how well this correlation holds by comparing S. paratyphi B and S. parutyphi B var. Odense and again by comparing S . typhimurium and S . typhimurium var. Copenhagen. At present, we are trying to identify chemically the definitive peaks in pyrochromatograms derived from various types of cellular matter. Simmonds and his associates have identified pyrolysis peaks from geological samples and from bacteria by means of mass spectrometry (11, 12). We conclude that PGLC may lead to a more direct, determinative classification and a more rapid means of cellular identification. In this (11) P. G. Simmonds, G. P. Shulman, and C. H. Stembridge, J. Chromatogr. Sci., 7, 36 (1969). (12) P. G . Simmonds, Appl. Microbial., 20,567 (1970).
respect the compatibility of the PGLC technique with modern computer methods could have practical significance. ACKNOWLEDGMENT
The authors thank W. H. Ewing, Consulting and Research Microbiologist, CDC, for his expert advice and encouragement. RECEIVED for review September 3,1971. Accepted December 14, 1971. Use of trade names is for identification only and does not constitute endorsement by the Health Services and Mental Health Administration or by the U S . Department of Health, Education, and Welfare.
Simultaneous Determination of Sample Concentration and Reagent Blank Max B. Kloster and Clifford C. Hach Water Analysis Research Laboratory, Hucli Chemical Company, Ames, Iowa 50010 THENEED FOR TESTING low concentrations of contaminants has increased in the areas of pure water and reagent grade chemicals. Although a great deal of work has been done on iron and a large number of good reagents are available for iron determinations, it remains difficult to separate the reagent blank from the iron content at low iron concentrations (less than 200 pg/l.). The same problem applies to silica. In many cases the reagent blank is larger or approximately equal to the iron or silica concentration being determined. In the case of ultrahigh purity water determinations, a sample of water is not available which has a lower iron or silica content than the sample being analyzed. It is then impossible to run a reagent blank without knowing the iron or silica content of the water being used. It is also impossible to determine the water concentrations without knowing the reagent blank. Formerly this dilemma could be resolved only by an independent analysis of either the sample or the reagent, but this procedure suffered from the inherent errors of a second analysis. A method has now been established for the simultaneous determination of reagent blank and trace iron in ultrapure water and is being extended to iron determinations in reagent grade chemicals using single buffer-reagent system and to trace silica determinations. EXPERIMENTAL
Apparatus. Colorimetric readings were taken with a Hach Model 1104 D R Colorimeter with a I-inch cell, a Bausch & Lomb Spectronic 20 spectrophotometer with a 1/2-inch cell, and a Hach Model 2400 Expanded Range Colorimeter. Reagents. Reagent grade chemicals were used to prepare the following solutions : ACID REAGENTSOLUTION.Dissolve 5.14 grams of disodium 3(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine (FerroZine) and 100 grams of hydroxylamine hydrochloride in a small increment of water. Add 500 ml of concentrated hydrochloric acid, allow to cool, and dilute to 1 liter with deionized water.
BUFFERSOLUTION.Dissolve 400 grams of ammonium acetate in water, add 350 ml of concentrated ammonium hydroxide, and dilute to 1 liter with deionized water. REAGENT-BUFFER SOLUTION.Dissolve 13.8 grams of disodium 3-(2-pyridyl)-5,6-bis (4-phenylsulfonic acid)-1,2,4triazine (FerroZine) in 1 liter of an iron buffer-reductant formulation (Hach Chemical Co. Cat. No. 2532-00). MOLYBDATE REAGENT.Dissolve 20 grams of ammonium molybdate [(NH4)6M01024.4H20] in deionized water, add 15 ml of concentrated sulfuric acid, and dilute to 100 ml. Do not store solution in glass as silica may leach out and cause high blanks. OXALIC ACIDSOLUTION.Dissolve 10 grams of oxalic acid (H2Cz04.2Hz) in deionized water and dilute to 100 ml. REDUCINGREAGENT.Dissolve 500 ml of l-amino-2naphthol-4-sulfonic acid and 1 gram of sodium sulfite (Na2SOa) in deionized water. Add 30 grams of sodium bisulfite (NaHS03) dissolved in deionized water. Dilute mixture to 200 ml and store in polyethylene. Procedure. DETERMINATION OF IRONIN NEUTRAL SOLUTIONS (1). To a 50-ml sample, add 1 ml of acid reagent solution followed by 1 ml of buffer solution and allow the color to develop for one minute; or to a 50-ml sample, add 1 ml of reagent-buffer solution and allow the color to develop for one minute. If iron is present, a purple color will develop which has a maximum absorbance at 562 nm. The color may be read and compared to a similarly treated standard. The concentration can then be determined by comparing the absorbance of the sample with that of the standard, or as an alternate method, a calibration curve may be prepared and the concentration of the sample may be read from it. The FerroZine-iron complex obeys the Beer-Lambert law between 5 pgjl. and 4 mg/l. If the sample contains magnetite (black iron oxide) or other highly refractory oxides, it may be necessary to heat the sample for 20 or 30 minutes after the addition of the acid reagent or the reagent-buffer solution to effect complete reduction. After heating, the sample is brought to its original volume with deionized water. (1) L. L. Stookey, ANAL.CHEM., 42, 779 (1970). ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
