Analytical application of the room and low temperature (77 K

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ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978

LITERATURE CITED (1) H. Budzikiewicz, C. Djerassi, and D. H. Williams, "Mass Spectrometry

of Organic Compounds", Holden-Day San Francisco, Calif., 1967, p 55. (2) A. J. v. Ferrer-Correia,K. R. Jennings and D. K. Sen Sharma, Org. Mass Spectrom., 11, 867 (1976). (3) F. Borchers, K. Levsen, H. Schwarz, C. Wesdemiotis, and H. U. Winkier, J . Am. Chem. Soc.. 99, 6359 (1977) and references herein. (4) H. D. Beckey "Principles of Fieid Ionization and Field Desorption Mass Spectrometry", Pergamon, Oxford, 1977. (5) J. H. Beynon. R. G.Cooks, J. W. Amy, W. E. Baitinger, and T. Y. Ridley, Anal. Chem., 45, 1023A (1973). (6) E. Stenhagen, S. Abrahamsson, and F. W. Mclafferty, "Registry of Mass Spectral Data", Wiley, New York, N.Y., 1974. (7) T. L. Kruger, J. F. Litton, and R. G. Cooks, Anal. Lett., 9, 533 (1976).

(8) H. D. Beckey and P. Schulze. 2. Naturforsch. A , 20, 1355 (1965). (9) F. W. Rollgen and H. J. Heinen, Int. J . Mass Spectrom. Ion Phys., 17, 92 (1975). (10) R. P. Morgan and P. J. Derick. Org. Mass Spectrum., I O , 563 (1975). (11) A. M. Fallick, P. Tecon, and T. Gciumann, Org. Mass Spectrom., 11, 409 (1976). (12) S.A. Rang, A.-M. A. Muurisepp,M.M. Liitma, and 0. G. Eisen, Org. Mass Spectrom., 13, 181 (1978).

RECEIVED for review May 19, 1978. Accepted July 5, 1978. Financial support by the Fonds der Chemischen Industrie and the Deutsche Forschungsgemeinschaft is gratefully acknowledged.

Analytical Application of the Room and Low Temperature (77 K) Phosphorescent Properties of Some 1,8-Naphthyridine Derivatives Clausius G. de Lima" and Ezer M. de M. Nicola Departmento de QGmica, Universidade de Braska, Bradia D. F., Brazil

Milch et al. ( 8 ) investigated the possibility of the use of ultraviolet spectrophotometric data for the characterization and determination of NA and its ethyl ester and naphthyridinic acid and its ethyl ester. As solvents HCl 0.01 M, NaOH 0.1 M, ethanol, and chloroform were evaluated. However, Milch et al. (8) concluded that these four naphthyridine derivatives cannot be determined in the presence of each other using this instrumental method. Dondi and Di Marco (5)determined NA after reaction with 2-naphthol; the compound produced has an absorption maximum a t 351 nm. Room temperature solution fluorimetry has been used by McChesney et al. (Z),Browning and Pratt ( 3 ) ,Milch et al. (81, and Staroscik and Sulkowska (9). McChesney et al. ( 2 ) demonstrated that NA became strongly fluorescent at a pH range of 0-1, using dilute sulfuric acid (ca. 2.5%) as solvent medium, showing an emission a t 375 nm after excitation a t 330 nm. Browning and Pratt (3) employed the method used by McChesney et al. (2) using, however, a sulfuric acid concentration of 60%, reading the fluorescence emission a t 408 nm and exciting at 325 nm. Using this method, Browning During an investigation of new synthetic antimicrobial and Pratt (3) were able to determine as low as 100 ppb of NA drugs, Lesher et at. (I) reported, in 1962, that some derivatives in chicken liver and in chicken muscle homogenates. Milch of l,&naphthyridine were highly effective against gram et al. (8) examined the fluorescence characteristics of some negative pathogens, in particular, one known by the trivial name of nalidixic acid (l-ethyl-1,4-dihydro-7-methyl-4-oxo- l,&naphthyridine derivatives and observed that NA, its ethyl ester, and naphthyridinic acid exhibited strong fluorescence 1,8-naphthyridine-3-carboxilic acid). Due to this fact, nalidixic in acid medium (0.01 M HCl), while the naphthyridinic ethyl acid (compound I in Table I) is at present commercially ester showed the effect in alkaline medium (0.1 M NaOH). available (2) for medical use and has also found application In the acid condition, NA showed an excitation and emission as an effective aid in the control of chicken infection (3). maximum a t 313 and 360 nm, respectively (8).Staroscik and Spectrophotometrically, nalidixic acid (hereinafter called Sulkowska (9) determined NA fluorimetrically in urine after NA) has been analyzed in tablets (4-6), in human urine, serum, extraction with chloroform, with a limit of detection of 5 pg and feces (2) and in animal tissues (2, 3) by ultraviolet abmL-'. sorption spectrophotometry and by room temperature solution In the present work we examined the application of room spectrofluorimetry. The application of ultraviolet spectrotemperature phosphorescence for the determination of NA photometry was suggested or has been used by several workers and other similar compounds (Table I) in comparison with (4-8). Salim and Shupe ( 4 ) used chloroform as a solvent, the low temperature (77 K) phosphorescence technique. Room reading the absorption either at 258 or 332 nm. Da Silva and temperature phosphorescence (RTP) is a relatively new effect Nogueira (6) employed NaOH 0.1 M as solvent, reading the which was first reported by Roth (10) and later independently absorption a t 258 nm, while Zubenko and Shcherba (7) deby Schulman and Walling (11, 12). Usually the effect is termined NA spectrophotometrically in methanol or in 0.1 M NaOH as solvent, a t 258 and 324 or 332 nm, respectively. observed with many salts of polynuclear organic compounds,

