Decantation as a Precision Step in Colorimetric Analysis HARRY GOLDENBERG D e p a r t m e n t o f Biochemistry, Hillside Hospital, G l e n
Oaks, N. Y.
While there is no alternative to volumetric precision in withdrawing fluid samples for gravimetric or volumetric analysis, the question is raised whether quantitative transfers are mandatory when analyses are based on the measurement of color or some other intensive property of solutions. It is shown in these cases that if the test samples are instead decanted, the erratic errors which would nominally occur may be circumvented by using small volumes of reagents in the subsequent steps of the analysis and by not diluting the mixtures to a prescribed mark. The decantation principle is illustrated by several biochemical colorimetric procedures which require precipitation of protein prior to analysis of the supernates; large losses in decanting the supernates are shown in each case to have virtually no effect on the final photometric readings. Use of the principle can greatly expedite routine operations in a busy laboratory. Attention is directed to the precautions that must be taken in the systematic use of decantation procedures, and to potential sources of error.
C
OSF’ENTIOS-1L quantitative methods require precision in transferring fluid samples that are being analyzed. The solutions may be carried along in toto or appropriate aliquots may be prescribed. I n either case a considerable economy in time and labor could be effected if it were possible to dispense (in part) with these quantitative transfers without sacrificing accuracy. The purpose of this paper is to show that, under appropriate conditions, solutions may be decanted rvith little attendant error in the final analysis when colorimetric methods are used. Specific applications are demonstrated by reference to representative colorimetric procedures that have been employed in this biochemical laboratory for the past three years. Other applications will doubtless occur to the reader. The phosphorus determination was the first case in which the test sample, a deproteinized solution, vas removed by decantation ( 2 ) . Attempts were made to minimize the relative decantation losses by using a large volume of protein precipitant. Subsequently a new approach was adopted wherein large losses can be sustained in transfer lvithout appreciable effect on the final photometric readings. DECANTATION PRINCIPLE
I t is instructive to consider why decantation losses produce analytical errors. Many methods are set up using volumetric flasks for mixing the sample with reagents and adjusting to mark. Consequently, variations in the sample volume incur similar variations in the final concentration of the component which is to be measured. The error would be automatically corrected if the total volume \yere t o be reduced in proportion to the sample loss. This can be readily accomplished. The first step is to discard volumetric flasks and instead mix the sample with color reagents without diluting t o the mark. When the amount of sample decreases owing t o decantation losses, the total volume m5ll decrease and tend t o compensate for the loss. But will it do so completely? Evidently not, unless all subsequent additions be decreased proportionately. For example, if the sample and color reagents are 5 ml. each, a sample loss of 1 ml. (or 207,) would cause the following: ( a ) a 207, error when the loss is made up with water (or other solvent) and the mixture
is taken to 10 ml.; ( b ) a smaller eiror (115;) when 5 ml. of rengents are added t o bring the mixture to 9 ml.; or ( e ) no erro: a t all n-hen the color reagents are scaled doxn to 4 ml. However, it is impractical to estimate the amount of decantate and adjust the color reagent volumes needed from sample to sample. A second change is therefore made by revising the procedure so as to require a minute volume of color ieagents. A 2OY0 sample loss now produces virtually no analytical error at all, for the total volume is approximately 207c less The desired objective has therefore been achieved by the simple expedient of curtailing all fluid additions beyond the decantation step. Analogous cases come t o mind in Jvhich decantation losses have no effect on the accuracy of the analyses. Khen the constituent to be measured already bears a color, fluoresces, or imparts a characteristic viscosity, it is obviously immaterial what the decantation error is, provided sufficient solution remains for the analytical measurement. I n each case an intensive property of the solution is measured, which is dependent on the solute concentration rather than the sample volume. By way of contrast, gravimetry and volumetry require precise measurement of the size of the sample. The fundamental rationale behind the