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 hypo-
chlorite reagent coiuld be added up to 30 minutes
later without change of absorbance, whereas if this
sample were mixed with a reagent containing hypo-
chlorite 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 tem-
perature is not recommended because the absorbance
decreased rapidly thereafter.
WITHINRECENT YEARS there has been enhanced interest in
the phenol-hypochlorite reaction for estimation of am-
monia. Use of catalysts, particularly sodium nitroprusside
( I ) , has improved the slmitivity and has made determinations
on ultramicro quantities of sample possible. This has in-
creased the practicability for such clinical biochemical applica-
tions 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 rea-
gents in other combinations have been proposed such as
alkali combined with phenol to form alkaline phenate, nitro-
prusside and hypochlorite as separate solutions (3), or nitro-
prusside and hypochlorite mixed before use (4). There are
contradictory views regarding the essential sequence of rea-
gents and the timing O F their addition and also there is varia-
tion in conditions of teinperature and time described for color
development. These factors prompted a study of the reac-
tion 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,
x
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.
(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).
(B) Alkaline hydrochlorite; 2.5 grams of sodium hydrox-
ide, 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,
Pa. ; precalibrated capillary tubing tolerance i1 x),
to tube. Tube covered with parafilm, shaken vigorously to
added
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 tempera-
ture 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 com-
partment of a Beckman DB spectrophotometer was con-
trolled 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 water-
bath 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 main-
tained at the basic concentrations as above. Color develop-
ment was at 37" C for 20 minutes.
Reagents, Sequence, Timing. Reagents were prepared as
follows: hypochlorite, nitroprusside, phenol plus nitro-
prusside, phenol plus alkali, alkali plus hypochlorite, hypo-
chlorite plus nitroprusside. Concentrations of these rea-
gents 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 phenol-
was used with similar time intervals. Color development was
for 20 minutes at 37 " C.
Phenol
Phenol reagents
+ nitroprusside
Hypochlorite reagents
+ hypochlorite
Alkali
Phenol + alkali Nitroprusside, hypochlorite (sep-
arate solutions added without
delay)
+
Phenol alkali +
Hypochlorite nitroprusside
(mixed immediately before use)
+
Phenol alkali, nitroprusside Hypochlorite
(separate solutions, added
without delay)
RESULTS AND DISCUSSION
The absorbance spectrum on a Beckman DB spectropho-
tometer 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
 also be accomplished by use of a smaller specimen volume-
Table I. Absorbance Maxima e.g., 1 pl-or by selection of a wavelength, such as 560 mp
Concn. (9,which is not at the maximum absorbance.
nitroprusside, Absorbance was affected by change in concentration of
rng/100 ml phenol, alkali, and hypochlorite (Figures 1 and 2). If it is
phenol-
nitroprusside Temperature, O C assumed that an absorbance of a t least 0.5 represents
soh. 20 25 37 50 75 loo satisfactory color development for this concentration of
0.4 ... o... 0.62 0.60 ammonium sulfate, equivalent to 4 pg of ammonia nitrogen
1 0.65 0:65 0:67 0.65 0.69 0.67 content, it is apparent that a range of concentrations for each
2 0.66 0.66 0.66 0.73 0.72 substance is possible: phenol 0.5 to 1.5 grams per 100 ml;
4 0.64 0.65 0.65 0:62 0.73 0.72 sodium hydroxide 0.2 to 1.0 gram per 100 ml; sodium hypo-
5 0.67 0.65 0.66 0.61 0.74 0.72
10 0.61 0.61 0.59 ,.. 0.75 0.73 chlorite 0.3 to 10.0 ml of 5 % solution per 100 ml. When the
15 0.62 0.60 0.58 0.77 0.76 concentration of hypochlorite was decreased very slightly
20 0.59 0.58 0.55 0150 o... 0.77 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,
Table 11. Time (Minutes) to Reach Maximum Absorbance
often abruptly. Preparation of fresh reagents, usually the
Nitroprusside hypochlorite, has corrected the problem. Use of a more con-
concn., centrated solution of hypochlorite-Le., containing up to 10
rngil00 rnl
phenol- ml of 5 x solution per 100 ml of reagent-could be used to
nitroprusside Temperature, "C extend the useful life of the reagent but fresh reagent may be
soh. 20 25 37 50 75 100 prepared easily. With change in concentration of alkali,
0.4 60+ 60+ 60+ 30+ 10 3a satisfactory color development occurred when the pH of the
1 56 44 34 20 10 5Q sample and reagents combined was within the range from 9.9
2 52 35 18 30+ 10 5'7 to 12.1.
4 28 22 14 20 20 5a
5 23 15 12 20 20 3a When the second reagent was added immediately after the
10 20 14 10 30+ 20 l a first, there was satisfactory color development regardless of
15 16 13 8 30+ 20 l a the sequence of reagents-Le., when phenol reagents were
20 11 9 6 20 30+ la added first and when hypochlorite reagents were added first.