The phosphorescence characteristics of some 1,8-naphthyrldlne derivatives both at room temperature (adsorbed on paper) or at low temperature (77 K ) in dilute alkaline solution have been investigated as a method of analysis. Attention was given principally to one of the derivatives-nalidixic acid-which is a potent antimicrobial drug produced on a commercial scale. For the room and low temperature phosphorescence study, a laboratory-assembled singlemonochromator spectrophosphorimeter was used in most of the experiments and a new paper chromatography accessory was tested, to be used directly in conjunction with the room temperature phosphorescence technique. Working curves, llmits of detection, lifetimes, and the effect of different parameters (such as the NaOH concentration, effect of irradiation, and effect of temperature in the sample compartment) were also determined.

0003-2700/78/0350-1658$01.00/0 1978 American Chemical Society


TABLE I 1,d-Naphthyridine Derivatives Studi ed. Compound 1 -Ethyl-l,L-dihydro7- met hy I- 4- ox o - 1,d napht hy rid in e-3carboxylic acid ( Nal i d i x I C a c i d)

Structure

L * CH3

NO

OOH

'

(1)

9%

1 -E 1hy I -1,4-d ihydr o -

4- oxo -1,8-nophthy r i d ine-3,7-d icar boxylic a c i d HOOC

e2H5 7-(Acelylamino)-lethyl-l,4-di hydro -4oxo-l,8-naphthy ridine-3-carboxylic acid

(111)

1 X t hyl-l,4 -d ihyd ro 7 h y d r o x y - 4 -oxo -1,8napht hyr I di n e - 3HO carboxylic acid

62% 7-Melhyl-l,8 n aph t hy r i d I n-4-01

in a solid substrate such as paper, silica gel, alumina, etc. in a dry atmosphere. RTP was also observed, with some molecules, using sodium acetate as substrate, by von Wandruszka and Hurtubise (13-15). In this case the method is relatively insensitive t o moisture. Seybold and White (16) demonstrated that the presence of sodium iodide enhances the RTP signal (a heavy-atom effect) of 2-naphthalene sulfonate adsorbed on filter paper. Recently, Vo Dinh e t al. (17) observed RTP with some non-ionic polycyclic aromatic hydrocarbons, by the use of external heavy-atom perturbers, such as sodium iodide and silver nitrate. Jakovljevic (18)showed that lead and thallium salts can also be used as external heavy atoms as a new technique t o enhance the RTP signal. RTP has been suggested by Schulman and Walling in their first papers (11,121 as a possible analytical technique and has been applied t o analytical problems by Winefordner and co-workers (17, 19-22), von Wandruszka and Hurtubise (13-15), and Jakovljevic (18) principally with biologicallyimportant compounds.

EXPERIMENTAL Apparatus. Luminescence Measurements. For the majority of the work, a laboratory-assembled single-monochromator spectrophosphorimeter was used. This instrument consisted of an ultraviolet illuminator with a mercury lamp source (85 watts, General Electric, Model H3FG) driven by a power supply (Model 14-031, Coleman Instruments, Oakbrook, Ill.). The radiation was focused through a quartz window (25 mm in diameter) using the lens system provided in the illuminator, with an angle of incidence of 90°, into an air and light-tight sample compartment (with dimensions of 150 X 170 X 150 mm) where a rotating can phosphoroscope was installed. In the preliminary experiments, the selection of the excitation wavelength was done by the use of absorption filters. Later the excitation was done without the use of filters, when better limits of detection were attained. To reduce the scattered light from the excitation source in the sample compartment, besides using a light trap and painting all surfaces

