values found by the spectrophotometric method were 0.012% (in duplicate) magnesium oxide for sample 81 and 0.022 and 0,02370 for sample 93. The fluorometrically determined values are 0.005 and 0.009% magnesium oxide for sample 81 and 0.022 and 0.023% for sample 03. The agreement by the present methods is thus considered satisfactory. COMPARISON OF FLUOROMETRIC AND SPECTROPHOTOMETRIC METHODS
The sensitivity of the spectrophotometric method is 0.0019 y of magnesium per sq. em., indicating that 15.5 x 10-5 pniole (3.8 X y) of magnesium per ml. can be detected using a 5-em. light path. The limit of detection of the fluorometric method is 7 X 10-6 pmole (1.7 X y) of magnesium per ml. The sensitivity of the fluorometric reaction is approxiniately double that of the spectrophotometric reaction. However, because of the extreme sensitivity of the fluorometric method, it magnifies slight variations in technique.
Effect of Other Ions. In t h e course of their work t o determine t h e stiucture and characteristics of t h e fluorescent metal chelates of t h e o,o’-dihydroxyazo compounds, Freeman and White (2) tested the following cations (2 pmoles per 25 ml.) for the formation of fluorescent or colored chelates in dimethylformamide: Al+++, Ga+&*, UOZ++, Th’+++, I i+, Be++, Si++, Cu++, Cd++, Co++, Ca++, Sn++++, P b + + , ZnAL, A h L L , F C ~ +La+++. +~ ZrO++, In+++, N d + + + , I+-++, and A&+. Results of their tests for fluorescent chelation with bissalicj lidene-ethylene diamine indicated that only Be++, I n + + + , and Zn++ formed fluorescent chelates in n.enk base and that yelloncolored complexes were formed by LT02++, Xi++, Cd+’, Co++, Ca++, En++++, Pb++, Zn++, Fe+++, and In+++. C U + T developed a decp blue color. These ions must be separated from magnesium when trace amounts of magnesium are determined. Because the separations necessary to remove
Be++, In+++, and Zn++ from magnesium nould separate most othcr cations as nell, no further studics ncre made n i t h other ions except for sodium chloride and 1 otassium chloride. Both of these salts arc’ practically insolublr in dimcthylformamide and had no vffrct on the msgnesium-l~i~~alicylidcnc-c.th~~1enediamine color or fluoresccnt $1 7tems. LITERATURE CITED
(1) Cuttith, Frank, IYliite, C. E., X i a i ~ . CIIEhI. 31, 208i (lclz!)). (2) Freeman, 11. C., Ktiite, C. E,, J . .1m. Chem. SOC.78, 2678 (1956). ( 3 ) Vosburph, IT-. C., Coopcr, G . R., Ibi(l., 63.437-42 f 1041). (4) fVhita, C: E., Hoifman, n.E,, 3Iagrv, J. S.,Jr., Spectiochim. ~ I c t a9 , 105 (1957).
RECEIVED for rf,viex J a n u : ~ r y29, 1950. .4cceptcd Geptemhcr 8, 1959. ‘rakrn from a thesis by Frank Cuttitta in partial fulfillment of the rrquirenieritP for t,he degree of master of science, Lniversity of Maryland, June 1958.
Spectrophotometric Study of the MagnesiumBissaIicy1ide ne-ethy le ned ia mine System FRANK CUTTITTA’ and CHARLES E. WHITE University o f Maryland, College Park, Md.
b Magnesium reacts with bissalicylene ethylenediamine in N,N’-dimethylformamide to yield a yellow complex. A spectrophotometric study was made on the reaction as used for the determination of trace amounts of magnesium. Maximum absorbance was obtained a t 355 mp when the solution contained 0.25 ml. of O.5M isobutylamine per 25-mi. volume. A complex having a metal to ligand ratio of 1 to 1 i s formed. The absorbance y obeys Beer’s law, and 3.8 X o f magnesium per mi. can b e detected i f a 5-cm. light path i s used. A value of 13,450 liter mole-l ern.-' was obtained for the molar absorptivity. The procedure of analysis i s simple and results show a standard deviation of less than 1%.
