The adamantane was separated from the distillate b y crystallization a t - 30 O C. and vas further purified by recrystallization from acetone. The identity of the material was established from its freezing point, 269" C., in good agreement with the values reported by Larda, and from a comparison of its infrared spectra with that of a synthetic sample synthesized by P. It. Schleyer, Department of Chemistry, Princeton University, Princeton, N.J.
0
L
-
7
1
L 8
1
9
Figures 1 and 2 show the infrared curves of the two samples of adaa_-mantane super-imposed on one another. -------1 ' ; L--____~_ - in the origiThe amount of adamantane 10 I/ 12 13 14 15 nal petroleum is estimated tobe0.0@04% Wcve Length in Microns b y volume.
Figure 2. Superimposed infrared spectra of samples of synthetic adamantane (solid line) and adamantane from petroleum (dotted line)
LITERATURE CITED
In CS?, 0.15 g./ml., cell 0 . 2 mm.
(1) Landa, S., HQa, S., Erddl u. Kohle
and H&la ( 1 ) isolated and estimated the amounts of adamantane in several crude petroleums by means of the solid molecular compound which it forms with thiourea. ISOLATION OF ADAMANTANE
=Idamantme has recently been isolated from the representative petroleum (4) of the -4PI Research Project 6. Iso-
lation of adamantane was effected as follows: At the beginning of a n azcotropic distillation, T\ ith the perfluoro chemical, jC4F9)31\7r of a concentrate of cvcloparaffins'having a normal boiling point near 190" C.. crvstals of adamantane collected in the condenser and in the tubing at the head of the distilling column. These crvstals \vere sublimed into the receiver biappropriate heating.
FIuo romet ric Stu dy of the ethy lenedia mine System
11,698 (1958). Landa, S., Mach&c'ek, V., Collection
(2)
Czechoslov. #3).
Chem.
Communs. 5 ,
1
elog, V., Seinwth, R., Ber. 74, (1941). mini, F. D., &lair, B. J., Streiff, A. J., "Hydrocarbons from Petroleum," Reinhold, Yew York, 1953. L
RECEIVED for review September 25, 1959. Accepted September 29, 1959. Investigation performed as part of the work of the .4merican Petroleum Institute Research Project 6.
M a gnesium-Bissa Iicy1ide ne-
CHARLES E. WHITE and FRANK CUTTITTA' Universify o f Maryland, College Park, Md. Magnesium ions combine with bissalicylidene-ethylenediamine in slightly alkaline N,N'-dimethylformamide to form a highly fluorescent complex which serves for the determination of The trace amounts of magnesium. yellow complex fluoresces blue when irradiated with ultraviolet light. The chelate shows maximum fluorescence excitation a t 355 mp and has a fluorescence emission maximum a t 439 mp. A complex having a metal to ligand ratio of 1 to 1 i s formed. The method i s sensitive to 7 X 10-6 pmole of magnesium per ml. The analysis of Bureau of Standards samples with the reagent showed excellent correlation between the fluorometric and spectrophotometric procedures.
I
of the structure and characteristics of the fluorescent metal chelates of o,o'-dihydroxyazo compounds Freeman and White ( 2 ) have i i .4 STUDY
shown that bissalicylidene-ethylenediamine reacted with a number of metals t o produce fluorescent or colored complexes. The magnesium complex in N,.2"-dimethylformamide is yellow in color and under ultraviolet radiation produces a bright blue fluorescence. A spectrophotometric study of this system has been made (1). The formula of the reagent is:
This study appraises the fluorescent system and presents data which are basic to the adaptation of the reaction to the quantitative estimations of submicrogram amounts of magnesium in complex materials. APPARATUS A N D REAGENTS
All spectrophotometric measurements
were made with a Beckman D U spectrophotometer equipped with photomultiplier and ultraviolet attachments. The spectral distribution of the fluorescence excitation and emission of the chelate were determined as described by Freeman and White ( 2 ) and White, Hoffman, and Magee (4). The spectral data were further substantiated with a n Aniinco-Bowman spectrophotofluorometer, after appropriate corrections for the light source and measuring units. The American Instrument Co. Polyfluorometer with a 90" horizontal arrangement was used for fluorometric measurenients. The source of exciting light was an H100A4 mercury lamp. The measuring unit, a 931A electron multiplier phototube, was used a t the same sensitivity for all measurements in this study. The potential applied to the phototube was set to produce a deflection of 0.0005 pa. for a 0.01--y per 1 Present address, U . S. Geological Survey, Washington, D. C.
