Anal. Chem. 1986, 58, 2337-2340
moted out of the ground state. The peak at 681 cm-' represents a new vibration produced by the triplet state of naphthalene and corresponds to Nishikida's results (I). The upward peak a t 535 cm-' arises from the triplet state of naphthalene-de and correlates with Clarke's work (2). The downward peaks a t 782 and 630 cm-' can be assigned to the bSuout-of-plane mode for naphthalene and naphthalene-d8, respectively (6, 3, and reflect the origins of the newly created absorptions at 681 and 535 cm-'. The ratioed spectrum then leads to the unambiguous assignment that naphthalene and naphthalene-de are present in the mixture. Clearly this simplified spectrum presents an advantage over that shown in Figure 2. The absorbance of the exicted triplet state bands depends upon the steady-state population of the molecules in the triplet state. To obtain a spectrum, usually files are ratioed against a background and then converted to an absorbance spectrum. However, due to the capabilities of our FTIR, we may obtain a final absorption spectrum by ratioing the triplet state file against the ground state file. This produces an absorbance spectrum with upward and downward peaks as seen in Figure 3. The ratioing and subtraction methods produce the same absorbance values for specific vibrational peaks (assuming the cell path length and molar absorptivity are constant). This is as expected since Subtraction:
A = [-log 10-(T1)]- [-log 10-(sO)]
(1)
Ratioing:
A = -log ~O(TI/SO) = [-log 10-(T1)]- [-log 1O-(s0)] (2) While the absorbance values are the same, the ratioing technique is preferred. This method eliminates ratioing data files against a background file, and the accompanying timeconsuming FT processing. A single beam instrument, such as the Nicolet 6000 FTIR, lends itself better to this form of spectrometry. For dual beam instruments, precision matched cells and dual cryogenic apparatus would be necessary for properly attained ETSIR spectra. The relative concentrations of molecules in the triplet state with our particular excitation conditions may be calculated from the ETSIR and ground state spectra using the following equation:
2337
% triplet concn = (area TIband)/(area So band) X 100 (3) with the ground-statepeak area being measured when the UV radiation is shuttered. Since the ground state and triplet state peaks differ in line width (Av = 2.0 cm-' for So;Av = 4.5 cm-' for Tl), the integrated intensities are used for this calculation instead of the absorbance a t the peak maximum. With our results, this leads to a relative triplet state concentration of approximately 2% for both the de and he naphthalene isomers. In summary, once triplet-accessible aromatic systems have been assigned triplet state infrared bands, ETSIR may be used to identify these specific molecules from mixtures of unknown composition with increased simplicity. The method lends itself particularly well to single-beam FTIR instruments because computer capabilities allow for ratioing of data files instead of subtraction, leading to shorter experimental time. Future work involves assignment of triplet state peaks for additional organic molecules and quantum mechanical calculations to correlate shifts of the triplet state infrared bands. Registry No. Naphthalene, 91-20-3;naphthalene-de,1146-65-2. LITERATURE CITED (1) Nishkida, K.; Kamura, Y.; Seki, K.: Iwasaki, N.; Kinoshita, M. Mol. phvs. 1983, 49, 1505-1507. (2) Clarke, R. H.;Kosen, P. A,; Lowe, M. A.; Mann, R. ti.: Mushlin, R. J . Chem. Soc.,Chem. Commun. 1973, 528-529. (3) Mitchell, M. B.; Smlth, 0. R.; Gulllory, W. A. J . Chem. phvs. 1981, 75, 44-48. (4) Baiardo. J.: Mukherjee. R.: Vala, M. J . Mol. Struct. 1982, 8 0 , 109- 112. ( 5 ) de Groot, M. S.; van der Waals, J. H. Mol. phvs. 1961, 4 , 189-190. (6) Scully, D. B.; Whiffen, D. H. Spectrochim. Acta 1960, 16, 1409-1415. (7) Wee, A.; Kydd, R. A. Spectrochim. Acta, part A 1989, 2 6 A , 1791-1803.
