Analytical Problems in the Determination of Evans Blue Caused by Absorption on Glass and Protein Surfaces W. 0. CASTER, ADA B. SIMON, and W. D. ARMSTRONG Physiological Chemistry Department, University o f Minnesota, Minneapolis, M i n n .
Beckman DC quartz spectrophotometer using either the stantlard 1 X 1 em. macrocells p-ith a 3-ml. liquid volume or 0.3 X 1 cm. microcells with a 150-gl. liquid volume.
So many difficulties were encountered in adapting Evans Blue methods to the microliter scale that a systematic study of error was undertaken. The major problems were due to the tendency for this dye to be adsorbed on glass and protein surfaces. In protein-free solutions as much as 20 to 50% of the dye was adsorbed upon the wall of the cuvette. This percentage could be even higher under varying conditions of pH and salt concentration. In solutions containing protein, the dye was adsorbed upon protein surfaces. In all cases low spectrophotometric results were obtained. 1 variety of analytical problems were observed as a direct result of adsorption errors. These problems are described and discussed. High concentrations of Zephiran prevented the adsorption of Evans Blue on glass surfaces, removed it from protein, and thus eliminated these adsorption errors.
E
ADSORPTION ON GLASS
The adsorption of Evans Blue on the walls of the 1 X 1 cm. quartz-faced cuvette and its slow elution with distilled water art' demonstrated in Table I. Both the rates and absolute amountof these absorbance changes are variable from cell to cell and were found to change from day to day within the same cell. The rate of adsorption is affected by small differences in thv character and cleaning on the glass surfaces. Scratches or cracks mav have a marked effect (8). I n Table I1 are shown the absorbance r e a d i n g s 01)tained when thret, different concentrations of dye were read / in microcells and in macrocells. I n a l l cases, there were s u b s t a n t i a 1 deviation. f r o m B e e r ' s lan. which became even 1 a r ge r \vi t h time. L 450 550 650 The column of expected results given MILLIMICRONS in Table I1 are the Figure 1. Evans Blue Absorpvalues obtained when tion Curve Zephiran was added to-the dye solutions to prevent the adsorption of the dye onto glass and quartz surfaces. The reason for using Zephiran is discussed shortly. U-ing these expected values as a basis for comparison, errors of 2 to 20% were encountered within the first minute after the solution was placed in the cuvette. Over a period of 24 hours these adsorption errors became as large as 49%. I n the last four lines of Table I1 are given comparable data for vblumetric flasks and Desicoted Erlenmeyer flasks. In these cases, larger concentrations of dye solution were allowed to stantl in contact with the flask surfaces. At the specified times, aliquots were rapidly pipetted into Zephiran solution (to prevent further adsorption) and spectrophotometric readings were made a t G20 mg. The changes caused by adsorption were very small in all cases, indicating that only trivial adsorption errors are involved in the storage of high concentrations of dye. I n the case of the Desicoted flasks, consistent increases were noted with time and The absorbance values continued t o increase for a few d a i ~ then progressively decreased. The increase WVBS apparentlv dut. to a turbidity set up in the solution as the Desicote film disintegrated and became suspended in the solution. As the Desicotr left the glass surface, adsorption of the d j e onto the glass progressed and the dye concentration decreased. Adsorption errors were frequently greater in microcells than in macrocells and much greater in these cells than in flasks. In part, this is due to the higher dye concentrations in the flasks. I n addition, however, the shape of the containers (surface area per unit of liquid volume) had a marked effect. The adsorption is depcndent upon the surface area involved. When compared 111
T'ASS Blue, or Tolidine-1824, is a blue dye with the absorption curve shown in Figure 1 and the structural forniulu:
SH, OH
OH S EL
Its synthesis and purification have been described ( 1 1 ) . Because it is rapidly and tenaciously bound to plasma albumin, t h i 3 dye is commonly used in phpiology for the determination of plasma volume. I n essence, this procedure involves iniecting a known amount of Evans Blue into the blood stream, and. after a suitable mixing period, withdrawing a sample of blood, removing the red cells by centrifugation, and determining the Evans Blue concentration in the plasma. From a knowledge of the amount of dxe injected and the final dye concentration in the plasma, it is possible to estimate the volume in rrhich the dye is distrihutecli.e.. the plasma volume of the animal. The physiological factors involved have recently hecn rrvien-ed ( 1 6 ) .
