
448
strong
420
moderate
320 312
465 510
moderate moderate
355
5 00
moderate
300
430
weak
290
430
weak
290
430
weak
290
430
weak
285 290
430 420
very weak very weak
285
Compounds not giving room temperature phosphorescence Sulfanilic acid
p-Aminophenyl- Ison ico tinic acetic acid acid
Terephthalic acid
p-Hydroxycinnamic acid rn-Hydroxybenzoic acid
pAminopheno1 0-Aminoben-
Aspirin
zoic acid Benzoic acid Aniline Benzocaine
p-Chlorobenzoic acid p-Bromobenzoic acid p-Nitrobenzoic acid
N, N-Dimethyl-4-
Halazone
aminobenzaldehyde Sulfanilamide 3-@-Hydroxyphenyl) propionic acid p-Hydroxyphenylacetic acid p-Aminobenzyl alcohol
Phthalic acid Potassium acid phthalate Isophthalic acid p-Toluic acid
Gallic acid p-Aminophthalic acid 4-Hydroxy-3methoxy benzoic acid 3-Hydroxy tyramine 7-Aminocephalosporanic acid l-Amino-2naph thol-4sulfonic acid Isocinchomeronic acid p-Aminosalicylic acid Serotonin Tryptophan Luminol
Cove Road, Greenvale, N.Y., using the BET method. RESULTS AND DISCUSSION Low T e m p e r a t u r e a n d Room T e m p e r a t u r e Phosphorescence. The room temperature phosphorescence behavior of a number of compounds adsorbed on sodium acetate was investigated (Table I). PABA gave the strongest signal at room temperature, but a t 77 K adsorbed samples of p-aminohippuric acid and p-hydroxybenzoic acid phosphoresced more strongly. A comparison of phosphorescence intensities at room temperature and a t liquid nitrogen
p-Aminobenzoic
Room temp/77 K
acid
*..
...
0.9
0.0038
6.8
0.18
43 6.5 262
0.32 0.116 0.218
32
0.160
39
0.186
77 K phosphorescence of PABA arbitrarily set a t 1000 units. All compounds 500 ng/lO mg NaOAc. temperature is given in Table 11. The data imply that differences in room temperature phosphorescence intensities are not due to inherent molecular effects, but rather to differences in rigidity of the adsorbed species. PABA appears to be adsorbed most strongly, since it retained relatively the greatest fraction of phosphorescence at room temperature. Molecular Requirements and Experimental Conditions. Consideration of molecular structures in Table I shows that additional ring substituents on PABA negatively affect room temperature phosphorescence. Examples include 3methyl-4-aminobenzoic acid, where the methyl group attached to the 3 position led to a 11.5-fold reduction in room temperature phosphorescence intensity compared to PABA. It also caused the phosphorescence to be more sensitive to moisture. Prolonged exposure to the atmosphere gave a 2-fold reduction in room temperature phosphorescence of 3methyl-4-aminobenzoic acid, while the PABA signal was not affected. Compounds in which the substituents led to a total absence of room temperature phosphorescence included p-aminosalicylic acid, 4-aminophthalic acid, and 3-methoxy-4-aminobenzoic acid. Benzocaine (ethyl-4-aminobenzoate) gave no room temperature phosphorescence when adsorbed on sodium acetate. The signal for N,N-dimethyl-4-aminobenzoic acid was reduced 7.9-fold as compared to PABA (Table 11). These considerations suggest a number of structural requirements for strong adsorption and room temperature phosphorescence of compounds similar to PABA. The presence of the carboxyl group attached to the 1 position appeared to be one requirement for compounds with the benzene nucleus. Compounds lacking this requirement are aniline, sulfanilic acid, and derivatives of benzaldehyde and benzyl alcohol (Table I). They did not show room temperature phosphorescence on sodium acetate. The same was true for most compounds in which the carboxyl group was displaced one or more carbon atoms from the ring. However, p aminohippuric acid was an exception, showing a strong phosphorescence signal. Attached to the 4 position on the ring, an electron-donating, hydrogen-bonding substituent appeared to be necessary. Table I shows several compounds lacking this requirement. The only suitable groups found were amino and hydroxyl groups. Interestingly, isonicotinic acid with nitrogen in the 4 position in the ring system gave no room ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
2165
Table IV. Room-Temoerature Phomhorescence Characteristics of Ionic- Organic Mol&ules Adsorbed on Sodium Acetate ExcitaLinear range of tion Emission Limit Peak, Peak of detec- c a1ibr a t i on Compound nm nm tion, ng curve, ng
Table 111. Surfaces Tested for Terephthalic Acid Room Temperature Phosphorescence Sodium acetate Sodium propionate Sodium butyrate Sodium valerate Sodium oxalate Sodiu m tartrate Sodium formate
0
1
I
3
LOGIhsOH1
