Anal. Chem. 1994,66,818-823
Inelastic Electron Tunneling Spectroscopic Study of Biological Compounds in Human Sweat Adsorbed on Alumina MorihMe Hlgo' and Satsuo Kamata Department of Appiied Chemistty and Chemical Engineering, Faculty of Engineering, Kagoshima University, Korimoto, Kagoshima 890, Japan Inelastic electron tunneling spectroscopy has been used for the detection and characterization of biological compounds in human sweat adsorbed on alumina surfaces. The sweat was collectedfrom students and distributed to aqueous and benzene phases by solvent extraction. Both solutions were doped onto alumina surfaces of tunneling junctions, and the vibrational spectra of the adsorbed speCies were measured by inelastic electron tunneling spectroscopy. Analysis of these tunneling spectra showed that lactic acid in the sweat is extracted with water and selectively adsorbed on alumina. Adsorbed fatty acids were detected from the benzene phase. These biological compounds in the sweat solutions were found to react with the alumina to give about a monolayer of adsorbed carboxylate anions on the surfaces. Inelastic electron tunnelingspectroscopy (IETS) is a unique technique using the phenomenon of electron tunneling through a metal/oxide/metal tunneling junction at cryogenic temperaturestoreveal thevibrational spectrumof theoxidesurface and species adsorbed on it.l The principle, apparatus, and applicationof IETS have been extensivelyreviewed by several authors.24 When a dc bias is applied across the junction, a vibrational mode of a frequency (v) appears as a peak in the second derivative (d2Z/dP) of the characteristic currentvoltage (I-V) function of the junction at a voltage of V = hv/e, where h is the Planck's constant and e is the electron charge. Both infrared the Raman-active vibrational modes appear in the tunneling spectrum. The normal spectral range extends from 50 to 500 meV (about 400-4000 cm-l, 1 meV = 8.065 cm-l). The high sensitivity and resolution inherent in IETS give the detailed vibrational spectrum of a fraction of a monolayer of adsorbed specieson the oxide surface. Thus, IETS is a powerful analytical technique for detecting adsorbed species and investigating their chemical reactions with the surfaces. Tunneling spectra of various compounds, for example, carboxylic acids,lsSl2 aldehydes,1°J2and esters of carboxylic (1) Jaklevic,R. C.; Lamb, J. Phys. Rev. Letr. 1966,17,1139. Lambe, J.;Jaklevic, R. C. Phys. Rev. 1968, 165, 821.
(2) Wolfram, T. Inelastic Electron Tunneling Spectroscopy; Springer: Berlin, 1978. (3) Hansma, P. K. Tunneling Specetroscopy; Plenum: New York, 1982. (4) Walmsley, D. G.;Tomlin, J. L. Prog. SurJ Sci. 1985, 18, 247. ( 5 ) Klein. J.; Uger, A.; Belin, M.;Defourncau, D.; Sangster, M.J. L. Phys. Rev. 1973, 87, 2336. (6) Lewis, B. F.; Moscsman, M.;Weinberg, W. H. Sur/. Sci. 1974, 41, 142. (7) Skarlatos,Y.; Barker, R. C.; Haller, G.L.; Yelon, A. Surf. Sci. 1974,43,353. (8) Shklyarevskii, 0. I.; Lysykh, A. A.; Yanson, I. K. Sou. J . Low Temp. Phys. 1976, 2, 328.