David E. Bugay
Willem R. Leenstra* Department of Chemistry University of Vermont Burlington, Vermont 05405 RECEIVED for review November 22,1985. Accepted April 28, 1986. This work was supported by grants from the Research Corporation (9326), the donors of the Petroleum Research Fund, administered by the American Chemical Society (13295-G6), and the University of Vermont (UVM PS-11).
Indirect Electrochemical Detection in Liquid Chromatography Sir: Considerable interest has recently been given to the development of so-called indirect detection approaches for use in liquid chromatography (1-5). In these systems, a suitable concentration of a species that can be readily monitored by a conventional detection scheme such as W-visible absorption or fluorescence is intentionally added to the mobile phase to generate a constant background signal. If the elution of sample components is accompanied by a displacement of the additive from the mobile phase as observed at the detector, a transitory decrease in the background level is thereby produced. The analyte species is thus monitored indirectly as a negative peak or trough in the steady-state absorbance or fluorescence signal. The primary advantage of this approach lies in the capability that it affords for the detection and quantitation of species which themselves possess no 0003-2700/86/0358-2337$01.50/0
strongly absorbing chromophore or other group readily lending itself to direct monitoring by one of the usual detection modes. Refractive index detection, utilizing measurement of the change in refractive index of the eluent plus analyte compared to that of the eluent alone, represents a familiar example of the indirect detection approach. Recently, schemes utilizing absorbance ( I ) , polarimetry (2,3), and fluorescence (4) have also been successfully demonstrated. However, the possibility of indirect amperometric detection of nonelectroactive analytes in an eluent containing an easily oxidizable additive has not yet been seriously considered for liquid chromatography. (Very recently, the use of such an approach for the determination of volatile hydrocarbons following gas chromatography (6)has been described.) Although direct electrochemical detection of analytes following liquid chromatography (LCEC) 0 1986 Amerlcan Chemlcal Soclety
2338
ANALYTICAL CHEMISTRY, VOL. 58, NO. 11, SEPTEMBER 1986
has been shown to produce analyses of extremely high sensitivity, its utility is clearly hampered by the paucity of compounds that undergo electrooxidation or -reduction at accessible potentials. Thus, it is of some interest to investigate the possibility of indirect LCEC for quantitation of nonelectroactive species. In this communication, we demonstrate the feasibility of this technique for detection and quantitation of simple, nonabsorbing organics following reverse-phase chromatography and characterize some of the principal experimental parameters governing its performance.
EXPERIMENTAL SECTION Instrumentation. The liquid chromatography system consisted of a Perkin-Elmer Series 10 pump, a Rheodyne (Berkeley, CA) Model 7125 injector with a 20-pL sample loop, and an IBM Model EC/230 amperometric detector. The LCEC electrode was a Bioanalytical Systems Model TL-5 thin-layer glassy carbon electrode assembly. The reference electrode was a Ag/AgCl electrode. Chromatography was performed with a 15-cm Econosphere C-8 5-pm column. The mobile phase consisted of 4060 CH,OH/pH 3.0 buffer that was 0.12 M in phosphate and 0.083 M in KNO,; in addition, a specified concentration of p-hydroquinone was also present. The flow rate was always 1.0 mL/min.
RESULTS AND DISCUSSION The first consideration in designing an indirect electrochemical detection scheme is the selection of an electroactive species for addition to the chromatographic mobile phase and generation of a steady-state background current. The choice for most of this work was p-hydroquinone, whose redox behavior consists primarily of a chemically reversible, pH-dependent oxidation to the corresponding benzoquinone. With this mobile phase additive, a steady anodic background was generated by the application of a modest positive potential. The analytes chosen for study consisted of mixtures of simple carboxylic acids and aliphatic alcohols for which the best available detection approach has usually been refractive index monitoring (7). These compounds were selected primarily because they lack a strongly absorbing chromophore and consequently are not well suited to detection via the usual spectroscopic methods. In addition, none of these compounds is oxidizable or reducible at a sufficiently low potential to make direct electrochemical monitoring a workable possibility. As expected, addition of hydroquinone to the chromatographic mobile phase produced an anodic background current that was dependent on the applied potential and the concentration of hydroquinone. Chromatograms obtained by recording the decrease in this otherwise steady-state background following injection and elution of the test mixtures are shown in Figure 1. In all cases, the sample mixtures injected were dissolved directly in the mobile phase solution (without hydroquinone) and were reasonably well resolved under the reverse-phase conditions employed. For both the alcohol (curves A) and the fatty acid (curves B) mixtures, distinct concentration-dependent troughs or inverse peaks were observed for all analytes. (Also shown for comparison are the identical chromatograms recorded by conventional UV absorption with no hydroquinone present in the mobile phase. No response was observed with absorption detection a t 210 nm even though relatively high alcohol and acid concentrations and the most sensitive instrumental settings permitted by the system noise level were employed.) The slightly different level of response that was obtained for each compound by indirect LCEC could not be accounted for simply by the concentration or retention differences in effect. Rather, each analyte appeared to be able to displace the hydroquinone from the mobile phase to a slightly different degree. The reasons for these differences are currently under investigation. It was natural to expect that changes in detector potential would directly affect the indirect LCEC response observed.