VOL. 31, NO. 12, DECEMBER 1959
2083
and no furt.her purification was needed. Solutions (0.5M) of isobutylamine and of glacial acetic acid in dimethylformamide v e r e used to alter basicit'y or acidity of the solutions. All other chemicals were of reagent grade quality.
ml. solution of quinine sulfate. Corning filter 5860 was used as a primary filter. -4 combination of Corning filters 3389 and 5113 was used to isolate the fluorescent iight (438 mp). Glass-stoppered borosilicate glass test tubes, graduated to 10 or 25 ml., were used for fluorometric measurements. A stock solution of quinine sulfate to be used as a fluorescence standard was prepared to contain 0.1000 gram of reagent grade quinine sulfate in 1 liter of 0.1N sulfuric acid. An aliquot of this solution was further diluted with 0.1N sulfuric acid to yield a solution containing 0.01 y per ml. This was used to set the fluorometer for comparable readings over the entire investigation time. Standard Magnesium Solutions. ..I stock standard solution of magnesium was prepared from high purity magnesium dissolved in distilled hydrochloric acid. T h e solution was evaporated to dryness on a steam bath and the resulting product dissolved in dimethylformamide and diluted so that 1 ml. contained 10 pmoles (243.2 y) of magnesium. Working solutions were prepared from the stock solution by dilution with dimethylformamide. Reagent. Bissalicylidene-ethylenediamine of high purity was obtained from Freeman and White ( 2 ) , who prepared the reagent according to the following procedure: "A solution of 20.0 grams of 707, ethylenediamine in benzene was refluxed until all the water had been removed in a Dean-Stark trap. After addition of 49.2 grams of salicylaldehyde, the solution was refluxed for 1 hour. The product crystallized on cooling and was recrystallized twice from methanol to give a iiyo yield of shiny yellow plates, melting point 124-5' C." A stock solution of bissalicylidene-ethylenediamine containing 10 pmoles per ml. was prepared in dimethylformamide. Working solutions were made from the stock solution by dilution with dimethylformamide. N,.V '-Dimethylformamide, Reagent grade or spectral grade dimethylformamide was found t o be suitable
Varying amounts of 0.5M solutions of isobutylamine and of glacial acetic acid in dimethylformamide were u s d as base and acid, respectively. Data prcsented in Figure 1 show that the point of maximum fluorescence occurs when 0.1 ml. of 0.5M isobutylamine is used. This is also the point of maximum difference between the solution and the reagent, blanks. Accordingly, 0.1 ml. of 0.5M isobutylamine per 10-nil. volume was chosen for the fluorometric study. This is also the optimum concentration of isobutylainine found for the spectrophot'ometric system. Effect of Magnesium Concentration. T h e effect of magnesium on fluorescence was tested with a series of solutions (10-ml. volumes) in which t h e magnesium concentration rvas raised to a level of great e x c m over that of reagent (2 pmoles). Figure 2 shows the fluorescence of these solutions plotted as a function of the magnesium concentration and reflects the progress of complex formation. The plateau of the fluorescence curve indicates the presence of a constant amount of complex \vith varying amounts of exccss magnesium. It also s h o w that amounts of magnesium up to 10 pmolcs have no apparent effect of interference or enhancement on the fluorescence of the complex. Therefore, solutions containing n mixture of the complex and a large cxcess of magnesium are in effcct solutions of the pure complex alone. Effect of Bissalicylidene-ethylenediamine Concentration. T h e effect of the bissalicylidene-ethylenediamine concentration on t h e fluorescence of the complex was determined b y use of tvio series of solutions. I n one series, each solution contained 1 pmoie of magnesium a n d amounts of reagent ranging from 0 to 16 pmoles per 10-ml. volume. Fiuorescence intensities were measured, and the fluorcscencp readings were plotted as a function of the total bissalicylidene-ethylenediamine added (Figure 3). The fluorescence increascs
EXPERIMENTAL
Effect of Experimental Variables on Sensitivity or Stability. Optimum conditions for t h e reaction between magnesium a n d bissalicylidene-ethylenediamine were established b y evaluation of t h e individual effects of t h e more important experimental variables. I n studying these effects, the reagents were added as indicated in the recommended procedure. Acidity. T h e effect of acidity on t h e fluorescent system was determined from fluorometric measurements on two series of solutions. I n t h e first series, each solution contained 2 pmoles of reagent alone in 10 ml. I n the second series, each solution contained 2 pmoles of bissalicylidene-ethylenediamine and also 1 pmole of magnesium as the chloride in dimethylformamidp.