818
AnaljltcalChemIsby, Vol. 66,No. 6,March 15, 1994
a ~ i d shave , ~ been ~ ~measured, ~ giving evidenceof the adsorbed structures. It has been found that carboxylic acids and aldehydes are adsorbed onto alumina surfaces as carboxylate anions. Esters of carboxylic acids react with the surface oxide OH groups to give adsorbed a n i ~ n s . ' ~ Since J ~ tunneling spectroscopy is very sensitive, it can be a suitable analytical technique for biological compounds. Tunneling spectra of some amino acids, nucleosides, nucleotides, and nucleic acids adsorbed on alumina surfaces have been measured to show the usefulness of this technique in biological research.16-19 However, no previous attempt was made to apply tunneling spectroscopy to the study of biological compounds in body fluids. Sweat is a dilute aqueous solution containing various kinds of inorganic and organic compo~nds.20-~3Lactic acid, the major organic component in sweat, is secreted by human eccrine glands and is derived from blood gluco~e.2~~2s Physiological studies have demonstrated that lactic acid concentration in sweat depends on such factors as sweat rate, heat exposure, exercise, and acclimatization.s28 The infrared spectra of sweat residues were measured and compared with that of sodium lactate.29 Analytical studies of lactic acid in sweat have been done by fluorescence micr~scopy~~and liquid ~~
~
~~~~~
(9) Cass, D. A.; Straw, H. L.; Hansma, P. K. Science 1976, 192, 1128. (10) Brown, N . M.D.; Floyd, R. B.; Walmsley, D. G.J. Chem. Soc., Faraday Trans. 2 1979, 75, 17. (1 1) Brown, N . M.D.; Nelson, W. J.; Walmsley, D. G.J. Chem. Soc., Faraday Trans. 2 1979, 75, 32. (12) Brown, N. M.D.; Floyd, R. B.; Walmsley, D. G.J . Chem. Soc., Faraday Trans. 2 1979, 75, 261. (13) Brown,N. M. D.; Nelson, W. J.;Turner,R. J.; Walmsley, D. G.J. Chem.Soc., Faraday Trans. 2 1981, 77, 331. (14) Bayman, A.; Hansma, P. K.; Gale, L. H. Sur/. Sci. 1983, 125, 613. (15) Kamata, S.; Higo, M.;Hida, A. Chem. Lett. 1993, 181. Kamata, S.; Hida, A.; Higo, M.;Inadome, H. J. Phys. Chem., in press. (16) Simonsen, M.G.; Coleman, R. V. Phys. Rev. 1973, B8, 5875. (17) Simonscn, M. G.;Coleman, R. V.; Hansma, P. K. J. Chem. Phys. 1974,61, 3789. (18) Hansma, P. K.; Coleman, R. V. Science 1974, 184, 1369. (19) Clark, J. M.;Coleman, R. V. Proc. Natl. Acad. Sei. LISA. 1976, 73, 1598. (20) Robinson, S.; Robinson. A. H.Physiol. Rev. 1954, 31,202. (21) Comar, C. L.; Bronner, F. Mineral Metabolism; Academic: New York, 1960. (22) Clarke, J. T.; Elian, E.; Chwachman, H. Am. J. Dis. ChiId. 1961, 101,490. (23) Sato, K. Rev. Physiol. Biochem. Pharmacol. 1977. 79, 51. (24) Wolfe, S.;Cage, G.;Epstein, M.; T i e , L.; Miller, H.; Gordon, R. S. J. Clin. Inuest. 1970, 49, 1880. (25) Gordon, R. S.;Thompson, R. H.; Muenzer, J.; Thrasher, D. J. Appl. Physiol. 1971, 31, 713. (26) Fellmann, N.; Grizard, G.;Coudert, J. J . Appl. Physiol. 1983, 51, 355. (27) Lamont, L. S. J . Appl. Physiol. 1987, 62, 194. (28) Pilardeau, P. A.; Chalumcau. M. T.; Harichaux, P.; Vasseur, P.; VayJsc, J.; Garnier, M. J . Sports Med. Phys. Fitness 1988, 28, 176. (29) Rousscau, D. L. Science 1971, 17I, 170. (30) Hannon, D.; Quinton, P. M. Anal. Chem. 1984, 56, 2350. 0003-2700/94/08660810$04.50/0
0 1994 Amerlcan Chemlcal Sockty
T O M . 1. SubJoctr a d P l a m for Swoal Colloctlon
subject
BBX
age
place
A
male male male
22 23 24
forehead forehead, chest, back forehead
B
C
ionization mass ~pectrometry.~~ Since sweat is connected with corrosion of metals32and sometimes the origin of accidental biological ~ontamination,~~*~3 it is interesting to investigate the components of sweat. In the present paper, we report the first attempt to detect and characterize biological compounds in human sweat using tunneling spectroscopy. The tunneling spectra of the adsorbed species doped from aqueous and organic solutions have been obtained and analyzed in order to clarify what sweat components are important in adsorption on the alumina surfaces of the tunneling junctions.
