Extraction Method for Colorimetric Determination of Phosphorus in Microgram Quantities NELSON S. GlNG University o f Kansas Medical Center, Kansas City, Kan,
Extraction of phosphorus as molybdophosphoric acid and subsequent development of the molybdenum blue color complex, either in the aqueous or the alcoholic phase, eliminate significant interference froni the major inhibitory ions and, because of the lower acidity employed, prevent hydrolysis of the labile phosphates. The use of hydroquinone as reductant is shown to be superior in producing greater color stability, in permitting the use of a low acid concentration, and in obviating the need for heating. Jlixtures of inorganic and organic phosphates can be determined separately with precision. The lower limit of sensitivity is about 0.5 y of phosphorus per ml. with an accuracy within =k2%.
T
HREE procedures for the colorimetric determination of phosphorus have been extensively described in the literature: The molybdenum blue complex is developed in the aqueous phase without extraction ( 2 , 3, 6)7 , 9-13, 16-17, 19); the phosphate is first precipitated as magnesium ammonium phosphate, and then the usual procedure is applied with modifications (1, 8); the phosphorus is converted to molybdophosphoric acid which is estracted with various alcohols, and the blue complex is developed in the alcoholic phase ( 4 , 14, 18). The greatest disadvantage of the first procedure is interference from inhibitory ions ( 4 , 10). The second procedure is time-consuming and requires a boiling water bath for development of the color. The third procedure requires a high acidity (1N) for reducing the molybdophosphoric acid to the blue complex, which can be measured only in the alcoholic phase (4,14, 18). The purpose of this investigation was (I) to develop a more accurate procedure for the determination of inorganic orthophosphates in the presence of labile phosphates, pyrophosphates, and the major inhibitory ions, ( 2 ) to increase the stability of the color complex, and (3) to eliminate the nonspecific blue color (4,23) which tends to develop in strong acid solutions with certain reducing agents. Five reducing agents have been employed in the biological field to develop the molybdenum blue complex-namely, hydroquinone ( 2 , 3, 6, 7, 10), l-amino-2-naphthol-4-sulfonic acid ( 1 , 6, 8, 9, i6),stannous chloride ( 4 , 11, 12, 24,18),ferrous sulfate (18, 19), and ascorbic acid ( 1 3 ) . Stannous chloride and ferrous sulfate require high final acid concentrations (1.1N and 0.7CiLV,respectively) in the methods of Kuttner and Cohen (11) and Rockstein and Herron (17). Three of these five reducing agents (hydroquinone, aminonaphtholsulfonic acid, and ascorbic acid) are relatively weak and can be used a t a lower acidity. This suggested their use for determination of the inorganic phosphate in the presence of labile phosphate. The color stability produced by these reducing agents was tested a t about pH 2.0. The observed data are plotted in Figure 1, Tyhere curves 5 and 6 clearly show that color intensity was much more stable \T+th hydroquinone than iT-ith the other reducing agents shown in curves I,2, 3 , and 4. Curves 7 and 8, Figure 2, Fhow that with hydroquinone, when the digestion time is doubled between the addition of sulfite mixture and the addition of hydroquinone, there is little change in color intensity and that the color is stable for many hours. Curves 9 to 12, Figure 3 , show that, when hydroquinone is used, color intensity increases only
&lightly n ith acid concentration. The acid concentration used to obtain these curves was 2 to 20 time. as much as used i n Method I1 (which follon-s). APPARATUS AND REAGESTS
A Beckman Model DU spectrophotometer and Cary recording spectrophotometer, Model 11 MS, m-ere used with 1.0-cm. cells for both models. The same amount of reagent was used i n the reference cell as in the sample cell for each experiment. The folloving reagents mere employed: approximately lo.\. sulfuric acid (280 ml. of concentrated sulfuric acid diluted to 1 liter), 2% hydroquinone (Coleman 8: Bell, c.P.), lEi’fZO sodium hydrogen sulfite, 207, sodium sulfite, sodium phosphate heptahydrate (dibasic), sodium hosphate (tribasic), 570 ammoniriin molybdate, n-butyl alcoho[ l-amino-2-naphthol-4-sulfonic acid (Ejmer and Amend, c.P.), ascorbic acid, glucose-6-phosphatr (Slgma Chemical), sodium P-glycerophosphate (Mallinckrodt), fructose-1,6-diphosphate magnesium salt and glucose-l-phosphate dipotassium salt (Schwarz Laboratories, Inc., New York), disodium phenyl phosphate for testing phosphatase activity
I IO
IO0
90
W V
z 4
m
80
K 0 v)
m 4
70
60
50
P
/
20
Figure 1.
