Anal. Chem. 1988, 60, 2317-2320
mh
MINUTES
Figure 3. Thermospray LC/MS in D,O of nucleosides from 2 0 pg of nucleic acids of Pyrodctium occunUm: (a) UV detection, 254 nm, of a portlon of the chromatogram. Major components: I, cytidine; 11, uridine; 111, deoxycytidine; IV, guanosine; V, deoxyguanosine; VI, thymidine. The principal constituent eluting at 3.05 mln is thymkllne (M, = 228, MD+ 232). Numerous minor constituents are not labeled. (b) Mass spectrum acquired at 3.19 min, scanned from m l z 250 to m / z 300. (c) Reconstructed ion chromatogram for m l z 277, corra sponding to approximately 2 0 ng of compound 2. The data in Table I suggest that, in the case of complete structural unknowns, caution should be exercised because of the possibility of unexpected exchange of slightly acidic hydrogen atoms that might not usually be considered sufficiently labile for simple exchange in D20. In such cases, the results obtained serve only to establish upper limits of the acidic hydrogen content. This approach is of greater general use in cases requiring differentiation of isomers that clearly differ in the number of exchangeable hydrogens, and for which exchange of slightly labile hydrogens is not an issue. An example is given by the differentiation of the isomers N4,2’-0-dimethylcytidine(1) and 5,2’-0-dimethylcytidine (2), possible constituents of transfer RNA of thermophilic archaebacteria that had been partly characterized by LC/MS of enzymatic digests of RNA (9). Compounds 1 and 2 were HNCHj
NH2
0
1
2
predicted to very nearly coelute, as later verified by using synthetic reference compounds, making chromatographic differentiation unreliable. The component in question also partially coelutes with a larger amount of thymidine arising
2317
from traces of DNA (Figure 3a) and was available in only nanogram-level quantities, making isolation impracticable. The mass spectrum taken at 3.19 min (Figure 3b) clearly shows MD’ at m / 2 277, in favor of structure 2 with four exchangeable hydrogens in the neutral molecule, vs three in 1 (9). Experience in applying this method to LC/MS analyses similar to that shown in Figure 3 involving 20- to 40-component mixtures, with many constituents chromatographically unresolved and in the nanogram range, has provided numerous examples of the utility of the exchange method in confirming and correcting assignments made on the basis of (protium) LC/MS data and in providing useful deuterium shift data on new or unexpected components (19).
ACKNOWLEDGMENT We gratefully acknowledge the following: R. Gupta for isolation of nucleic acid from cells provided by K. 0. Stetter; T. Hashizume and R. J. Goebel for preparation of synthetic nucleosides; and P. F. Crain for helpful suggestions. The provision of specially prepared D20 by Suraj Manrau, Merck Frosst, Canada, is gratefully acknowledged. LITERATURE CITED (1) Budzlklewlcz, H.; Djerassi, C.; Williams, D. H. Shrcture ElucMeMon of Natural Products by Mass Spectrometry, Vol. 1 . Alkakeldds; HoldenDay: San Franclsco, CA, 1964; Chapter 2. (2) Thomas, A. F. Deuterium Lebellng in W n l c Chemistry; Appleton Century Crofts: New Yo&. 1971. (3) Harrison, A. G. Chemlcai Ionkation Mass Spectrometry;CRC Press: Boca Raton, FL, 1983; pp 129-131. (4) Sethi, S. K.; Smlth. D. L.; McCloskey. J. A. Blochem. Blqhys. Res. Commun. 1983, 112, 126-131. (5) McCloskey, J. A. In Mihods h Enzymology, Vol.XIV;Lowensteln, J. M., Ed.; Academic: New York, 1969; p 438. (6) Reference 2, Chapter 4. (7) Henlon, J. D. J . Chromatogr. Scl. 1981. 19, 57-64. (8) Hsu, F. F. Ph.D.Dissertation. Unhrerslty of Utah. 1986. (9) Edmonds, C. G.; Crain, P. F.; Hashirum, T.; Gupta, R.; Stetter, K. 0.; McCloskey, J. A. J . Chem. Soc.,Chem. Commun. 