EXPERIMENTAL SECTION Sweat Samples. Sweat was collected from three healthy students after running outside at 15 OC. The threesubjects, their ages, and the places for the sweat collection are shown in Table 1. The sweat (-60 mg) was collected from clean foreheads with pipets, taking care to assure the absence of toilet goods or cosmetics. In the case of the subject B, sweat (- 1 g) was also collected with small plastic cups (diameter 95 mm) fitted to his chest and back. About 60 mg of forehead sweat and an equal volume (2-3 mL) of distilled water and benzene were mixed and shaken for -10 min at -20 OC in order to distribute biological compounds in the sweat to the aqueous and benzene phases. In the case of sweat from the chest and back of the subject B, 300-500 mg was used. The mixture was left for 5-60 min, and the phases were separated by centrifugation (3000 rpm, 5 min). Reagents. Lactic acid (90.6% aqueous solution), n-capric acid (99.9%), lauric acid (99.1%), myristic acid (99.0%), palmitic acid, stearic acid, and oleic acid were purchased from Wako Pure Chemical Industries and used without further purification. Benzene (99.5%, Nacalai Tesque) and distilled water were used as thesolvents. Aluminum (99.999%, Mituwa Chemicals) and lead (99.999%, Wako) were used as the electrodes of the tunneling junctions. Apparatus and Procedures. The method of junction preparation and the apparatus for measuring the tunneling spectrum have been described in detail pre~iously.~'3~Aluminum was evaporated from a molybdenum boat on a clean glass slide to form three strips (1 mm wide) at a pressure of Torr (1 Torr = 133.322 Pa). The surfaces of the strips were oxidized in an oxygen dc glow discharge in the bell jar. The slide was removed from the vacuum system and set on a spinner. One drop (-60 pL) of the aqueous or benzene sweat solution was dropped on the strips, and excess solution
-
~
~
~
~~
~~~~
(31) Yokoyama, Y.; Aragaki, M.; Sato, H.; Tsuchiya, M.Anal. Chim. Acrn 1991, 246, 405. (32) Bailey, R.A.; Zaccardi, J. A. J. Elecrroana1. Chcm. 1983, 144, 443. (33) Lowry, R. K.;Linn, J. H.; Grove, G. M.;Vicroy, C. A. Scmicond. Inr. 1987, 73. (34) Kamata, S.; Higo, M. Chem. Lcrr. 1984, 2017. (35) Higo, M.;Mizutaru, S.; Kamata, S. Bull. Chcm. Soc. Jpn. 1985, 58, 2960; 1989, 62, 1829. (36) Higo, M.;Kamata, S.J. Phys. Chcm. 1990, 94, 8709.