1659

with a dull-black nonreflecting finish, black felt baffles were used, aligned in such a way that they lightly touched the revolving can. The rotating can phosphoroscope constructed was 45 mm in the height and 30 mm in diameter. The can was driven by a small, variable speed, dc motor (Malitron, S. Paulo, Brazil, maximum speed 10000 rpm) controlled by a dc power supply (0-12 V, Ceteisa, S. Paulo, Brazil). The compartment was flushed continuously during the experiments with nitrogen either from a cylinder or from a Dewar with liquid nitrogen, which was evaporated by the use of an immersed Nichrome wire coil, heated by passing a low ac current through it. To reduce possible moisture present in the nitrogen, the gas flowed through a glass tube (200 mm in length and 20 mm in diameter) packed with silica-gel. During the temperature effect study, the nitrogen was heated by passing through a coiled 4.5-m copper tube (6.3 mm in 0.d.) which was heated by using a heating tape, as described by Wellons (23). The temperature variation was continuously followed by using an electronic thermometer (Yellow Springs Instruments Co., Yellow Springs, Ohio), the probe cable being introduced through a hole in the paper holder in such a manner that the probe was located near the paper substrate. The luminescence produced was focused with a silica lens (28 mm in diameter and 150 mm in focal length) onto the entrance slit of a Czerny-Turner mounting monochromator with 0.45-meter, f / 8 , reciprocal linear dispersion of 3.5 nm mm-', (McKee-Pedersen Instruments, Danville, Calif., model MP-1018). A half band-width of ca. 12 nm was used in most of the experiments. The monochromator wavelengths were calibrated by using a mercury pen-light and according to the instrument manual (29). 2-Naphthol and Eosin Y were also used as standards, for the RTP preliminary experiments, and coronene in n-heptane as standard for the low temperature measurements as the spectral characteristics of these compounds are known in the matrix employed (11, 12, 24). A front window photomultiplier tube (RCA 1P28, spectral response S-5) was attached a t the exit slit of the monochromator and operated at 900 V, by using a Keithley Model 146 power supply (Keithley Instruments, Cleveland, Ohio). The signal from the photomultiplier, in the preliminary experiments, was fed to a photometer (Atomic Laboratories, Chicago, Ill., model 86407). Later, a single channel photon counting system (Model INS-11, Elscint LTD, Haifa, Israel), was used as an amplifier. The amplified signal was recorded using a strip-chart recorder (Model SR, Sargent-Welch, Skokie, Ill.). A paper holder, similar to one used by Paynter et al. (19)and a paper chromatography accessory were also constructed. For the low temperature (77 K) measurements, a commercial silica Dewar flask was employed, together with sample tubes, constructed from silica tubing (Vitreosil, Jencons, Hemel-Hempstead, Hertz, England), with 200 mm in length, 3-mm i.d. and ca. 1-mm wall thickness, sealed at one end, and liquid nitrogen was used as a coolant. To minimize frosting in the silica Dewar exterior walls, a coil of Nichrome wire was used as described elsewhere ( 2 4 ) . Lifetime measurements were done by recording the decay of the photomultiplier signal using a storage oscilloscope (Tektronix, model R5103N, Beaverton, Ore.) as described by Parker (25). U'ith the kind permission of Prof. L. Campos de Souza (Department of Chemistry, Federal University of Pernambuco, Brazil), a few spectra were run in a double monochromator spectrofluorimeter (American Instrument Co., Silver Spring, Md.) using the instrument phosphoroscope accessory and a laboratory-constructed sample holder. Absorption Spectra. A Zeiss RPQ 20 A spectrophotometer and 5-mm silica cells (10-mm optical path) were employed to record some spectra in the ultraviolet and visible range. Paper Chromatography and Multiple Sample Accessory. A laboratory-constructed paper chromatography accessory which can also be employed as a multiple sample (6-8) accessory was tested in a series of preliminary experiments. Recently Vo Dinh et al. (22) have described the design and use of an automated phosphorimeter instrument which can deal with several samples, having however, a different design. Figure 1 shows our design of the accessory. A driven rubber band runs around the two brass pins (b and c in Figure l),which have been machine-grooved in order to give better traction. The band is driven manually through

1660


Figure 1. Design of the paper chromatography accessory. (a) Helipot dial; (b) brass traction pin; (c) lower brass pin; (d) rotating can phosphoroscope; (e)sample compartment wall. (All measurements in mm)

two paper substrates were those with the lowest phosphorescence located far from the analytes' phosblank and with the A,, phorescence emissions. The Whatman chromatography paper and the Whatman filter paper used have weak phosphorescence blank emissions, located at 504 and 520 nm, respectively. When necessary, phosphorescence signals were corrected for blank phosphorescence. A 10-pL syringe (Hamilton, Whittier, Calif.) was used for delivering the sample solution on the paper substrate. R T P Procedure. The usual experimental procedure consisted of spotting 20 p L of the sample solution on a 20 X 30 mm piece of paper, by using a microliter syringe. The use of less than 20 pL produced a weaker signal, which we attribute to the illumination system employed. Primary drying was accomplished by warm air (50 "C) using a warm air blower, drying for 2 min, and then placing the paper in a heated vacuum oven for at least 1 h, at 40 k 2 "C. After that, the pieces of paper were stored in a desiccator with P,On, until used. Measurements were taken only after 15 min in the sample compartment with nitrogen flushing, which was then maintained during the whole experiment. A quicker and safer method of drying, by using infrared lamps has been used by Wellons et al. (20), which undoubtedly could be used in our case. Low Temperature Phosphorescence Procedure. Ca. 200 pL of the analyk solution was introduced in the silica sample tube, which was then plunged into the liquid nitrogen contained in the Dewar flask, and the flask was then placed in the sample compartment for the determination of the low temperature data. Cracked glasses were formed when the NaOH concentration was higher than 4M. At lower NaOH concentrations, cracked polycrystalline frozen solutions were obtained.

