A PICRIC ACID METHOD FOR THE SIMULTANEOUS
DETERMINATION OF LACTOSE AND SUCROSE
IN DAIRY PRODUCTS 1
N. A. P E R R Y ' AND F. J. DOAN
The Pennsylvania Agricultural Experiment Station
State College
For the determination of lactose in milk, "Official Methods" of the A.O.A.C.
(1) recognizes a polarimetrie method (Double Dilution) and a copper reduction
method (Munson-Walker). With sweetened condensed milk the Munson-Walker
method is specified for lactose, while a polarimetric method, requiring r e a d i n ~
before and after hydrolysis, is the only procedure mentioned for sucrose. No
sugar methods are included for such products as cream, ice cream, and dry milk.
White (18) has developed a procedure based on the Munson-Walker technique
which can be used for lactose and sucrose when present together. This method
corrects for certain irregularities in the unmodified Munson-Walker method and
has been found more accurate and convenient than "Official Methods" for sweet-
ened condensed milk (13, 10).
At best, the recognized methods for lactose and sucrose in dairy products are
tedious, lengthy, or require specialized equipment and for this reason sucrose
determinations often are avoided.
Colorimetric methods for certain carbohydrates, based on the reduction of
picric acid, have been developed and used for many years, particularly for glucose
in blood and urine (2, 5, 6, 9, 13, 15), but later for carbohydrates in plant materials
(16, 17, 19) and for lactose in milk (3, 10, 12) and other dairy products (3).
These methods have proven simpler than the official ones and on the whole quite
satisfactory. Most of them, however, were evolved using visual eolorimeters (less
sensitive and less accurate than present photoelectric instruments) and in many
cases the color development was not strictly proportional to the concentration of
sugars, thereby necessitating correction factors, standard curves or standard
colors close in color density to the unknowns.
h i the belief that a satisfactory eoIorimetric technique, based on the reduction
of picric acid, for the simultaneous determination of lactose and sucrose in dairy
products would be a very useful tool in dairy products control and in dairy prod-
ucts research, the study reported here was undertaken.
EQUIPMENT A N D METHODS
A Klett-Summerson photoelectric colorimeter was used for all color compari-
sons, employing a no. 52 filter (520 m~) which was found to be slightly more
sensitive than the no. 54 recommended by the maker of the instrument for picric
Received for publication Nov. 21, 1949
1 Authorized for publication Oct. 17, 1949, as paper no. 1551 in the J o u r n a l Series of the
Pennsylvania Agricultural Experiment Station.
Present address: Mid-west Dairy Products Corp., Cape Girardeau, Missouri.
176
LACTOSE AND SUCROSE D E T E R M I N A T I O N 177
ereatinine determinations. The scale of this instrument is graduated logarith-
mically and the readings are proportional to color density and hence to concen-
tration of the substance responsible for the color.
A water bath equipped with a peripheral perforated steam pipe and an over-
flow made it possible quickly to obtain boiling water at a constant level for heat-
ing flasks and tubes. In this bath (14" × 8" × 8") the introduction of a series of
tubes or flasks did not lower significantly the temperature of the water.
In the early studies, flasks (100 ml.) and various tubes were. used, as called for
by the method or modification under investigation, but later the " s u g a r tubes"
suggested by Meyers and Bailey (14) were adopted because of their greater con-
venience. These are straight-sided, 200 × 15 mm. tubes graduated at 3, 4, 10, 15
and 20 ml.
Standard lactose solutions were prepared from dried C.P. lactose monohydrate
on which triplicate polarimetric determinations indicated a purity of 99.88 per
cent.
Standard sucrose solutions were made from a supply furnished by the National
Bureau of Standards (lot 4602, standard sample 17) analyzing less than 0.003
per cent moisture, 0.003 per cent ash and less than 0.002 per cent invert sugar.
A portion of this was subjected to twice-repeated crystallization with absolute
alcohol from concentrated water solution, followed by vacuum drying over PzO~.