1061
~~~~~~~
~~
~
~
~
Table I. Examples of FerroZine Determinations. Reagent formulation Reagent-buffer solution undiluted 50 ml reagent-buffer solution plus
250 ml concd ammonium hydroxide diluted to 1 1. 50 ml reagent-buffer solution plus 50 ml concd ammonium hydroxide, diluted to 11. 50 ml reagent-buffer solution plus 500 ml glacial acetic acid diluted to 11.
Applications Neutral salts or neutral solutions (sugar, urea, NaCl, etc) Weak acids; e.g., H3PO4, acetic Weak acids; acetic
e.g.,
50
Reagent vol., ml 1
0-10 mg/l. Fe in acid
5
20
0-50 mg/l. Fe in acid
1
24
5
20
Measurement rangeb 0-2 mg/l. Fe
H3PO4,
Sample vol., ml
0-10 mg/l. Fe in
All strong alkalis; e.g., KOH, LiOH, NatC03, Na3P04,etc
alkali 0-50 mg/l. Fe in 1 24 alkali 0-10 mg/l. Fe in 5 20 50 ml reagent-buffer solution plus Strong mineral acids; e.g., acid HCI, HzS04, HClOa, 500 g ammonium acetate di0-50 mg/l. Fe in 1 24 luted to 1 1. "01 acid a The FerroZine-iron color complex exhibits a stable maximum absorbance over the region between pH 4 and pH 9. Therefore, the quantity of any of the pH adjusting materials in the above formulations may be adjusted up or down to neutralize the sample in the desired dilution range. The desired pH in the final solution is between pH 4 and pH 6. * The measurement ranges are applicable to a Hach DR Colorimeter with a 1-inchcell or a Bausch & Lomb Spectronic 20 spectrophotometer with a l/t-inch cell.
e25
I
I I .25
Section
Section
2
+
3
Section
.15 mg/l Fe determined
Figure 1. Reagent-to-sample ratio us. iron content
0
25
5 20
Soaking or rinsing glassware in hydrochloric acid will remove traces of iron which would interfer with this test. Initial determination with new glassware may result in high readings due to the leaching of iron from glassware. DETERMINATION OF IRON IN STRONG ACIDS AND BASES. FerroZine may be formulated in stable, highly basic or acidic solution for use in trace iron determination. The formulations are added to the strong acid or base to neutralize it. The FerroZine color is allowed to develop for five t o ten minutes and is then read at 562 nm to determine the mg/l. iron in the solution. By multiplying this value by the dilution factor, the mg/l. iron is determined in the acid or alkali sample solution. If it is desired, the per cent iron may then be determined in the original sample. Table I shows some represen1062
ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
10 15
15 10
20 5
25 ml acetic acid 0 m l reagent
tative formulations for the determination of iron in various solutions. RESULTS
Determination of Reagent Blank and Iron Content of the Sample. If the ratio of the volume of reagent to the volume of sample is varied and the iron content of the mixtures is determined, a plot such as that shown in Figure 1 will be found. In this case, the iron was determined in glacial acetic acid using a reagent formulation containing 250 g/l. ammonium acetate. Section 3 of the plot is the area where the p H of the final solution is less than 4 and is below the range of maximum absorbance of the FerroZine-iron complex. The curve may
also fall off as it approaches the pure sample because of a shortage of reagent to the point that incomplete color development occurs. The drop-off in section 1 may or may not occur. If the pH of the reagent is between 4 and 9, maximum color will develop, and the curve will be linear to 0 ml reagent. However, if the pH of the reagent is outside this area, maximum color will not develop, and the line will curve off the point where the pH falls below 4 or rises above 9. If the linear section of the curve, section 2, is extrapolated to the points corresponding to 0 ml sample and 0 ml reagent, both the iron content of the sample and the reagent blank may be determined. In Figure 1, the reagent blank is 0.008 mg/l. and the sample contains 0.169 mg/l. iron. When the linear section of the curve has been determined for a given buffer and sample, the iron concentration of the reagent or sample may be determined from only two points on the curve. The only criteria is that the two points be taken in the area where the curve is linear. In the linear section of the curve : Where V s = volume of sample (ml) V R = volume of reagent (ml) VT = total combined volume (ml), V , = V s V, Cs = iron concentration of sample (mgil.) CR = iron concentration of reagent (mgil.) CT = iron concentration of total solution (mgil.) Then the following relation ship exists:
30
20
t
i I 0
I
1
I 2
I
I
3
4
ml of molybdate reagend25 ml sample
Figure 2. Volume of molybdate reagent us. silica value Sample contained 4.5 p g / l . SiO, and molybdate blank is 5.0
pg/l.