T O M 2. Typlcal Condttlons of Tunmllng Mocrrurrmonta
resistances of the junctions modulation frequency modulation voltage spectral range accuracy resolution time constant trace time
30-3000 D 500 Hz 3 mV peak-to-peak 260-4000 cm-1 +4 to +8 cm-1 20 cm-1 3s 60 min
was immediately spun off in order to adsorb the biological compounds onto the alumina surfaces. The slide was returned to the vacuum system, and the junctions were completed with an evaporated Pb cross strip (1 mm wide). Resistances of the measured junctions were in the range 90-1750 s2. Tunneling spectra were obtained by measuring the second derivativeof the tunneling current through the junction at 4.2 K with a lock-inamplifier. Typical conditions of the tunneling measurements are shown in Table 2. The peak positions were obtained by averaging those of five spectra and corrected by -1 meV (-8 cm-l) for the energy gap of the superconducting Pb electrodes3 The accuracy was estimated to be i 4 or &8 cm-l in the frequency range 250-2000 or 2000-4000 cm-', respectively. The transmission infrared spectra of lactic acid and the fatty acids listed above as well as their sodium salts were measured in KBr pellets with a Jasco A-3 infrared spectrophotometer.
RESULTS AND DISCUSSION Sweat is a dilute aqueous solution (-99% water), involving many inorganic and organic compounds.2G23These biological compounds are considered to have different solubilities to solvents used. Therefore, we tried to distribute these compounds in the sweat to aqueous and organic phases by solvent extraction. In the extraction with water and nitrobenzene, tunneling spectra doped from aqueous solutionswere identical; however, those doped from nitrobenzene solutions lacked reproducibility. Finally, we used the extraction with water and benzene and obtained good tunneling spectra. Adsorbed Species from Aqueous Phase. The tunneling spectra of the aqueous sweat solutions doped onto the alumina surfaces of the electrodes are shown in Figure 1. The sweat was collected from the foreheads of the subjects A-C and extracted with equal volumes of water and benzene. The tunneling spectrum of the alumina doped from water is also shown and is identical to that of the undoped alumina,35136 showing that water is a good solvent for measuring tunneling spectra of sweat. The spectrum has broad peaks at 3600 and 930 cm-l due to the stretching modes ( v ) of surface OH groups and A10, respectively. The peak near 280 cm-1 is caused by the A1 phonon of the A1 ele~trode.~sJ6The peak positions and their relative intensities in the tunneling spectra of the aqueous sweat solutions agreed well with one another within experimental uncertainty. The tunneling spectra have strong characteristic peaks at 2960 and 2910 cm-1, due to v(CH) of the adsorbed specieson the alumina surfaces. Peaks caused by deformational modes (6) of C H are present at 1440 and 136Ocm-l. Aweakandbroadpeakat 253Ocm-l ispresent in the sweat tunneling spectrum of subject A. The tunneling Analyticel Chemistty, Vol. 86, No. 6, Mer&
15, 1994
819
I
I
CHqCH (OH I COOH
I
Tabla 3, Vlbratloml Fr.qmnd.l (om-')and Mod. Ambmmts tor Adsorbed Spec108 on AbO, D0p.d from Aquooua PhaH of SwsaV
sweat 3606 w br
A
IR lactate*
3624 w br
2956 va 2911 s 2858 w 2531 w br 1855 w br 1646 w br 1586 sh 1440 8
2957 vs 2913 8 2858 w
1357 8
1361 s
1284 w
1280 w
1101 m 1048 m 923 s
1101 m 1042 m 930 s
875 w 771 vw 654 w br 430 m 286 m
/I
IETS lactic acid
1885 w br 1656 w br 1604 sh 1442 s
864W
782 vw 658 w br 435 m 281 m
3300 s br 2990 w 2945 w 2890 ah
1590 s br 1455 m 1422 m 1364 w 1315 m 1268 m 1121 8 1083 m 1040 8 929 m 856 m 779 m
assignment v(0H) (surface) v(0H) (alcohol) v~CHS) vs(CHd v(CH)? ? v(AlH) (surface) vM(Co@)
vM(coo-)
6JCHa) V,(COo-)? &@Ha) 6(CH) ?(alcohol) v(C0) (alcohol) v(C-CHs) ?(alcohol) VWO) &OH) (acid) ? ? ? ?
Al phonon
*
0 The sweat was corrected from the forehead of subject A. Na salt in KBr pellet: vat very strong; 8, strong; m, medium; w, weak; vw, very weak; sh, shoulder; br, broad; v , stretching; 6, bending or deformation;88, asymmetric;8, symmetric.