Samples of the original and " p u r i f i e d " sugar tested by the procedure of Willa-
man and Davison (19) gave identical readings.
The picric acid employed (Baker's, C. P., Analyzed, Crystalline, Special for
blood test) was found satisfactory by the Folin and Doisy test (7).
The first part of the work consisted of a reexamination of the picric acid
methods of Myers and Bailey (14), Benedict and Osterberg (2), Bierman and
Doan (3) and Willaman and Davison (19) in an effort to select a procedure
most satisfactory as a basis for determining lactose and sucrose in dairy products.
The methods were modified in various ways (largely by altering the amount and
concentration of reagents and by varying the heating periods) in an effort to
obtain color development in conformity to Beer's law which none of them ex-
hibited initially.
The second portion of the work consisted of comparative determinations of
lactose and of lactose and sucrose irt milk, lactose adjusted milk, simulated
sweetened condensed milk and ice cream mix. The methods compared were
the official Munson-Walker, the official polarimetric and the picric acid method
developed in this study, where lactose alone was being determined. Where lac-
tose and sucrose were being determined, White's (18) copper reduction method
only was used as the standard for comparison with the picric acid method. Ten
replicate determinations were made by each of the above methods on each of
the 19 samples analyzed and the data were examined statistically.
RESULTS
Development of Method. The detailed studies made in tiais portion of the
work are not reported nor are data shown, but a number of important observa-
178 Y. A. PERRY AND F. J . DOAN
tions should be mentioned, inasmuch as they affect the determination by the
picric acid method and since they influenced the selection of a procedure. The
color development, on which the method is based, is substantially complete when
lactose or invert sugar is heated in the presence .of picric acid and sodium car-
bonate for 20 min. However, it is not absolutely complete and therefore the
heating period must be controlled. Blanks or standards preferably should be
heated along with unknowns. Color, after development, is relatively stable (con-
stant for 25 rain.) but it does fade slowly. Consequently, samples should be
limited to a number which can be read in the colorimeter in a 20-min. period. I n
this study 20 samples were found to be the maximum.
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FIG. I . R e g r e s s i o n curves f o r lactose, sucrose a n d i n v e r t e d sucrose.
Lactose. While none of the original methods produced color density in pro-
portion to the concentration of sugar for the full. length of the curve, a few of
the modifications did. In general, increased heating periods increased the slope
of the curve (concentration of sugar = X and color density = Y) and also shifted
the point of origin in a positive direction. A modification of the method of
Willaman and Davison finally was selected as the most suitable, of those giving
results conforming to Beer's law, for use in analyzing milk. A calibration curve
and regression equation for lactose (fig. 1) was established from the analysis of
LACTOSE AND SUCROSE D E T E R M I N A T I O N 179
seven series 9f samples each containing six concentrations of pure lactose varying
from 0.6 to 1.6 mg. in the reaction mixture. This equation is as follows:
¥ = - 0.4 + 189.9 X, where Y is the colorimeter reading, - 0 . 4 the origin of the
curve, and 189.9, the increase of reading for each milligram of lactose (slope).
Lactose and sucrose. The literature (16, 19) states that sucrose is easily and
quickly inverted by heating in picric acid and, further, that for reasonable per-
iods at room temperature picric acid does not invert sucrose even in saturated
solution (3, 16, 17). These facts were verified. Sucrose was found to be com-
pletely inverted in sugar tubes heated in the boiling water bath for 4 rain. and
saturated picric acid caused no measurable hydrolysis of sucrose at room tem-
peratures over a period of 30 rain. These observations are important because
in the determination of lactose and sucrose in a medium where both are present
colorimeter readings are made twice, once without inversion of sucrose and
once a f t e r sucrose inversion.