a Helipot dial (a) installed in the upper brass pin which at the same time serves to locate the spot. A previously dried chromatography paper strip or the strip with sample spots (250 mm in length and 14 mm in width) is attached to the band using a small piece of Celotape, in such a manner that the chromatography starting point (or the first sample) corresponds to the zero position of the Helipot dial. Because of the elastic character of the band, there is some small variation in the determination of the position. The use of a flexible metal band and appropriate gears will correct this inconvenience. Also, it is possible that by using a point-like source and a better lens system in place of the system employed (which produced a broad illumination), a larger number of sample spots or chromatography spots with closer R, values, could be analyzed. For testing the accessory, some paper chromatography experiments were made using Whatman No. 1 chromatography paper, ascending solvent flow technique, and multiple runs (3 runs). The application of the compound solutions and the development were made in conditions of semidarkness and without heat. For the detection of the spots and determination of the R, values, ultraviolet light (using a 366-nm filter) was used, after spraying with 0.1 M NaOH. The paper strip was then dried as described in the RTP Procedure. Drying Apparatus. As demonstrated by previous workers (11, 12, 16-22), drying of the sample is necessary for the observation of RTP. Primary drying was done by using an air blower (Span-Jet, SBo Paulo, Brazil) and final drying by using a vacuum oven (National Appliance Co., Model 5850, Portland, Ore.). Materials and Reagents. Samples of pure l&naphthyridine derivatives used were kindly provided by Dr. F. C. Nachod, Sterling-Winthrop Research Institute, Rensselaer, N.Y. Analytical grade reagents, sodium hydroxide and sodium iodide (BDH Chemicals Ltd., Poole England); n-butanol, (Carlo Erba do Brasil S.A. Silo Paulo, Brazil); and concentrated ammonium hydroxide (Ecibra, SBo Paulo, Brazil); and distilled water were used without further purification. Substrate. According to previous works (11-15), it is possible to observe RTP on a variety of substrates, such as silica, paper, sodium acetate, asbestos, and glass fibers. We chose paper as the substrate, although with some of the other substrates similar signals could be obtained; however, this has not been examined in the present work. To choose the best paper substrate, we examined the phosphorescence blank of the papers available, as suggested by Paynter et al. (19). In most of our work, Whatman chromatography paper (No. 1) was used as substrate. In some experiments Whatman filter paper (No. 40) was also used. These

RESULTS AND DISCUSSION Influence of the N a O H Concentration. Previous studies of room temperature phosphorescence (RTP) indicated that several ionic molecules show intense phosphorescence signals when dissolved in aqueous solvents containing a strong acid or base, most frequently 1 M NaOH (11,17,194'2). Recently, von U'andruszka and Hurtubise (13-15) demonstrated that p-aminobenzoic acid and several other molecules were found to phosphoresce at room temperature when adsorbed on sodium acetate. Also, Schulman and Parker (26) showed that a mixture constituted of 0.1 M NaOH/5.56 M HzO in methanol could be used as a solvent for the observation of the RTP signal of sodium 4-biphenylcarboxylate adsorbed in Whatman No. 1 filter paper. In our work we examined the influence of the NaOH concentration on the phosphorescence signal of the compounds studied. Figures 2A, 2B, and 2C show the results using chromatography paper as substrate. With KA (Figure 2A) using solutions with an analyte concentration of 1 X M and 5 X M (which give an amount per spot of 4.64 and 2.32 pg, respectively, after adsorption and drying on the paper) the best signals were obtained a t 0.15 M NaOH. Using a lower solution concentration (2.5 X M of NA, which corresponds to 1.16 pg per spot) 0.1 M NaOH is slightly better. With a solution concentration of 1 x M of NA (0.46 pg per spot), there is no significant variation between 0.05 M and 0.25 M NaOH. In consequence, during most of our experiments 0.15 M NaOH was used as a solvent. Compounds I1 and I11 (Figure 2B) have nearly the same characteristics, showing better signal a t 2.5 X M NaOH. Compound IV (Figure 2C) presents a better signal at 0.75 M NaOH by using a solution with a concentration of 1 X M (4.68 pg per spot). Only a slight variation is observed between 0.25 M and 1.0 M NaOH when using a lower solution M (0.46 pg per spot). With compound concentration, 1 X V, 1.0 M NaOH appears to be an optimum concentration, in the range of NaOH concentration examined (0.01-1.0 M), either using a compound solution concentration of 1 x M or 1 X lo-*M (which correspond to 3.2 and 0.32 pg per spot) although the curve with 3.2 pg per spot suggests that NaOH concentration higher than 1.0 M would be better. These

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Flgure 2. Effect of NaOH concentration on the RTP emission, with different compound concentration (in the solution, before adsorption),using Whatman No. 1 chromatography paper as substrate. The phosphorescence signal intensities are adjusted between curves arbitrarily: (A) NA (at 472 nm) -0-, 1.0 X M; -A-, 5 X M; -0-, 2.5 X M; and -A-,1 X M. (B) Compound I1 -0-, 1 X M; -0-, 1 X M; and compound 111: -A-, 1 X M; and -A-,1 X M. (C) Compound IV: -0-, 1 X M; and -0-, 1 X M; and compound M V: -A-, 1 X 10-3 M; and -A-, 1 X