4 ; MAGhESUM
CLiELATE
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-
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0
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ACE-:
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Figure 1. cence
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Effect of acidity on fluores-
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6 MAGhESIUM
5
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Figure 2. Effect on fluorescence of increasing amounts of magnesium a d d e d to a constant amount of reagent (2 pmoles) 2084
ANALYTICAL CHEMISTRY
1
,-A I3
HCP3'*CLES
3F
REASELIT
FEE
F
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Figure 3. Effect o n fluorescence on increasing amounts of reagent a d d e d to a constant a m o u n t of magnesium ( 1 pmole)
n ith increasing reagent concentration until a maximum point is reached. Then, the curve breaks sharply and the fluorescence decreases as the amount of bissalicylidene-ethylenediamine increases. I n the second series, each solution contained 0.01 pmole of magnesium and varying amounts of reagent ranging from 0 to 2 pmoles. Again the fluorescence intensities were measured. Figure 4 shows these fluorescence readings plotted as a function of the reagent added. RIaximuni fluorescence is obtained from solutions containing 0.25 to 1.0 pmole of reagent per 10-ml. volume. The curve shown in Figure 4 indicates that relatively large amounts of free hissalicylidene-ethylenediamine are needed to form the complex. As a compromise toward attaining a balance between the opposing effects of decrease in fluorescence caused by absorption of exciting light by unreaeted bissalieylidene-ethylenediamineand the quenching effect wident M ith increase in reagent and the increase in fluorescence with reagent as a result of the Ian of mas< action, 0.5 pmole of reagent per 10 ml. was chosen for the recommended procedure. Effect of Temperature on Fluorescence. T h e fluorescence of t h e magnesium complex is affected by temperature. T h e fluorescence of t n o solutions Containing the equivalent of 0.5 and 0 . i pmole of magnesium and 2 pmoles of reagent per 10 ml. was nieasurcd between -lo and 50' C. Results of the tests are shonn in Figure 5 . I n the range from 21' to 50' C., the intensity of fluorescence decreases only very slightly with increasing temperature. The readings decreased 6.8% when the temperature was increased from 21' to 50' C. However, the differences in fluorescence corresponding
to the differences generally found in room temperature are small and can usually be neglected. Effect of Exposure to Ultraviolet Radiation. T h e effect of ultraviolet radiation on fluorescence was determined b y periodic measurement of fluorescence intensities of eight solutions. The first solution contained 0.05 pmole per ml. of bissalicylidene-ethylenediamine alone. In addition to the 0.05 pmole of reagent, the other seven solution, contained magnesium varying from 2 X 10-4 to 15 X 10-4 pmole per ml. The fluorescence intensities for each solution was plotted as a function of the elapsed irradiation time. The data show that the fluorescence of the magnesium chelate is markedly affected by ultraviolet light. I n the first half hour, thc fluorcscmcc decreased approxi-
Table
I.