I ""
. I
0 100, 200 300 . 400 500 ElmeV 0 1000 2000 3000 4000 G/cm-' Figwr 1. Tunneling spectra of aqueous phases of sweat and lactlc acld (CH3CH(OH)COOH) on AIz03. The sweat (68.0, 49.2, and 67.0 mg) waa collected from the foreheads of subjects A-C and extracted wlth 3, 2, and 3 mL of water and benzene, respedtvely. Lactlc acld was doped from an aqueous solution (1.1 mg/mL). The tunnellng spectrum of A1203 doped from water Is also shown. ,
spectra ofthe aqueous sweat solutions suggest that a relatively simpleorganic compound is adsorbed on the alumina surfaces. The tunneling spectrum of lactic acid ( C H 3 C H ( O H ) COOH), the major organic component of is also shown in Figure 1. Lactic acid was adsorbed onto the alumina surfaces from an aqueous solution (1.1 mg/mL). A detailed analysisand comparison of the sweat tunneling spectra showed good agreement with that of lactic acid except for the weak and broad peak at 2530 cm-l. The tunneling spectrum of urea, the second major organic component of doped from its aqueous solution (9.7mg/mL) was measured, but it disagreed with the sweat tunneling spectra. As did spectra of other organic components of sweat, for example, phenol, pyruvic acid, uric acid, and creatinine. These compounds are minor components of sweat and their concentrationsare a few orders of magnitudes smaller than that of lactic acid.2s23 Thus, we conclude that lactic acid in the sweat is distributed to the aqueous phase and selectively adsorbed onto the alumina surfaces. The peak positions of the tunneling spectrum of the sweat collected from subject A and that of lactic acid, and the infrared 020
Analytlcel Chemism, Vol. 66,No. 6,Much 15, 1994
spectrum of sodium lactate are shown in Table 3, along with their assignments. The assignmentswere made with reference to those of the infrared spectra of lactic acid and its saltseJ7-39 The tunneling spectrum of lactic acid has peaks due to the asymmetric stretching mode of the carboxylate group (vai(COO-)) at 1656 and 1604 cm-* and no v ( C 4 ) peak, showing that it is adsorbed as lactate anion on the alumina surface. The tunneling spectrum of the sweat also has the vas(COO-) peaks at 1646 and 1586 cm-l. This finding is consistent with the fact that many carboxylic acids studied by tunneling spectroscopy are adsorbed on the metal oxide surfaces as carboxylate anions.l-12 The peaks of the OH (alcoholic) group of lactic acid are obscured in the tunneling spectrum, which is considered to be mainly due to the effect of the top Pb electrode as discussed previously.3 Peak broadening caused by hydrogen bonding may be also involved. The weak and broad peak at 2530 cm-l in the sweat tunneling spectrum of subject A is not present in the spectrum of lactic acid. The presence of this weak peak may suggest other adsorbed species on the surfaces. Though the identification of this peak is difficult, the quantity of the adsorbed species is considered to be much smaller than that of the adsorbed lactic acid. The tunneling spectra of sweat collected form other places were measured and compared in subject B. The tunneling spectra of the sweat from the chest and back agreed with that of lactic acid and showed that lactic acid is also detected from the aqueous phases of sweat collected from these places. (37) Bolard, J. J. Chim. Phys. 1965, 62, 887. (38) Ranadc, A. C.; Biswas, A. B. J. Indian Chem. Soc. 1967, 44, 314. (39) Snvastava, P. C.; Bancrjee, B. K. Ferr. Technol. 1979,16, 141.
