trode is recommended for use with the pH meter. The variatisn with time and buffer solution of the standard potential (EO) of such a n electrode is shown in Figure 3. Ten successive measurements in pH 4.01, p H e.87, and pH 9.18 buffer solutions were miide over a period of 2 weeks. Again, the measurements in pH 4.01 buffer show the largest standard deviation although palladized electrodes were used. Moreover, a slightly lower value for the Eo was observed. Similarly, 10 successive measurements were made on the KO. 39071 saturated calomel reference electrode. Figure 4 shows the stability of such an electrode in p H 6.87 buffer over a time interval of 2 weeks. The standard
deviation obtained verifies the good reproducibility and stability of the carborundum frit junction of the reference electrode. The standard potential of the reference electrode could not be determined accurately in the hydrogen electrode cell assembly since the electrode compartment was too short to accommodate the entire electrode. This caused temperature gradients to exist in the reference electrode internal and its electrolyte. To overcome this diificulty, a modified cell assembly as shown in Figure 5 was used. This cell assembly was immersed in a deeper constant temperature bath to keep the entire electrode at the desired temperature. The results
obtained with this modified cell assembly are presented in Figure 6. The average E ” value is 0.43 mv. higher than the corresponding potential of Figure 4. LITERATURE CITED
(1) Bates, R. G., “Electrometric pH Determinations,” p. 160, Wiley, New York,
1954.
(2) Bates, R. G., Ibid., p. 166. (3) Bates, R. G., Ibid., p. 190. (4) Bates, It. G., Ibid., p. 313.
(5) Bates, R. G., J. Research N B S 66A,
179 (1962). (6) Bates, R. G., Pinching, G. D., Smith, E. R., Ibid.,. 45, 418 (1950). (7) Guggenheim, E. A,, J. Am. Chem. Soc. 5 2 , 1315 (1930). RECEIVEDfor review March 25, 1963. Accepted July 11, 1963. r
Sensitive Method for the Determination ot Submicrogram Quantities of Manganese and its Application to Human Serum ALBERT0 A. FERNANDEZ, CHARLES SOBEL, and S. L. JACOBS Bio-Science laboratories, 7 2330 Santa Monica Blvd., Los Angeles 25, Calif.
b Oxidation of leucomalachite green with periodate in the presence of manganese i s a catalytic type reaction which has been used for the quantitation of submicrogrcim amounts of manganese. There is not, however, linear proportionality between the absorbance at any given time during this reaction and the content of manganese in the system. Experimental data indicated that this reaction i s complicated b y a sutlsequent reaction which destroys the dye. Oxidation of malachite green with periodate in the presence of manganese follows a first order reaction catalyzed b y manganese. This subsequent reaction explains the behavior of the leucomalachite green oxidation curves. A method for determination of manganese in the milliniicrograrn range based on the oxidation of malachite green i s proposed and applied to human serum. The normal level of serum manganese was found to be 0.36 to 0.90 rnHg. per rnl.
T
HE OCCURRENCE of minute amounts of manganese in certain biological materials has prompted the development of a variety of procedures (4-10, 12). The most sensitive of these employs neutron activation analysis, a procedure not practical for most analytical laboratories (2,R, IO). An e vcellent review by Cotzias (S) provides a comprehensive background for this su‘sject. The more sensitive of the chemical
methods involve oxidation of colorless Ieucomalachite green (LMG) to the dye, malachite green (MG). Manganous ions are converted by periodate to a higher state of oxidation and then oxidize the leuco base to the dye. Many times the manganese present would necessarily be consumed if the reaction proceeded through etoichiometric reactions involving Mn+2 or its oxidation products. This then would imply a catalytic effect of manganese upon the reactions which may be written Mn+’> -.