Under these conditions it is necessary to know whether uninverted sucrose ex-
hibits any reducing properties when heated in alkaline picrate solution. The
literature suggests that it does not but repeated, careful trials in this work
demonstrated that it does have a slight but significant effect. F o r accurate
work, therefore, lactose determinations on products containing sucrose must
be corrected. Since the reducing power of uninverted sucrose was found to
increase with the alkalinity and to be proportional to the amount of sucrose
present, this phenomenon a p p a r e n t l y is the result of a slight degradation of the
sucrose brought about by heating in alkaline solution, giving rise to fragments
with reducing power. White (18), in his study of the Munson-Walker method
for sweetened condensed milk, found the same error in that method and his
modification corrects for it.
Previous workers f o u n d that the heat treament in picric acid, used for invert-
ing sucrose, does not affect the reducing power of lactose and other reducing
sugars if also present. This fact was confirmed.
A considerable amount of time and effort was expended in attempting to find
conditions for the reduction of picrate by inverted sucrose which would result
in a color regression curve originating at zero and still not complicate the pro-
cedure or change it too radically from the simple procedure found satisfactory
with lactose. This effort was not successful and it was finally decided to use the
lactose procedure and adjust the concentration of sucrose in the sample to the
portion of the color curve away from the lower readings.
A calibration curve and regression equation was established from data ob-
tained in a similar m a n n e r as with lactose alone, but for both u n i n v e r t e d sucrose
(correction on lactose) and inverted sucrose. These curves also are plotted
in figure 1. F o r u n i n v e r t e d sucrose, Y = 1.7 +6.1 X. F o r inverted sucrose,
Y = 8.9 + 246.6 X.
METHOD
Reagents. Saturated picrie acid is p r e p a r e d by heating 16 g. of the wet, C.P.,
reagent (special for blood test) per liter of distilled water until dissolved, then
180 N . A. P E R R Y A N D F . J . DOAN
cooling it slowly at room temperature for several days until the excess solute has
crystallized in the f o r m of large, blade-like crystals. Quick cooling and agitation
are conducive to fine, p o w d e r y crystals diffieult to keep out of the supernata~t
liquid which is the reagent.
Twenty-five g. of C.P., anhydrous Na~CO3 are dissolved and made u p to 100
ml. with distilled water. This solution should not be allowed to cool much u n d e r
65 ° F. or some of the solute will crystallize.
Where lactose is the only sugar. Approximately 1 g. of milk, cream, skim-
milk or a proportionate amount of other product, is weighed accurately into a
100-ml. volumetrie flask and diluted to volume with saturated picric acid. The
flask contents are shaken and filtered through a fluted filter. Two ml. of the
filtrate are t r a n s f e r r e d to a Myers and Bailey sugar tube containing 1 ml. of the
Na~CO~ reagent, stoppered lightly, shaken and placed in a bath of boiling
water for 20 rain. The tube then is cooled to room temperature (approx. 20 ° C.),
diluted to 20 ml. with distilled water and mixed by inverting. A portion is
t r a n s f e r r e d to a colorimeter tube and a reading obtained within 20 rain, of re-
moval from bath. A blank consisting of 2 ml. of picric acid and 1 ml. of Na2C03
is heated, cooled and diluted along with the unknown for adjustment of the
zero point of the colorimeter scale.
The calculation is simple where lactose is the only sugar present. The value
Y + 0.4
of X is first determined from the regression equation for lactose, X = 189.-'---~9'
where X = rag. of lactose in 2 ml. of filtrate and Y = photoelectric colorimeter
reading. F r o m this, the amount of lactose in the unknown is calculated as
X " 100" 100 5X
follows: % lactose ~-
1000" 2" wt. of sample = wt. of sample
Where lactose and sucrose are both present. Approximately 5 g. of sweetened
condensed milk, 13 g. of ice cream or an amount of other product giving a com-
parable amount of sucrose are weighed into a 100-ml. volumetric flask and diluted
to volume with distilled water. The contents are well mixed and a 5-ml. portion
t r a n s f e r r e d to another 100-ml. flask and made to volume with saturated picric
acid, mixed and filtered. Then 2 ml. of the filtrate are introduced into a " s u g a r
t u b e " containing 1 ml. of Na2CO~ reagent, mixed and lightly stoppered.
