Phenol-Hypochlorite Reaction for Determination of Ammonia M. W. Weatherburn Laboratory of Hygiene, National Health and Welfare, Ottawa, Canada
The Berthelot color reaction has been investigated with the particular aim of presenting a simple, reliable analytical procedure. The combination of two reagents prepared readily, phenol plus nitroprusside and alkali plus hypochlorite, gave excellent reproducibility with great convenience. After the ammonium sulfate sample was mixed with the phenol reagent the hypochlorite reagent coiuld be added up to 30 minutes later without change of absorbance, whereas if this sample were mixed with a reagent containing hypochlorite or with alkaline phenate plus nitroprusside, there was a decrease in absorbance unless the second reagent was added immediately. Satisfactory color development with minimal precautions was given by a variety of reagent concentrations and by reaction temperatures of 20°, 2 5 O , 3 7 O , and 7 5 O C. Sensitivity was increased at 7 5 O C although the time to reach maximum absorbance was longier than at 3 7 O C. The absorbance maximum was reachied quickly at looo C but this temperature is not recommended because the absorbance decreased rapidly thereafter.
WITHINRECENT YEARS there has been enhanced interest in the phenol-hypochlorite reaction for estimation of ammonia. Use of catalysts, particularly sodium nitroprusside ( I ) , has improved the slmitivity and has made determinations on ultramicro quantities of sample possible. This has increased the practicability for such clinical biochemical applications as the estimation of ammonia and urea in biological fluids. Color development i i S described by Chaney and Marbach involves a simple technique using two reagents-nitroprusside with phenol and hypochlorite with alkali (2). Similar reagents in other combinations have been proposed such as alkali combined with phenol to form alkaline phenate, nitroprusside and hypochlorite as separate solutions (3), or nitroprusside and hypochlorite mixed before use (4). There are contradictory views regarding the essential sequence of reagents and the timing O F their addition and also there is variation in conditions of teinperature and time described for color development. These factors prompted a study of the reaction in some detail, particularly with the aim of recommending a reliable yet simple technique for a busy laboratory. ESXPERIMENTAL
Reagents for all studies were phenol, sodium nitroprusside (sodium pentacyanoniixosyloferrate 111), sodium hydroxide, sodium hypochlorite (2; available chlorine), and ammonium sulfate; all were reagent grade. Solutions were prepared from water which had been passed through a cartridge o f ion exchange resin, Illco-way Ion X changer, for removal of ammonium ion. Basic Reagents (2). (A) Phenol plus nitroprusside; 5 grams of phenol with 25 mg of sodium nitroprusside per 500 ml of solution. !;tore in amber bottle in refrigerator, 1 month.
x
(B) Alkaline hydrochlorite; 2.5 grams of sodium hydroxide, 4.2 ml of sodium hypochlorite to 500 ml of solution. Store in amber bottle in refrigerator, possibly 1 month. The basic procedure is a modification of the method of Chaney and Marbach (2): Reagent A, 5.0 ml measured from automatic pipet (Schuco Polypette) into test tube. Specimen, 20 pl of standard solution of ammonium sulfate, measured in a Microcap (Drummond Scientific Co., Broomall, added Pa. ; precalibrated capillary tubing tolerance i1 to tube. Tube covered with parafilm, shaken vigorously to mix. Reagent