Sodium adipate Soaium pimelate Magnesium hydroxide Barium hydroxide Calcium hydroxide Lithium acetate Quaternary ammonium i o n exchange resin (AG 50W-X8, Bio-Rad)
4
ItblRLI
I
I
I
2
LOG
p-Aminohippuric acid 5-Hydroxyindoleacetic acid FrHydroxytryptophan
O
nciI
Figure 1. Room temperature phosphorescence intensity of PABA evaporated onto sodium acetate from ethanolic solutions of different
acid and base strengths temperature phosphorescence. All molecules investigated showed low temperature phosphorescence when adsorbed on sodium acetate. Terephthalic acid, with two carboxyl groups para to each other, was investigated on a number of surfaces (Table 111). In no case could more than extremely weak room temperature phosphorescence be detected. Dissolving the compound in an alkaline solution and then evaporating onto the surfaces gave no improvement. Two compounds with the indole nucleus showed room temperature phosphorescence on sodium acetate, 5hydroxyindoleacetic acid (5-HIAA) and 5-hydroxytryptophan (5-HTP). The signals were observed only when alkaline ethanolic solutions of these compounds were evaporated on sodium acetate. Both 5-HIAA and 5-HTP were unstable in alkaline solutions and formed green and brown decomposition products, respectively, when !eft for more than 20 minutes (8). These products did not give room temperature phosphorescence. To avoid decomposition, aqueous stock solutions of pH 2 were prepared. Small amounts of these solutions were added to an excess 0.04 N ethanolic NaOH less than a minute before evaporation. No decomposition products were observed in this short period and the systems showed analytically useful room temperature phosphorescence. Tryptophan and serotonin (5-hydroxytryptamine) were treated similarly but did not give room temperature phosphorescence. The effect of pH on the phosphorescence of PABA adsorbed on sodium acetate was investigated. PABA was dissolved in a series of ethanolic NaOH and HC1 solutions and each was evaporated onto sodium acetate. The room temperature phosphorescence intensities obtained are shown in Figure 1. The decrease of the signal in more strongly acid solutions was probably due to protonation of the carboxyl and possibly the amino groups, preventing strong interaction with the sodium acetate surface. In the alkaline solutions, the reduced intensities could be due to precipitation of NaOH upon evaporation. A similar effect was noted when PABA was evaporated onto sodium acetate mixed with sodium hydroxide. 2166
ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
318
448
5
0-140
312
510
50
0-200
320
500
50
0-150
The determination of PABA, using room temperature phosphorescence on sodium acetate has been described in detail ( 5 ) . Analytical results for p-hydroxybenzoic acid, folic acid, and benzocaine were reported (6). Analytically useful results have now been obtained for p-aminohippuric acid, 5-HIAA, and 5-HTP (Table IV). There was some overlap between the fluorescence emission and the phosphorescence emission spectra for 5-HIAA and 5-HTP. However, the signal measured at 510 nm was 92% phosphorescence for 5-HIAA and at 500 nm was 98% phosphorescence for 5-HTP. Solvents. Ethanol was the solvent routinely used for room temperature phosphorescent compounds. Water could be used as a solvent for PABA, although it dissolved the sodium acetate adsorbent. After evaporation of the water, the crystalline residue showed room temperature phosphorescence. The intensity of the signal was reduced 3-fold compared to a similar ethanolic sample. This was probably due to the formation of the trihydrate of sodium acetate, a less suitable adsorbent. The same reduction in intensity was observed when an ethanolic PABA solution was evaporated onto the trihydrate instead of onto anhydrous sodium acetate. n-Propanol could be used as a solvent in room temperature phosphorescence work, but this was not commonly done because of the longer time required for its evaporation. Other solvents that could be used were isopropanol and isobutanol. Aprotic solvents such as ether, acetone, dimethylformamide (DMF), and cyclohexane gave no room temperature phosphorescence of PABA on sodium acetate. Adsorption Mechanism. Adsorption of PABA on sodium acetate is preceded by partial neutralization with dissolved sodium acetate in alcoholic solutions. The PABA anion formed has a strong tendency to adsorb on the surface, forming the sodium salt. This is supported by the strong room temperature phorphorescence of the sodium salt of PABA (Na-PABA) when it