Anal. Chem. 1988, 60.2314-2317
2314
self-CI might be observed. For example, a rate constant for the reaction of 1 X lo4 cm3/(molecule s) under these conditions would yield that ratio [MH+]/[M+’], = 0.05. This would lead to a [ (M 1)+]/ [M+’] ratio in the mass spectrum of ~0.05above the fraction expected on the basis of the 13C contribution, provided the ions are trapped and detected with equal efficiency. At longer reaction times, such as would result from a scan starting at 20 amu, the [MH+]/[M+’], ratio would be 0.13. The purpose of this discussion is to show that products from self-CI (or other ion/molecule reaction) can be observed for suitable compounds under conditions that can be readily obtained, in the ITD and ITMS. The likelihood that this may occur can be anticipated in a straightforward manner. As has been demonstrated, space charge can lead to peak broadening which can cause the data system to misinterpret signals as arising from (M + l)+ions in mass spectra obtained from large sample sizes (greater than about 50 ng depending upon the compound (1,lO)). While the automatic gain control mode of operation recently developed by Finnigan (11)addresses the space charge problem, this does not reduce the potential for self41 since it is the concentration of the neutral, not the ions, along with the reaction time that determines the relative degree of self-CI. The segmented scan function used in the ITMS (and ITD) data system reduces the reaction time and thus reduces the likelihood of the ion/molecule reactions being observed. This paper has shown that “large” sample sizes can lead to the observation of significant concentrations of protonated species from self-CI in systems with fast reaction rates. The two experimental variables that can be controlled to avoid self-CI are reaction time and sample concentration. (In GC/MS, the sample concentration is inversely related to the chromatographic peak width, i.e. narrower peaks give higher instantaneous concentrations at the peak maximum than wider peaks, for the same quantity of sample.) The Finnigan data system is designed to reduce the reaction time to the minimum practical so it is up to the experimentalist be aware of the approximate concentration of the analyte, to prevent self-CI, or a t least to be cognizant of its possible occurrence. In general, self-CI or other ion/molecule reactions are expected
+
to be observed in the ITD or ITMS mainly with samples in which it is observed in other types of mass spectrometers, such as, for example, esters. The observation of self41 will likely require a rate constant 21 x cm3/(molecule s).
ACKNOWLEDGMENT The authors thank Don Hoekman and Michael WeberGrabau of Finnigan for providing experimental software and for helpful discussions and Henry S. McKown of ORNL for construction of the dc pulsing circuit used in these experiments. LITERATURE CITED (1) Eichelberger, J. W.; Budde, W. L.; Slivon. L. E. Anal. 0”.1987, 59, 2730. (2) Stafford, G. C.; Kelley, P. E.: Stephens, D. R. US. Patent 4540884, 1985. (3) Stafford, G. C.; Kelley, P. E.; Syka. J. E. P.; Reynolds, W. E.; Todd, J. F. J. I n t . J . Mass Spectfom. Ion Processes 1984, 60,85. (4) Olson, E. S.; Diehl, J. W. Anal. Chem. 1987, 59,443. (5) Todd, J . F. J. Dynamic Mass Spectrometry; Price, D., Todd, J. F. J., Eds.; Heyden: London, 1981; Vol. 6, Chapter 4. (6) Kelley, P. E.; Stafford, G. C.; Syka, J. E. P.; Reynolds, W. E.; Louris, J. N.; Todd, J. F. J. Adv. Mass Spectrom. 1986, 106, 869. (7) Louris, J. N.; Cooks, R. G.; Syka, J. E. P.; Kelley, P. E.; Stafford, G. C.; Todd, J. F. J. Anal. Chem. 1987, 59, 1677. (8) Henchman, M. Ion-Molecule Reactions; Franklin, J. L., Ed.; Plenum: New York, 1972; Vol. I, Chapter 5. (9) Lawson, G.; Bonner, R. F.; Mather, R. E.; Todd, J. F. J.; March, R. E. J. Chem. SOC.. Faraday Trans. 1 1978. 72, 545. (10) Eichelberger, J. W.; Budde, W. L. Blamed. Environ. Mass Spectrom. 1987, 14. 357. (11) Stafford, G. C.; Taylor, D. M.; Bradshaw; S. C.; Syka, J. E. P.; Uhrich, M. Presented at the 35th ASMS Conference on Mass Spectrometry and Allied Topics, Denver, CO, May 24-29, 1987.
Scott A. McLuckey* Gary L. Glish Keiji G. Asano Gary J. Van Berkel Analytical Chemistry Division Oak Ridge National Laboratory Oak Ridge, Tennessee 37831 RECEIVED for review April 29, 1988. Accepted July 22,1988. Research sponsored by the U.S. DOE Office of Basic Energy Science under Contract DE-AC05-840R21400 with Martin Marietta Energy Systems, Inc.