Another 1-ml. portion of the same filtrate is t r a n s f e r r e d to a sugar tube con-
taining I ml. of saturated picric acid, mixed well and placed in the boiling water
bath for 5 rain., after which it is cooled to room temperature. This second tube
now contains inverted sucrose. One ml. of Na2C03 is added to it and both tubes
are placed in the boiling water bath for 20 min., along with a blank for each of
them consisting of 2 ml. of saturated picric acid plus 1 ml. of carbonate for the
first and 1 ml. of each reagent for the second. All the tubes then are cooled
to room temperature, diluted to 20 ml. with distilled water and t r a n s f e r r e d to
colorimeter tubes for reading. The instrument scale is set at zero using the
p r o p e r blank before the respective readings are made.
The calculations required when both sugars are present are considerably more
involved, even when results for only one are desired, inasmuch as a correction
LACTOSE AND SUCROSE DETERMINATION 181
must be made for the effect of u n i n v e r t e d sucrose on the lactose reading. The
following symbols are used in explaining these calculations: X~ = rag. lac-
tose in 2 ml. of filtrate, X2 = rag. sucrose in 1 ml. of filtrate, YI = colorimeter
reading before inversion of sucrose, Y, = colorimeter reading after inversion
of sucrose and Ys = portion of Y1 reading due to action of uninverted sucrose.
Using the regression equation for inverted sucrose, Y = 8 . 9 + 2 4 6 . 6 X or
Y-8.9
X = 246.-~6-' an estimated value for X,, sufficiently accurate for calculating the
value Y~, is obtained, thus:
y= _ ~1 _ 8.9
X2 (est.) 246.6
The amount of sucrose present in the reaction mixture when the color due to
lactose is being developed is twice this amount or 2X~ (est.). The regression
equation for uninverted sucrose, Y = 1.7 + 6.1X, then becomes Ya = 1.7 + 6.1
[2X2 (est.)]. Substituting the value of X~ (est.), one obtains Y s = l . 7 + 6 . 1
2 ~ , which may be simplified to Ya = 20.2
Y + 0.4
The regression equation for lactose, Y = -0.4 + 189.9 X or X = 189.9 ' now
Y1 - Ya + 0.4
is employed and X 1 - 189.9 Consequently, the lactose in the sample
can be calculated as follows :
X1 • 100. 100" 100 100 X1
% lactose =
1000- 2- 5- wt. o~ sample wt. of sample
The regression equation for inverted sucrose again is used for calculating
accurately the value of X2, and hence the amount of sucrose in the sample:
X~=Y~ Y 1 - Y s 8.9
2 X~. 100. 100" 100 200 X2
246.6 , and % sucrose - 1000- 1- 5- wt. of sample = wt. of sample
The regression curves and methods of calculation used in the described
method should be applicable to any colorimeter using a scale similar to the Klett-
Summerson (logarithmic). F o r other colorimeters, calibration curves obtained
with pure sugar solutions can be employed. Such curves (fig. 1) also may be
used, if desired, with logarithmic scales.
ACCURACY OF ~£ETHOD
Lactose. The picric acid method, as described, was employed in analyzing
nine samples of milk and lactose-adjusted milk for lactose alone and the results
compared with those obtained by means of the official l~Iunson-Walker method
and the official polarimetric method. Ten replicate determinations were made by
each method on each sample. The results are summarized in table 1. These
show excellent agreement of the average results b y all three methods. The picrie
182 N. A. PERRY AND F. J. IX)AN
a c i d m e t h o d gives r e s u l t s i n t e r m e d i a t e b e t w e e n t h e t w o official m e t h o d s b u t t h e
r e s u l t s a r e s l i g h t l y Ihore v a r i a b l e t h a n f o r e i t h e r o f t h e m .