60
.
I
,
,
100 I40 T I M E IN M I N U T E S
,
,
,
200
Stability of blue color roniplex produced by three reducing agents
l-Amino-2-naphtho1-4-sulfonic acid: 16.5 -;of piiospliorus per 25 ml. l-Amino-2-naphthol-4-sulfonic acid: 174 j of phosphorus per 2 5 ml. 3. l-.lmino-2-naphtliol-4-sulfonic acid; 103 y of phosphorus per 25 ml. 4. .4scorbic acid. 6 2 y of phosphorus per 2.5 ml. 5 , 6 . Hydroquinbne; 165 y of pliosi~horubper 25 71.1. 1.
2.
1330
r
V O L U M E 28, NO. 8, A U G U S T 1 9 5 6
a 0 In
""' m
a .400
2
Fig. 2.
6
4
8 TIME IO
LN 12HOURS 14
16
22
20
18
Effect of digestion time on color intensity and stability 7. 8.
Id-minute digestion ::O-ininute digestion
1331
aqueous layer is drained off and discarded. Fifteen milliliters of water is added t o the alcoholic extract remaining, and the mixture is shaken for a few seconds. At this stage a fairly stable emulsion, lasting 5 t,o 10 minutes, is observed. The format'ion of this emulsion is desirable because it serves as an indicator of lowacidity. To the emulsion 1 ml. of 2y0 hydroquinone is added and the solution is again shaken and allo7ved to stand 5 to 6 minutes. B light green emulsion is n o x observed. Finally 1 ml. of sodium sulfite-sodium hydrogen sulfite solution (95 ml. of 15% sodium hydrogen sulfite plus 5 ml. of 20% sodium sulfite) is added, and the mixture is shaken until the yellow complex and the emulsion have disappeared. The solution is alloived t o stand for 5 t o 6 minutes. Then the blue aqueous layer is transferred to a 25-ml. volumetric flask and diluted to t'he mark with water. The absorbance is measured a t i20 nig in a 1-em. cell. -4 calibration curve is shown in Figure 4. The unknown sample and the blank are prepared in t'he same manner as the standards, except that no phosphate is added. I s PRESENCE OF MAJORINHIBITORY IONS.If the major inhibitory ion in the sample is beyond the normal range shown in Table I, the concentration of sulfuric acid is increased 5 to 10 times (5 ml. of 0.2,V sulfuric acid is replaced by 5 ml. of I!\- or 2h7, and the volume of molybdate is increased from 2 to 5 ml. In this case it is convenient t o remove excess acid and molybdate from the alcoholic extract by washing once Kith 5 ml. of water and then adding 15 ml. of water and shaking until the emulsion forms. The procedure then follows that just described. The blue complex can also be measured in the alcoholic phase by drawing off the blue aqueous layer before diluting t o volume and adding approximately 8 ml. of n-butyl alcohol, followed by 5 ml. of 1N sulfuric acid. When the solution is shaken, the molybdenum blue complex is extracted quantitatively into the alcoholic phase. At this point there is no visible color observed in the aqueous phase and it can be discarded. The alcoholic phase is transferred t o a 25-ml. volumetric flask. The funnel is washed twice with alcohol and the vashings are also transferred. -4 perfectly clear solution is obtained. A calibration curve for this procedure is also shown in Figure 4. Method 11. I N L4BsESCE O F EITHERhI.1JOR ~ H I B I T O R YIOSS OR h B I L E PHOSPHORTS. -1standard solution of phosphorus is
,700
I/
0.025 2
3
MG. OF P 0 050 0.075 0.100 0.125 4 5 6 7 6 9 ML. OF 2N HsO4/25 ML
,600 1
0
Figure 3. Comparison of color intensities produced at various acid concentrations with hydroquinone 3314 hours after hydroquinone addition; phosphorus, 0.075 mg. per 25 ml. 18 hours after hydroquinone addition; phosphorus, 0.075 mg. per 25 ml. 11, 12. Standards containing indicated mg. of phosphorus per 25 ml. 9.