Table I. Demonstration of Dje Adsorption by Elution of Adsorbed Di-e from the Walls of a 1 X 1 Cm. Curette
Cell
KO. 2
x
4
5 min. 0.366 0.356 0.362
Absorbance a t 620 A I p ( 5 y Dye/Ml. H20) 20 min. 30 min. 2 hours 0.350 0.342 0.341 0.351 0.338 0.337 0.357 0,353 0.352
Absorbance a t 620 hlp after Rinsing and Refilling Cuvettes with Water 6 min. 24 hours 0 005 0.023 0.008 0.011 0.006 0 012
EXPERIIIENT.4 L
The dye used in this work was obtained from Eastman Kodak Co. and checked for purity by the method of Hartwell and Fieser (11). The spectrophotometric values were determined with a
713
ANALYTICAL CHEMISTRY
114
Table 11. Direct Estimation of Extent of Dye Adsorption on Surfaces of Containers Commonly Used in Analytical Work Container in Which Soln. Was Stored Microcell (quartz faced)
.4bsorbance a t 620 Mp 1 min. 30 min. 24 hr. 0.184 0.130 0.205
Dye Concn.. y/ML Stored Read 4 4 6 6 8 8 4 4 6 6 8 8 12 6 100 6 12 6 100 6
Macrocell (Corex glass) Volumetric flask (borosilicate glass) Desicoted Erlenmeyer flask
0.297 0.472 0.184 0.326 0.460 0.373 0.386 0.384 0.383
0.323 0,482 0.205 0.348 0.500 0.386 0.382 0.381 0.376
0:42s 0.173 0.301 0.433 0.375 0.384 0.407 0.408
Difference betw. Obsvd. a n d Expected, % 30 min. 24 hr. -20 28 49 - 16 23 ... - 16 -6 -8 - 20 - 28 - 32 15 - 22 -9 -2 10 - 15 -3 -2 0 -1 +6 -2 0 +6
Expected Results 0.256 0.384 0.512 0.256 0,384 0.512 0.384 0.384 0.384 0.384
1 min.
--
-
-
i:
+A
Table 111. Effect of pH on Tendency for Evans Blue to Be Adsorbed and on Shape of Its Absorption Curve Wave Lengths, ,\I$
2
480 540 620
0.057 0.191 0.385
4
6
7
0.40
PH 8
1
0
1
2
Absorption 0.055 0.186 0.377
0.057 0 . 0 5 6 0.188 0.182 0.394 0.381
0.36
0.054 0.179 0.375
0.061 0.190 0.346
0.110 0.254 0.143
24 HOERSLATER 480 540 620
0 . 0 2 5 0.039 0,090 0.141 0.180 0.292
0.047 0.161 0.340
0,174 0.361
0.32 m
0.053 0 , 0 5 1 0.054 0.107 0.173 0.336
W
0.183 0.354
0.248
0.153
[z
8
028
m
a
0.24
terms of square centimeters of glass surface per milliliters of liquid, ratios of 10 to 5 to 3 are found for microcells, macrocells, and flasks, respectively. Thus, each unit of liquid in a microcell is exposed to twice as much glass surface as if it were in a marrocell and three times as much as if i t were in a flask, I n all containers the adsorption is similar when expressed in terms of dye adsorption per unit of glass surface. I n this basis, the average adsorption in Table I1 amounted to 0.067 of Evans Blue per sq. cm. of glass surface a t 1 minute and 0.167 of Evans Blue per sq. cm. of glass surface a t 24 hours. I n Table I11 is seen the effect of p H upon the tendency of Evans Blue to be adsorbed on the glass surface. The more acidic the solution, the greater is the adsorption on glass. At a p H of 8 there is a substantial reduction in the tendency for the dye to be adsorbed on glass, but little change in the extent of its binding to plasma protein (Table IV). At pH’s more alkaline than pH 8 the shape of the absorption curve of this dye changed and the absorption maximum moves progressively toward shorter wave lengths as the p H increases. From the point of view of this analytical procedure such a shift would be undesirable. Greater difficulty would be encountered in correcting the values for the presence of hemoglobin and the exact shape of the curve would be dependent upon pH.
Table IV. Effect of Diluent on the Discrepancy between Protein-Containing and Protein-Free Dye Solutions Dye S o h . Containing
Diluent Buffer ( P H 8)
Concn., y Dye/Ml.