1987, 909-910. (10) Edmonds, C. G.; Vestal, M. L.; McCloskey, J. A. Nucleic Aclds Res. 1985. 13, 8197-8206. (11) Verma, S.; Pomerantz, S. C.; Sethi, S. K.; McCioskey. J. A. Anal. Chem. 1986, 58, 2898-2902. (12) Hsu, F. F.; Edmonds, C. G.; McCloskey, J. A. Anal. Lett. 1986, 19, 1259-127 1. (13) Shapiro. R. H.; Djerassi, C. J . Am. Chem. Soc. 1984, 86. 2825-2832. (14) Vestal, M. L. Int. J . Mass Spectrom. Ion Phys. 1989, 46, 193-196. (15) Hunt, D. F.; Sethi. S. K. J . Am. Chem. Soc.1980, 102, 6953-6963. (16) Organic Chemistry of Nucleic AcMs, Part B ; Kochetkov, N. K., Budovskii, E. I., Eds.; Plenum: New York, 1972; pp 282-284. (17) Pang, H.; Schram, K. H.; Smlth, D. L.; Gupta, S. P.; Townsend, L. B.; McCloskey, J. A. J . Org. Chem. 1982, 4 7 , 3923-3932. (18) Vis, E.; Fletcher, H. 0.. Jr. J . Am. Chem. SOC. 1958, 79, 1182-1 185. (19) Edmonds, C. G.; Pomerantz, S. C.; Hsu, F. F.; McCloskey, J. A. 36th ASMS Conference on Mass Spectrometry and Allled Topics, Sen Francisco, June, 1988.
RECEIVED for review April 7,1988. Accepted June 17,1988. This work was supported by the Institute of General Medical Sciences through Grant GM 21584.
Porous Fiber-optic Sensor for High-Sensitivity Humidity Measurements Quan Zhou, Mahmoud R. Shahriari, David Kritz, and George H. Sigel, Jr.* Rutgers University, Fiber Optic Materials Research Program, P.O. Box 909,Piscataway, New Jersey 08854 Several approaches have been reported for fiber-optic sensors to determine the relative humidity of air. Typically, either light absorption or fluorescence of chemical indicators is monitored on a real-time basis by using fiber-optic waveguides to deliver and transport signals. Such devices have the advantage of higher sensitivity and quantitative precision 0003-2700/88/0360-2317$01 S O / O
compared to the traditional visual indicator method (I)as well as offering the potential for remote monitoring. The use of an optical fiber sensor for humidity measurements has been previously described by Russell and Fletcher (2). In their device, a cobalt chloride/gelatin film is immobilized on a 12 cm long silica optical fiber as the humidity probe. Ballantine 0 1988 American Chemical Society
2318
ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1988 Porous segment oa8 out
GAS
CHAMBER
CHART COMPUTER
LOCK-IN
RECORDER
CHOPPER GAS
PHOTODIODE
CHAMBER
-
MONOCHROMATOR
Figure 1. Schematic diagram of the experimental arrangement of the humidity sensor.
and Wohltjen described an optical waveguide humidity sensor that employed the same colorimetric reagent/polymer system on a 9-cm glass capillary (3). In contrast, Zhu and Hieftje utilized a fluorescence fiber-optic sensor for atmospheric humidity (4). They employed a fluorescent dye entrapped within 9 polymer matrix. However, all of these previous sensor devices were limited to the determination of the relative humidity above 40%. There is a need for development of moisture sensors at very low levels. The purpose of this paper is to present the initial efforts in the development of a new optical fiber humidity sensor that is smaller than previous devices while at the same time exhibiting greatly enhanced sensitivity. In this work, CoClz as a colorimetric reagent was directly employed in conjunction with a porous optical fiber, a unique chemical substrate recently developed by the authors. The high surface area and direct absorption of light passing through the porous glass sensor segment result in a device with the demonstrated capability to measure relative humidity levels below 170at room temperature.
