Phosphorescence Study of Excited Triplet State Properties of Some K Vitamins and Their Analytical Usefulness J. J. Aaron' and J. D. Winefordner2 Department of Chemistry, University of Florida, Gainesuille, Fla. 32601 Phosphorescence excitation and emission spectra and lifetimes of Vitamins K1, K3, and Ks have been determined at 77 O K in several solvents (n-hexane, methanol, ethanol, and mixtures methanol-water). Phosphorescence bands are attributed to n, R* triplet states for vitamin K5. Phosphorescence analytical characteristics of those vitamins are given for six different solvents. Limits of detection range between 0.07 and 1.5 Hg/ml, according to the structure of the vitamin, but are not significantly influenced by the nature of the solvent. The usefulness of phosphorimetry for the quantitative determination of vitamins K1 and Ks is compared with other analytical methods and is shown to compete favorably with those methods.
THEIMPORTANCE of the physiological, biological, and bacteriological properties of the different K vitamins is evident by the number of studies devoted t o these vitamins in the past years ( I ) . I n spite of this increasing interest, quantitative determination of the naturally-occurring vitamin K1 (also called phylloquinone or 2-methyl-3-phytyl-l,4-naphthoquinone) and water-soluble vitamin K 5 (2-methyl-4-amino-1 naphthol hydrochloride) has been the object of only a few analytical procedures (2-7). Most of the analytical studies and assay methods have concerned vitamin K, (also called menadione and 2-methyl-l,4-naphthoquinone),a potent fatsoluble vitamin K analog; these analytical studies include colorimetry (2, 8-11), spectrophotometry ( 2 , 12-15), fluorimetry (16), polarography (2, 17, Is), coulometric titration (19), On leave from Laboratoire de Chimie Organique Physique, Paris, France. Author to whom reprint requests should be sent. (1) R. A. Morton, Biol. Rev. Cambridge Phil. SOC.,46, 47(1971). (2) P. Sommer and M. Kofler, Vitam. Horm. (New York), 24, 349 (1966), and references therein. (3) D. A. Libby, A. R. Prosser, and A. J. Sheppard, J . Ass. O$c. Anal. Chem., 50, 806 (1967). (4) W. Wetter, M. Vecchi, H. Gurman, R. Rueeg, W. Walther, and P. Meyer, Helo. Chim. Acta, 50,1866 (1967). (5) G. H. Dialameh and R. E. Olson, Anal. Biochem., 32,263 (1969). (6) K. Schilling and H. Dam, Acta Chem. Scand., 12, 347 (1958). (7) A. J. Sheppard and W. D. Hubbard, Methods Enzymol., 1812, 461 (1971). ( 8 ) J. S. Conticello, Rev. Fac. Cinc. Quim., Unil;.Nac. La Plata, 19, 29 ( 1946). (9) T. K. Pradman, J. Znst. Chem. (India),40, 144 (1958). (10) F. J. Bandelin and R. E. Pankratz, Drug. Stand., 27, 36 (1959). (11) S. Baczyk, K. Baranonska, W. Jakubowska, and I. Sobisz, Mikrochim. Acta, 1970,1306. (12) F. C. G. Hoskin and C. Von Eschen, Biochem. Biophys. Acta, 67,669 (1 963). (13) C. Levorato, Boll. Chim. Farm., 107,184(1968). (14) V. Sathe, J. B. Dave, and C. V. Ramakrishnana, ANAL.CHEM., 29, 155 (1957). (15) V. J. Vajgand and F. S. Uvodic, Glas. Hem. Drus. Beograd, 30, 185 (1965). (16) F. Veronese, A. Meani, and E. Boccu, Acta Vitamiriol. Enzymol., 22,203 (1968). (17) V. J. Vajgand and F. S. Uvodic, Glas. Hem. Drus. Beograd, 30, 89 (1965). (18) K . Burger, Acta Pharm. Hung., 32, 180 (1962). (19) G. J . Patriarch and J. J. Lingane, Anal. Chim. Acta, 49, 241 ( 1970). 2122