compounds, IV and V are the compounds with hydroxyl substituents. These differences, observed when KaOH concentration is changed, suggest that this effect could be useful during an analysis of a mixture, as at certain NaOH concentration the room temperature signal of one compound can be enhanced when the other is quenched. However, the utilitv of the NaOH concentration effect is deDendent on the amount of compound present; as can be seen from the curves (Figures 2A, 2B, and 2C) with a lower compound amount (with M), the NaOH effect is solution concentrations of 1 X negligible. We also tested this effect using Whatman No. 40 filter paper as substrate. The curves obtained after plotting signal intensity vs. NaOH concentration were similar in shape although the best signals were obtained in most cases a t slightly different concentrations of NaOH with the exception of NA where the best signals were found a t the same NaOH concentrations. The other compounds were tested by using a solution concentration of 1 x M, which corresponds to an amount per spot of 5.24 pg (II), 5.50 pg (III), 4.68 pg (IV), and 3.20 pg (V). The best signals were obtained a t NaOH solution concentration as follows: compound 11, 5 x M; compound 111, 2 X M; compound IV, 0.40 M; and compound V, 0.75 M. The effect of NaOH concentration shows that the R T P signal intensity appears to be somewhat dependent on the paper substrate used and much more dependent on the NaOH concentration, the analyte structure and concentration, and moisture. It is possible that the differences observed between papers are somewhat related with the type of physical and chemical treatment, which the paper has been subjected to during manufacture. For each analyte, there seems to exist, a t room temperature, an optimum range of NaOH concentration where the interaction between the analyte and the NaOH-paper substrate provides the best condition of adsorption and consequent rigidity, with appearance of the strongest R T P signal. No effect on the signal was observed on the low temperature phosphorescence spectra of all compounds examined between 1 x lo-* M and 12 M NaOH. Effect of Drying Conditions. Different methods of drying were tested by Wellons et al. (20) using other compounds as analytes, when these workers observed that hot air blowers and ovens proved to be too destructive to the paper or sample. Some attempts were made by us to accelerate the drying steps using a horizontal Pyrex tube, where the paper pieces were hooked in a wire support and where warm air was blown. In such cases. the phosphorescence signal intensity fluctuates,

using NA as analyte (4.64 pg per spot), given a relative standard deviation of 49% in place of ca. 6.0%, usually obtained when the drying procedure described in the Experimental section was employed. As this result suggested a thermolability of the compound examined (NA), this procedure was abandoned and a careful drying was exercised, as described in the ExDerimental section. The effect caused by the drying conditions was then examined. In some experiments, using NA as analyte, drying was performed a t room temperature, either a t the primary step (air blower, 15 min) or a t the final step (vacuum oven, ca. 3 h). Under such conditions, the maximum of the RTP emission was located at -438 nm, which was a smaller value compared with that obtained (472 i 2 nm) when warm air (ca. 50' C) and vacuum oven (at 40 f 2' C) drying procedures were used. With the other compounds, no differences were observed. However, as soon as the paper, dried a t room temperature, with NA adsorbed in it, is introduced in the spectrophosphorimeter, a continued red shift on the wavelength maximum (from the original value of 438 nm) was observed, until stabilized, after 15-20 min at 472 nm. At the same time the signal intensity decreased as the A,, shifted to a higher wavelength. After stabilization the spectral characteristics are similar to those when the paper is dried using the adopted procedure. From the practical point of view, the drying procedure using room temperature either in the primary step (blower) or in the final step (vacuum oven) is time consuming; therefore, the method of drying using minimum heat as described in the Experimental section was employed with all compounds. Although with this latter procedure the signal intensity of NA is reduced by nearly 30%, the wavelength is at the most stable position. Effect of Irradiation. To investigate whether the shift in the wavelength and decrease in the R T P signal of NA can be produced by irradiation, experiments were made (using samples dried a t room temperature) where the infrared component of the excitation source was eliminated by the use of a continuous flow, cool water filter (a cylinder quartz cell with 25-mm 0.d. and 100-mm length) between the excitation source and the quartz window of the sample compartment. Figure 3 shows the result obtained with NA which confirmed the behavior previously observed, and demonstrated that the shift in wavelength and decrease in signal were due, in this case, to a photochemical modification in the molecule adsorbed in the paper substrate. Together with the change in the

1662

ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978 I

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Figure 3. Effect of irradiation on the RTP spectral characteristics of NA: 4-,variation of wavelength vs. time; -A-,variation of signal intensity vs. time no1

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a

ul

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Figure 4. Effect of irradiation on the RTP signal intensity of compound V: 4-;compound IV: -A-:compound 11: -0-: and compound 111: -A-

phosphorescence maximum, a blue fluorescence appears. Figure 4 shows the behavior observed with the other compounds. Compounds I11 and V are slightly affected by irradiation and there is no sensitive effect of irradiation observed with the compound 11. Compound IV is, however, very sensitive to irradiation, suffering an irreversible suppression of ca. 40% after 20 min of irradiation, although there is no shift in the wavelength maximum. To compensate for this effect, all analytical data of the compound IV were collected after 1 minute of irradiation. Effect of Temperature during Measurements. As described in the Experimental section, the sample compartment was gradually heated for the study of the effect of and on the signal intensity. Figure temperature on the A,, 5 shows the behavior of derivatives of 1,8-naphthyridines examined. To reduce a possible concomitant effect of the irradiation to a minimum during the experiments, with all compounds the introduction of the excitation light was done only at the instant of the measurements. With all compounds, the signal intensity decreased as the temperature increased. With NA, the suppression is irreversible. With the other compounds, the signal intensity returns to the original as soon as the temperature is lowered to room temperature. Compound 111 has shown the highest suppression on the signal intensity (ca. 80%) and the lowest suppressions (ca. 30%) were observed with NA and compound V. Probably the reversible suppressions are due to a radiationless conversion of the triplet state (25). In the case of NA, it is possible that an irreversible thermal reaction is occurring. Although heating the sample compartment seems beneficial to a large number of compounds tested by Wellons et al. (20)