adsorbs on sodium acetate suspended in an ethanolic solution of Na-PABA. No signal was observed for PABA and Na-PABA dissolved in acetone or DMF. This is probably due to ion pairing and the formation of conjugated species in these solvents (9). It may be hypothesized that the formation and adsorption of the anion also occurs in other compounds that phosphoresce on sodium acetate. In 5 - H I M and 5-HTP solutions, the dissolved acetate does not give the required neutralization of the solute. Addition of a stronger base such as NaOH is therefore necessary. This is in agreement with the generally much smaller acid dissociation constant of molecules with an indole nucleus (IO). After evaporation of the solvent, ethanol, samples of Na-PABA and K-PABA gave phosphorescence signals that were 16% stronger than the signal of a similar PABA sample. When the PABA and Na-PABA samples were cooled down to 77 K, their phosphorescence intensities were equal. The data strongly suggest that the PABA is not completely ionized on sodium acetate. It was calculated from the phosphorescent data that 84% of PABA is converted to the sodium salt on the sodium acetate surface. Further evidence for ionization
Table V. Reflectance Maxima of Compounds Adsorbed on Sodium Acetate, Starch, and Talc
Compound
-~ 150
150
300
400
WAVELENGTH nm
-
Maximum on NaOAC, nm
Maximum on starch or talc, nm
250, 290 240, 270 27 5
250, 290 240, 275 325
27 0
300
3 20 33 0 250
330 350 27 5
255 260
255 295
Aniline Benzoic acid N, N-Dimethyl-4-aminobenzoic acid 3-Methyl-4-aminobenzoic acid m-Aminobenzoic acid o-Aminobenzoic acid p-Hydroxybenzoic acid Sodium salt of PABA PABA
Figure 2. Reflectance spectra of PABA adsorbed on sodium acetate (-),
starch (- - -) and talc (-
-
-)
was provided by reflectance data discussed below. An important consequence of the ionized adsorbate strongly adsorbing on sodium acetate is the previously noted relative insensitivity of the phosphorescence signals to moisture. Apparently, the chemisorption interactions between adsorbent and adsorbate are so favorable that the adsorbate effectively displaces adsorbed water from the surface and the predominant interactions are directly between adsorbent and adsorbate. Water thus cannot effectively compete for occupied sites on the adsorbent. Reflectance Spectra. The mode of adsorption of PABA and other compounds was studied with the aid of reflectance spectroscopy. The surfaces chosen were sodium acetate, talc, and starch (Figure 2). Sodium acetate is a polar surface which interacts strongly with many of the compounds investigated. It was the only surface found that gave room temperature phosphorescence of adsorbed molecules. Talc and starch are relatively less polar (11)and the compounds studied did not give room temperature phosphorescence when adsorbed on them. The reflectance spectra of PABA on lithium acetate and potassium acetate were found to be the same as the PABA spectrum on sodium acetate. This is in contrast to Zeitlin's and Van Lieu's results with mononitrophenols adsorbed on alkali metal carbonates (12). They found a hypsochromic shift in the reflectance spectra with increasing effective nuclear charge of the adsorbent cation, i.e., Li' > Na+ > K+. Their interpretation of the results was that the smaller cations polarized the adsorbate more effectively, thus causing a hypsochromic shift. PABA probably formed the carboxylate anion on all three acetate surfaces, resulting in the same reflectance spectra. There was a hypsochromic shift of 35 nm in the reflectance maximum of PABA on sodium acetate as compared to PABA on talc or starch (Figure 2). This indicated strong interactions between sodium acetate and adsorbed PABA, suggesting the formation of the sodium salt of PABA upon adsorption. This was supported by the reflectance spectra of the sodium salt of PABA evaporated onto sodium acetate and starch. The same reflectance maximum was obtained on the two surfaces and this maximum was the same as that of PABA adsorbed on sodium acetate. When PABA was evaporated onto sodium acetate from an acetone solution, no room temperature phosphorescence resulted. The reflectance spectrum of this sample had a 282 nm maximum, indicating that the PABA molecule was the adsorbed species. Phosphorescence did appear when a little moisture was introduced to the sample. This was probably due to the ionization of the adsorbed species and consequently its strong chemisorption to the surface. Subsequent extensive drying did not reduce the phosphorescence intensity.