Saline (0.9%)
4 6 8
0.219 0.322 0.426
0.221 0.332 0,438
0,266 0.377 0.507
5 % serum albumin
4 8
0.237 0.458
0.232 0.465
0.255 0.525
10% serum albumin
4 8
0.239 0.484
0.236 0.479
0,263 0.509
Distilled water (protein-free)
4 6
0.218 0,340 0.465
0.262 0,391 0.508
0,266 0.395 0.493
Plasma
8
Zephiran (12%)
ADSORPTION ON PROTEIN
The absorptivity of Evans Blue is lower in plasma or proteineontaining solutions because of the adsorption of this dye upon the surface of protein particles, The absorptivity varies from one plasma sample to the next, is not directly proportional to
0.20L “
0
2
’
4
PER CENT
6
8
IO
12
ZEPHIRAN
Figure 2.
Effect of .4dding Various Concentrations of Zephiran to a Dyed Plasma Solution
At concentrations below 6 70,turbidity prevented useful readings; attempts t o remove this turbidity by centrifugation were n o t consistently successful
protein level (Table and is reported to vary with dilution ( 2 ) . Any procedure \\-hich would eliminate these problems would give this method wider applicability. The authors have found that high concentrations of a cationic detergent will remove the dye from its protein binding and hold it in true solution. Zephiran, an alkyldimethylbenzylammonium (ahloride (available in 12.8% aqueous solution from Winthrop Stearns Co., New York 13, N.Y.) is very satisfactory for this purpose. In the presence of Zephiran, protein-free Evans Blue solutions obey Beer’s law and give absorbance readings which are constant with time, and there is no difference in abRorptivity between aqueous standards and dyed plasmas. This eliminates the problem of variable absorptivity normally encountered in work with protein-containing solutions and allows dyed plasma to be compared directly with protein-free dye standards. It is necessary to use high concentrations of Zephiran because of its effect upon protein. The addition of low concentrations of Zephiran to dyed plasma samples resulted in a marked turbidity attributable to the precipitation of protein. This may account for a previous failure (6) to obtain a useful result with cationic detergents. As seen in Figure 2, this turbidity can be eliminated by the addition of higher concentrations of Zephiran. I t is also to be noted in Figure 2 that the absorbance a t 620 mp was greater after adding Zephiran than i t was in the absence of Zephiran, indicating that the Zephiran had broken the dye-protein binding and had brought the dye into true solution. In Table IV is seen a direct comparison of results obtained when saline, p H 8 buffer, or Zephiran was used to dilute aqueous dye standards and dyed protein solutions. I n saline solution all of the values are low because of adsorption errors. When p H 8 phosphate buffer was used as the diluent, the samples containing plasma and serum protein gave low results, but the standard solutions of dye in water gave higher results which obeyed Beer’s law
V O L U M E 26, NO. 4, A P R I L 1 9 5 4 fairly well, With Zephiran as a diluent all of the values agreed with the predicted values within the manipulative variation of this procedure. In preparing all of the solutions in Table IV it was necessary a t some point in the procedure to pipet and work with Evans Blue standards made up in distilled water. The larger reading variations observed with the saline solutions were associated with a difficulty in repeating spectrophotometric readings on rapidly changing solutions. When all manipulations were carried out in Zephiran-containing solution, duplicate dilutions prepared and read on different days agreed within u = 0.003 absorbance, or O . i % relative error, and all values showed excellent agreement with Beer’s law.
715 Vierheller (18) reported the dependence of absorption values upon the shape of the spectrophotometer cell. It seems possible that these results are analogous to those in Table I1 and are explainable in the same fashion. Errors of 30 to 40% were occasionally observed by Gregersen ( 7 ) in his work with a visual color comparator. If one compares dyed plasma with a protein-free dye solution in a Duboscq-type colorimeter, adsorption effects might conceivably account for errors of this size because of the relatively large glass surface involved. .illen and Gregersen ( 2 ) report that when dyed plasma is diluted the absorptivity of the dye changes. Wherever deviations from Beer’s law are observed in Evans Blue work, adsorption effects should be studied for the pwsihle cause.