B, 5.0 ml measured from automatic pipet, added, mixed thoroughly. Color development at 37" C, for 20 minutes. Absorbance measured at room temperature at 625 mp. Rate of Reaction. Using the concentrations of reagents as above, with the exception of nitroprusside which was varied from 0.4 to 20 mg per 100 ml of solution, the rate of reaction was investigated at six temperatures ranging from 20" to 100" C. By use of a Haake thermostatically controlled circulating water bath the temperature of the cuvette compartment of a Beckman DB spectrophotometer was controlled to 20", 25", and 37' C ; absorbance at 625 mp was charted directly on an attached Sargent SRL recorder. At 50", 75", and 100' C, tubes were left in a Magni Whirl waterbath for controlled time intervals ranging from 1 minute to 60 minutes; tubes were rapidly cooled under cold running water to room temperature for absorbance readings at 625 mp. Concentration. Depth of color at 625 mw was studied using varying concentrations of phenol, 0.1 to 5.0 grams per 100 ml; hypochlorite, 0.1 to 100 ml per 100 ml; and alkali, 0.05 to 5.0 grams per 100 ml. Other reagents were maintained at the basic concentrations as above. Color development was at 37" C for 20 minutes. Reagents, Sequence, Timing. Reagents were prepared as follows: hypochlorite, nitroprusside, phenol plus nitroprusside, phenol plus alkali, alkali plus hypochlorite, hypochlorite plus nitroprusside. Concentrations of these reagents were calculated to be such that the quantity in each test, 10-ml volume, would be the same as that when basic reagents were used as above. Reagents were used in the combinations listed below adding the hypochlorite reagent immediately after the phenol reagent and also allowing an interval of time-2, 5, 10, 20, and 30 minutes-before adding the hypochlorite. The reverse sequence-i.e. hypochlorite reagents followed by phenolwas used with similar time intervals. Color development was for 20 minutes at 37 " C.
x),
Hypochlorite reagents + hypochlorite Nitroprusside, hypochlorite (separate solutions added without delay) Phenol alkali Hypochlorite nitroprusside (mixed immediately before use) Phenol alkali, nitroprusside Hypochlorite (separate solutions, added Phenol Phenol
Phenol reagents nitroprusside alkali
+ +
Alkali
+
+
+
without delay)
RESULTS AND DISCUSSION (1) B. Lubochinsky and J. P. Zalta, Bull. Sti Chim. Biol., 36,1363 (1954). (2) A. L. Chaney and E. P. Marbach, Clin. Chem., 8, 130 (1962). (3) J. K . Fawcett and J. E. Scott, J . Clin. Path., 13, 156 (1960). (4) T. W. MacFarlane, r'roc. Assoc. Clin. Biochem., 3, 21 (1964).
The absorbance spectrum on a Beckman DB spectrophotometer showed a maximum at 625 mp. On this instrument Beer's law was followed in a concentration range from 0.5 to 6 pg of ammonia nitrogen. VOL 39, NO. 8, JULY 1967
971
Table I. Absorbance Maxima Concn. nitroprusside, rng/100 ml phenolnitroprusside soh. 0.4 1 2 4 5 10 15 20
Temperature, 20
25
37
... 0.65 0.66 0.64 0.67 0.61 0.62 0.59
0:65 0.66 0.65 0.65 0.61 0.60 0.58
0:67 0.66 0.65 0.66 0.59 0.58 0.55
O C 50
o...
0.65 0:62 0.61 ,..
0150
75 0.62 0.69 0.73 0.73 0.74 0.75 0.77
o...
loo 0.60 0.67 0.72 0.72 0.72 0.73 0.76 0.77
Table 11. Time (Minutes) to Reach Maximum Absorbance Nitroprusside concn., rngil00 rnl phenolnitroprusside Temperature, "C soh. 20 25 37 50 75 100 30+ 10 3a 0.4 60+ 60+ 60+ 1 2 4 5 10 15 20 Q
56 52 28 23 20 16 11
44 35 22 15 14 13 9
34 18 14 12 10 8 6
20 30+ 20 20 30+ 30+ 20
10 10 20 20 20 20 30+
5Q
5'7 5a
3a l a l a
la
Absorbance decreased thereafter.