and chromatography (2,20-23). In the colorimetric methods, the concentration of menadione is evaluated indirectly, after preparation of a highly-colored derivative and colorimetric (or spectrophotometric) measurement of this derivative. I n the other techniques, menadione (or its sodium bisulfite derivative) is determined directly. From a photochemical point of view, several K vitamins are also very important, as they are involved in naturallyoccurring light-induced reactions life photosynthesis processes ( I ) and oxidative phosphorylation (24). I n addition, the mechanism by which vitamin K controls prothrombin synthesis is still not elucidated ( I ) . For these reasons, a renewal of interest has recently appeared in the photochemistry of vitamins K1 and K, and the identification of their photoproducts (25-27). Nevertheless, little research has been undertaken on the excited states of those vitaminsespecially those excited states possibly involved in photochemical reactions occurring in aqueous solution (the medium for biological systems). In fact, two significant studies have been concerned with only the luminescence of menadione (vitamin K:,) and other quinones (28, 29). These spectroscopic studies described the phosphorescence bands of a series of quinones including menadione, in different rigid solutions of hydrocarbons or alcohol at 77 OK. So far, no luminescence study of vitamins K1 and K 5 have been previously reported. However, recent improvements in analytical phosphorimetry, which have been developed in this laboratory (30, 31), have permitted an investigation of matrix effects o n the phosphorescence characteristics and have facilitated analytical measurements of phosphorescence in aqueous solution and snowed, rigid solvents. Therefore, one of the purposes of this report was to evaluate the phosphorescence characteristics of the previously mentioned K vitamins in aqueous and nonaqueous media, in order to obtain more precise information about the lower excited triplet state of these compounds. Another purpose was to estimate the usefulness of the phosphorimetry for the quantitative determination of the K ~
(20) A. J. Sheppard and W. D. Hubbard, Methods Enzymol., 1% 465 (1971). (21) A, J. Sheppard and W. D. Hubbard, J. Ass. Ofic.Anal. Chem., 53, 1093 (1970). (22) D. A. Libby and A. B. Sheppard, ibid., 48,973 (1965). (23) M. H. Hashmi, F. R. Chughtai, and M. J. D. Chughtai, Mikrochim. Acta, 1969, 53. (24) E. Lederer and M. Vilkas, Vitam. Horm., (New York), 24, 409 (1966). (25) H. Werbin and E. T. Strom, J. Amer. Chem. Soc., 90, 7296 (1968). (26) C. D. Snyder and H. Rapoport, ibid., 91,731 (1969). (27) G. Leary and G. Porter, J . Chem. Sor. A, 1970,2273. (28) A. Kuboyama and S. Yabe, Bull. Chem. SOC.Jup., 40, 2475 (1967). (29) N. A. Shcheglova and D. N. Shigorin, Russ. J . Phys. Chem.,38, 684 (1964). (30) R. Zweidinger and J. D. Winefordner, AUAL.CHEM.,42, 639 (1970). (31) R. Lukasiewicz, P. Rozynes, L. B. Sanders, and J. D. Winefordner, ibid., 44, 237 (1972).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 13, NOVEMBER 1972
vitamins and to compare phosphorimetry with other analytical methods. EXPERIMENTAL
--d 0
Apparatus. Phosphorescence spectra are measured at 77 “K on a n Aminco-Bowman spectrophotofluorometer with an Aminco-Keirs phosphoroscope attachment (American Instrument Co., Inc., Silver Spring, Md.). A Harrison (HewlettPackard, Palo Alto, Calif.) constant-current dc power supply is used to power the 150-W xenon arc lamp, and phototube signals were measured with a low noise nanoammeter (32). A rotating capillary tube, approximately 1-mm i.d., and 6-mm 0.d. made of synthetic high purity optical grade quartz (Quartz Scientific Inc., Eastlake, Ohio) is used as the sample cell (31). Polarized excitation is used to decrease the phosphorescence background of the quartz tube (31), by means of a thin-film, quartz plate UV transmitting