.5OC
w
.40C
10.
a
m LL
0
Laboratories. Pllilwaukee, Kis.), sodium arsenate, ammonium oxalate, sodium acetate, sodium sulfate, sodium fluoride, sodium citrate, and sodium silicate. i Paul-Lewis
PROCEDURES
hfter the appropriate reducing agent and the role of acid concentration had been determined, an extraction method was used to eliminate the effect of the inhibitory ions and the labile phosphates. Method I. INPRESETCE OF LABILEPHoSPH-~TES. About 10 nil. of distilled water is transferred t o a separatory funnel (50 t o 100 ml,), and the following are added in the order given: a standard solution containing 0.025 to 0.125 mg. of phosphorus as orthophosphate (equivalent t,o 1 t o 5 y of phosphorus per ml. in the final dilution), 5 ml. of 0 . 2 s sulfuric acid, and 2 ml. of 5% mimonium molybdate solution, This Polution is mixed well by ,shaking a few seconds and allowed to stand at room t'eniperature for 1 minute. To extract, t,he yellow molybdophosphoric acid which forms, 7 to 8 ml. of n-butyl alcohol is added and the mixture is shaken vigorously for 30 to GO seconds. After the alcoholic and aqueous layers have distinctly separated (1 to 2 minutes), t h e
.300
a .2oc
.IO0
Figure 4. 13.
Calibration ciirtes for IIethods I and I1
Method I, color deieloped In aqueous phase 14. hlethod I1 15. Method I, coloi del eloped in alcoholic phase
ANALYTICAL CHEMISTRY
1332
Tahle I. Effect of JIajor Inhibitory Ions on Determination of Orthophosphate (Each solution contains 10 ml. of 1.V sulfuric acid, 2 ml. of 5% ammonium molybdate, and 125 y of orthophosphorus per 25 ml.)
Inhibitor Xone Sodium arsenate
Absorbancea 0.551 0,553 0,592 0.663 0.538 0.530 0.547 0.570
Sodium fluoride Ammonium oxalate Sodium citrate Sodium silicate (I
Ratio, Inhib. Anion t o Orthophosphate
...
2.8 8.3 10.1 15.2 70.0
151
2.2
Measured 20 to 30 minutes after final mixing.
Table 11. Effect of Labile Phosphates on Determination of Orthophosphate (Each solution contains 5 ml. of 0.2.V sulfuric acid, 2 ml. of 5 % ammonium molybdate, and 62 y of orthophosphorus per 25 ml.) m /c
Labile Phosphate
Absorbance None 0.286 Sodium 0-glyceropbos- 0 . 3 2 2 c phate Glucose-1-phosphate 0.36OC 0 293 Disodium phenyl phos- 0 , 6 3 0 phate 0.525 Glucose-6-phosphate 0.326C 0.286 ~~
a
~
Ta
Fb
2iOO
23
..
I150 1.6 987 Kegligible 2480 3860 774 9 , Segliyible
.
Hydrolyzed Ratio, or Labile to Impurity Ortho
..
i.’i
34
1.4
..
18.5 16 0
3 0 1,s 1.1
40 62 12.5
.,
..
T. phosphorus concentration (y/25 ml.) added in addition t o ortho-
phosphorus. b F orthophosphorus determined in excess of t h a t initially added. c Ahueous solutions remained a t room temperature for several days before measurements were made.
pipetted into a 25-m1. volumetric flask and washed t o the lower portion of the flask with about 10 ml. of distilled water. This is followed in succession by 5 ml. of 0.2117 sulfuric acid, 2 ml. of 5y0 ammonium molybdate, and 1 ml. of 2y0 hydroquinone. The solution is mixed and allowed to stand a t room temperature for 15 t o 20 minutes. Then 1 ml. of the sodium sulfite-sodium hydrogen sulfite solution is added and mixed, and the solution is diluted to 25 ml. It is allowed t o stand for 10 to 15 minutes before the absorbance is measured a t 720 mp. The unknown sample and the blank are prepared in the same manner as the standards, except that no phosphate is added. A calibration curve is given in Figure 4. The same amounts of phosphorus were used to prepare this curve as were used for the curves prepared by both modifications of Method I.