TECHNICAL NOTES Thermospray Liquid Chromatography/Mass Spectrometry in Deuterium Oxide Charles G . Edmonds, Steven C. Pomerantz, Fong Fu Hsu, and James A. McCloskey* Departments of Medicinal Chemistry and Biochemistry, University of Utah, Salt Lake City, Utah 84112 Deuterium labeling of organic compounds plays an important role in mass spectrometry in studies of ionic reaction mechanisms, and in applications involving structural characterization. In addition to conventional methods of deuterium introduction including direct exchange in the mass spectrometer inlet system (I, Z ) , gas-phase exchange under chemical ionization conditions (3)and exchange in conjunction with fast atom bombardment (FAB) (4)have been studied. The on-line exchange of deuterium for protium during combined chromatography/mass spectrometry, fist reported using GC/MS (5), is advantageous because both there is direct applicability to components in mixtures and high exchange levels of acidic hydrogen atoms with minimum back exchange
can be obtained. Although the introduction of deuterium can be effectively carried out by a variety of novel preparative liquid and gas chromatographic procedures (6),the direct combination with mass spectrometry obviates the necessity of isolation of individual components and permits direct comparison of mass spectra of labeled constituents of a multicomponent mixture with spectra of unlabeled components obtained in conventional fashion from a separate chromatographic analysis. Directly combined liquid chromatography/mass spectrometry (LC/MS) provides particular advantages in the analysis of polar compounds for which GC/MS is less well suited. Deuterium-protium exchange using LC/MS was first reported
0003-2700/88/0360-2314$01.50/00 1988 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1988
by Henion, who obtained the chemical ionization mass spectrum of [2H3]sulfadimethoxine acquired by the "direct liquid introduction" method, using CD&N-D20 eluants with a microflow chromatography system (7). We presently report some experimental details and results of on-line H/D exchange using thermospray LC/MS, with CH&OzND4-Dz0 mobile phase and conventional flow reversed-phase HPLC, which has been used in this laboratory for several years (8). The application of this method to the structure elucidation of new nucleosides from RNA has been previously reported (9). EXPERIMENTAL SECTION Materials. The tripeptide Leu-Trp-Met was obtained from Chemical Dynamics Corp. (South Plainfield, NJ). N4,2'-0-dimethylcytidine and 5,2'-O-dimethylcytidine were prepared as earlier reported (9). 8-[2H]Guanosine was obtained from D. Phillips. &Bromoadenosine was from the Cancer Chemotherapy National Service Center, National Cancer Institute. Other reference materials were obtained from Sigma Chemical Co. (St. Louis, MO). Cells from the bacterium Pyrodictium occultum were a gift from K. 0. Stetter, Regensberg University, Federal Republic of Germany. Preparation of Transfer RNA Nucleosides. Nucleic acids from Pyrodictium occultum were previously isolated for an earlier study (9). Hydrolysates of mixed tRNA8 were prepared by a two-stage method consisting of incubation with nuclease P1 followed by treatment with alkaline phosphatase. The use of this method in the LC/MS analysis of transfer RNAs has been described (10). Instrumentation. A Beckman (Berkeley, CA) 322M liquid chromatograph including a Beckman Model 400 2.8-mL mixing chamber was interfaced in series through a dual-wavelength (254 and 280 nm) Waters (Milford, MA) Model 440 UV absorbance monitor to a noncommercial quadrupole mass spectrometer, as previously described (IO),using a Vestec Corp. (Houston, TX) thermospray interface. Vaporizer exit temperatures were controlled in the range 240-270 OC, with ion source temperature maintained so as to maximize thermospray ionization yield, corresponding to approximately 290 "C vapor temperature at the point of ion sampling. The mass spectrometer was controlled by a Teknivent (St. Louis, MO) 29K or a Vector/One data system. Sample Introduction and Chromatography. Sample solutions generally consisting of 1-2 pg of material dissolved in microliter volumes of HzO were introduced with a Beckman 210 injedor fitted with a 100-pL injection loop, with chromatographic separation using a 4.6 X 75 mm 3-pm Beckman Ultrasphere ODS column. Deuterium Oxide Mobile Phase. DzO (glass distilled, low conductivity, 99.8% minimum isotopic purity) was obtained from MSD Isotopes (St. Louis, MO). Ammonium acetate buffer was prepared in DzOwith pD adjustment using CF,COZD (prepared by hydrolysis of trifluoroaceticanhydride in DzO). Mobile phase was 0.1 M ammonium acetate HPLC grade (J.T. Baker, Phillipsburg, NJ), pD 7.25, with acetonitrile (0-40%) (American Burdick and Jackson, Muskegon, MI) at a flow rate of 2 mL/min. Dilution of deuterium enrichment by protium in the buffer salt was calculated as 0.8%. For those experiments where the maximum possible enrichment was desired, residual protium was fist removed from buffer salt by successive evaporation from D20 solution, with storage of the crystalline residue under dry nitrogen. Preliminary experiments with D20 buffer solutions resulted in rapid occlusion of the thermospray vaporizer. Although the exact nature of the occluding deposits (likely to be siliceous) was not determined, the problem was alleviated by use of D20stored in polyethylene bottles. Calculations of Deuterium Content. Extent of incorporation, isotopic enrichments, and estimates of acidic hydrogen content were obtained by methods described previously (11). RESULTS A N D DISCUSSION Changeover to Buffered DzO and Extent of Deuterium Exchange. Thermospray mass spectra can be acquired by direct injection of samples dissolved in buffered DzO. However, in the absence of a column or precolumn the resulting
00 60 -
%D
-
I
1
I
0
4 0 12 16 20 TIME AFTER CHANGEOVER, minutes
24
Figure 1. HPLC column equilibration following substitution of H20buffer with D20 buffer calculated from the isotopic composition of the ammonium adduct ion (M.NH,+) of 1-0-methylgalactopyranoside(M, = 194).