NaIO4
MnOX
LAlG
Mnox -+ Mn+l
and the observed effect would be appearance of RIG. If periodate and LMG are in large excess, the reaction should be zero order and, if Beer’s law is obeyed, absorbance plotted us. time should yield a straight line. This, however, is not the case (1%’). The reaction, therefore, seems t o be somewhat complicated and provided the impetus for this investigation leading to the development of a new method for determination of trace amounts of manganese of the order of 1 to I5 mpg. A simple test with this sensitivity would be desirable for the determination of manganese in human blood serum. EXPERIMENTAL
Reagents. All reagents were rragent grade or the purest commercially available. Water used in preparing all reagents was redistilled from all-
glass apparatus. T h e solutions were stored in acid-washed polyethylene bottles. PHOSPHATE ACETATEBUFFER. NaH2P04.H20(27.6 grams) in water and 12.0 ml. of glacial acetic acid were diluted to 100 ml. with water. Then 21.0 ml. of 1.ON NaOH was added. The pH of a 1 to 4 dilution of this solution was 3.6 to 3.7. LEUCOMALACHITEGREEN (p,p’-
tetramethyldiaminotriphenylmethane), obtained from Hopkins and Williams, Ltd. Fifty milligrams was dissolved in 100 ml. of 0.5% (v./v.) HCI. The reagent was stored in a refrigerator and discarded after one week. MALACHITE GREEN, obtained from Matheson, Coleman and Bell. Ten milligrams was dissolved in 100 ml. of water, and discarded after one week.
MANGANESE STANDARD
SOLUTION,
5 pg. per 100 ml. MnS01.H20 (30.8 mg.) was dissolved in 1 liter of 0.1N H2S04; 0.5 ml. of this solution plus 0.5 ml. of 0.1N H2S04 were freshly diluted to 100 ml. with water. Methods. All glassware used was treated with 6N HC1 a t 60’ C. for about 30 minutes and then washed with redistilled water. Experiments designed t o study the kinetics of t h e reaction were performed as follows. Test tubes were set up to contain 0, 0.02, 0.05, and 0.10 pg. of manganese. The folloning then were added in order: 1 ml. of 1N HC1, 7.5 ml. of phosphateacetate buffer, a volume of 1 N NaOH determined to give a final pH of 3.6, and 1.0ml. of leuco-base or dye. Water was added to 14.5 ml. and then 0.5 ml. of 1% sodium periodate solution was added to start the reaction. The tubes VOL 35, NO. 11, OCTOBER 1963
1721
MII.IU~ES
Figure 1. Catalytic oxidation of leucomalachite green by periodate in the presence of manganous ions
MINUTES
Figure 2.
were mixed after the addition of reagents, stoppered with plastic covered corks, and placed in a water bath at 26' C. Readings were taken hourly a t 620 mp with water as a reference blank in a Beckman Model B spectrophotometer equipped with a test tube adapter. Treatment of Serum. Blood obtained by venipuncture was allowed t o clot and centrifuged. For analysis, 3.0 ml. of serum was placed in 25X 100-mm. T'ycor test tubes (Corning Glws KO.7915) which contain less than 1 p.p.m. of manganese. (Boroqilicate glass contains too much manginrse to be used i n this part of the procedure.) For recovery experiments, additional tubes were set up containing qerum, and 0.2 ml. of manganese solution (equivalent to 10 mpg. of manganese) was added. The tubes were placed in an aluminum heating block and the samples were dried at 116" C. in about 2 hours. Bshing of dried residues was performed in the same tubes in a muffle furnace at 540" C. ilfter 1 hour the cooled residues were moistened with 1 drop of water, redried, and reashed. After a n additional wetting, drying, and reashing treatment, the ashes were completely free of visible carbon. They were dissolved in 0.3 ml. of LL' HCl; the tubes mere stoppered with Saranwapped corks and heated in a TO" C. water bath for 5 minutes. After the tubes cooled to room temperature, 2.30 ml. of buffer, 0.85 ml. of water, sodium hydroxide (0.45 ml. was used to achieve a p H of 3.6), 0.3 ml. of MG, and 0.3 ml. of 0.5% sodium periodate were added as described in the general method above. -1 blank, devoid of sample ash, was prepared in the same way (including the HCl), and standards containing 5, 10, and 15 mpg. of manganese were set up by substituting 0.1, 0.2, and 0.3 ml. of manganese standard for part of the water. -4 sufficient volume of each solution was transferred t o the cuvette tubes u ed for spectrophotometric reading. These tubes were stoppered with Sarann-rapped corks and placed in a 26' C. water bath. The hourly spectrophoto1722
ANALYTICAL CHEMISTRY
Test for a first order loss of manganese
metric readings were commenced 3 hours after the addition of sodium periodate and were taken for 5 hours. The absorbances were plotted on semilogarithmic paper us. time. The slopes of these lines mere used in calculating the manganese concentrations, The total volume of solution for the catalytic reaction using serum was reduced to 0.3 of that employed in the preliminary investigations. This was necessary to achieve the sensitivity required in assaying levels of manganese found in convenient volumes of serum. Concentrations of all reagents in the final solutions were not altered.