nating the nonspecific blue color and the catalytic effect of molybdate on the labile phosphates. p H is increased to I (measured after full color development and dilution), which prevents the interference of acid-labile phosphate. This method also provides for removal of excess reductant, which is much more soluble in alcohol than in water. Thus, reduction due to the nonspecific types can further be prevented. Lastly, the advantages of the fundamental procedures are retained, such as validity of Beer’s law, stability of color, sensitivity, accuracy, high pH, and elimination of the major inhibitory ions. If only very small samples are available, the volume and the concentrations of different reagents, of course, can be proportionally reduced and the addition of distilled water, molybdate, and acid also combined as one step. A series of samples can be run almost simultaneously if a mechanical shaker is used. Hydroquinone solution \vas found to be much more stable in n-butyl alcohol than in aqueous solution. Method I1 is essentially the same as that of Ging and Sturtevant ( 7 ) with a few modifications (change in molybdate added from 5 ml. to 2 ml., increase in hydroquinone concentration, and a change in the wave length from 810 to 720 mp). The reasons for these changes are to permit measurement by both the Cary recording and the Beckman spectrophotometers and to enable samples to be run a t pH 2. At this pH other published methods (6, 11-15) were found to be unsatisfactory, because a nonspecific blue color develops either immrdistely or fairly rapidly in the absence of orthophosphate. n-hich is The final acid concentration of Method I1 is 0 04 from 7 to 27 times lower than other authors have reported (3-6, 8, 11, 12, 17-19). At such low acidity (final pH after dilution is approximately 2), no significant hydrolysis of the labile phosphates is observed, but when the inhibitory ions, arsenate, oxalate, acetate, and fluoride, reach the concentrations given in Method I or, in fact, are even lower, satisfactory results cannot be obtained. The following concentrations of added ion cause only negligible change in color development by Method 11. y
Sulfate as sodium sulfate Kitrate as potassium nitrate Ferric as ferric chloride Citrate as sodium citrate
per 25 ml. 116,500 3,900 540 94,500
In the presence of major inhibitory ions Method I1 and all of the other published methods are found to be unsatisfactory
DISCUSSIOS
All data in the tables and points on the curves are the average of two runs with the exception of curves 5 and 6, Figure 1, which are from a single determination. Table I shows that, when arsenate is present, the ratio of inhibitory anion to phosphorus can be raised to 3 before inhibition occurs; for the other anions considerably larger ratios may be tolerated (15 to 150). Higher ratios-oxalate = 1060, fluoride = 220, citrate = 2 3 0 G o f anions to phosphorus have also been tested. No significant error was introduced by any of these except arsenate and silicate. Data on the effect of labile phosphates on this determination are given in Table 11. The applicability of this method t o the analysis of biological fluids is demonstrated by curves 16 and 17, Figure 5 , which were obtained by diluting urine to the proper strength. This extraction method presents several advantages. The molybdenum blue complex is developed in the aqueous phase after extraction of the molybdophosphoric acid. Any appreciable amount of molybdate or molybdic acid is removed, thereby elimi-
d
.400
U
ML. OF URINE/25 ML. Figure 5.
Dilutions of urine
0.04N
V O L U M E 28, N O . 8, A U G U S T 1 9 5 6
1333
when the blue complex is developed in the aqueous phase without prior extraction. This point has been thoroughly discussed by several authors (4, 10,18). If total phosphoms in the biological materials is desired, the organic matter is first destroyed with various oxidizing agents (9,9). Then the described procedure is followed with proper strength of acid and molybdate reagent.
(8) Griswoid, B. L., Humoiler, F. L., McIntyre, A. R., ANAL.CHEM.
23, 192 (1951).
King. E. J., Biochem. J . 26, 292 (1932). (IO) Kitson. R. E., Mellon, M. G., IND.ENG.CHEM.,ANAL.ED. 16, (9)
466 (1944).
(11) (12)
(13) (14) (15)
LITERATURE CITED
(16) (1)
Beil, F. K.. Carr, C. J., Kranta, J. C..