-
A
ISVCSTIGATION of hissalicylidene-ethylenediamine as a fluorometric reagent for the determination of magnesium has been reported (10). This present papm appraises the system
N
1 Present address, u. s. Geological Furvey, Washington 25, D. C.
from a colorimetric standpoint. Coupled n-ith suitable separations, the usefulness of the reaction can he estended t o permit the determination of microgram amounts in complex materials. The apparatus and reagents are detailed in (10). STUDY OF EXPERIMENTAL VARIABLES
Studies m r e made of the factors considered in selecting the conditions recommended in the gcneral procedure. The data shon-ing the effect of the esperimental variables on the rcnction between magnesium and bissalicylidene-ethylenediamine n-ere obtained by adopting a standardized order for the addition of reagents. Acidity. To study t h e effect of acidity on t h e absorbance of the reagent and of the niagncsiuni complex a t 355 mu, varying amounts of 0.5.U solution in A-,.~’-dinieth!-lforrnaniide of isobutylamine and of glacial acetic acid were used as base and acid, respectively. The data, plotted in Figure 1, show that the reagent has a relatively constant absorbance (curve A ) while the absorbance of the solution
containing the niagncsiuni chelste (curve B ) is markedly affected by changes in acidity. The point of maximum difference in absoibance between the solution and the reagent blanks occnrred v-1ic.n 0.25 ml. of 0.5M isobutylamine per 25 nil. w : i s nsed. Accordingly, this concentration of base was selected for a11 suhscqumt work. Effect of Concentration of Magnesium. T o test the effect of magnesium on absorbance: a series of solutions m s prrpnred in which t h e magnesium concentration was increased t o a point of p e n t excess over t h a t of hissalic!-lidenc-ethylcnedinminc. I n Figure 2, t h e a h x b a n c e of thrsc solutions is shown ns a function of t h e magnesium concentration. Tiir, horizontal portion of the ahsorhancc curve is represent:itive of solutions t h a t arc? niistures of a constant amount of complex n-ith mrying amounts of mngncsiuni. When the ningnc?iiim content reached about 1.5pmoles. further increase up t o 6 pmoles of magnesium rcsultcd in less than a 37, increase in the absorbance a t 355 nip. Effect of Bissalicylidene-ethylenediamine Concentration. T h e effect of VOL. 31, NO. 12, DECEMBER 1959
2087
I". /
i
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, A C E T C ACl3-
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3
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.
IO3
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2
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Figure 1. Effect of acidity on absorbance at 355 mp
variation of reagent concentration was studied by preparing two series of solutions. I n the first series, the magnesium concentration of each solution was kept constant (1 pmole of magnesium as the chloride in D h l F per IO-ml. volume) while the reagent concentration varied, ranging from 0 t o 5 pmoles per 10-ml. volume. I n the second series, each solution contained varying concentrations of reagent alone ranging from 0 to 5 pmoles per IO-ml. volume. The differences in absorbance a t 355 mp (net absorbances) between the magnesium solutions and the corresponding reagent blanks were used to prepare the curve in Figure 3. They reached a constant value, indicating a constant amount of complex, when 2.25 pmoles or more of reagent were present. On the basis of these data, 2.5 pmoles of bissalicylideneethylenediamine was selected for use in the recommended procedure. Temperature. D a t a show t h a t changes in temperature d o not noticeably affect t h e absorbance readings over t h e range from 1' t o 49O C. Therefore, t h e effect is negligible for such variations as generally occur in room temperature over a period of a few hours under laboratory conditions. However, at 49" C., a 1-minute exposure to ultraviolet light decreased the absorbance readings of the reagent blank from 0.075 to 0.045, of the 0.5 pmole magnesium solution from 0.335 to 0.305, and of the 1.0 pniole magnesium solution from 0.582 to 0.545. Exposure to Ultraviolet Radiation. Exposure t o ultraviolet light affects t h e stability of the color. A t room temperature, absorbance measurements were essentially constant for the first 30 minutes. However, after the initial 30 minutes, the absorbance of the chelate decreased almost linearly a t a n average rate of 0.026 absorbance unit per hour. The absorbance of the rea2088
ANALYTICAL CHEMISTRY
gent blank was also essentially constant for the first 30 minutes. Then, the absorbance of the blank (2 pnioles of reagent) increased linearly at the rate of 0.002 absorbance unit per hour. Stability of Complex. Having established t h e conditions for t h e recommended procedure, t h e stability of t h e color was determined by periodic readings (absorbance at 355 mp) on four stock solutions. T h e stock solutions were left on the laboratory table exposed to the ertificial light of the room. Fresh aliquots were used for each absorbance measurement (355 mp) . These results indicate that the yellow color develops almost instantly. The greatest total change in absorbance during 4 hours was less than 1'%, which is within the accepted experimental error for spectrophotometric work. The complex is stable, provided the solutions are not exposed to ultraviolet light. I n the recommended procedure, 15 minutes were allowed for the color to reach equilibrium before the absorbance was measured.
