instrument the fluorescence emission passes through a monochromator ,maximum intensity a t 500 mp) onto a phototube. Rlultiplier phototubw R.C.A. Xos. 1P28, lP21, and iP22 nere used in these tests. The tube response wna measured GII a microImmeter 07% .worded on a Moseley recorder. Sinw the phntotubcs have I response curve which -iaries n i t h wive length, the recorded fluorescence emission spectra must be c o r r e c t d . To obtain the standard c'irres for these tubes a previously standardized 500n a t t 11I:izda projection bulb was placed about 16 feet from the instrurner!t eo that the light passed directly through the curette compartment to the emission monochromator. A '/er-inch slit 'ras used before the monochromator and the response of the tubes was read on the microammeter a t 5-mp intervals Irom 350 to 700 mp. Since the photorubes have glass envelopes, no d u e s '!ere recorded below 350 mp. The corrections become so large with the lP28 and 1P21 tubes over 610 rnp and the lP22 tube over 660 mp that
the results be>onri t i P.P u ints ire :lot considered reliaijie. r h P vaiitlity of the correction f:,ctorc 6 r the exciting source is ao cein and nianv
'iic
LITERATURE CITED
Because all xf of the nrc, all gratings, and all phototubes may give i s e r e n t results, the correction factors must lie determined for each spectrophotofluorometer. Samples of the correctim factors obtained by the describaa nethods are recorded in rabies I and I1 to give an klea of the order of corrections involved. S o attempt should be made to use these figures with other instruments, because of the variable conditions indicated above. ACKNOWLEDGMENT
The authors express their appreciation to the Nntionai Science Foundation
(1) Furtune, W. B., 1fe11011, M. G., I h D . E X G . CHEhI., . i N A L . ED. 10, 60 (19381. ( 2 ) Hatchard, C. G., Parker, C. A., Proc.,Roy. Soc. A 235, 518 (1056). ( 3 ) h r k e r , c. .I.,.valure 182, 1003 (1958). ( 4 ) Parker, C. d.,Proc. Roy. Soc. A 220, 104 (1953).
(5) Parker, C. A., Barnes, W. J., Analyst
82, 606 (1957). ( 6 ) Weber, G., Teale, W. J., T~ans. Faraday SOC.54, 640 (1958). (7) Vhite, C. E., Ho, ,M., Weimer, E. Q . , Spectrochzm. Acta, in press.
CHARLES E. WHITE MAYHo E. Q. WEIMER Department of Chemistry University of Maryland College Park, Md.
Separation of Halogenated Phenols by Paper Chromatography of p-nitrobenzenediazoniurri fluoborate is), prepared by diazotizing p-nitroaniline and reacting rhe diuonium salt with 40% fluoboric acid. The fluoborate was dried and made into 1% solution in water as needed for use. A second spray solution was 5tT potassium hydroxide in methanol. Individual samples of the six phenols were prepared (1 gram of phenol per liter of benzene) and 2 - 4 . samples were applied to a sheet of Whatman No, 1 chromatography paper. The
SIR: TTTo-dimensional paper chromatography m s successfully used for the separation of the ortho and para isomers sf chloro-, bromo-, and iotlophencls. TKO developing solvents were used: Solvent 1 consisted of benzene, acetic m d , and water in 2:2:1 proportions by volume (I), and solvent 2 mas hydrochloric acid, 20% wv./v. Color development was produced by the use 1 Present address, University of Kansas, Lawrence, Xan.
Table I.
Compound o-Chlorophenol p-Chlorophenol o-Bromophenol p-Bromophenol o-Iodophenol p-Iodophenol
440
0
RI Values of Solvent 1 0,671). 70 0.07-0.16
0.45-0,48 0.31-0.32
0.91-0.94 0.90-0.94
ANALYTICAL CHEMISTRY
LITERATURE CITED
Halogenated Phenois
Rf Range
Solvent 2 0.72-0.83 il .70-O,83 0 . 8 7 - 0 .88 0.80-O. 83
0.8&0.90
9.54-0.57
system was subjected to two-dimensional chromatography, first with solvent 1, then at right angles with solvent 2. The individual spots were made visible by spraying with the w-nitrobenzenediazonium fluoborate solution, drying, and then spraying with potassium hydroxide in methanol. It was difficult to record the color of the spots photographically, because the spots faded rapidly. The range of RI values for several trials and the colors of the spots are recorded in Table I. Separations were made a t 25' C.
Color of spot Light red Light red Dark red Dark red Dsrk red Dark red
(1) Bray, H. G., Thorpe, M. V., White, K., Biochem. J . 46, 271-5 (1950). (2) Freeman, J. H., ANAL. CHEM. 24, 958 (1952).
HAROLD S. CHOQUILL E. BISSINQ~ DONALD Chemistry Laboratory Fort Haya Kansas State College Hays, Kan.