In the absence of nitroprusside, color development was minimal, a n absorbance reading of only 0.05 for a solution containing 4 pg of ammonia nitrogen; nitroprusside increased the sensitivity more than 10-fold. Tables I and I1 show that a number of conditions of temperature and nitroprusside concentration gave absorbance readings which did not change with longer periods of time and, hence, could be selected conveniently for the reaction-e.g., concentrations of nitroprusside from 1 to 20 mg per 100 ml a t temperatures of 20", 25", and 37" C for times ranging from 56 t o 6 minutes; nitroprusside from 0.4 to 15.0 mg per 100 ml a t 75' C for 10 to 20 minutes. The differences in reaction rates at 20" and a t 25" C should be noted because any temperature within this range might ordinarily be regarded as room temperature. A temperature of 50" C would not be desirable for the reaction because with most concentrations of nitroprusside there was a slight but continuing increase in absorbance. A reaction temperature of 100" C would require critical timing and rigid duplication of standards because the absorbance reached a maximum rapidly and decreased abruptly. At both 75" and 100" C, the absorbance was considerably higher than a t lower temperatures. This effect a t 75" C which has not been reported, is remarkable because with one exception-the highest concentration of nitroprusside-the absorbance remained constant a t that temperature for an additional 10 or 20 minutes after reaching the maximum. Consequently, if utmost sensitivity from these reagents is desired, a temperature of 75" C could be used without critical timing. The sensitivity a t lower temperatures is more than adequate for many purposes, for instance in the measurement of urea nitrogen in serum. With elevated values of urea, it is frequently necessary to use a means of lessening the depth of color such as by dilution with water or with blank. This may 972
ANALYTICAL CHEMISTRY
also be accomplished by use of a smaller specimen volumee.g., 1 pl-or by selection of a wavelength, such as 560 mp (9,which is not at the maximum absorbance. Absorbance was affected by change in concentration of phenol, alkali, and hypochlorite (Figures 1 and 2). If it is assumed that an absorbance of a t least 0.5 represents satisfactory color development for this concentration of ammonium sulfate, equivalent to 4 pg of ammonia nitrogen content, it is apparent that a range of concentrations for each substance is possible: phenol 0.5 to 1.5 grams per 100 ml; sodium hydroxide 0.2 to 1.0 gram per 100 ml; sodium hypochlorite 0.3 to 10.0 ml of 5 % solution per 100 ml. When the concentration of hypochlorite was decreased very slightly below the minimum level, there was a substantial decrease in absorbance. In this laboratory and in others, the intensity of color development from the same reagents has decreased, often abruptly. Preparation of fresh reagents, usually the hypochlorite, has corrected the problem. Use of a more concentrated solution of hypochlorite-Le., containing up to 10 ml of 5 x solution per 100 ml of reagent-could be used to extend the useful life of the reagent but fresh reagent may be prepared easily. With change in concentration of alkali, satisfactory color development occurred when the pH of the sample and reagents combined was within the range from 9.9 to 12.1. When the second reagent was added immediately after the first, there was satisfactory color development regardless of the sequence of reagents-Le., when phenol reagents were added first and when hypochlorite reagents were added first. When there was an interval of time before addition of the second reagent, there was frequently a substantial decrease in absorbance. Thus, in all instances when hypochlorite reagents were added first and in one instance with a phenol reagent-Le. phenol with both alkali and nitroprusside-there were decreases in absorbance of up to 8.5 % after a 2-minute interval, 8.2 to 17.0% after a 5-minute interval, and 33.9 to 6 4 S x after a 30-minute interval. On the other hand, when the first reagent was phenol with nitroprusside or phenol with alkali, there was no decrease in absorbance after any of the time intervals studied-up to 30 minutes before addition of hypochlorite. The observation of Fawcett and Scott (3), that the color reagents should be added promptly after each other and that for every minute interval between the addition of nitroprusside and hypochlorite the final optical density is diminished by about 1.5 %, should be quoted only in relation to reagents used by these authors and should not be quoted, as it has been, in relation to procedures using other reagents. The decrease in absorbance in using alkaline hypochlorite first has been attributed to volatilization of ammonia caused by alkalinity of the hypochlorite reagent (6). Because the combination of alkali and phenol at pH 11.85 did not result in a decrease in absorbance except when nitroprusside also was present, it would appear that some factor additional t o that of alkalinity is responsible for the decrease of absorbance. A reagent sequence which does not require critical timing would be preferred in a busy laboratory. If using either phenol plus nitroprusside or phenol plus alkali, it would be possible to mix specimens with the reagent in a series of tubes, then add the hypochlorite reagent conveniently with a time interval up to 30 minutes. In carrying out the various tests
(5) A. Kaplan, "Standard Methods of Clinical Chemistry" S. Meites, ed., Vol. 5, p. 250, Academic Press, New York, 1965. (6) R. L. Searcy, N. M. Sirnrns, J. A. Foreman, and L. M. Bergquist, Clin. Clzirn. Acta, 12, 170 (1965).