-
I
6-
I
,
l
,
l
,
l
,
l
1 2 3 4 5 MICROMOLES MAGNESlllM P E R IO M L VOLUME
,
I
6
Figure 2. Effect of magnesium concentration on absorbance at 355 rnp
Effect of Water. T h e possibility of a shift in t h e absorption peak of dimethylformamide solutions of bissalicylidene-ethylenediamine n-hen water is present was investigated. Absorbance measurements for wave lengths between 300 and 420 mp were made on a series of solutions containing 2 pmoles of reagent and amounts of water ranging from 0 to 70% (v./v.). The absorbance data are presented as a family of curves in Figure 4. An isoabsorptive point (1, 2, e), evident a t approximately 335 mp, indicates the presence of two lightabsorbing components in equilibrium (8,9)-in this case bissalicylidene-ethylenediamine and a complex containing wa t er . The solutions varied from almost colorless to a canary yellow; the color becomes proportionately more intense with increasing water content. The
yellow color of the more dilute solutions [5, 10, and 20% water (v./v.)] suffered no apparent decolorization on standing. -4solution containing 70% (v./v.) water a a s allowed to stand until some decrease in color became evident. Absorbance measurements were taken over the aforementioned wave-length range and the data are shown as curve G, Figure 4. The maxima of both original peaks have shifted from 318 and 396 mp to 330 and 391 nip, respectively. -411 of the absorbance measurements for curves A through F , Figure 7 , were taken before any sign of decolorization mas apparent in freshly prepared solutions. Absorption Spectrum. The absorption spectrum of t h e magnesiuni chelate was determined for a solution n hich contained 10 pmoles of niagnesium for each 2 pmoles of reagent per 15-ml. volume. An excess of the metal was chosen so as to ensure reasonably complete chelation of the organic reagent. Figure 5 shows a spectrophotometric curve for the magnesium chelate (curve B ) and one for a. solution of pure bissalicylidene-ethylenediamine (curve A ) . The data presented in Figure 5 show that the absorption peaks for the magnesium complex (355 mp) and for the reagent (317 mp) are well separated. The region of each of those peaks is particularly suitable for relating absorbance measurements to magnesium concentration. The increase in absorbance due to complex formation can be measured a t 355 mu. The decrease in absorbance due to disappearance of reagent can be measured a t 317 mp, Howevrr, the absorbance measurements presented in Figures 5 and 6 indicate considerably greater sensitivity a t 355 mu. Accordingly, all subsequent work n-as done a t 355 mp. Curves C and C' (identical) show the absorption spectra of diniethylformamide alone and of dimethylforniamide plus 10 pmoles of magnesium per
i
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4
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5
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Figure 3. Effect of bissalicylideneethylenediamine concentration on absorbance at 355 mu
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360 WAVE
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-
absorbance at 355 mp against dimethylformamide in the reference cell. 2. Absorbance measurements with a 1-cm. light path. a. Add 2.5 pmoles of bissalicylidene-ethylenediamine in dimethylformamide. b. Add the dimethylformamide solution of magnesium chloride containing not more than 1.0 kmole (24.32 7)of magnesium. c. The remaining operations are as described in I Cto If.