EXPERIMENTAL SECTION Optical fibers of high-transparency alkali borosilicate glasa were drawn using a commercial fiber drawing facility, the typical diameter of the fiber ranging from 150 to 300 pm. Then a small section of the fiber was phase separated by heat treatment at about 500 OC, one phase is an alkali borate rich phase, another is a silica rich phase. The soluble alkali borate phase is leached away by suitable acid solution to leave a silica rich porous skeleton. A similar process of making Corning Vycor glass is described in ref 5. The bared optical fiber,with a 0.5 cm long porous core section, was then immersed in a cobalt chloride (CoCl2.6HZ0,Fisher ScientificCo.) aqueous solution. Then the fiber was dried in dry air overnight at room temperature. The treated porous optical fiber was placed in a small flowing gas chamber, which both held the fiber in place and served to provide the appropriate gas environment. A gas flow system was designed and constructed to control the introduction of water vapor. A digital hygrometer with 0.1% resolution (Fisher Scientific Co.) was monitored on-line to detect the relative humidity of the gas flow and serve as a calibration device for the fiber sensor. For the lower percent relative humidity measurements, the relative humidity of diluted vapor streams was calculated by the flow rates of dry air and high humidity air, which were mixed before introduction into the chamber. Figure 1 shows the experimental arrangement used for humidity measurements. The measurements are simple and straightformrd. Chopped light from a quartz halogen lamp passed through
Flgure 2. Typical transmission spectra measured on a CoCI,-treated porous optical fiber saturated by moisture (A) and dried by alr (B).
a monochromator (Jarrell Ash Mono Spec-27, ARIES,Inc.) and was focused onto one end of the optical fiber. Light passing through the fiber was picked up by a photodiode (FO-WE, United Detector Technology). Finally, the signal was amplified by a lock-in amplifier (Model 5207, EG&G Princeton Applied Research) and was plotted out by a chart recorder (Fisher Recordall Series 5000). Both the monochromator and the amplifier were interfaced to an IBM personal computer.
RESULTS AND DISCUSSION The structural nature of the porous fibers used in this work has been characterized by a scanning electron microscope, a BET surface area measurement, and a mercury porosimeter. The results indicate an interconnective porous structure with pore size between 20 and 1000 A and a total surface area of about 200 m2/g. Although most of the light launched into the porous optical-fiber segment will be influenced by the absorption of the indicator, some will be scattered out by the porous structure of the glass host. An integrating sphere was used to determine the light scattering loss of the porous glass fiber; the typical scattering loss is about 0.7 dI3/cm at 633 nm. Inside the porous optical fiber, the light can interact with CoClz or other chemical reagents introduced into the waveguide and very strong light absorption occurs just as in the case of light passing a sample absorption cell. This process is called in-line optical absorption. It is well-known that CoClz can form a hydrated salt having six molecules of bound water with the octahedral structure, which has a absorption peak around 500 nm and appears pink (6). When it is dried well, CoClz changes color to bright blue and has high optical absorption between 550 and 750 nm. Figure 2 shows the typical transmission spectra of CoClz incorporated into the porous glass optical fiber (without taking into account wavelength dependence on the intensity of lamp and on the response of photodetector). It is clear to see that the maximum absorption lies near 690 nm, so the light signals are monitored at this wavelength for a continuous recording of the humidity-induced intensity changes in the experiments. Figure 3 depicts a series of typical response curves for a porous fiber sensor, which was exposed to a number of step changes in humidity. When the moisture is admitted into the chamber, the device responds rapidly, and after a couple of minutes the output signal approaches a stable value. Conversely, when the flow of water vapor is stopped and only dry air is admitted into the gas chamber, the signal rapidly decays back to the base line observed before the introduction of water vapor. The response time for this device (from Figure 3) is about 2-3 min, which also includes the time needed for flow system and gas chamber to reach the equilibrium after a change in humidity. The chemical species diffusion into the porous optical fiber may be the kinetic controlling step. In-
ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1988
75
i
I
5
TIME
[ mins
Flgure 3. Typical response curves of porous glass sensor at 690 nm
at a continuous flow of water vapor air: A, 8.2% RH; B, 10.4% RH; C, 12.1% RH; D, 12.6% RH; E, 14.2% RH; F, 15.7% RH. The flow rate is about 1 L/min. at 25 OC.
601
./*
,
20 73 43 F e l s t i v e H u m i d i t , 1%) and experimental (*) humidity sensor re-
10
Figure 5. Simulated (-) sponse curves.
10
I
J
C
3
2310
There is a dynamic equilibrium involved in the response of the moisture probe. Free water vapor first diffuses into porous glass fiber and adsorbs onto the porous glass surface until equilibrium is reached. Meanwhile, the adsorbed water combines with CoCl,, which is already attached on the porous structure, to form a CoC12.6Hz0complex. The relationship between the water vapor concentration and optical intensity of CoCl2 at 690 nm can be derived as follows:
Ke = N C o C 1 ~ 6 H 2 0 /[H2016NCoC12
(1)
where K , = 1