1.3
20 CONCENTRATION
?Lo
4.0
I
5.0
(G.1 IOOMLJ
in this work, it was found that the precision of results using the phenol plus nitroprusside combination was noticeably superior to that of any of the alkaline phenate combinations. The phenol plus nitroprusside reagent has an additional advantage in that it requires only two solutions, both of which are reasonably stable, whereas the phenol plus alkali combination requires either three solutions or a second solution which is a rather unstable combination of hypochlorite plus nitroprusside. Recommendations for a simplified procedure would therefore be as follows. Reagents. Phenol plus nitroprusside; alkaline hypochlorite. As described in Experimental, basic reagents. Procedure. Place in each tube the specimen or standard containing 0.5 to 6 pg of ammonia nitrogen. For ammonia in readily available form, as in urine, reagents are added directly to the specimen. The specimen is measured conveniently in a Microcap, 1- to 20-11 capacity depending on
concentration. For ammonia in blood a preliminary diffusion (7, 8) or ion exchange resin treatment (9, IO) is necessary. For urea nitrogen in serum (2), the specimen, 1 to 20 p1, is incubated with 0.2 ml of solution of urease in ethylenediaminetetraacetic acid buffer (EDTA) at 37" C for 15 minutes before addition of colorimetric reagents. Using an automatic pipet, measure 5.0 ml of phenol plus nitroprusside into each tube. Cover the tubes with parafilm. Shake vigorously to mix well. Add 5.0 ml of alkaline hypochlorite to each tube. Cover with parafilm. Mix well. Prepare a reagent blank of 5.0 ml of phenol p'us nitroprusside and 5.0 ml of alkaline hypochlorite. (7) R. H.Brown, G. D. Duda, S. Korkes, and P. Handler, Arch. Biochern. Biophys., 66,301 (1957). (8) J. L. Ternberg and F. B. Hershey, J . Lab. Clin. Med., 56, 766 (1960). (9) J. C.B. Fenton, Cfin.Chirn. Acta, 7,163 (1962). (10) G. E. Miller and J. D. Rice, Am. J. Clin. Puthof., 39,97(1963).
Q 0.1
IQO
5.0
20.0
15.0
CONCENTRATION CMLS%SOL" PER 100 ML>
VOL 39, NO. a, JULY 1967
973
Set the tubes in a rack for color development in a water bath at 37' c for 15 minutes. Alternatively Set at room temperature (20" C or more) for 30 minutes. Measure absorbance at 625 mp. Subtract absorbance of blank and calculate concentration from absorbance of standard. ACKNOWLEDGMENT
The author thanks Margot Pickard and Thomas Chung for technical assistance in carrying out this work, and R. H.
Allen and J. E. Logan for helpful suggestions in preparation ofthe paper and for interest in the work. RECEIVED for review January 9,1967. Accepted April 3, 1967. Part of this work was presented as a paper at the Conference of the Canadian Society of Clinical Chemists, Winnipeg, Manitoba, June 1966. Work conducted in The Medical Research Section, Ottawa Civic Hospital, Ottawa, Canada.