0 4 8 6
HABA
T
i
f
,
L
i
~
L -
Figure 3. Relative energy levels of PABA, maminobenzoic acid (MABA), and oaminobenzoic acid (OABA) on sodium acetate (l),in cyclohexane (2),and on starch (3). Fluorescence wavelengths are shown in nanometers
The reflectance maxima of other compounds adsorbed on sodium acetate, talc and starch are listed in Table V. It can be seen that the compounds expected to interact strongly with the surface, by forming the sodium salt, had hypsochromic shifts when adsorbed on sodium acetate. The table includes the compounds that exhibited room temperature phosphorescence. T h e para arrangement of the substituents in PABA enhances the electron-withdrawing character of the carboxyl group and the electron-donating character of the amino group (13). The radiation-absorbing process can be represented by an intramolecular charge transfer from the amino group to the carboxyl group (13, 14):
The species adsorbed on sodium acetate is the PABA anion. This makes the charge transfer a more energetic process, leading to the observed blue shift in the reflectance spectrum (Figure 2). Similar arguments can be invoked for other compounds showing hypsochromic reflectance shifts on sodium acetate. The shift for m-aminobenzoic acid was rather small, only 10 nm. It is interesting to note that the charge transfer mechanism cannot be applied to this molecule, because of its meta configuration. The fluorescence spectra of PABA, m-aminobenzoic acid, and o-aminobenzoic acid, dissolved in cyclohexane, adsorbed on sodium acetate and on starch were obtained. It was assumed that the solventsolute interactions in cyclohexane were ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
2167
csoo
2ocoo
I ~ C O O A M O L V -
I
35oc
IO00
2500
:do0 WIYiNUMBLR
l5OC
PABA
minimal, so that there was relatively little disturbance of the molecular energy levels of the solute. When adsorbed on sodium acetate and starch, the energy levels of the molecules were altered to varying degrees. This led to different maximum fluorescence emission wavelengths (Figure 3). The molecular energy levels of PABA will be lower in the ground state and the excited singlet state compared to PABA in cyclohexane wher. PABA forms the sodium salt on sodium acetate because of greater interaction of the PABA anion with sodium acetate (Figure 3). A 19-nm bathochromic shift in the fluorescence maximum was observed for the sodium acetate adsorbed species compared to the molecular species in cyclohexane. This indicates that the interaction between PABA and sodium acetate is greater in the excited singlet state than in the ground state. Strong excited triplet state interactions are supported by the room temperature phosphorescence of the PARA-sodium acetate system. The fluorescence maximum of PABA on starch was at a longer wavelength than the emission of the cyclohexane solution. However, on starch the adsorbed species will be the PABA molecule, rather than the anion. Thus, the relative lowering of energy levels in the ground and excited singlet states will not be as great compared to lowering of energy levels with the anion. This is indicated in Figure 3. Adsorption probably involves hydrogen-bonding between adsorbent and adsorbate, but this does not lead to room temperature phosphorescence. rn-Aminobenzoic acid and o-aminobenzoic acid show little or no room temperature phosphorescence and their fluorescence emissions on sodium acetate were at shorter wavelengths relative to their cyclohexane solutions. These compounds probably form the sodium salts on the surface, but it appears the positions of the amino and carboxyl groups in the molecule prevent them from adsorbing as strongly and rigidly as PABA. Especially excited singlet state adsorption is weaker leading to the hypsochromic fluorescence shift on sodium acetate (Figure 3). The fluorescence maxima on starch show a bathochromic shift and the interactions should be similar to those of PABA on starch. However, the interactions are weaker as indicated by the smaller bathochromic shift compared to PABA on starch. I n f r a r e d Spectra. The mode of adsorption of PABA on sodium acetate was further investigated by infrared spectroscopy. The spectra of PABA, of sodium acetate and of PABA adsorbed on sodium acetate are shown in Figure 4. Strong sodium acetate bands obscured much of the adsorbate spectra, but a number of observations and conclusions could be made. The N-H stretching vibrations of PABA a t 3350-3450 cm-l disappeared for PABA adsorbed on sodium acetate. They were probably shifted to longer wavelengths and broadened, due to hydrogen-bonding between the amino 2168
I 0 3 O C
Figure 5. Room temperature phosphorescence intensity of PABA on 10 mg sodium acetate
,000
C r n ?