+
VsCs
+ VRCR
=
VTCT
By determining the concentration of iron at two different ratios or reagent to sample, the values of V s , V R ,V,, and CT are determined at two different points. The values may be used in two equations following the one above and when these equations are solved simultaneously, the iron concentration of either the reagent blank or the sample may be determined. For example, using the values found in Figure 1, the calculations would be: Ratio 1
Ratio 2
10-ml sample Vsl = 1 0 m l VRl = 15 ml v T 1 = 25 ml CT1 = 0.0725 mg/l.
5-ml sample Vsz = 5/ml V,, = 2 0 m l VT2= 25 ml CT2 = 0.04 mg/l.
VSlCS lOCs
=
CTlVTl
+ VRZCR
=
CTZVTl
+ 20cR
=
(0.04) (25)
or CR
=
(1-.5C,,-/20)
=
Figure 3. Possible blank determination cases
(1)
+ 15CR = 25(.075) = 1.81
VS2cS
5cs
+ VRlCR
Increasing reagent added
(2)
(3) 1.0
(4) (5)
Substituting C R of Equation 2 and solving for C s yields The iron content of the reagent blank can now be determined by substituting the value found for Cs in either Equation 2 or 4 and solving for CR. Cs = 0.169 mg/l.
Equation 2
10(.169)
+ 15CR = 1.81 CR = 0.008 mg/l.
Determination of Silica (2). To a 50-ml sample, add 1 ml of the molybdate reagent and allow the sample to stand ten (2) “Standard Methods for the Examination of Water and Wastewater,” A.P.H.A., A.W.W.A., W.P.C.F., 13th ed., 1971, p 552.
minutes for complete formation of the silica-molybdate complex. If silica or phosphate is present, a yellow color will develop. After ten minutes, add 1 ml of the oxalic acid solution to eliminate the phosphomolybdate complex. Two minutes after the addition of the oxalic acid, add 2 ml of the reducing reagent to form the heteropoly blue form of the silica-molybdate complex. Beer’s law is obeyed between 650 and 850 nm with greater sensitivity at the higher wavelengths. Determination of Reagent Blank and Silica Content of the Sample. In the silica reagents, the blank of the reagents is contributed by the molybdate reagent. The silica was measured using variable quantities of molybdate reagent, and the data were plotted as in Figure 2. At very low concentrations of molybdate reagent, color development is incomplete due to the shortage of the molybdate. At high concentrations of molybdate reagent, color development is incomplete because the acidity of the solution becomes too high for the silicamolybdate complex to form. If the straight section of the plot is extended to the point of zero reagent, the value at this point is the silica content of the water. The difference between the silica value for 2 ml of molybdate reagent and l ml of molybdate reagent is the molybdate reagent blank. ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
1063
DISCUSSION
Previously there has been no direct and relatively easy way of determining the blank of very pure, low-blank reagents. In the case of silica, the molybdate reagent contains a small quantity of silica, but this cannot be determined directly because it is necessary t o know the silica content of the water being used for the blank determination. Conversely, the silica content of the water cannot be easily determined unless the silica blank of the reagent is known. By the system described, these two unknown factors may be determined simultaneously, either mathematically or graphically, after a calibration curve has been prepared for the reagents involved. This method may be easily extended to include iron determinations for reagent grade chemicals. After the preparation of a calibration curve, the iron concentration of the material being tested may be determined simultaneously with the reagent blank by running only two rapid and direct colorimetric tests. There are three possible cases which may result in the blank determinations. See Figure 3. In case 1, the reagent blank
is positive and adds to the apparent concentration in the determination. The reagent blank may be greater, equal to, or less than the sample content. In case 2, the reagent blank is zero. In case 3, the reagent blank is negative indicating that the reagent has a demand for the substituent being tested. This may result in the determination of free chlorine, oxidizing or reducing agents. After the preparation of a calibration curve, the use of this method will give a simultaneous check on the reagent while running an analysis. This provides a continual check on the reliability of a reagent. ACKNOWLEDGMENT
The authors acknowledge J. P. Sickafoose for his assistance during the preparation of this manuscript. RECEIVED for review July 16, 1971. Accepted December 14, 1971.