The effect of such changes as typified by the indirect peak observed for isopropyl alcohol (peak 1in Figure la) is shown in the hydrodynamic voltammogram (HDV) in Figure 2. Also shown for comparison is the HDV obtained conventionally for hydroquinone itself by direct LCEC. Clearly, the magnitude of the isopropyl alcohol signal-actually, the extent of the background current decrease-had exactly the same potential dependence as the background current generated by the hydroquinone oxidation. Thus, the potential dependence of the indirect LCEC signal was essentially the same as that of the oxidation/reduction of the electroactive species added to the mobile phase. This observation was further confirmed by the fact that alteration of the current-potential behavior of the background signal by changing the mobile phase pH or by switching to a different mobile phase additive (such as uric acid in place of hydroquinone) produced exactly equivalent changes in the potential dependence seen for the indirect response toward the alcohol and acid mixtures. Note that behavior similar to that described for isopropyl alcohol was observed for nearly all of the nonelectroactive analytes examined. The results shown in Figure 2 indicate that larger indirect LCEC peaks were obtained at high potentials where the background current due to the mobile phase additive was larger. This was confirmed by chromatograms a and b in Figure 3, which were obtained at different applied potentials for injection of three different isopropyl alcohol concentrations. Changing the detector potential from +0.26 V vs. AgjAgC1 to midway up the hydroquinone oxidation wave at +0.38 V produced a corresponding increase in both the steady-state background level and the indirect response for isopropyl alcohol. An alternative means of increasing the background current (i.e., instead of increasing the applied potential) was simply to increase the hydroquinone concentration in the mobile phase while keeping the detector potential constant. This possibility is illustrated in curve c where increasing the hydroquinone concentration to 1.0 X M generated the identical response as in curve b but at a potential of only +0.26 V. In fact, chromatograms obtained for hydroquinone concentrations between 1.0 X lo-, and 1.0 X lo4 M showed that the magnitude of the indirect LCEC response for all analytes examined was determined only by the magnitude of the background current. Any combination of applied potential and hydroquinone concentration that produced a given background current level always produced exactly the same analyte response. Unlike with conventional LCEC, selection of optimum conditions for performing quantitation by the indirect electrochemical approach involved more than just the selection of a potential that produced maximum electrolysis current for the desired analyte. Rather, for indirect detection, maximum signal levels could be generated by a variety of applied potentialladditive concentration combinations; and selection of conditions that minimized the system noise level became the limiting factor. In the present case, periodic current fluctuations related to pump pulsation often represented the dominant noise contribution-especially a t high potentials in the mass transfer limited regions for the hydroquinone oxidation. Accordingly,optimum sensitivity for indirect LCEC was usually obtained by operation at relatively low potentials in the activation controlled region at the foot of the hydroquinone HDV where pump pulsations exercised virtually no effect on the observed currents. Of course, operation at these low potentials required the use of a very high hydroquinone concentration for the generation of a correspondingly high background current. Thus, lowest detection limits were obtained here by a judicious selection of low applied potential and high hydroquinone concentration. Under these conditions
ANALYTICAL CHEMISTRY, VOL. 58, NO. 11, SEPTEMBER 1986
2338
I*=0.016
I
a
Az0.048
r 1
1
3
,IA
8 nA
1
I
I
I
I
I
1
0
1
2
3
4
5
Retention Time (min)
’
1
4
1
1
1
1
1
1
1
1
2
3
4
5
6
7
8
9
.