concentration of 1In. This was not observed (Figure 2 ) and the hypothesis of a n irreversible first order loss of Rfn was abandoned. A second possible explanation for the behavior of the system as described by the curves in Figure 1 may be made with the assumption of a secondary reaction culminating in destruction of dye. In that case, the low concentration of dye justifies the assumption of a first order reaction. Then, using PIIG instead of LRIG, there ought to be decolorization of the dye. If the reaction is catalyzed by manganese, then
RESULTS A N D DISCUSSION
Kinetics of Reactions. If the concentrations of LMG and NaIO, are in large excess relative t o products formed, the reaction LRlG
+ 103-
Mn+2
+ RIG
+ other products
should be zero order. and the plot of absorbance (which in the case of hIG is proportional to concentration) against time must he linear. The plot of experimental data of absorbance v s . time, however, was not linear (Figure 1). There wa4 an apparent loss of velocity in the formation of the LIG color with time. This deceleration in the reaction might be logically attributed to an irreversible loss of manganese in its catalytically active form. Because of the low concentration of manganese, that supposed loss should be a first order reaction, and in that rase it may be shown that
\There z is the absorbance in a test solution (to which has been added) at the time t , x, is the absorbance in the blank at the iaiiie time, and 01 and kl aic cwnztaiit-. 'l'hen, plotting log (z - 2,) t us. t should give a straight line for each
where x is the absorbance of the solution and ICl a constant proportional to manganese concentration. Actually, kr should be proportional to the active form of manganese, ?\Inox-i.e., the osidized form. A t the outset, only &Ini2 is present and it is therefore necessary that an initial lag period pass until a constant value for the relationship [?.In0"]/ [Mn+2] be attained. Equation 2 is valid only after the lag period and when integrated beta een t, and fB
therefore
Equation 4 describes the slope, k,, of the straight line obtained by plotting the readings taken periodically 2)s. the corresponding times. These slopes should he proportional to the Mn content of the system and also, therefore, to b 6 [Mnl where b ia the slope when no manganese has been added to the system. This
+
1 0 L 09 -
08 07 -
06
-
-- 0 5 0)
8 m 04 01
303
v
\
\m
8 33 0 i
\
M Mn W e d
02 -
0,
J2
4
,
\, 0
1
2
C:
4
'
s
6
I
7
8
9
10
Figure 3. Catalytic oxidation of malachite green by periodate in the prescsnce of manganous ions
conclusion was confirmed experimentally as shown in Figure 3 and by the linear relationship, shown in Figure 4. The deceleration in catalytic 0: idation of LMG can now be explained by this subsequent reaction. Furthermore, after a certain period :Ldecrease in absorbance may be expected, this period being shorter for higher concentrations of manganese. This was experimentally demonstrated (Figurc 5 ) . A similar subsequent reaction ca,n also esplain the observations of Single (11) using tetrabase in place of LMG. The method now proposed depends on the first order reaction MnOr
MG -+ coloi~lessproducts
and is suited to analytical determinations in the range of 0.2 to 3.0 mgg. of manganese per ml. of final solution read. An operating tempera ure of 26' C. and pH of 3.6 were chosen because higher temperatures and pH's increased the rate, thus limiting the range over which the reaction could he followed. Reaction velocities were too low at lower temperatures and pH";.