decantation principle is non apparent. By not appreciably diluting the sample following decantation, the analysis is geared to measure the concentration of solute rather than its absolute magnitude in the final mixture. In the phosphorus analysis, it is apparent that, though phosphate does not possess a suitable physical property that permits its direct determination, the molybdenum blue color can be produced by adding a minute volume of acid molybdate plus reducing agent to the protein-free decantate. I n this manner the concentration of phosphorus in the decantate, which wns established a t the outset of the analysis by mixing prescribed amounts of serum and trichloroacetic acid, is not significantly altered by the color reagents and consequently the color intensity produced, under ideal conditions, is independent of hoa much deproteinized solution is lost. In practice, the extent to which the color reagents can be scaled down in volume is determined by the solubility of the reagent components as well as by other considerations. Fortunately, it is unnecessary to use exceedingly minute amounts of concentrated solutions. Acceptable results may often be obtained when the total color reagent volume is about one tenth t h a t of the supernate or decantate, provided of course the mixture is not subsequently brought to mark. This is demonstrated belolv. A general expression for the analytical error incurred by decantation losses is readily derived by reference to a symmetrical three-sided figure such as an isosceles triangle (Figure 1) or a circular sector. Let V, = volume of supernate, VI^ = volume of decantate, V, = total prescribed volume of color reagents. The parallel bases, V,and V,,are cut into proportional segments by the pointer ivhich swings from the apex a t 0. Hence, the final color mixture has the same intensity Jvhether V, be mixed with V , or Vd be mixed with y. Because V , is, in practice, not decreased to compensate for the decantation loss ( V , - Vd), a corresponding error (x) is made whose absolute magnitude is determined from the relation
The analytical error produced Lvhen t o (vd V,) is
+
1003
(V, +
y ) i s diluted by
x
ANALYTICAL CHEMISTRY
1004
detail elsewhere. All readings were taken with a Beckman llodel
DU spectrophotometer (using 1-cm. matched cells), because the
Table 1. Theoretical Analytical Errors Incurred by Decantation Losses Supernate ( 1’s)
MI.
Decantate (Vd),
I
Mi.
10 10 10
Color Reagents ( V c ) , MI.
9.5 9.5 5,O 4.5 4.5
; 5 3 3
5
1.0 0.5 0.1 0.5 0.2 0.1 0.2 0.05
3.0 2.5 ‘7.0
yc analytical error
Error. % DecantaAnalytical tion J
50 10
10 40 16.7 33.3
= T’d
A-+ vc x
0.5 0.3 1 .o 1.0 0.4 1.3 1.2 0 8
100
+
[The volumetric error introduced by x-viz., (lOOx)/(Vd y)should not be confused with the resultant analytical error. For example, when Vd = 4, y = 1, and x = 1, the volumetric error is 1 part in 5 , but the analytical error is 1 part in 6, because the final absorbance is five sixths of its correct value.] Equation 2 is based on the following t x o assumptions: (1) Beer’s law is obeyed, and ( 2 ) the sole effect of the excess reagent ( 2 )is to dilute the color on a calculable volumetric basis. The second assumption is not justified when the concentration of color reagent is a critical factor in the analysis (see below). From Equations 1 and 2, T’ va - x 1003 (3) % analytical error = -5
+
v c
absor tion differences between solutions prepared from decantates and tiose prepared from aliquoted samples were anticipated to be too small for accurate measurement w-ith clinical instruments. Determinations were made in duplicate. Phosphatase (#-Nitrophenol). A convenient way to determine the concentration of a hydrolytic enzyme is to measure its release of dye from a colorless chromogenic substrate under standard conditions of incubation. The esters of p-nitrophenol are \yell suited for this purpose, the phosphate being attacked by either acid or alkaline phosphatase a t the appropriate pH. Enzyme acltion is stopped by adding tungstic acid. The colorless supernate is mixed with potassium carbonate solution to develop the stable yellow p-nitrophenolate color. whose intensity is related to the phosphatase activity. I n this investigation an aqueous p-nitrophenol solution was substituted for the buffered substrate. This was done in order to confine the study to purely volumetric considerations by isolating the decantation step from the enzyme-substrate reaction. Serum \vas also substituted for the m-ater regulai-1:- used in the blank so as to correct for any traces of bile pigment not removed during protein precipitation. REAGESTS.p-Nitrophenol, 0.1 pmole per ml. Dilute 2 nil. of a 5-pmole stock standard containing 0.348 gram of p-nitrophenol ( 1 ) per 500 ml. of solution to 100 ml. with distilled water.