,
1 0 1 1
Retention Time (min)
Flgure 1. Chromatograms of mixtures of (A) alcohols and (B) aliphatic carboxylic acids. Lower traces were obtained by indirect LCEC at E = +0.27 V vs. Ag/AgCi with a mobile phase hydroquinone concentration of 1.0 X M; upper traces were obtained by UV absorbance at 210 nm with no hydroquinone added. The alcohol mixture contained (1) isopropyl alcohol, (2) 1-propyl alcohol, (3)1-butyl alcohol, and (4) isobutyl alcd.loi; the acid mixtwe contained (1) propionic acid, (2) 1-butyric acid, (3)isovaleric acid, and (4) l-hexanoic acid. Ail concentrations were 1.0% by volume.
500
i
-
(n$
400
-
300
-
6.
Flgurr 3. Indirect LCEC of isopropyl alcohol for (a) E = +0.26 V vs. Ag/AgCi and 5.0 X lo-’ M hydroquinone in mobile phase, (b) E = +0.38 V and 5.0 X lod M hydroquinone, and (c) E = +0.26 V and 1.0 X lo4 M hydroquinone. Isopropyl alcohol concentrattons injected were (1) 0.25%, (2) 0.50%, and (3) 1.0% by volume.
d
.? POTENTIAL
I
- 0.0 V
vS
AdAaCI
Hydrodynamicvottammogemsobtakredforisopropylalcohol by the lndirecl approach (A)and for hydrosuklone by direct LCEC (0). Chromatographic conditions were similar to those in Figure 1 except -2.
that the hydroquinone response was obtained directly with no eiectroacthre additive in the mobile phase. Current scale on left applies to the hydroquinone signal (I,) while that on the right corresponds to isopropyl alcohol (la).
(which were essentially those employed to generate the chromatograms in Figure l),limits of detection observed for
both the aliphatic alcohols and fatty acids were uniformly in the 0.01% range (or a few micrograms for the 20-pL injection volume used here). Furthermore, response was linear, for concentrations up to at least 2 orders of magnitude higher. The analytical capabilities demonstrated here for indirect LCEC detection do not rival in sensitivity the results usually obtained by direct amperometric detection. However, for compounds not possessing an electroactive group or a strongly absorbing chromophore, indirect LCEC may represent an attractive detection approach. In particular, for applications currently carried out with refractive index detection, this approach would appear to offer distinct advantages. For the families of compounds examined in this study, the indirect LCEC technique provides a detectability that compares well with that typically seen for refractive index monitoring (7). In addition, the instrumentation required and its ease of operation are far simpler. Further characterization and ap-
Anal. Chem. 1886, 58,2340-2342
2340
plication of indirect LCEC are continuing in this laboratory. Registry No. Isopropyl alcohol, 67-63-0; 1-propyl alcohol, 71-23-8;1-butyl alcohol, 71-36-3;isobutyl alcohol, 78-83-1;propionic acid, 79-09-4; 1-butyric acid, 107-92-6;isovaleric acid, 503-74-2; 1-hexanoic acid, 142-62-1;p-hydroquinone, 123-31-9.
(7) Snyder, L. R.;Kirkland. J. J. Introduction to Modern Liquid C b r m t o g raphy, 2nd ed.; Why: New York, 1979; pp 140-145.
Jiannong Ye Richard P. Baldwin* Department of Chemistry University of Louisville Louisville, Kentucky 40292
LITERATURE CITED (1) Small, H.; Miller, T. E., Jr. Anal. Chem. 1982, 5 4 , 462. (2) Bobbltt, D. R.; Yeung, E. S.Anal. Chem. 1964, 56, 1577. (3) Bobbltf, D. R.; Yeung, E. S. Anal. Cbem. 1985, 57, 271. (4) Mho, S . 4 . ; Yeung, E. S.Anal. Chem. 1965, 57, 2253. (5) Banerjee, S.Anal. Chem. 1965, 57, 2590. (6) Mills, A.; GMdings, S. L. Anal. Chem. 1986, 58, 153.