Figure 4. Relationship between the slopes kl and manganese concentration
Table I. Normal Values for Manganese in Human Blood
Author Bowen ( 8 )
Method Activation analysis
Papavasiliou and Cotzias (IO)
Activation analysis
Bentley, Snell, and Phillips (1) Kehoe, Cholak, and Storey ( 7 )
Microbiological Spectrographic
Normal Levels in Human Serum. Normal values were determined on 12 laboratory workers. T h e 95% normal range obtained by calculation of the mean and standard deviation was 0.36 t o 0.90 mpg. per ml. Data for males and females showed no significant difference by the t test (57% level); therefore, the results were pooled. A comparison of these normals with those obtained by other methods is shown in Table I. The divergence of these data from those published by others may be due to a variety of causes, including 0.8
To calculate the level of manganese in a sample of serum, Equation 4 was used in the following form Kz - b Ka - b
x
8
where K , is the slop(?for the sample, K , is the slope for the standard containing S mpg. of manganese, and b is the slope of the blank. Recovery. Mangrtnese equivalent to serum concentrations of 3 mpg. per ml. was added t o each of 7 serums. The mean recovery was 76T0 (range 74 to The above normal values were not corrected for recovery.
Mn/mLa
14-34 (B) 11-25 (S) 9-15 (B) 2-3 (S) 50-80 (€3) 0-240 (a)
0-100 (PI 35-120 (P) 0 36-0.90 (S)
Miller and Yoe ( 9 ) Spectrophotometric Fernandez, Sobel, and Jacobs (This work) a B = whole blood; S = serum; P = plasma.
APPLICATION TO SERUM
mpg. Mn =
Mpg.
specificity of methods and contamination of sample preceding and during anzlysis. Interference by Other Ions. The following ions were tested in t h e method for interference. T h e figure after each ion indicates the concentration of the element in micrograms per milliliter equivalent t o one milliliter of serum. These ions are not likely to be found in serum in concentrations greater than those given. Therapeutic levels of certain elements ale included. Fe+2, 10; Cu+*, 5 ;
1
Figure 5. Reaction of leucomalachite green with periodate in the presence of manganous ions over an extended period
HOURS
VOL. 35, NO. 1 1 , OCTOBER 1963
1723
Sn+2,0.6; AsOe-, 3; Ba+*, 3; Ag+2,3; SbCS,15; 0.6; F-, 15; I-, 30; 0.6; Ca+2, 300; VOS-, 0.6; Br-, 300; Ni+Z, 1.5; Sri-2, 0.6; 1.5; Li+, 0.6; Zn+*, 15; Pb+*, 15; .41A3,15; Bi+3, 108. Yo interference was observed except with iodine, where rapid partial decolorization was observed during the lag period after which the interference ceased-i.e., curves in Figure 3 are displaced downward without changing their slopes. Levels of 0.3 pg. of I - per
ml. of serum (normal total serum iodine is less than 0.1 p g . per ml.) do not produce any effect. LITERATURE CITED
(I) Bentley, 0. G., Snell, E. E., Phillips, P. H., J . Biol. Chem. 170,343 (1947). (. 2.) Bowen, H. J. M., J . Nucl. Eneralr -3 , l S (r956). (3) Cotzias, G. C., Physiol. Reo. 38, 503 (1958). (4) Djaniond, J. J., ANAL.CHEM.28, 328 (1956). (5) Fore, H., Morton, R. A,, Biochem. J. 51 , 594 (1952).
(6) Gates, E. M., Ellis, G. H., J. Biol. Chem. 168, 537 (1947). (7) Kehoe, R. A., Cholak, J., Storey, R. V.,J. Nutr. 19,579 (1940). (S) Meinke, W. m7., Science 121, 177 (1955). (9) Milfer, Mdfer, D. O., Yoe, J. H., Anal. Chim. Acta 26, 224 (1962). (10) Papavmiliou, P. S., Cotzias, G. C., J . BWZ. Chem. 236,2365 (1961). (11) Single, W. V., Nature 180,250 (1957). (12) Yuen, S. H., AnaEyst 83, 350 (1956).