Improved Tissue Solubilization for Atomic Absorption Andre J. Jackson, Leslie M. Michael, a n d H e r b e r t J. Schumacher Department of Encironmental Health, Unicersity of Cincinnati, College of Medicine, Cincinnati, Ohio 45219
ATOMICABSORPTION SPECTROMETRY has proved to be a very useful analytical tool in the study of the trace metal content of animal tissues. Despite its many applications to metal analysis, there are persistent problems such as sample preparation and reproducibility of results. Organic matter present in the sample is a potential source of interference to the aspiration, atomization, and detection of elements in the flame because of nonspecific matrix effects (I). In order t o eliminate this problem the organic material is usually destroyed or disrupted before the sample is analyzed ( 2 ) . Several methods are currently in use, including wet oxidation, dry oxidation, and oxidative fusion (3). In our laboratory, we have been investigating the use of Soluene-100 (purchased from Packard Scientific), a quaternary ammonium hydroxide tissue solubilizer specifically formulated for use with toluene and xylene based scintillation counting solutions. Employing this product, one can prepare tissue samples for atomic absorption quickly and with minimal handling thus reducing sample loss or possible contamination from excessive sample preparation. Another major advantage of this procedure is that the organic based solubilizer enhances the sensitivity for the metals investigated. Furthermore, the same sample can be used for liquid scintillation counting ( 4 ) as well as gas chromatography ( 5 ) . (1) G. D. Christian and F. J. Feldman, “Atomic Absorption Spectroscopy,” Wiley-Interscience,New York, N.Y., 1970, p 177. (2) T. T. Gorsuch, “The Destruction of Organic Matter,” Pergamon Press. New York. N.Y.. 1970.. D_ 11. (3) Zbid.,pp 19-41. 14) Packard Technical Bulletin. Downers Grove. Ill. 60515. 1970. (5j Joseph MacGee, Veterans Hospital, Cincinnati, Ohio 45220,
private communication, 1971. 1064
ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
Table I. Comparison of Aqueous and Soluene Sensitivities Sensitivity,* ppm at primary absorbing line (6) Aqueous Soluene method Zn 0.025 0.01 cu 0.10 0.05 Fe 0.20 0.05 Mn 0.01 0.005 absorption. a Concentration that will give a 1
EXPERIMENTAL
Reagents. Reagent grade chemicals were used. The Soluene contains a 2 % (WjV) solution of ammonium-lpyrrolidene dithiocarbamate. Procedure. Weigh the tissue sample into a 50-ml volumetric flask. Add 0.5-1.0 ml of Soluene per 100 mg of tissue. Stopper the flask and let it stand at room temperature for 24 hours. Heating to 60 “C will speed solubilization. All tissues investigated gave a clear, homogeneous, and aspiratable solution suitable for atomic absorption. A threeto fourfold dilution of the preparation is made with toluene. The sample was analyzed by the method of additions (6). Similar results were obtained by plotting absorbances of the metal standards in the Soluene matrix against concentration. All determinations were corrected for any reagent metal content by using a reagent blank. Of the metals investigated, only zinc gave a measurable signal. All samples were analyzed on a Perkin-Elmer Model 303 atomic absorption spectrophotometer using instrument settings suggested for organic (6) “Analytical Methods for Atomic Absorption Spectrophotometry,” Perkin-Elmer, Norwalk, Conn., 1971, pp 2-5.