Ion Pair Extraction of Pharmaceutical Amines Role of Dipolar Solvating Agents in Extraction of Dextromethorphan Takeru Higuchi, Arthur Michaelis, T. Tan, and Arthur Hurwitz Laboratory f o r Pharmaceutical Analysis, School of Pharmacy, University of Wisconsin, Madison, Wis. 53706 Although the ready transfer of ion pairs formed between several simple anionic species and nitrogenous cations from aqueous solution to lipoidal solvent is well recognized, the special solvating role of protondonating molecules has not been seriously considered. Since the phenomenon affords a particularly useful technique in separation and analysis of amines, the physical chemistry of these systems has been studied. Data are presented which suggest that ion pairs such as dextromethorphan hydrochloride can be extracted into organic solvents only as complexes containing of the order of five molecules of proton donors such as chloroform or phenol. The nitrate, bromide, and iodide behave similarly, except that the latter appears to require only four molecules of the proton donor. Less hydrophilic anions yielded more readily extractable ion pairs.
ALTHOUGH EXTRACTION OF PHARMACEUTICAL AMINES and quaternary ammonium compounds as ion pairs with anionic dye molecules has received a great deal of attention, investigations into the physical chemical basis of the observed phenomena have been relatively limited until recently. The extraction of quaternary ammonium compounds has been reviewed by Ballard et ai. ( I ) . Biles et al. have studied the partitioning behavior of amines paired with aromatic sulfonates and made an attempt to relate some physical parameters to the magnitude of the extraction constants (2, 3). Schill has recently reported on an extensive study of the extraction of amines and quaternary ammonium compounds with bromothymol blue (4-6) and also on the extraction of some high molecular weight amines with inorganic anions (7). A hypothesis based on the possible role of solvated ion pair species in enhancing the extractive process, proposed recently by Higuchi (2, S), assumes that the free energy involved in the (1) . , C . W. Ballard, J. Isaacs, and D. G. W. Scott. J. Pharm. Pharmacol., 6,971 (1954). ( 2 ) L. R. Hull and J. A. Biles. J . Pharm. Sci.. 53.869 (1964). . , (3j G. J. Divatia and J. A. Biies, Ibid.,50,916 (i961). (4) G. Schill, Acta Pharm. Suecica, 1, 101 (1964). (5) Zbid.,p. 169. (6) Ibid.,2, 13 (1965). (7) Ibid.,p. 99. (8) T. Higuchi and E. Roubal, Division of Analytical Chemistry, ACS, Abstracts of Papers, 149th Meeting, 28 B (April 1965).
transference of the ionic components from the water phase to form simple ion pairs in the organic phase would in many cases be far too unfavorable to yield useful partition coefficients. The addition of suitable solvating species to the organic phase would be expected to enhance markedly the extractive process through solvation of the formed ion pairs. Some evidence for this has already been presented by Biles (2). Experimental evidence presented here confirms the theoretical prediction that greater ion pair extraction results when suitable solvating agents are added to the nonpolar organic phase. It appears that formation of stoichiometric complexes between the ion pair and solvating species in the organic phase is more responsible for favorable distribution coefficients in most instances than are parameters such as dielectric constant (9). The studies were performed with dextromethorphan, d-3-methoxyN-methylmorphinan,
CH,O
as an example of a pharmaceutically important monoprotic organic base. GENERAL CONCEPT
The concept of the role of the solvating agent and its affinity for the ion pair has been considered by us in the following generalized manner. Ion pair solvation, or, equivalently, masking of the ionic character of the ion-ion bond, would be a significant factor in the extractive equilibrium if the ion pair structure was such as to expose strongly charged surfaces. These lipophilic ion pairs may, from a somewhat oversimplified viewpoint, be classified into three possible cases:
'
974
ANALYTICAL CHEMISTRY
case I
Case 11
Case 111
In the first case it is assumed that the cation is large and lipophilic except for the positively charged center. The small ex(9) P. Mukerjee, ANAL.CHEM., 28, 870 (1956).