Figure 4. IR spectra of sodium acetate ( l ) , PABA (2),and adsorbed on sodium acetate (3). KBr pellets were used
40lDC P L B l
ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
group and the carboxyl groups of the surface sodium acetate. The peaks possibly disappeared under the strong methyl stretch band of sodium acetate a t 3000 cm-'. The spectrum of a similarly prepared o-aminobenzoic acid sample retained its N-H peaks when adsorbed on sodium acetate. Because there is an intramolecular hydrogen bond between the amino group and carboxyl group in o-aminobenzoic acid, this compound apparently is not held to the surface by strong intermolecular hydrogen-bonding. o-Aminobenzoic acid does not show room temperature phosphorescence. p-Hydroxybenzoic acid on sodium acetate gives room temperature phosphorescence. Its infrared spectrum lacks the phenolic 0-H stretching frequency at 3300 cm-'. Again, this is probably due to hydrogen-bonding between the hydroxyl group and carboxyl group of the surface. The 0-H stretching frequency of m-hydroxybenzoic acid, which does not show room temperature phosphorescence, remained in the infrared spectrum of the adsorbed system. The C-N stretching frequency of PABA a t 1280 cm-' still appeared in the adsorbed system, as did a number of peaks in the finger-print region. The formation of the sodium salt of PABA upon adsorption on sodium acetate should lead to the disappearance of any carboxyl-OH frequencies. This cannot be seen in the spectra because of broad adsorbent infrared bands in the regions of interest. Surface areas. Kortiim and Oelkrug (15) and Kortiim (16) have described a method of determining surface areas of adsorbents by reflectance measurements of the adsorbate. Their systems consisted of sodium chloride adsorbent and an adsorbed lactone dye. The color of the dye was different in the primary adsorbed layer, as compared to further adsorbed layers. No such color change occurred in the PABA-sodium acetate system. The reflectance values decreased with increasing amounts of adsorbed PABA. A plot of the Kubelka-Munk function vs. amount of PABA adsorbed gave a curve that was not well defined and the point at which monolayer coverage occurred could not be determined. However, chemisorbed PABA molecules on the sodium acetate surface are distinguished from physically adsorbed molecules on top of the primary layer by their room temperature phosphorescence behavior. Only molecules that strongly and directly interact with the sodium acetate surface are held rigidly enough to show room temperature phosphorescence. PABA molecules in the second and subsequent adsorbed layers will not phosphoresce but rather decrease the signal by absorbing exciting radiation as well as emitted phosphorescence from the chemisorbed layer. A series of room temperature phosphorescence measurements of PABA on sodium acetate were taken (Figure 5). The maximum signal was obtained for 6100 ng PABA on 10 mg sodium acetate. The surface area occupied by a flatly adsorbed PABA molecule was calculated to be 68.8 A* by Snyder's method (17),
-COON=
\\\\\\\\\\\
-COONa
2
r1 \
\
-COON=
\\ \
-COONa l
l
Figure 6. Adsorption of PABA (1) and 5-HIAA (2) on a sodium acetate surface
which has been proven to give reliable results in adsorption chromatography. If it was assumed that the PABA molecule was adsorbed flatly and that the maximum phosphorescence signal corresponded to completion of the monolayer, then the calculated surface area value for the sodium acetate adsorbent was 1.8 m2/g. This value was identical with the value obtained by the BET method (see Experimental). This indicates that the above mentioned assumptions were correct. Snyder's method was also used to calculate the flat surface area of one sodium acetate molecule and it was found to be 34.1 A*. So the molecular surface area ratio between adsorbate and adsorbent was 68.8 A'134.1 A* or 2.0211. At the point of maximum room temperature phosphorescence, the molar surface ratio was 1.9711. This shows that 1 mol of PABA would adsorb on two surface moles of sodium acetate. Such an arrangement can lead to good overlap