T a w 4. Vfkatknal Froqwncha (cm-’) and Mod. AedgmwW for A d r o r k d Speck on 4 0 , D0p.d from b.nrw Phrw ol Swrat.
sweat
IETS palmitic acid
3619 m br
IR palmitate0
3635 m br
2862 vs 2833 s 2721 w 1856 vw br 1647 vw br 1592 w br
2867 vs 2833 s 2714 w 1869 vw br
1438 s 1368 w
1429 s 1365 w
1282 m 1254 m
1288 m
1121 w 1054 m 928 s
1119 w 1084 vw 1049 w 935 8
883 sh
877 sh
719 vw
726 w
286 m
279 m
1588 w br
2960 m 2925 s 2880 vw 2860 8
1560 8 1472 m 1446 m 1422 m 1342 vw 1324 vw 1302 vw 1279 vw 1258 vw 1236 vw 1212 vw 1189 vw 1094 vw 925 w
100
200
300
400 500 ElmeV I 1000 2000 3000 4000 3lcm-l Flguro 2. Tunneling spectra of benzene phasesof sweat and palmltlc ac#(CH3(CHZ),,CO0H) on A1203. The sweat (63.4,49.2, and 67.0 mg) was colkoted from the foreheads of subjects A-C and extracted with 3,2,and 3 mL of water and benzene, respectively. Palmitic acid was doped from a benzene solutlon (0.1 mg/mL). The tunneling spectrum of A1209doped from benzene is also shown.
In the present study, the combination of tunneling spectroscopy and solvent extraction showed that lactic acid in the sweat is easily and selectively detected from the aqueous solution. The lactic acid is adsorbed on the alumina surfaces of the tunnelingjunctions as the lactate anion. It is concluded that lactic acid reacts with surface OH groups on alumina to give adsorbed lactate anion, as in the case of many carboxylic acids on the alumina surfaces studied by tunneling spectroscopy.1-12 Because the tunnelingjunctions are prepared in high vacuum and the lactic acid is adsorbed as thecarboxylate anion on the surfaces, the adsorbed lactate is considered to be present at about a monolayer thickness on the alumina surfaces of the tunneling junctions. The tunneling spectra of lactic acid doped from the concentration in the range 0.1-1.1 mg/mL showed no spectral change, suggesting the saturated monolayer coverage of the surfaces. Adsorbed Species from Benzene Phase. The tunneling spectra of the separated benzene sweat solutions doped onto the alumina surfaces of the electrodes are shown in Figure 2. The sweat was collected from the foreheads of subjects A-C and extracted with equal volumes of water and benzene. The
780 vw 720 m 700 m 593 w 538 vw 494 vw
The sweat was corrected from the forehead of subject A. b Na salt in KBr pellet: v8, very strong; s, strong; m, medium; w, weak; vw, very weak; sh, shoulder; br, broad; v, stretching; 6, bending or deformation; y , scissoring; w, wagging; T , torsion; p, rocking; as, asymmetric; 8, symmetric.
tunneling spectrum of the alumina doped from benzene is also shown and is identical to that of the undoped alumina,35*36 showing that benzene is also a good solvent for measuring the tunneling spectra of sweat. The peak positions and their relative intensities in the tunneling spectra of the benzene sweat solutions agreed well with one another within experimental uncertainty. The tunneling spectra have strong characteristic peaks due to u(CH) of the adsorbed species at 2860 and 2830 cm-l. The peaks caused by the deformational modes of CH are present in the frequency range 1250-1440 cm-*. The peaks of u(CC) are present at 1050 and 880 cm-l. The tunneling spectra of the benzene sweat solutions suggest that organic compoundshaving long alkyl chains are adsorbed on the alumina surfaces. We measured tunneling spectra of many organic compounds and found that fatty acids with long alkyl chains (CH3(CH2),2COOH) give tunneling spectra similar to that of the benzene sweat solution. The tunneling spectrum of palmitic acid (n = 16) doped from a benzene solution (0.1 mg/mL) is shown for example in Figure 2. The peak positions of the tunneling spectrum of the sweat collected from subject A and that of palmitic acid, and the infrared spectrum of sodium palmitate, are shown in Table 4. The assignments of the tunneling spectrum of the sweat were made with reference Analytcal Chemktty, Voi. 66, No. 6, March 15, 1994
021
2oFt
q
r 10 c)
-
5
0
5
10
15
20
Number of carbon atoms Flgure 5. Intensity ratio of v8(CHS)(-2870 cm-l) and v,(COO-) (- 1590 cm-l)(l(v,(CH3))/I(v,(COO-))) vs numberof carbonatoms (n) of adsorbed fatty acids on Al2OS. The values obtained in the present work (0)are shown, along with those by Brown et ai. (0).