A n a l y s i s of v a r i a n c e of t h e o r i g i n a l d a t a i n d i c a t e s t h a t differences b e t w e e n
replicates are not significant. Variations among methods, using the interaction
b e t w e e n m e t h o d s a n d r e p l i c a t i o n s , a r e s i g n i f i c a n t . T h e difference b e t w e e n t h e
a v e r a g e p e r c e n t o f lactose b y t h e p i c r i c a c i d a n d t h e p o l a r i m e t r i c m e t h o d s is n o t
TABLE 1
Accuracy and variability of the picric acid method as vompared with the Munson-Wal#er and'
polarimetriv methods on milk where ~actose only is being determined
Standard Coefficient of
Method Laetosea deviation variation
(%) (%)
Picric acid 5.929 0.055 0.93
Munson-Walker 5.904 0.044 0.74
Polarimetrie 5.932 0.044 0.74
a Av. of 90 determinations by each method.
s i g n i f i c a n t ; t h a t b e t w e e n t h e p i c r i c a c i d a n d the M u n s o n - W a l k e r m e t h o d s is
s i g n i f i c a n t ; t h a t b e t w e e n t h e p o l a r i m e t r i c a n d t h e M u n s o n - W a l k e r m e t h o d s is
h i g h l y s i g n i f i c a n t . F o r o r d i n a r y a n a l y t i c a l p u r p o s e s these differences a r e be-
l i e v e d to be i n c o n s e q u e n t i a l .
F o u r o f t h e s a m p l e s a n a l y z e d in t h i s c o m p a r i s o n w e r e o b t a i n e d b y a d d i n g lac-
tose to two n o r m a l s a m p l e s of milk. T h e r e c o v e r i e s of these a d d e d q u a n t i t i e s o f
t h i s s u g a r b y a l l t h r e e m e t h o d s u s e d a r e i n d i c a t e d i n t a b l e 2. These a r e excep-
t i o n a l l y close to t h e e x p e c t e d values.
TABLE 2
~eeovery o f added lactose in four samples o f milk by the three methods of analysis
Sample
Method
Normal Added lactose Normal Added lactose
Pieric acid method
% lactose found 4.83 6.23 7.62 4.74 6.04 7.33
% lactose expected 6.24 7.60 6.05 7.32
% of expected 99.8 100.3 99.8 10(/.1
Munson-Walker method
% lactose found 4.78 6.17 7.54 4.68 6.03 7.29
% lactose expected 6.19 7.55 5.99 7.26
% recovery 99.7 99.9 100.7 100.4
Polarimetrie method
% lactose found 4.81 6.21 7.62 4.78 6.07 7.37
% lactose expected 6.22 7.58 6.09 7.36
% of expected 99.8 160.5 99.7 100.1
Lactose and sucrose. W h i t e ' s c o p p e r r e d u c t i o n m e t h o d f o r l&ctose a n d su-
crose was t h e m e t h o d u s e d f o r c o m p a r a t i v e p u r p o s e s w i t h t h e p i c r i c a c i d m e t h o d
where both sugars were determined. Ten replicate determinations were made
on each of t e n d i f f e r e n t s a m p l e s of ice c r e a m m i x , s i m u l a t e d s w e e t e n e d c o n d e n s e d
m i l k a n d s w e e t e n e d c o n d e n s e d m i l k . T h e r a t i o s of sucrose to lactose in the ice
L-&CTOSE AND SUCROSE DETERMINATION 183
cream mix samples and the simulated sweetened condensed milk were of such a
range as to cover all possibilities likely to be encountered in commercial products.
The results obtained for lactose in these products are summarized in table 3.
The data indicate excellent agreement between the two methods but, as in the
previous comparison, the picric acid method exhibits results which are slightly
more variable than the method used for .comparison. Analysis of variance re-
veals no significant difference between the mean values obtained by the two
methods.