sample ion profiles are relatively narrow if full scans are desired, and this approach has limited applicability to mixtures. If a column is employed, the use of 3-pm packings is desirable in many cases because of generally shorter analysis times. For experiments in which the column has not been previously equilibrated with D20,the equilibration can be followed with suitable test substances containing exchangeable hydrogens, such as carbohydrates, which exhibit excellent thermospray response a t the nanogram level (12). In the DzO LC/MS experiments, a reservoir containing buffered DzO is located a t a manual changeover valve a t the inlet of the A pump of a conventional liqid chromatograph. Dry acetonitrile is contained in the B pump of the system to provide organic modifier for isocratic and gradient elution liquid chromatography. Figure 1 shows the deuterium exchange of the ammonium molecular adduct ion of l-o-methylgalactopyranoside, containing eight exchangeable hydrogens, after switching of H 2 0 buffer to DzO buffer. The time for changeover is determined by the volume of this system (Le,, mixing chamber, injection loop, column, and connecting lines), and exchange is better than 95% complete, as measured by shift of the M.NH4+ion (m/z 212) to M.ND4+ (m/z 220), after 16 min at a flow rate of 2 mL/min. Equilibration to an exchange level of 90% D was achieved in about 14 min. This level of exchange, when applied to molecules having up to six acidic hydrogen atoms, provides an easily interpretable isotopic pattern (11). Alternatively, the equilibration level of the system can be empirically followed without use of a reference compound by observation of the mass shifts of ions in the thermospray background. For example, the cluster ion at m/z 119, CH3CO2NH4CHSCN.H+,may be observed to shift to m/z 124, although in specific cases these ions may be obscured by contributions from other ions in this low mass region. The maximum extent of exchange, using a fully equilibrated column and mobile phase made from preexchanged ammonium acetate, was measured by using the disaccharide trehalose, Figure 2. The availability of a relatively large number of acidic hydrogens in the M-NH4+ion provides an isotopic pattern from which the deuterium content can be accurately measured. The observed exchange level of 97 f 1% D (n = 3) is approximately the same as can be obtained with FAB from deuteriated glycerol (4, 11),although the FAB method requires care to avoid back-exchange during sample handling. Exchange levels using the LC/MS method are probably equivalent to the best levels obtained from chemical ionization with labeled reagent gases (3). In general, exchange levels of greater than 95% are required for analysis of isotopic patterns
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1988
Table I. Extent of Deuterium Exchange from Thermospray LC/MS in Deuteriated Mobile Phase MD+
no. of acidic hydrogens exchanged"
?np
compound (mol wt)
(re1 int)
16-c~-hydroxytestosterone(304) 1-0-methylgalactopyranoside (194)
308 220 379 125 456 250 250 274 157
prostaglandin El(354) threonine (119) Leu-Trp-Met (448) uridine (244) cytidine (243) adenosine (267) guanosine (283)
no. of D addedb
% DC
2 4f 3f 4 6 4
(100) (100)f (100)f (100) (100) (100) (100) (66) (1)g
additional exchange m/z (re1 int)d % De 309 221 380 126 457 251 251 275 158
5 5 48
0 0
(28) (7) (51) (7) (57)
22
0 20
(11)
0
(32) (100) (100)
17 59 99
a Protium bound to 0 or N in the principal tautomeric form. One D in the case of MD+, four D in the case of M.NDIt ion. Calculated as percent of maximum exchange of heteroatom-bound hydrogen, &3% estimated error. Values in parentheses are assumed levels. Intensity values not corrected for contributions from natural isotopes. e For exchange of one additional protium bound to C in the principal tautomeric form. Calculated assuming 100% exchange of heteroatom-bound hydrogen; corrected for natural isotopes. fM-ND,+ ion. #Guanine base fragment ion, BD,+; MD+ ion not observed (ref 10).