to those of palmitate anion on alumina made by Brown et al.12 The tunneling spectrum of palmitic acid has the weak and broad peak due to v,,(COO-) at 1588 cm-l and no v ( C 4 ) peak, showing that it is adsorbed as palmitate anion on the alumina surface. The tunneling spectrum of the sweat has also the va,(COO-) peaks at 1647 and 1592 cm-1 and no Y(C=O) peak. Thus, we conclude that fatty acids in the sweat are distributed to the benzene phase and selectivelyadsorbed onto the alumina surfaces. At this stage, however, it is not clear which fatty acid in the sweat plays the most important role for the adsorption. The tunneling spectra of the sweat collected from the chest and back of subject B were similar to that of palmitic acid and showed that fatty acids are also detected from the benzene phases of the sweat collected from these places. In order to determine which fatty acid in the sweat is adsorbed on the alumina surfaces of the tunneling junctions, we attempted to measure the tunneling spectra of fatty acids (CH3(CH2),2COOH, n = 10, 12, 14, 16, 18) doped from their benzene solutions (0.1-1 .O mg/mL) to compare the spectral features. The tunneling spectra showed that the positions of the two strong v(CH) peaks near 2870 and 2830 cm-1 have a tendency to shift slightly toward lower frequencies as n increases. The positions of the two v(CH) peaks in the tunneling spectra of the benzene sweat solutionswere compared with those of these tunneling spectra of the fatty acids. However, we could not determine the adsorbed fatty acid because of the accuracy ( f 8 cm-l) of the tunneling spectra. A good correlation between the intensity ratio of v,(CH3) (-2870 cm-1) and va,(COO-) (- 1590 cm-l) (Z(vS(CH3))/ Z(vas(COO-))) with the number of carbon atoms (n) of adsorbed fatty acids has been found by tunneling spectroscopy.12 The measured intensity ratios for the fatty acids (n = 10, 12, 14, 16, 18) are shown in Figure 3, along with those reported by Brown et a1.12 These intensity ratios agree well with those reported, suggesting the possibilityfor determining the adsorbed fatty acids doped from benzene sweat solutions. Figure 3 shows that the intensity ratio increases as n increases. This is due to the fact that since the surface is saturated with adsorbed fatty acid, and the acid interacts with the surface through the carboxylate group, the v,(CH3) mode gives the increase in intensity relative to that of the vag(COO-) mode with increasing CH2 group. The obtained values of the 822
Analytical Chemistry, Vol. 66,No. 6,March 15, 1994
intensity ratios in the tunneling spectra of the sweat from the foreheadsofsubjects A-Care 14.8,11.3,and 15.1,respectively. Two of the values agree well with each other and suggest that palmitic acid (n = 16) plays an important role for adsorption on the alumina surfaces from benzene sweat solutions. For the other result, however, the intensity ratio suggests a major contribution of a smaller fatty acid (n = 9). It seems that decomposition of fatty acids on the skin surface of subject B may occur and yield the smaller fatty acid. Tunneling spectroscopy clearly shows that fatty acids are selectivelyand sensitively detected from the benzene phase of the sweat. These fatty acids are considered to be collected with the sweat. However, fatty acids are not involved in the organic components of sweat2G23nor have they been detected in the analytical ~tudies.