TABLE 3
Accuracy and variability of the picric acid method as compared with W h i t e ' s modification
o f the Munson-Wal~er qnethod for lactose in dairy products containing sucrose
Method Lactosea Standard Coefficient of
deviation variation
(%) (%)
Picrie acid ............................ 5.409 0.062 1.15
White's .................................. 5.396 0.053 0.98
a Average of 100 determinations by each method.
The results obtained for sucrose on these same samples are presented in sum-
marized form in table 4. Analysis of variance of these data does indicate a
significant difference between the mean values obtained by the two methods.
Most analysts probably would consider even the widest variations obtained in
the s t u d y (0.33 per cent for a sample of commercial, sweetened condensed milk)
a reasonably small error for sucrose determination. As the data show, the picric
acid method gives somewhat more variable results than the method of White.
TABLE 4
Accuracy and variability of the pieric acid ~nethod as compared with Whitc's modification
of the Muuson-Walker method for sucrose in dairy products
Standard Coefficient of
Method Suet°sea deviation variation
(%) (%)
Pieric acid ............................. 16.609 0.114 0.69
White Js ....................................... 16.694 0.096 0.58
a Average of 100 determinations by each method.
Six of the ten samples analyzed for both lactose and sucrose were samples
of ice cream mix and simulated sweetened condensed milk, where the lactose con-
tent of the milk and condensed milk used in their preparation was known and
the amount of sucrose added was weighed carefully. Consequently it was pos-
sible to compare the values obtained by the picric acid method and W h i t e ' s
method with expected values for sucrose. These comparisons are presented in
table 5. The results show excellent agreement except for one value by W h i t e ' s
method.
184 N. A. PERRY AND F. J . DOAN
TABLE 5
Recovery of added sucrose in six samples of ice cream mix and simulated
sweetened condensed milk by two ~aethods of analysis
Picric acid method White's method
Ratio of Sucrose
sucrose to expected Sucrose Per cent of Sucrose Per cent of
lactose found expected found expected
(%) (%) (%)
2.40-1.0 10.43 10.49 100.6 10.57 101.3
2.65-1.0 1 L39 11.43 100.4 11.35 99.6
2.90-1.0 12.33 12.31 99.8 12.28 99.6
3.30-1.0 12.93 12.88 99.6 12.99 100.5
3.65-1.0 14.11 14.08 99.8 14.15 100.3
4.00-1.0 15.25 15,19 99.6 15.27 100.1
CONCLUSIONS
As a result of detailed studies of the procedures recommended in the litera-
ture for the colorimetric determination of sugars by the reduction of picrie acid
or sodium picramate, a method employing this technique but utilizing a photo-
electric colorimeter was evolved for the simultaneous determination of lactose and
sucrose in dairy products. The method also is applicable to the determination
of either sugar alone.
The picric acid method is empirical and time factors must be controlled
carefully. The technique is relatively simple as only two easily prepared re-
agents are required and, compared with commonly used methods, it is less tedious
and less time consuming. The calculations, where both sugars are present, are
somewhat involved but not difficult to apply.
Comparative studies indicate that the picric acid method probably is as ac-
curate as the Mtmson-Walker, the polarimetric or White's modification of the
~unson-Walker, although analysis shows that the variation of replicates is
slightly wider.
It is felt that the method offers distinct advantages over currently used pro-
cedures for sugar determinations in a number of dairy products. It also appears
simpler, especially insofar as reagents are concerned, than the modified ferri-
cyanide procedure of ttites and Ackerson (8) and at least as convenient as the
saecharimetric-refractometric method of Browne (4).
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(3) BI~aMA~, H. R., AND DOAN, F . J . A Colorimetrie Picric Acid Method for Determining
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1945.
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LACTOSE AND SUCROSE DETERNIINATION 185
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