RECEIVED for review July 5, 1962. Resubmitted May 21, 1963. Accepted July 17, 1963.
Fast Qua Iita tive a nd Qua ntita tive Microanalysis of Plasticizers in Plastics by Gas Liquid Chromatography JAVIER ZULAICA' and GEORGES GUIOCHON France Ecole Polytechnique, Laboratoire du Professeur 1. Jacqui, 17,rue Descartes, Paris (S),
b Heavy esters used as plasticizers, such CIS dioctyl phthalate or dioctyl adipate and related compounds, may be analyzed on gas chromatographic columns at 200" to 240" C. using SE30 or poly(neopentylg1ycol adipate) on glass beads. Use of retention indices greatly helps in identifying the compounds eluted from the column. A special technique of mild pyrolysis of the sample in the injection port of the apparatus allows analysis of the plasticizers without any kind of extraction step. Good qualitative and semiquantitative results may be obtained.
P
are very commonly used t o increase flexibility, workability, or distensibility of plastics or elastomers. The more important compounds used are esters of aliphatic dicarboxylic acids (oxalic, succinic, adipic, suberic, and sebacic acids), phthalic and phosphoric acids, and of aliphatic alcohols from C1to GO, mainly n-butyl and 2-ethylhexyl alcohols. The analysis of these compounds must be made on elaborated materials which may contain from a few per cent to as much as 50% of plasticizers. Conventional chemical methods of analysis which include solvent extraction, saponification, and identification of acids and alcohols are time consuming and quite difficult. Little information is available for the analysis of plasticizers by mass spectrometry. Infrared (IR) spectrometry is quite useful for identifying the major component of a mixture, but like all these methods 1 Fellow of Centre d'Etudes des Matibres Plastiques, 21, rue Pinel, Pails, France. LASTICIZERS
1724
ANALYTICAL CHEMISTRY
does not seem efficient enough to distinguish between mixtures of closely related compounds such as n-butyl and n-hexyl phthalate, or n-butyl suberate and n-hexyl adipate (4, 10, 11). However, it is sometimes possible to use this method without the lengthy extraction step. Polarography (9) and fluorescence (2) were limited until recently to phthalic esters, and were also time consuming. Paper chromatography does not seem to have given good results (3, 6, 7, 8 ) ; it is not specific enough to allow direct identification, and does not give enough sample to get a n IR spectrum. Column chromatography separates sufficiently large samples but is slow and needs a large amount of material. Gas chromatography theoretically allows the separation and identification of all these esters, but many difficulties arise and no study of the whole mass of cited esters rvaa made until recently. Bassemir (1) gave some analyses of phthalates as example of an application of gas chromatography to the separation of high molecular weight compounds; Lewis and Patton ( l a )
C A
I
gave retention data for a number of phosphates, phthalates, and related compounds on Apiezon K a t 283' C., using short columns with high load (30% on Celite 545). A large volume of solute must be used (10 pl.), and the low vapor pressure of these components results in important leading of the peaks because of over-loading of the column, Cook et al. (6) used a very short column (9 inches) of 25% silicon grease to obtain the separation of dibutyl-, butylbenzyl-, and dibenzyl phthalates. Zulaica and Guiochon discussed (15, 16, 18) the use of low-loaded columns to analyze the cited esters. Using 0.5% of silicon gum SE30, or of poly(neopentylglycol adipate) , on glass beads etched by hydrofluoric acid, they showed that the elution of heavy esters boiling up to 400' C., under atmospheric pressure, was possible at 200" to 240" C.; no loading is naticed, provided a sufficiently small sample is introduced (0.05 to 0.20 pl.). The behavior of these columns, and especially the reproducibility of retention data, was studied. Retention volumes and retention indices
E $'
\
G
Figure 1.
Diagram of electrical circuit for pyrolysis unit