"_, .
~ A L CHEM. . 24,
1184
,,OCI)\ \I
( 2 ) Beii, R. D., Doisy, E. A., J . Bioi. Chem. 44, 55 (1920). (3) Benediot. S. R., Theis, R. C., Ibid., 61, 63 (1924). (4) Berenbium, I . , Chain, E., Biochem. J . 32, 286-95 (1038). (5) Briggs, A. P.. J . Bioi. Chem. 53, 13 (1922). (6) Fiske, C. H.. Subbarow, Y., Ibid., 66, 375 (1925). (7) Gmg, N. S., Sturtevant, J. M.. J . A n . Chem. Soc. 76, 2087 (1954).
(17) (18) (19)
Kuttner, T., Cohen, H. R.. J . Bid. Cheni. 75, 517 (1027). Kuttner, T.. Liohtenstein, L.. Ibid., 86, 671 (1930). Lowry, 0. H., Lopeu. J. A.. Ibid., 162, 421 (1946). Mlitrtin, J. B., Doty, D. M.. ANAL.CHEM.21, 065 (1040). Martiand. M., Robison, R., Biochem. J . 20, 847 (1920). Xiakarnurs. G. R., ANAL.CHEX.24, 1372 (1952). Rookstein, M., Herron, P. W., Ibid., 23, 1500 (1951). Sohaffer, F. L., Fong, J.. Kirk. P.. Ibid., 25, 345 (1953). Sumner. J. B., Science 100, 413 (1944).
RECE~V-ED for revlew November 5 , 1955. Accepted March 19, 1956. Supported in Dart by &n Institutionnl Grant from tire American Canoer Society and in part by Grant No. CS 9051 from the National Cancer Institute Xationd Institutes of Health. Public Heelth Servioe, Bethesds, Md.
Analysis of ixtures of slllucose and M a n n o s e by Paper Electrophoresis D. R. BRIGGS, E. F. GARNER, REX MONTGOMERY,
and FRED SMITH
Department of Agricultural Biochemistry, University of Minnesota, St. Paul, M i m .
A simple method is proposed for analyzing mixtures of D-glucose and D-mannose in which there is a high ratio of glucose to mannose. The sugars are separated by paper electrophoresis and determined by the phenolsulfuric acid method. The construction of a simple inexpensive apparatus for carrying out paper electrophoresis is described.
Paper Electrophoretic Apparatus. The apparatus is a simole
it f r i m 'the &r components,. rests on a-piece of foam rubber, I/, x 12 x 18 inches. These components are placed between
extendine from'the ends of the's;nd&&.
1
N STUDIES on certain wood pulps and polysaccharides contzunmng . ' glucose and mannose, the problem of analyzing
a mixture of these two sugars arises. Although a separation of glucose and marnose can be effected using paper partition chromatography there are in general two disildvantageswhicb, depending upon the solvent system used, render this technique impractical for quantitative work. Either the difference in R, values is not great enough or the R, value8 are 80 small that an undesirable length of time is required for the separation of the sugars. This paper describes a simple apparatus (f, 4 ) and a relatively rapid method for the analysis of mixtures of >glucose and o-mannose by paper electrophoresis.
diod into wide b6rdsilir
piwe; supply bavlngan out ut Gf 0 to 1500 volts a t 0 to 300 ma. Procedure. A known v&me of a solution containing a mix-
EXPERIMENTAL
WhatmanNo. 1filterpaper (11 X 22.5 inches) was used in all experiments as the supporting medium. Reagents. Sodium tetraborate @orax), 0 . l M buffer solution, pH = 9.2. p-Anisidine tricbloroacetate. Dissolve 0.1 gram of recrystallized p-anisidine in 20 ml. of water containing 3.0 grams of trichloroacetic acid (6). Methanolic hydrogen chloride. Prepare a 1% solution of anhydrous hydrogen chloride in anhydrous C.P. methanol. Phenol. Saturate freshly distilled phenol with water. T h e solution contains SO% phenol (w./w.). Sulfuric acid, concentrated C.P. reagent grade.
Figure 1. Apparatus for paper electrophoresis
Figure 2.
End view showing assembly of components for paper eleetrophoresis