polarizer (Polacoat. Inc., Cincinnati, Ohio) mounted in the excitation beam. For the measurements with low surface-tension solvents (e.g., methanol, ethanol, and hexane), a specially-designed Teflon (Du Pont) cap closed the bottom part of the quartz capillary tube, in order to prevent solution leaking out prior to freezing or flushing of the solution by liquid nitrogen when the capillary tube is introduced into the liquid nitrogen in the dewar. By this improvement, the very small samples (*20 PI) stayed at the same level of the capillary tube during freezing, and the reproducibility of‘the phototube signals is increased. The short lifetimes of phosphorescence ( 5 1 msec) are measured by the combination of the rotating shutter and an oscilloscope type 545 A (Tektronix, Inc., Portland, Ore.) directly connected to the Aminco phototube IP28, with a highgain differential amplifier-input resistance 30 to 100 kn; longer phosphorescence lifetimes (ryl sec) were measured by recording the nanoammeter output as a function of time, after complete termination of the exciting radiation by a guillotinetype shutter. UV irradiation of the solutions of vitamin K is performed by means of a 140-W Hanovia medium pressure mercury quartz lamp with a 350-nm (half-band width of 20 nm) filter (Optics Technology, Inc., Palo Alto, Calif.). Reagents. Vitamin K1 (2-methyl-3-phytyl-l,4-naphthoquinone): vitamin KB (2-methyl-l,4-naphthoquinone),and vitamin K5 (2-methyl-4-amino-1-naphthol hydrochloride) 0
VITAMIN
KI
0 VITAMIN
K3
NH2HCI VITAMIN
KS
Structure of vitamins K1, Ks, and Kj (32) T. C. O’Haver and J. D. Winefordner, J. C/zem. Educ.. 46, 241 (1969).
,? 6 0 0
50
WAVELENGTH
(nm)
Figure 1. Phosphorescence excitation and emission spectra of vitamin K L _--_ in Hexane (Aern = 570 nm; A,, = 346 nm) - - - - in Ethanol (Aern = 564 nm; A,, = 348 nm)
-- -
were purchased from Nutritional Biochemical Corp., Cleveland, Ohio. Vitamin K, was used as received. Its UV absorption spectrum was found to be identical with literature data ( 2 ) . Vitamin K3 was recrystallized from petroleum ether (mp 105 “C). Because of its great sensitivity to oxidation, vitamin K5 was purified by recrystallization from alcoholether in an atmosphere of nitrogen (33) with an especially designed apparatus ( 3 4 ) ; white crystals were obtained, while colored oxidation products stayed in the mother liquor. Solvents used were: methanol (Matheson, Coleman and Bell, Manufacturing Chemists, Norwood, Ohio, “spectroquality” grade), ethanol, hexane, and deionized water. Ethanol and hexane were purified by distillation and methanol was used as obtained from the manufacturer. Procedure. Solvents used for the preparation of the solutions of the K vitamins were different mixtures of methanolbater (10, 20, and 30 volume per cent), methanol, ethanol. and n-hexane. For the phosphorimetric study, solutions of different concentrations were prepared by successive dilutions. Solutions of vitamin Ks were prepared with solvents continuously flushed by dry nitrogen in a dry box, under atmosphere of nitrogen. These solutions were constantly bubbled with a nitrogen flow and used immediately (within 30 minutes). Otherwise, the formation of oxidized phosphorescent products was observed and could possibly have distorted the analytical curves. Solutions of vitamins K1and K 3were prepared under attenuated daylight (No significant difference of the intensity of the phosphorescence signal was observed in preparing the solutions in the darkroom illuminated with weak red light.) and stored in brown bottles in order to prevent any photodecomposition or photoreaction