between the carboxyl group on sodium acetate and the functional groups of PABA (Figure 6). This overlap allowed for the strong interactions necessary for room temperature phosphorescence. When the same geometric model was used for rn-aminobenzoic acid, a far less favorable overlap of the groups resulted. A similar study was done with p-aminohippuric acid and with 5-HIAA, both of which give room temperature phosphorescence on sodium acetate. In both cases, it was found that the molecular surface area ratio of the flatly adsorbed molecule with sodium acetate corresponded to the molar surface area ratio at the point of maximum phosphorescence. As mentioned earlier, a small part of the signal measured for 5-HIAA was due to fluorescence. This may introduce a slight error, but it would not significantly affect the correspondence between the surface area ratios. So it is again indicated that the molecules are adsorbed flatly. Good overlap of functional groups was also possible with these compounds. For instance, the molecular surface area of a flatly adsorbed 5-HIAA
molecule was calculated to be 105.4 A2. The molecular surface area ratio between adsorbate and adsorbent was 105.4 A2/34.1 A* or 3.0911, indicating the adsorption of one 5-HIAA molecule on three sodium acetate species (Figure 6). A possible reason for the absence of room temperature phosphorescence of PABA adsorbed on potassium acetate and lithium acetate surfaces is the different molecular sizes of these adsorbents. The surface area of a potassium acetate unit is 37.7 A' and of a lithium acetate unit 31.5 A2, compared to 34.1 A* for sodium acetate. This could lead to less favorable overlaps of the functional groups of adsorbate and adsorbent and hence a less rigid adsorption on the potassium and lithium salts. Surface adsorption data could not be obtained from fluorescence measurements because both chemisorbed and physically adsorbed species can fluoresce. The fluorescence of 4-aminosalicylic acid samples adsorbed on sodium acetate was measured. The intensity was found to increase beyond 40 pg p-aminosalicylic acid on 10 mg sodium acetate. There was no break in the curve to indicate monolayer coverage.
CONCLUSIONS Room temperature phosphorimetry of compounds adsorbed on sodium acetate has the analytical advantages of selectivity and insensitivity to moisture. Sensitivity is good and low limits of detection have been achieved. Certain molecular requirements for room temperature phosphorescence have been established. They may be used as a guideline for investigation of other molecules and surfaces in order to give the technique a still broader analytical scope. LITERATURE CITED (1) M. Roth, J . Chromatogr., 30, 276 (1967). (2) E. M. Schulman and C. Wailing, Science, 178, 53 (1972). (3) E. M. Schulman and C. Wailina. J . Phvs. Chem.. 77. 902 11973) i4j R. A. Paynterl S. L. Wellons, axdJ. D.'Winefordner, Anal. Chem.: 46, 736 (1974). (5) R. M. A. von Wandruszka and R. J. Hurtubise, Anal. Chem., 48, 1784 (1976). (6) R. M. A. von Wandruszka and R. J. Hurtubise. Anal. Chim. Acta, in press. (7) J. D. Winefordner and M. Tin, Anal. C,bim. Acta, 31, 239 (1964). 18) Shaw and Morris. Biochem. Preo.. 9 , 92 (1962). (9) S. Patai, "The Chemistry of Carboxylic Acids and'Esters", Interscience, New York, N.Y., 1969, p 236. (10) W. J. Houiihan, Ed., "Indoles", Part One, Wiley-Interscience, New York, N.Y.. 1972. - ~. D 11. (1 1) H. Zeitlin, N. Kondo and W. Jordan, J . h y s . Chem. Solids, 25, 641 (1964). (12) H. Zeitlin and Van Lieu, J . Catal., 4, 546 (1965). (13) H. H. Jaff6 and M. Orchin, "Theory and Applications of Ultraviolet Spectroscopy", John Wiley and Sons, New York, N.Y., 1965, p 260. (14) N. Mataga, Bull. Chem. SOC. Jpn., 36, 654 (1963). (15) G. Kortum and D. Oelkrug, Z.Phys. C'hem. (Frankfurt am Main), 34, 58 (1962). (16) G.Kortum. "Reflectance Spectroscopy", Springer-Verhg, New York, N.Y., 1969, p 270. (17) L. R. Snyder, "Principles of Adsorption Chromatography", Marcel Dekker. New York, N.Y., 1968, p 199.
RECEI\-ED for review August 3, 1977. Accepted September 19, 1977.
ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
2169