mp
Figure 4. Spectrophotometric curves showing effect of water on absorbance of the reagent A, C, D, E, F, and G contain zero, 10, 20, 40, 60, 70% water (v./v,), respectively
1 5 ml., respectively. All absorbance measurements were made a t 5 mk intervals in 1-cm. silica cells with water as a reference. Standard Procedure for Determination of Magnesium in P u r e Solutions. On the basis of the results of t h e foregoing tests, the following standard procedure was adopted for the determination of magnesium in pure solutions. T h e solutions were generally prepared in 25-ml. glass-stoppered volumetric flasks. The procedure is also applicable to the determination of trace amounts of magnesium in complex substances. After appropriate separation, the solution containing the magnesium as the chloride is evaporated to dryness on a steam bath, and the re-
__~_
sulting chlorides are dissolved in dimethylformamide. Interfering ions and a comparison of analytical results are shown in the companion paper (IO). 1
Absorbance measurements with a
a. Add 1 micromole of bissalicylidene-ethylenediamine in dimethylformamide. b* Add the solution of magnesium chloride containing not more than 0.20 Pmole (4.864 7) magnesium* c. Add 0.25 ml. of 0.5jfisobutylamine in dimethylfomamide. d. Adjust volume to exactly 25 ml. with dimethylformamidea e. Stopper and mix thoroUghlY* f. After l5 minutes, measure the Of
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I 13
Reaction Characteristics. Absorption spectra d a t a were also used to resolve the reaction stoichiometry of the magnesium color system and to determine the number of complexes formed. These d a t a were obtained from the absorbances of eight solutions for wave lengths between 280 and 700 mp. Each solution contained 2 pmoles of reagent and amounts of magnesium ranging from 0 to 30 @molesper 25-m1. volume. A group of related spectrophotometric curves were plotted and the spectra from 280 to 400 mp are shown in Figure 6. Curve d , nhich represents the absorption spectrum of the reagent alone, has a maximum at 317
5-ci. light path,
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Figure 5. Absorption spectra of reagent and complex in DMF
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Spectrophotometric curves showing isoabsorptive
Curve
A 8 C
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point Magnesium, pmoles None 1 2
Curve
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Figure 7. Determination of composition of complex by the method of continuous variations VOL. 31, NO. 12, DECEMBER 1959
2089
mp. Curves B through E are for various levels of magnesium. When the magnesium concentration reached about 10 pmoles, a single and identical curve ( F , Figure 6) resulted from 10, 20, and 30 pmoles of magnesium. Since excess‘ magnesium has no absorbance at these wave lengths, curve F represents the absorption spectrum of pure magnesium complex which has a maximum a t 357 mp. I n Figure 6, there are isoabsorptive points a t 265, 330, and 410 mp which indicate that equilibrium exists between a single magnesium complex and the reagent (8,9).
YICROMOLES
(
Slope-Ratio Method. T h e sloperatio method of Harvey and Manning (3) was uscd further to support the evidence indicating a 1 t o l combining ratio of metal to ligand in the complex. For this stud). of thc complex, two series of solutions wiw, preparcd for absorbance measuwminnts a t 355 mp. I n the first scries, the magnesium concentration of e:xh solution n-as kept constant and a t a grcxt eyccss (SO pmoles per 25ml. volume) in orrlcr t o ensure complete reacbion of tlis bissalie!-lidene-ethylenediamine i m s r n t to form the chelate. The reagent contrnt of these solutions ranged from 0 to 1 pnio!e per 25 ml. The
2090
ANALYTICAL CHEMISTRY
MAGNESIUM
FER
Slope 1 -Slope 2
LITER
CdfliE 2 )
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JFIVE I :SS MAGNES UV U’CROMOLES I
50
DETERMINATION OF METAL-LIGAND RATIO
Method of Continuous Variations. T h e composition of t h e complex (metal-ligand ratio) n-as determined by the method of continuous variations ( 7 ) . rlhsoibance measurements at 315, 355, and 3iO mQ irere made on 33 solutions. The total Concentration of magnesium and reagent in each solution was kept constant (3 pmoles) TI hile the mole fractions of magnesium and bissalicylidene-ctlI\-leacdlamine were varied. Figure 7 shons values for the function Y ( 7 ) plotted as a separate curve for each of the three nave lengths-315, 355, and 370 mp. The maxima of all the curves in Figure 7 occur whcre the mole fraction of magnesium is essentially 0.5. This indicates that the moles of ningncsium in the complex are equal to thc moles of bissalicyliIt has been dene-ethvlencdiamiiic. proved by x-ra!. cvidcnce (4) that the covalent salicglidelletliomine complexes have a bond from rach nitrogen to the metal atom. This is probably true in the case of this complex. Accordingly, a possible structure for the magnesium chelate 1i:iving a 1 to i metal to ligand ratio is:
OF
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LITER
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Figure 8. Determination of metal to ligand ratio by t h e slope ratio method data resulting from absorbance measurements (355 mp) on these solutions were plotted against the different concentrations of reagent (in terms of micromoles per liter) and are shoxn as curve 1 in Figure 8.