h,nm

Figure 6. Room temperature phosphorescence spectra of some of the compounds examined. (Roman numerals refer to compound numbers). All spectra are uncorrected and relative intensities were adjusted arbitrarily and Vo-Dinh et al. (21), in the case of the compounds examined by us, it seems better to work at room temperature. Spectral and Analytical data. Table I1 and Figures 6, 7 , 8, and 9 show the spectral and analytical characteristics of the compounds examined using room (at the optimum NaOH concentration) and low temperature (at 0.10 M NaOH) phosphorescence techniques. The limits of detection a t low temperature are smaller if compared with the limits obtained a t room temperature although the comparison is not exact, as different sample handling systems, with different geometries, were employed. Lifetimes were longer a t low temperature in comparison with the lifetimes observed at room temperature. Absolute limits of detection are also reported in Table 11. Typical analytical growth curves of the compounds are plotted logarithmically in Figures 7 and 8. Concentrations in molarity refer to the analytes' concentrations in the solutions before adsorption on the paper. Relative standard deviations (in percent) of the data presented showed a range from 35% at low concentrations to ca. 6% a t


0.17-

Figure 7. Room temperature phosphorescence analytical calibration curves of the compounds examined, at the best NaOH concentrations. Compound V -0-; compound IV, -A-;NA, -H-; compound 11, -0-: and compound 111, -A3. P

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CONCENTRAT ION ( M o l a r ) Figure 8. Low temperature (77 K) phosphorescence analytical calibration curves of the compounds examined in 0.1 M NaOH solution. NA, -0-: compound 11, -0-: compound I V , -A-;compound V, -W-: and compound 111, -A-

1,””

Flgure 9, Comparison of room temperature emission and excitation spectra of nalldlxlc acid adsorbed on Whatman No. 1 chromatography paper as substrate: (A) using room temperature drying procedure; (B) after heat and lrradlatlon treatment; (C) same as B, after chromatography, wlth a R, = 0.0 (modifled NA). All spectra uncorrected and relatlve intensitles were adjusted arbltrarlly

the higher solution concentration. With the exception of compound V, the other compounds differ only by the substituent in position 7 (Table I). By comparing the R T P intensity of NA and compounds 11,111, and IV, it can be seen that substitution of the hydroxyl in position 7 of compound IV by a methyl group (NA) produced a compound with a weaker signal (Figure 7 ) ;the substitution by a carboxyl (compound 11) weakens the signal intensity even more. The 7-acetylamino derivative (compound 111) showed the weakest signal, either a t room temperature or at low temperature. The introduction of an hydroxyl substituent

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* 1663

1664


(compounds IV and V) seems to provide the compound with the most rigid condition and, consequently, with the strongest signal at room temperature. At low temperature (Figure 8), the most intense compounds are NA and compound 11, followed by compounds I\' and V. Compound I11 shows an abrupt inflection in the calibration M, curve (Figure 8) a t concentrations higher than 1 X suggesting some kind of interaction between the analyte molecules a t these high concentrations, probably before or during the freezing, quenching the phosphorescence signal. As observed by Seybold and White (16),Jakovljevic ( I B ) , and Vo Dinh et al. (21),an external heavy atom effect is also observed in RTP. In our case, using sodium iodide (1 M), compounds IV and V, which are the compounds with the 7-hydroxyl substituent, showed an enhancement on the order of 7 and 2 times in the phosphorescence signal intensity, respectively (Table 11). With the other compounds, a suppression in the signal appears; a similar effect has been observed in the phosphorescence emission of 6-methylmercaptopurine at room temperature (21). In most of the cases, the emission maxima occur nearly at the same place either at room temperature or at low temperature, with the exception of NA and compound IV. The emission maximum of NA at low temperature (438 nm) is similar to that observed a t room temperature when neither ultraviolet irradiation or heat had been applied to the analyte adsorbed in the paper substrate. In the case of compound IV no differences result if the compound is carefully dried at room temperature and in absence of light. Attempts were made to understand the behavior of NA. The increase of the NaOH concentration from 1 X lo-' M to 1 2 M did not produce any effect in the absorption spectrum or in the low temperature phosphorescence spectrum. Using an instrument capable of determining the excitation and the emission room temperature spectra, we examined the spectral modifications caused by heat and irradiation using NA as analyte and Whatman No. 1 chromatography paper as substrate. Figure 9A shows the excitation and emission spectra of NA before heat and irradiation treatment where we can observe an emission maximum a t 438 nm and excitation maxima a t 254 nm and 336 nm. After treatment (Figure 9B), there is a red shift of 30 nm in the emission maximum (468 nm) while in the excitation spectrum, the maximum a t 254 nm disappears and the maxima a t 265 nm and 388 nm appear, suggesting that the NA molecule has been modified. Recently, Detzer and Huber (27) examined the photochemical and thermal behavior of NA. When NA is photolized (in oxygen-free 0.1 M NaOH solution) a t 80 "C a decarboxylation product, carbon dioxide, ethylamine, and a new diketone appear. Thermolysis (in melt and in paraffin oil solution a t 390 "C) produces, besides the decarboxylation product, a dimer. Although the reaction conditions are different, we tested the possibility of decarboxylation in our conditions; however, no evolution of carbon dioxide was detected or formation of carbonate on the paper substrate (except the carbonate present, by absorption of carbon dioxide from the air), using barium hydroxide as reagent. Some few attempts were also made to isolate the modified compound, using paper chromatography of a heated and irradiated spot at the starting point (with 1 X M of NA) and using N H 4 0 H (concentrated):n-butanol 12/88 (v/v) as eluent. These attempts showed that a small amount of a compound with a R, value of 0.67 was present, which was later identified as the nonmodified nalidixic acid either by the similar R, value or by the R T P spectrum. The modified NA was strongly bound to the paper substrate and did not move from the application point even after other eluents were tested (such as benzene,