SAMPLE VOLUME COLOR REAGENT VOLUME
v,
J’d
v s
and therefore,
% analytical error
I’
= --..-i x c/c decantation error (4)
Ve f
v d
When V , is very small relative to the decantate, Equation 1 may be approximated by the expression analytical error =
V ~
4
X
% decantation error
v d
(5)
+
The ratio F’Jl’d or V J (V, V,) may be regarded as a reduct,ion or “telescoping” factor, for when V , < Vd the analytical error is seen to be but a fraction of the decantation error. If the decantate is 100 times the volume of the color reagents, the analytical error is about one hundredth of t,he decantation loss. If ITcis one tenth of v d , as i t might well be under average working conditions, the decantation error is telescoped elevenfold; thus a 10% sample loss causes a colorimetric error of less than 1%. Table I demonstrates the applicability of the decantation principle a t several volumetric scales of analysis. COMMENTS ON EXPERIMENTAL TECHNIQUE
The decantation principle does not permit the chemist to dispense with all precautions. Steps leading to the preparation of the supernates must be carried out with analytical precision. It is only a t this juncture, when the concentration of the test component in the supernate has been fixed, that rigor may be relaxed in t,ransferring the supernate for further treatment. Reasonable care must be exercised in adding the reagents for color development. The photometer is set t o zero with a method blank prepared in the usual way, generally with water substituted for the biological sample. The standards are processed in similar fashion. I t requires little effort to carry through a method blank and standard(s), because the reagents are mixed in a single tube; absence of debris makes decantation or filtration unnecessary. METHODS
The decantation principle is illustrated by application to the determination of inorganic phosphate, phosphatase, and iron in pooled human serum. The first, two methods will be presented in
Figure 1.
Diagraniniatic representation of decantation principle
Tungstic acid, stabilized. Add 2.1 ml. of concentrated sulfuric acid with cooling to a 1-liter volumetric flask containing 1 gram of finely powdered poly(vinv1 alcohol) (Elvanol, Du Pont, grade 51-05) previously dissolved in several hundred milliliters of hot distilled water. Add with shaking a solution of 11.1 grams of sodium tungstate dihydrate in about 200 ml. of water and bring the contents to the mark. Potassium carhonate, saturated. A 5-ml. quantity of tungstic acid mas pipetted PROCEDURE. into a 13 X 100 mm. test tube containing 1 ml. of p-nitrophenol solution and 0.1 ml. of serum. The blank tube contained serum, tungstic acid, and 1 ml. of water. The tubes were centrifuged, the contents decanted into clean tubes, and 0.1 ml. of carbonate solution was added. Readings were taken a t 400 mp. No effort was made in decantation to remove the last drop or two of liquid clinging to the mouths of the tubes, nor were the decantate volumes measured. T o determine the analytical error resulting from known decantation losses, as well as to obtain a reference value with which
V O L U M E 2 8 , NO. 6, J U N E 1 9 5 6
lo05
to compare all absorbances, precise aliquots of the tungstic acid supernate were withdrawn ranging from 6.1 to 2 ml. Each sample was mixed with 0.1 ml. of carbonate. All aliquots were drawn from a common supernate prepared from 20 ml. of p-nitrophenol, 2 ml. of serum, and 100 ml. of tungstic acid; this was done to minimize experimental variations. Centrifugation was carried out in 250-ml. bottles. Inorganic Phosphate. The phosphorus determination is based on measurement of the molybdenum blue coloration produced when ferrous ion is used as the reducing agent. REAGENTS. Ferrous-trichloroacetic acid reagent, stabilized. Add successively t o a 500-ml. volumetric flask 50 grams of tricahloroacetic acid, 200 to 400 ml. of distilled water, 5 grams of thiourea (recrystallized from R-ater if necessary to remove t'races of phosphorus), and 15 grams of ferrous ammonium sulfate hexahydrate (Mohr's salt). Shake the mixture to dissolve the solids and bring to the mark. Acid molybdate, regular. Add 45 nil. of concentrated sulfuric acid with cooling to 200 ml. of distilled water in a 500-ml. volumetric flask. Transfer a solution of 22 grams of ammonium molybdate, (S114)J10i024,4H20, in 200 ml. of water to the volumetric flask, mix the contents, and dilute to the mark. Acid molybdate, concentrated. Prepare as above bj- doubling the specified amounts of acid and molybdate. PROCEDURE.Five milliliters of ferrous-trichloroacetic acid reagent were pipetted into a test tube containing 0.2 ml. of serum. The contents were shaken initially t o prevent clumping of protein. Distilled water was substituted for serum in the blank tube. The tubes were centrifuged a t 3000 r.p.m. for 15 minutes and the supernates decanted without regard to small physical losses. The color was developed by adding either 0.5 ml. of the regular molybdate reagent or 0.25 ml. of the more concentrated one. Readings were taken after 30 minutes a t 750 mp. ( A 660-mp setting is also permissible.) The source of deproteinized phosphate solution for the aliquot studies was a large supernate pool prepared by mixing 4 ml. of serum with 100 ml. of the precipitant in a bottle and centrifuging a t high speed. Portions of 5.2, 5.0, 4.5,and 3.0 ml. were removed and treated with either 0.5 or 0.25 ml. of molybdate. Iron. T h e method used is essentially that of Schales (3);modified to afford a smaller VJV, ratio. REAGESTS. Hydrochloric acid, 0 . 