1.35 X = -~
lo4 - 1 1.42 X lo4 1.06 Or essentially 1 :1
This indicates that the molar ratio of magnesium to bissalicylidene-ethylenediamine is 1 to 1. This ratio was further substantiated (10) by a fluorometric modification of the method of continuous variations. Conformance to Lambert-BouguerBeer Law. A plot, presenting absorbance values as a function of t h e magnesium concentration, shows a linear standard curve for both t h e 1-em. and 5-em. light paths. T h e sensitivity of the reaction, as obtained from t h e curve (I-cm. light path) using Sandell’s critrrion (5) of an absorbance difference of 0.001 as the limit of detection, is 0.0018 y of magncsium per sq. cm. This means that 0.00036 y of magncsium per ml. can be detected using a 5-cm. light path. A value of 13,450 liter mole-i em.-’ was obtained for the molar absorptivity of the complex a t 355 my. Reproducibility. Table I shov s reproducibility d a t a based on six standard curves prepared according t o t h e recomniended procedure over a period of 51/2 months. I n each case, reagents less than a month old were used. LITERATURE CITED
Reproducibility of Standard Curves Xet Absorbance ( 1 pmolc ilIg./25 MI.) Difference 0.504 -0.007
Table I.
0.515
+0.004
0.508 -0.003 0.515 +0.004 0.512 - t o .001 +0.001 0.512 Mean = 0.511 Av. deviation = 0.003 Standard deviation = 0,004 % std. deviation = 0 . 8
I n the second series, each solution contained varying concentrations of magnesium ranging from 0 to 1 pmolc per 25 ml. The reagent content of these solutions was kept constant and a t a great excess (50 pmoles per 25 ml.) in order to ensure complete reaction of magnesium present to form the chelate. The absorbance data for the second series of solutions w r c plotted against the different concentrations of mynesiuni (in terms of micronioles per liter) and are presented as curve 2 in Figure 8. The ratios of metal to ligand in the complex may be determined by taking the ratio of the slopes of ciwves 1 and 2 in Figure 8.
(1) Boltz, D. F., “Spectzophotometric
Analysis,” in “Selected Topics in Modern 1nst)rumental Analysis,” ed. hy D. F. Boltz, pp. 155-6> Prentice-Hall, New York, 1962. (2) Brodc, W. R., “Chemicsl Spectroscopy,” pp. 248-50, Wilpy, Yew York, 1943. (3) Harvey, -4.E., Jr., Manning, D. L., J . d m . Chem. Sac. 72, 4488-03 (1950). (4) hfartell, A . E.. Calvin, AI., “Chemistry of the hletal Chelate Compounds.“ p. 266, Prentice-Ilall, S e w York, 1952. (5) Sandell, E. B., “Colorimetric Determination of Traces of 3Ictsls,” 2nd ed., p. 49, Interscience, Xew Yorli, 1950. (6) Steams, E. A , , “i2pplicntions of Ultraviolet and, Visual Spectrophotometric Data,” in “Annlyt,ical Ahsorption Spectroscopy,” ed. by M.G. Mellon, p. 318, Wiley, K e r York, 1950. ( 7 ) Voshurg, W. C., Cooper, G. R., J . Ani. Chem. SOC.6 3 , 437-42 (1941). (8) Weigert, F.: Ber. deut. chem. Ges. 49 (lo), 1496-532 (1951). (9) West, W.,::Spectroscopy and Spectrophotometry, in “Physical Methods of Organic Chemistry,” ed. by A . Weiesberger, Vol. I, Part 11, pp. 1298-9, Interscicnce, Xew Yorli, 1945. (10) White, C. E., Cuttitta, F., ANAL. CHEM.31, 2083 (1959). RECEIVEDfor review January 29, 1959. Accepted Se tember 8, 1959. Taken from a thesis by $rank Cuttitta in partizl fulfillment of the requirements for the degree of master of science, Universit,y of Maryland, June 1958.