ethyl acetate, dimethyl sulfoxide, NaOH 0.1 M, CHC13 preceded by HC1 0.1 M, etc.). This strong binding process is not, however, a condition for the observation of RTP as the nonmodified NA, which migrates, shows phosphorescence emission. The R T P spectrum of the modified NA ( R f = 0.0) after chromatography is shown in Figure 9C. The small excitation peak at 336 nm, which was present in the spectrum of Figure 9B, disappeared and excitation maxima can be seen a t 268 and 388 nm and an emission maximum at 470 nm. Probably the excitation peak at 336 nm present in Figure 9B belonged t o the nonmodified NA molecule which was separated by the chromatographic process. Effect of Interferences. Nalidixic acid was used as a model compound to study the effect of interferences. No effect was observed with some of the cations and anions usually present in urine (28) such as potassium, calcium, magnesium, chloride, oxalate, urate, and phosphate, in the molar ratios of 1:1,1O:l and 1OO:l (interference:NA, using a concentration of NA of 5 x M). Longer drying time in the vacuum oven was, however, required. Human urine was also treated as a solvent in place of water and no interference was observed. Because of its similarity in the spectral characteristics of the l&naphthyridines examined, obviously the presence of each other will cause a direct spectral interference or a severe overlap (see Figure 6). In some cases, the differences observed in relation to the experimental parameters (such as NaOH concentration, ultraviolet irradiation behavior, temperature, presence of heavy atom) eventually may help when it is necessary to deal with real samples. P a p e r Chromatography Accessory Results. Previous separation of the compounds is evidently the best answer when direct spectral interference is present. Using the solvent system N H 4 0 H (c0ncentrated):n-butanol (12/88 v/v) the following R f values were obtained: NA, 0.67; compound 11, 0.026; compound III,0.14; compound IV, 0.023; compound V, 0.95. Other solvent systems were tested to improve the separation between compounds I1 and IV without success. Because of the close proximity of the R, values of compounds I1 and IV, test of the paper chromatography accessory was done in the absence of one of them. Because of the excitation optical system employed, however, it was necessary to use strong concentrations of the analytes ( 5 x 10-4-1 X M) and to spot the samples solution in a large zone (4 x 4 mm) of the paper strip a t the starting point. Together with the R f value, the determination of the R T P spectra using the paper chromatography accessory confirmed the identification of the compounds, showing the utility of the accessory. The R T P signal intensities were different if compared with the intensities obtained by spotting the sample solution on a chromatography paper strip or a piece of paper. After chromatography, NA and compound I11 showed more intense signals (ca. 3 and ca. 2 times, respectively) and compounds 11, IV, and V were less intense (0.7, 0.2, and 0.1, respectively). In the case of compound IV, it seems reasonable to believe that the reduction is due t o the action of the ultraviolet irradiation used during the determination of the R, value in the development of the chromatography. In the other cases, the effect of the molecules being previously adsorbed with a different solvent system (NH,OH/n-butanol) may explain the differences observed in the signal intensities. Further experiments are undoubtedly necessary to explain such differences. With some improvements, such as the use of a better excitation (a point-like source) and a driven band with gears, the chromatography accessory may provide a simple tool for collecting R T P data directly from a paper chromatogram or for the determination of a number of samples under the same


sample compartment conditions.

ACKNOWLEDGMENT The authors express their thanks to D. Tucker for doing some of the preliminary R T P experiments when a t the Department of Chemistry, Imperial College, London, F. C. Nachod for the donation of the samples of pure naphthyridines studied, K. Skeff Net0 for the loan of the photomultiplier power supply and the channel photon counting instrument, and L. Styer Caldas for linguistic advice.

LITERATURE CITED G. Y. Lesher, E. J. Froelich, M. D. Gruett, and J. H. Bailey, J. Med. /?arm. Chem., 5, 1063 (1962). E. W. McChesney, E. J. Froelich, G. Y. Lesher, A. V. R. Crain, and D. Rossi, Toxicol. Appl. fharmacol., 6, 292 (1964). R. S. Browning and E. L. Pratt, J . Assoc. Off. Anal. Chem., 53,464 (1970). E. F. Salim and I. S. Shupe. J . Pharm. Sci., 55, 1289 (1966). G.Dondi and M. Di Marco. Boll. Chim. Farm., 105,491 (1966). M. J. J. V. da Silva and M. T. C. Nogueira, Rev. f o r t . Farm., 15,290 (1966). V. G. Zubenko and I. A. Shcherba, Farm. Zh. (Kiev), 30,28 (1975); Chem. Abstr.. 83.330624 (1975). Gy. Milch, I. Aninger, and K. Kaloy, h o c . Conf. Appl. fhys. Chem. 2nd, I, 397 (1971). R. Staroscik and J. Sulkowska, Acta Pol. Pharm., 30,499 (1973); Chem. Abstr., 81, 1524372 (1975).