K. Ravichandran Department of Chemsitry University of Georgia Athens, Georgia 30602
RECE~VED for review January 27,1986. Accepted May 1,1986.
AIDS FOR ANALYTICAL CHEMISTS Digestion of Bldogkal Materials for Mineral Analyses Uslng a Combination of Wet and Dry Ashlng A. D. Hill,* K. Y. Patterson, C. Veillon, a n d E. R. Morris
US.Dewartment of Agriculture.. Aaricultural Research Service, Beltsuille Human Nutrition Research Center, Beltsuilie, Maryland 20705 Periodically in our laboratory, it is necessary to analyze large numbers of diet samples and other biological materials from human studies for a wide variety of essential minerals. Atomic absorption spectrometry is accepted as the preferred method (1). Atomic absorption analysis requires the sample be in an aqueous form and this usually requires the destruction of the organic material. Some common procedures for digestion are low-temperature dry ashing in an oxygen plasma (2-6))high-temperature dry ashing (6-13))and low-temperature wet ashing in an acid solution (13-19). None of these methods is without problems. The equipment for low-temperature ashing in an oxygen plasma is expensive. Most ashers can handle only a few small samples a t one time. Low-temperature ashing can be very time-consuming when high concentrations of salts are present. Some minerals have been reported to be lost during hightemperature ashing through either volatilization or adsorption onto the walls of the container (20-22). Contamination from the ashing vessel has also been reported (23). Wet ashing with perchloric acid is widely used but potentially explosive conditions are needed to totally digest the lipid portion of the sample. Large volumes of acids are sometimes needed and constant operator attention is required. Sulfuric acid, which is used extensively in wet ashing procedures, has been shown to interfere in atomic absorption analysis (24) particularly with calcium. The procedure described in this paper utilizes dry ashing a t 375 "C in a muffle furnace for 24-48 h. This temperature will char the sample and burn off the major portion of the organic matrix including the lipid material without causing loss of the seven biologically important minerals investigated in this study. The ash is suspended in small amounts of deionized water and nitric acid. The samples in borosilicate tubes are then placed in a heating block and hydrogen peroxide is added to complete the digestion. Very little operator time is required. Only small amounts of reagents are used lowering the chance
Table I. Recovery of Added Metals
amt recovered," amt added, Kg
cu
1.0
Fe
6.5 5.4
4.0 2.0 4.0
4.01
2.8
2.07 4.07
7.2
8.0
8.52
10.0 20.0
9.80 19.5 40.3 4.96
40.0
5.0
Zn
10.0 20.0
35
Ca
70
75
M€!
re1 std dev, %
0.99 2.03
2.0
Mn
bg
150
10.2
19.9 36.7 70.8 81 155
2.0 2.8 9.4 4.4 2.1
6.5 2.8
%
recovery 99
102 100 104 102
107 98
98 101
99 102 99
1.6 12.7 6.9
105 101
8.4
108
9.3
103
Amount recovered is the averaze of tridicate determinations. ~
of contamination and resulting in low blanks and allowing for more accurate analysis a t low concentrations in samples. EXPERIMENTAL SECTION Procedure. Homogenized, dried samples of about 1 g are weighed into 20 X 150 mm borosilicate test tubes. The tubes are placed in 1000-mLglass beakers, covered with a watch glass, and placed in a muffle furnace. Furnace temperature is increased 50 deg/h to 375 "C and held at that temperature for 48 h. After cooling, samples are removed from the furnace and 0.20 mL of deionized water and 0.20 mL of Ultrex nitric acid (Baker, Phillipsburg, NJ) are added to each. Tubes are placed in heating blocks (ThermolyneDri-Bath,Dubuque, IA) and the temperature is raised to 90 "C.Hydrogen peroxide (50%) is added in 0.1-mL aliquots at 10-15 min intervals until all black carbon particles are digested. Samples are allowed to evaporate to dryness and
This article not subJect to US. Copyright. Published 1966 by the Amerlcan Chemlcal Society