of the vitamins with the solvent. Measurement of phosphorescence and excitation spectra and measurement of the analytical phosphorescence signals were performed immediately (within 30 minutes) upon the freshly-prepared solutions. For the photochemical measurements, solutions of the K vitamins (concentrations of about 10- 3M; more dilute solutions resulted in the same phosphorescence spectra indicating the improbable formation of dimers) were irradiated by UV light in a 1-cm quartz rectangular cuvettes. After each threeminute period of illumination, a sample was taken with the quartz capillary tube, and the phosphorescence signal at the (33) E. Knobloch, Collect. Czech. Chem. Commun., 14,508 (1949). (34) R. S . Tipson, “Techniques of Organic Chemistry,” A. Weissberger Ed., Interscience, New York, N. Y . , 1956, Vol. 111, Part I, p 529.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 13, NOVEMBER 1972
2123
z80[ E 70
- 60
t J 50
z 2 v,
40
W
30 0
w
g 20 n. 0
E
IO
0200
250
300
350
400
450
500
WAVELENGTH
550
600
650
750
700
(nm)
WAVELENGTH
Figure 2. Phosphorescence excitation and emission spectra of vitamin K3 in Hexane
550 nm; Xex = 335 nm) 545 nm; Ae, = 338 nm) in Methanol (Aern = 546 nm; Aex = 339 nm) in Methanol/water v/v l0/90 (hem = 546 nm; A,, nm) (Aern = =
_ _ - _ _ _in_ Ethanol (Aern
....... -. - .- .-
WAVELENGTH
=
338
(nrn)
Figure 3. Phosphorescence excitation and emission spectra of vitamin KS
- _ _ _ _ _ in- Ethanol (Aern -. -. - .-
= 500 nm; A,, = 315 nm) in Methanol/water v/v 20/80 (Aern = 500 nm; A,, = 317 nm) in Methanol/water v/v 50/50 (Aern = 499 nm; X,, = 315 nm)
emission maximum wavelength of the photoproduct was determined at 77 OK. No further change of the phosphorescence signal with the time was observed at the liquid nitrogen temperature, which indicated that the photoreaction is inhibited at that low temperature. RESULTS AND DISCUSSION Phosphorescence Characteristics. I n Figures 1-3, the excitation and emission phosphorescence spectra of the different K vitamins are given. All the spectra are uncorrected for instrumental response. A spectral halfband pass of about 15 nm was used. The shapes of the phosphorescence bands of vitamins K1 and K S seem to be independent of the type of solvent used. Band wavelengths and lifetimes of phosphorescence are reported in Table I. 2124
(nm)
Figure 4. Influence of illumination by UV light (350 nm) on phosphorescence bands of vitamin KJ in ethanol 1 and 2: Excitation and emission spectra of Vitamin K3 after UV irradiation. 3 and 4: Excitation (Aern = 450 nm) and emission (Aex = 305 nm) spectra of photoproduct obtained after W illumination of Vitamin K3 at room temperature. Compare spectra 1 and 2 with spectra of non-irradiated Vitamin Ka in Figure 2
Several common characteristics of the phosphorescence bands of vitamins K1 and K 3 (both have quinone structures) can be noticed. The phosphorescence main peaks occur a t shorter wavelengths in alcoholic and aqueous solvents than in hexane; this blue shift of 150-200 cm-1 induced by a polar solvent like methanol, ethanol, or water is consistent with an hypsochromic displacement of about 200-300 cm-l, observed for other quinones when a nonpolar solvent is replaced by a polar one (28, 35). This change is typical of n,a* phosphorescence. Another proof for the assignment of the phosphorescence bdnds of vitamins K1 and K3 is that the emission from a n,T* triplet state has very short lifetimes, and the observed lifetimes range from 0.4 to 0.8 msec, depending upon the rigid solvent used (see Table I). Indeed, it is generally estimated that a lifetime in the range