Effect of Water on Fluorescence of Chelate
92 1 1 1 1 ~ (V./V.)
Ii,o (V./\-,)
20
80
Fluorescence, Meter Readings Blank Chelate 17
mately 35% a t the 15 X 10 -4pn101( Iirr ml. of magnesium lcvel, 17y0 a t t l i t i 8 X 10-41evel, and 15y0a t the 2 X level. The fluorescenee of the reagcmt blank was esqcntially constant, thc meter readings gradually decreasing from 12 to 11 ovcr a period of 1 hour. Hoivever, reasonable clxposurc to ultraviolet in radiation during the time required for readings showed no appreciable effect. Time of Standing. T h e stability of t h e fluorescence n i t h time n a s determined by periodic readings on t n o stock solutions prepared according to t h e recommended procedure. T h e first was a reagent blank and t h e second solution contained 0.01 pniolc of magnesium. The fluortwxnce dt%veloped immediately and reproduciblc readings nere obtained over a period of 2 hours. The stock solutions \\ere protected from ultraviolet radiation a t all times. Fresh aliquots were used for each measurement. The differmce botween the initial and the final reading was less than 2% in 2 hours. Tests I , I showed that reagent solutions wcrc 35 3 I5 stable for as long as 3 months if s t o r d UZ~OMO.ES OF R E A S ~ N T PER o M L in the dark. Figure 4. Effect on fluorescence of Effect of Water. Preliminary increasing amounts of reagent added screening tests indicated t h a t Rater to a constant amount of magnesium interfered with t h e fluorescence of (0.01 pmole) t h e magncsium chelate. T o determine t h e extent of t h e effect of water, a series of solutions containing 2 pmoles of reagent, 2 pmoles of magnesium, and varying amounts of Ivater were prepared. Table I presents the fluorescence of these solutions in terms of meter readings. The d a h
Y
-____I
A
Figure 5. Effect of temperature on the fluorescence of the magnesium-bissalicylidene-ethylenediamine chelate A. 6. C.
0.7 pmole magnesium per 10 ml. 0.5 pmole magnesium p e r 10 ml. Reagent blank
:.A\
ELEhC'r
- 'il
II-
-
crc-s
Figure 6. Absorption (A), fluorescence excitation ( B ) , and fluorescence emission (C)spectra of the magnesium chelate in dimethylformamide VOL. 31, NO. 12. DECEMBER 1959
2085
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( N o 81)
SAND
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$2
I 005 VICROMOLES
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KAGNESIUM
010 PER
10
ML.
Figure 7. Standard fluorometric curve show that water has a. marked effect upon the system, the fluoresc,ence of the chelate decreasing with increasing water contcnt and that of the reagent blank increasing n.ith increasing water content. Tlirrefore the procedure for the final analysis is designed to avoid the presenci. of w t e r other t,han the small amounts con(-aincd in the organic reagents. The effect of the water on the fluorescence of the reagent blank niay be due to H-bonding. This may result in a configuration which allows fluorescence. Fluorescence Excitation and Emission Spectra. T h e excitation spect r u m represented b y a plot of t h e exciting wave length against t h e intensity of t h e fluorescent light a t 439 mp is shown in Figure 6. The spectrum shows a n excitation maximum a t 353 mp. Data for the excitation spectrum were not extended into the region of the fluorescence emission (beyond 390 mp), as they would have no meaning beyond this point. Fluorescence Emission Spectrum. A t room temperature, the fluorescence spectrum of the complex under excitation with a 355-mp source was a continuous band of about 150 mp n.idth with a maximum a t 439 mp. The data shown in Figure 6 represent the relative intensity of the fluorescence light over the range of 380 to 560 mp. Figure 6 shows the absorption, the fluorescence excitation, and fluorescence emission spectra of the magnesium chelate. The data confirmed the results obtained with the arrangement of the Beckman DU spcctrophotometer previously described. Standard Procedure for Fluoro-
hfg., pmole/lO M1.
None
0,004
0.006 0.010
2086
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