~~-31 Human skin surface lipid secreted by sebaceous glands comprises triglycerides, wax esters, squalene, and free fatty acids.m3 The major fatty acids found in skin surface lipids have chain lengths from 14 to 18 carbon atoms. They include odd- and even-numbered carbon atoms; they may be branched or unbranched and contain saturated or unsaturated chains. Of these fatty acids, palmitic acid (n = 16) is the major component of skin surface lipid. Kosugi and UetaN have reported the separation and detection of fatty acids in the lipids from faces of several young Japanese men using chromatography. Though a relatively large quantity of branched chain and odd-carbon-number fatty acids have been found, the major component is palmitic acid. An important contribution of this fatty acid has been also reported in the lipids from scalp hair b~lbs.~l,42 Tunneling spectroscopy is now recognized to be a very sensitive tool to detect adsorbed species on the oxide surface. The sensitivity is high enough to detect less than 1013 molecules/cm2, which corresponds to a fraction of a monolayer of the ~ u r f a c e . ~The ~ .selective ~~ detection of the fatty acids in the benzene phase of the sweat is because of this high sensitivity for carboxylic acids. Recently, analytical study for the detection of a carboxylic acid has been done by IETS; Mazur et al.46 have utilized Al/A1203/Pb/Au junctions externally doped with formic acid vapor and reported that the tunneling spectra of the formate ion can be observed over a wide range of the concentration, with a lower limit below 50 PPm. CONCLUSIONS We have reported for the first time that IETS is applicable to the detection and characterization of the biological compoundsin humansweat. Onlyonedropofthesweat (-60 mg) is sufficient and no complex pretreatment of the sample is necessary. The combination of solvent extraction and tunneling spectroscopy enables us to analyze rapidly and selectively the biological compoundsin thesweat. Theanalysis of the tunneling spectra doped from the separated aqueous and benzene solutions of the sweat provided important (40) Kosugi, H.; Ucta, N. Jpn. J. Exp. Med. 1977, 47, 335. (41) Gcnhbein, L.L.; Baburao, K.Fetre, Seven, Atutdchm. 1984, 86, 121. (42) Goctz, N.; Burgaud, H.; Berrcbi, C.;Bore, P. J . Soc. Comet. Chem. 1984, 35,41 1. (43) Ricgcr, M. M. C o w ” Toiletries 1987, 102, 36. (44) Langan, J. D.; Hansma, P. K. Surf. Sei. 1975, 52, 21 1. (45) Cederberg, A. A. Surf. Sci. 1981, 103, 148. (46) Mazur, U.; Wang, X. D.; Hipps, K. W. Anal. Chem. 1992,64, 1845.
information on the adsorbed species and their chemical interaction with thealuminasurfaces. It was found that lactic acid is distributed to the aqueous phase and selectivelyadsorbed onto the surfaces as lactate anion. Fatty acids with long alkyl chains are distributed to the benzene phase and adsorbed as carboxylateanions. These adsorbed species are considered to be present at a thickness of about a monolayer on the surfaces. The high sensitivity of IETS enables us to detect and characterize these biological compounds.
ACKNOWLEDGMENT We thank Messrs. M.Yazaki, M. Isobata, and H. Hayashi for their cooperation. We also thank Professor S.Ikeda of
Ryukoku University for his valuable comments and encouragement. The present study was partially supported by CibaGeigy Foundation (Japan) for the Promotion of Science and the Light Metal Educational Foundation. Preliminary reports of this work were presentedat the 54th meeting of the Chemical Society of Japan, held in Tokyo, April 1987, and at the 40th meeting of the Japan Society for Analytical Chemistry, held in Yokohama, November 1991. Received for revlew August 31, lQQ3. Accepted January 3, 1994.@ Abstract published in Aduuncc ACS Abstruczs, February 1, 1994.
AnalyticelChemistry, Vol. 66, No. 6, March 15, 1994
825