3 S . Dilute 25 nil. of concentrated acid to 1 liter with distilled water. Trichloroacetic acid, lOyc (w./v.). .Ammonium acetate, 50% (w./v.). HydroTinone-phenanthroline reagent. Dissolve 0.05 gram of o-phenant rohne in 0.5 ml. of glacial acetic acid in a 10-ml. volumetric flask. Dilute the solution with a little distilled water, add 0.15 gram of hydroquinone with shaking, and dilute to the mark. PROCEDURE. A mixture of 2 ml. of serum and 1 ml. of 0 . 3 5 hydrochloric acid was permitted to stand for 1 hour in a test tube. Then 2 ml. of lo%;,trichloroacetic acid % w eadded. The centrifugate was decanted into a clean tube; 0.2 ml. of hydroquinonephenanthroline reagent and 0.3 ml. of ammonium acetate were added with shaking. Readings n-ere taken a t 510 mp after 30 Table 11. Application of Decantation Principle to Determination of Phosphatase, Inorganic Phosphate, and Iron in Human Serum (I.a Determination Pliospliatase ip-Kitrophenol)
=
l-- measured sample (compare the first and last lines for each determination). It also demonstrates the close agreement between the theoretical and observed analytical errors, and so tends to confirm the validity of Equation 4 as well as the nnderlying principle. Some question may be raised as to the justification for assuming that the supernate, V,,is equal to the sum total of all its coniponente, because a volu:iie c-ontraction is knonn to orcur \\hen protein separates from solution. The volume c1i:inges ran be readily computed. Assuming a protein content for serum of 0.07 gram per ml. and a partial specific volume equal to 0.73 nil. per gram of protein ( d ) , the decrease in volume approsiniates 0.05 ml. per milliliter of serum. The contraction for e:ic,h system is therefore as folloxs: phosphatase, 0.005 ml. pel' 6.1 nil. (0.1:;); phosphate, 0.01 nil. per 5.2 ml. (0.2%); iron. 0.1 nil. per 5.0 ml. (2%). Only in the iron analysis is the c.h:iiige a t all significant. Here the maximum obtainable TY3 is 4.!1 nil. I n practice, however, it is impossible to remove 4.9 nil. of the iron extract, as a verj- considerable amount of liquid is enti.apped by the protein precipitate. Smaller amounts are lost in the other t n o analyses. Because the losses are variable, depending on how well the precipihte is packed, it seemed brst to define V,arbitrarily in the manner indicated. This procedure does not invalidate the conclusions drawn in the paragraph above, for the data collected in the aliquot studies were derived on a comparative basis from a common supernate, and consequently any value for V . could have been chosen. T h e correct value for T', was important here only in selecting an aliquot to establish thc refc~enceabsorbance for estimating the decantate recoveries. But liecause the decantation principle has already been proved by the aliquot studies, it is almost irrelevant whether T', (in the iron determination) is taken as 5.0t 4.9, or possibly even 4.5, Reference to the absorbance readings in Table I1 bears this out. Absolute standards wwc- not necessar>- to prove the tiecanta~ t ~ tion thesis and n-ere accordingly not run. I n an actual analysis, a blank and standard(s) arc processed in essentially the same fashion as the sera and the sera unknowns calculated by comparing their absorbances with the standard(s). When the proportion of serum used is high, the volume contraction accompanying protein precipitation tends to concentrate the serum components in the supernate. .4s this effect is not duplicated in the standard tubes, erroneously high values result. The magnitude of the error is about 27, in the iron determination; a correction should therefore be applied \?-hen maximum accuracy is required. Considerations of volume contraction are, of course, not peculiar to analyses incorporating the decantation principle but are rather applicable t o all biochemical analyses requiring relatively large amounts of serum. CRITERIA FOR INVOKIVG DECANTATION PRINCIPLE
There are several criteria for deciding whether the decantation step is applicable to a given colorimetric analysis. These are as follom-s:
ANALYTICAL CHEMISTRY