I
2
I
40-
;20 20e
LT
. . . . , . . . . , . .! . , . . . . ,
0360
365
370
375
380
m /z Figure 2. Mass spectrum of the ammonium adduct Ion (WND,') region of trehalose (M, = 342) following exchange by thermospray LC/MS in D20, showlng 97% exchange of acklic hydrogen. The m l z 372 ion contains 12 deuterium atoms.
only if the number of exchangeable hydrogens is relatively large, e.g., greater than 15. The results of deuterium exchange from a variety of substances are shown in Table I, emphasizing cases in which some hydrogens nominally bound to carbon, and having slightly acidic character, have undergone partial exchange. In such cases, accurate calculation of deuterium exchange levels for heteroatom-bound hydrogen is difficult and is therefore arbitrarily listed as 100% in Table I. Exchange levels below the estimated level of accuracy (&3% D) are listed as zero. 16-a-Hydroxytestosteroneundergoes no significant exchange of A-ring hydrogen, even though such exchange can be effected under strongly basic conditions in solution (13). Some exchange of enolizable hydrogen (22%) is observed in prostaglandin E,, although no exchange was observed a! to the carboxyl group of threonine. Some exchange of carbon-bound hydrogen observed in the tripeptide Leu-Trp-Met may arise from the imidazole portion of the molecule. In recognition of the occurrence of chemical ionization processes that occur in the thermospray jet (14),the exchange of aromatic hydrogens would mirror earlier reports of similar exchange during chemical ionization with labeled reagent gases (3, 15). The most significant exchange of slightly acidic hydrogen was observed in the purine nucleosides adenosine and guanosine (Table I) and is attributed to H8, which can be quantitatively exchanged in solution by heating for several hours at neutral pH (pD) (16). As expected, Sbromoadenosine undergoes no analogous exchange, and under conventional thermospray conditions using protium solvent, 8-[2H]guanosine quantitatively loses the deuterium label (data not shown). Partial exchange of H8 of adenosine was found to be generally reproducible from run to run and relatively insensitive to changes of pD over a range suitable for re-
versed-phase HPLC: 58% D8 at pD 5.7, and 52% at pD 7.25. Three nucleosides showing partial exchange at H8 (adenosine; 1-methyladenosine, 65% D; 9-8-D-ribofuranosylpurine, 38% D) were also studied as a function of vaporizer temperature. Over the range effective for ion production (vaporizer tip 255-296 "C, vapor 280-302 "C), no significant variation in incorporation levels was observed. Uridine and ita derivatives undergo no exchange of H5,6 in the pyrimidine ring, but some partial exchange of one hydrogen in cytidine takes place, as anticipated from the slightly acidic character of H5 in solution (16). It is of interest to note that in some cases the extent of H8 exchange showed significant variation between the molecular species and the corresponding protonated base fragment ion, further pointing to an exchange m e c h a n i i specifically in the thermospray jet. For example, in 1-methyladenosine 65% of H8 is exchanged in the nucleoside and 51% in the base ion, while in 2'-0-methylcytidine, the H5 exchange levels are 30% and 68% ,respectively. In all casea, the fragmentation patterns of the nucleosides show no deuterium exchange at carbon in the sugar moiety. Applications. The D20-thermospray exchange method can be applied principally in two areas: in studies of reactions associated with thermospray ionization and as an element of structural characterization. A n example of the former is given by the characteristic sugar (S) fragment ion, (S-H).NH4+, formed in the presence of ammonium acetate buffer (IO), which permits distinction between the three sugars that naturally occur in nucleic acids ribose, 2-0-methylrib, and 2-deoxyribose. An analogous ion, (S-H)+, is formed in the electron ionization mass spectra of hydroxyl-blocked ribonucleoside derivatives (In,in which case the hydrogen lost is derived specifically from C2'. However, when mass spectra of nucleosides me acquired under thermospray (Ts)ionization conditions with deuteriated solvenb, mechanisms involving loss of H from carbon were excluded by observation of the sugar ion at m/z 169 (eq 1) rather than m/z 170, pointing to an ion isomeric with that shown in eq 1in analogy to stable neutral molecules having such bicyclic structures (18). 0
'\, W
DO
+--+
I / '
Z
I
'
DO
1
OCH3
OCH3
m/z 169
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