DI scus SIOY
Alany of the problems and anomalies in the Evans Blue literature (1, 2, ?-10, 18) may be the direct result of a failure to appreciate the analytical significance of these adsorption effects. This may not he surprising, for in most other cases in analytical cheniistry (4, 12-24, 27) adsorption errors have proved to be negligiblv small. Gregersen et al. report that Evans Blue is unstable in phyyiological saline solution (9, 10) and that its absorbance decreases progressively with increasing salt concentrations (8). Allen (1) criticizes IIorris’ (16) method on the basis that Evans Blue is unstable in acid solutions. The present work also confirms that lower results are obtained in solutions containing salt (Table IFr) and acid (Table 111). Recovery experiments have demonstrated, however, that these effects are due to increaspd adsorption rather than to a destruction of the dye. C‘ontrary to the views expressed by A y e s (S), the fact that a standard curve does not obey Beer’s law indicates that there is an uncontrolled and usually an unknown factor operating in the anal>-tical system Tvhich may be a source of analytical error. This factor may be chemical, physical, or instrumental ( 5 ) . In the present case this deviation from Beer’s law was one of the inost marked and easily measured indicatois of this adsorption ePirct.
LITERATURE CITED
Allen, T. H., Proc. SOC.Exptl. B d . X e d . , 76, 146 (1951). -illen, T. H., and Gregersen, XI. I., A n i . J . Physiol.. 172, 377 (1953).
.%ires,G. H., =isa~. CHEM.,21, 652 (1949). Bird, L. H., Sew Zealand J . Sci. Technol., B30, 334 (1949) Caster, W. O., As.iL. CHEM.,23, 1229 (1951). Chinard, F. P., and Eder, H. A , , J . Exptl. M e d . , 87, 4 i 3 (1948). Gregersen, 31. I., J . Lab. Clin. Med., 23,423 (1938). Gregersen, 31. I.. and Gibson, J. G., Jr., A m . J . Phusiol.. 120, 494 (193i).
Gregersen, 31. I., and Rawson, R. -I., Ibid., 138, 698 (1943). Gregersen, 31. I., and Stewart, J. D., Ibid., 125, 142 (1939). Hartwell. J. L., and Fieser. L. F., Oig. Syntheses, Coll. T-ol., 2, 145 (1943).
Hensley. J. W., Long, 11. 0..and Willard, b. E., I d . Eng. Cheni., 41,1416 (1949).
Hershenson, H. 31., and Rogers, L. E.. A S ~ L C. H n r . , 24, 219 (1952).
Horn. D. IT., A m . J . Pharm., 108,324 (1936). lIorris, C . J. 0. R., Biochem. J . , 38, 203 (1944). van Porat. B., Acta M e d . Scand., Suppl.. 140, KO.256, 1 (1951). Schoonover, I. C . , J . Research S a t l . Bur. Standards, 15, 377 (1935).
Vierheller, F., Seniana mdd. (Bztenos dires), S u p p l . diario, 51, S o . 2651. 885 (1944).
RECEIVEDfor review Xovenibes
10, 1952, -4ccepted Janriary 10, 1954. Work carried o u t under Contract AT(ll-1)-106 between the Pnivessity of Minnesota and the .4tomic Energy Commission.
Microscopic Identification of Wustite In Presence of Other Oxides of Iron RALPH G. WELLS Research and Development Laboratory, Pittsburgh, Pa., United States Steel Corp.
In the microscopic examination of partly reduced iron ores, wustite is often overlooked because of its resemblance to magnetite. RIicroscopic methods of identifying wustite, hematite, and magnetite are described. . i n etch solution of saturated alcoholic stannous chloride will attack wustite in 1 or 2 minutes, but it does not affect the otheroxides. Wustite is easily scratched with a steel needle, but magnetite is scratched with difficulty o r not at all. Wustite has a Knoop hardness of 155; magnetite, 361. Powdered wustite is black, isotropic, and nonmagnetic. Powdered magnetite is black, isotropic, and magnetic. Hematite has a greater reflectivity than the other oxides; it is anisotropic, and forms a red nonmagnetic powder when scratched. The described techniques used with reflected-light microscopy of partly reduced iron ores offer simple and accurate means of determining the types and amounts of the oxides present.
I
S STVDI-ING the reducibility of iron ores, it is desirable to know which of the three forms of iron oxide-wustite (FeO), hematite (Fe2O8),and magnetite (FesOl)-are present in partly reduced ore or sinter. Chemical analysis will show whether the iron is ferrous or ferric, hut will not reveal which of the oxide phases are present. X-ray diffraction analysis will show the major constituents present, but will not detect oxide phases below certain concentration thresholds. Xcroscopic methods are the easiest, cheapest, and quickest; n i t h these methods, it is possible not only t o identify the oxide phases but also to determine the quantity and distribution of each phase in a given specimen. However, in a microscopic examination of partly reduced iron ores or sinters, wustite is often overlooked because of its resemblance t o magnetite. Methods by which wustite can be distinguished from magnetite microscopically are described in the present paper.