1006 Is V , Small Relative to Vd or V,? The necessity for maintaining a reasonably low P, to Vd ratio has already been demonstrated. V, is understood t o mean the sum total of all additions to the decantate. When V , cannot be constricted by increasing the color reagent concentrations, V, is enlarged by suitable adjustments of sample and precipitant volumes to yield V,/V, equal to about 10 or more. An incidental point is t h a t Vd must not be appreciably altered, whether by dilution (as with washings) or evaporation, prior t o addition of color reagents. Consequently the decantation principle cannot be applied to the Schoenheimer-Sperry method for blood cholesterol, because the alcohol-ether blood extract must first be taken to dryness. Is Concentration of Color Reagents Critical? Because of the lack of control over the ratio of decantate to color reagents, appreciable variations may occur in the final concentration of the color reagents as well as in pH. Hence, systems whose ultimate coloration is sensitive t o such variations are usually excluded from consideration unless they can be redesigned. This is one of the reasons for using ferrous iron in preference to aminonaphtholsulfonic acid in the phosphorus method just described. The effect of p H fluctuations may often be minimized by the judicious use of buffers-e.g., carbonate in the phosphatase determination and acetate in the iron analysis. What Is Nature and Origin of Blank Color? I t is unnecessary to dwell here on the need for running blanks to correct the
standards and unknowns for extraneous color introduced in the course of analysis. One may well inquire, however, as to the validity of the blank correction nhen T.'d is not quite constant from one run to another. T h e answer to this query rests on the nature of the color system. Generally, there need be little concern --hen the extraneous color is carried in the supernate, for the same rationale applies here as in the formation of the color proper. However if the blank colors arise in the color reagent, whose final concentration is not constant, the absorbances may be subject to error. Fortunately V C is small relative to Vd, so that extraneous color generally undergoes a dilution of about tenfold or more, which may obviate much of the expected error. When the variable blank contribution of the color reagent cannot be ignored or eliminated in some way, as, for example, b y transferring the malefactor to the supernate. then decantation is contraindicated. LITERATURE CITED
(1) Goldenberg, H., AXAL.CHEM.26, 690 (1954). (2) Goldenberg, H., Sobel, A. E., unpublished data, 1952. (3) Schales, O., unpublished method, taken from Klett-Summerson
Clinical Manual, Klett Manufacturing Co., Sew York, N. Y. (4) Schmidt, C. L. A,, "Chemistry of the Amino Acids and Proteins," 2nd ed., p. 516, C. C Thomas, Springfield, Ill., 1945. RECEIVED for review Jrtnuary 16, 1956. Accepted March 21, 1966. Presented at the Meeting-in-Miniature of the Rletropolitan Long Island Subsection, New York Section, ACS, Brooklyn, N. Y., February 26, 1956
Colorimetric Determination of Bisphenol-Type Epoxy Resins and Their Fatty Acid Esters M. H. SWANN and G. G. ESPOSITO Paint a n d Chemical Laboratory, A b e r d e e n Proving Ground,
No quantitative method is available for measuring epoxy resins that is not affected by curing or ester formation w-ith fatty or rosin acids. A modified Blarquis reagent develops with bisphenol-type epoxy resins a blue color which is specific and can be used to obtain quantitative measurement of the resins in their unmodified form, in esters and silicone blends. The color is due to the etherified bisphenol grouping and is independent of molecular weight of the polymer. Epoxy resins can be measured in coating applications in solution or in dried films.
Md.
purpose. Unmodified epoxy resins are also used in special applications r i t h small amounts of curing agents such as amines to form films by solvent evaporation. The only analytical method available to date measures the unreacted epoxide group or oxirane ring, which disappears as the resins are esterified or cured and varies with molecular weight or polymer length ( 1 ) . The colorimetric procedure descrihed is based on reaction with the etherified diphenol grouping CH3
-
O
-
~
b
1- ~ - O CH3
p,p'-iso-propylidenediphenol and p,p'-sec-butylidenediphenol v i t h epichlorohydrin have the following typical structure
h
which is not affected by curing of the resin film or esterification with acids, and so is applicable to the unmodified epoxy resins, their esters, and dried films.
T h e resins are used in coating vehicles, either unmodified or esterified with fatty acids, resin acids, or polybasic acids. If esterified with drying oil acids, the resulting vehicle may be airdried. If the fatty acids of nondrying oils are used, the resulting ester can be used in baking enamels in combination with nitrogen resins. When unmodified, the epoxy resins are generally used in baking finishes in mixture with a resin that will react with the hydroxyl and epoxide groups to cross-link the polymer on drying. T h e nitrogen and phenolic resins are most widely used for this
Marquis reagent, which is used for the qualitative identification of alkaloids, develops an intense blue color n