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(10) M. Roth. J . Chromatogr., 30,276 (1967). (11) E. M. Schulman and C. Walling, Science, 178, 53 (1972). (12) E. M. Schulman and C. Walling, J . Phys. Chem., 77, 902 (1973). (13) R. M. A. Von Wandruszka and R. J. Hurtubise, Anal. Chem., 48, 1784 (1976). (14) R. M. A. Von Wandruszka and R. J. Hurtubise, Anal. Chem., 49, 2164 (1977). (15) R. M. A. Von Wandruszka and R. J. Hurtubise, Anal. Chim. Acta., 93, 331 (1977). (16) P. G. Seybold and W. White, Anal. Chem., 47, 1199 (1975). (17)T. Vo Dinh, E. Lue Yen and J. D. Winefordner, Talanta, 24, 146 (1977). (18) I. M. Jakovljevic, Anal. Chem., 49, 2048 (1977). (19) R. A. Paynter, S.L. Wellons, and J. D. Winefordner, Anal. Chem., 46, 736 (1974). (20) S.L. Wellons, R. A. Paynter, and J. D. Winefordner, Spectrochlm. Acta, Part A, 30,2133 (1974). (21)T. Vo Dinh. E. Lue Yen, and J. D. Winefordner, Anal. Chem., 46, 1186 (1976). (22)T. Vo Dinh, G. L. Walden, and J. D. Winefordner, Anal. Chem., 49, 1126 (1977). (23) S. Wellons, W.D. Dissertation, University of Florida. Gainesvilk, Fh., (1974). (24) G.F. Kirkbright and C. G. de Lima, Ana/yst(London), 99,338 (1974). (25) C. A. Parker, "Photoluminescence of Solutions," Elsevier Publishing Co., Amsterdam, 1968. (26) E. M. Schulrnan and R. T. Parker, J . Phys. Chem., 81, 1932 (1977). (27) N. Detzer and B. Huber, Tetrahedron, 31, 1937 (1975). (28) R. J. Henry "Clinical Chemistry Principles and Technics", Hoeber-Harper International Reprint, New York, N.Y., 1964 (29) "MP-Systems 1000, Operations and Applications", McKee-Pedersen Instruments, 3rd ed., Danville, Calif., 1968,1-16,

RECEIVED for review May 8, 1978. Accepted July 14, 1978.

Repetitive Determinations of Amylase, Maltose, Sucrose, and Lactose by Sample Injection in Closed Flow-Through Systems D. P. Nikolelis' and Horacio A. Mottola" Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74074

Conditions have been developed for the repetitive determination of amylase and some disaccharides by coupling enzyme-catalyzed reactions yielding glucose as a product with the glucose oxidase catalyzed oxidation of this sugar. Determinations have been performed by sample injection into a continuously circulated reagent mixture and monitoring of oxygen depletion with a three-electrode amperometrlc system. Maltose, sucrose, and lactose in the range of 50 to 500 mg/100 mL, 10 to 250 mg/100 mL, and 25 to 250 mg/100 mL, respectively, and amylase in the range of 50 to 500 units/100 mL have been determined with relative errors and relative standard deviations (population) of about 2 % The maximum determination rate is 240 injections/h for maltose, 700 injectionslh for sucrose and lactose; and 120 injectiondh for amylase at room temperature. Applications to real samples (a variety of food products, human blood serum, and serum calibration references and controls) are reported.

.

The usefulness of repetitive determinations using injection of the sample containing the sought-for species into a continuously circulated reagent mixture and based on the general reaction scheme: %substrate)

+ HzO + O2

E

Product(s1 + H 2 0 2

+

(1)

Permanent address, L a b o r a t o r y of Analytical Chemistry, University of Athens, Athens, Greece. 0003-2700/78/0350-1665$01 .OO/O

in which E is an appropriate enzyme, has been recently demonstrated ( I ) . The work reported here was designed to exploit the use of reaction 1 (with glucose as substrate and glucose oxidase as enzyme) as an "indicator reaction" for the determination of other chemical species entering into enzyme-catalyzed reactions producing glucose as a product. Detection is based on the amperometric measurement of oxygen decrease according to the reaction of Equation 1 by means of a three-electrode nonmembrane system reported previously (2). This paper describes the determination of maltose. sucrose, and lactose by their enzyme-catalyzed hydrolysis t o glucose and the estimation of the enzyme amylase by its catalytic effect on the hydrolysis of starch to glucose. In situations found in industrial process and quality control as well as in clinical determinations, the use of closed flow-through systems, such as the one used in the reported studies, affords decreased operating cost per determination by decreasing the number of manipulations, shortening the time for determination, and providing a better utilization of reagent solutions. Maltose levels are seldom measured as a clinical test but as interest in maltose metabolism increases, its determination in biological fluids will gain in relevance (3). On the other hand, disaccharides are regularly determined in several food products ( 4 ) . The determination of a-amylase in serum is of help in the diagnosis of acute pancreatitis, occlusion of the pancreas, and parotitis ( 5 , 6 ) . The sample injection procedures reported here have been applied to real samples: corn syrup and malt syrup for maltose, a variety of food products for sucrose, milk products for lactose, and human blood serum @ 1978 American Chemical Society

Analytical application of the room and low temperature (77 K

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