Q Absorbance decreased thereafter. 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 rea-
gents were added first and in one instance with a phenol
In the absence of nitroprusside, color development was reagent-Le. phenol with both alkali and nitroprusside-there
minimal, a n absorbance reading of only 0.05 for a solution were decreases in absorbance of up to 8.5 % after a 2-minute
containing 4 pg of ammonia nitrogen; nitroprusside in- interval, 8.2 to 17.0% after a 5-minute interval, and 33.9 to
creased the sensitivity more than 10-fold. Tables I and I1 6 4 S x after a 30-minute interval. On the other hand, when
show that a number of conditions of temperature and nitro- the first reagent was phenol with nitroprusside or phenol with
prusside concentration gave absorbance readings which did alkali, there was no decrease in absorbance after any of the
not change with longer periods of time and, hence, could be time intervals studied-up to 30 minutes before addition of
selected conveniently for the reaction-e.g., concentrations of hypochlorite. The observation of Fawcett and Scott (3),
nitroprusside from 1 to 20 mg per 100 ml a t temperatures of that the color reagents should be added promptly after each
20", 25", and 37" C for times ranging from 56 t o 6 minutes; other and that for every minute interval between the addition
nitroprusside from 0.4 to 15.0 mg per 100 ml a t 75' C for 10 of nitroprusside and hypochlorite the final optical density is
to 20 minutes. The differences in reaction rates at 20" and a t diminished by about 1.5 %, should be quoted only in relation
25" C should be noted because any temperature within this to reagents used by these authors and should not be quoted,
range might ordinarily be regarded as room temperature. A as it has been, in relation to procedures using other reagents.
temperature of 50" C would not be desirable for the reaction The decrease in absorbance in using alkaline hypochlorite
because with most concentrations of nitroprusside there was a first has been attributed to volatilization of ammonia caused
slight but continuing increase in absorbance. A reaction by alkalinity of the hypochlorite reagent (6). Because the
temperature of 100" C would require critical timing and rigid combination of alkali and phenol at pH 11.85 did not result
duplication of standards because the absorbance reached a in a decrease in absorbance except when nitroprusside also
maximum rapidly and decreased abruptly. At both 75" and was present, it would appear that some factor additional t o
100" C, the absorbance was considerably higher than a t lower that of alkalinity is responsible for the decrease of absorbance.
temperatures. This effect a t 75" C which has not been re- A reagent sequence which does not require critical timing
ported, is remarkable because with one exception-the highest would be preferred in a busy laboratory. If using either
concentration of nitroprusside-the absorbance remained phenol plus nitroprusside or phenol plus alkali, it would be
constant a t that temperature for an additional 10 or 20 possible to mix specimens with the reagent in a series of tubes,
minutes after reaching the maximum. Consequently, if then add the hypochlorite reagent conveniently with a time
utmost sensitivity from these reagents is desired, a tempera- interval up to 30 minutes. In carrying out the various tests
ture 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 fre- (5) A. Kaplan, "Standard Methods of Clinical Chemistry" S.
Meites, ed., Vol. 5, p. 250, Academic Press, New York, 1965.
quently necessary to use a means of lessening the depth of (6) R. L. Searcy, N. M. Sirnrns, J. A. Foreman, and L. M. Berg-
color such as by dilution with water or with blank. This may quist, Clin. Clzirn. Acta, 12, 170 (1965).

972 ANALYTICAL CHEMISTRY

 1.3 20 ?Lo
CONCENTRATION (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 combina-
tion requires either three solutions or a second solution which
is a rather unstable combination of hypochlorite plus nitro-
prusside.
Recommendations for a simplified procedure would there-
fore be as follows.
Reagents. Phenol plus nitroprusside; alkaline hypo-
chlorite. 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 con-
veniently in a Microcap, 1- to 20-11 capacity depending on
Q
0.1
5.0
CONCENTRATION CMLS%SOL" PER 100 ML>
4.0
I
5.0
concentration. For ammonia in blood a preliminary diffu-
sion (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 ethylenedi-
aminetetraacetic 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 hypo-
chlorite to each tube. Cover with parafilm. Mix well.
Prepare a reagent blank of 5.0 ml of phenol p'us nitro-
prusside 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).
IQO 15.0
20.0
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 tempera-
ture (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.
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 be-
tween several simple anionic species and nitrogenous
cations from aqueous solution to lipoidal solvent is
well recognized, the special solvating role of proton-
donating 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 extract-
able ion pairs.
ALTHOUGH EXTRACTION OF PHARMACEUTICAL AMINES and qua-
ternary 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 magni-
tude of the extraction constants (2, 3). Schill has recently re-
ported 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. Phar-
macol., 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).
974 ANALYTICAL CHEMISTRY
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, Mani-
toba, June 1966. Work conducted in The Medical Research
Section, Ottawa Civic Hospital, Ottawa, Canada.
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 respon-
sible for favorable distribution coefficients in most instances
than are parameters such as dielectric constant (9). The
studies were performed with dextromethorphan, d-3-methoxy-
N-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 over-
simplified viewpoint, be classified into three possible cases:
case I Case 11 Case 111
In the first case it is assumed that the cation is large and lipo-
philic except for the positively charged center. The small ex-
(9) P. Mukerjee, ANAL.CHEM.,
28, 870 (1956).