Journal of Dairy Research (1974), 41, 31-35 31

Further studies on the estimation of diacetyl by the methods

of Prill and Hammer and Owades and Jakovac
BY B. WALSH AND T. M. COGAN
National Dairy Research Centre, The Agricultural Institute,
Fermoy, Co. Cork, Irish Republic

(Received 18 April 1973)

SUMMARY. Both the rate of formation and the total amount of dimethylglyoxime
formed from diacetyl in the presence of hydroxylamine were increased on omission
of phosphate from the Owades & Jakovac (1963) hydroxylamine reagent. Sodium
acetate was also unnecessary and slightly decreased the rate of dimethylglyoxime
formation. The optimum concentration of hydroxylamine lay between 0-5 and
0-75 M. The heating time necessary for formation of dimethylglyoxime can be
reduced from 60 to 20 min at 75 °C. Based on these findings a shortened and more
sensitive method for the estimation of diacetyl is recommended.

Prill & Hammer (1938) described a colorimetric method for the estimation of
diacetyl in milk cultures, using a tedious steam distillation procedure, and Owades &
Jakovac (1963) described a similar method for the determination of diacetyl in beer
except that diacetyl was released from the sample by purging with Na instead of bj r
steam distillation. This latter method was successfully applied to the estimation of
diacetyl in cheese cultures by Pack et al. (1964). Cogan (1972) has shown that rapid
methods of steam distillation are effective in releasing diacetyl from milk and that
time and temperature are critical in the formation of dimethylglyoxime. The basis
for these methods is that diacetyl is converted to dimethylglyoxime in the presence
of buffered hydroxylamine and converted to a pink ammono-ferrous dimethyl-
glyoximate complex by reaction with PeSO4 in alkaline solution.
This paper is concerned with a study of several factors affecting dimethyl-
glyoxime formation from diacetyl with a view to increasing the overall sensitivity
of the test.
MATERIALS AND METHODS
Diacetyl (BDH Chemicals Ltd, Poole, Dorset, England) was redistilled in an all-
glass apparatus at 88-92 °C. 5 ml of an aqueous solution (20 /ig/ml) was routinely
assayed using the reagents recommended by Owades & Jakovac (1963) except that
the samples were heated at 75 °C for 1 h to form dimethylglyoxime (Cogan, 1972).
The extinction was read at 530 nm in an SP 800 spectrophotometer (Unicam Instru-
ments Ltd, Cambridge, England). In initial experiments, the reagents recommended
by Prill & Hammer (1938) were used.
Analytical grade hydroxylammoniuni chloride (BDH Chemicals Ltd) was used
in the preparation of the various hydroxylamine reagents.
3 DAR4I
32 B. WALSH AND T. M. COGAN

Table 1. Colour formation by diacetyl using different systems of analysis

System Changes in procedure Extinction at 530 ran
Prill & Hammer None 0-47*, 0-48, 0-49
Ovvades & Jakovac None 0-36, 0-36, 0-36
Owades & Jakovao H2O used instead of K 2 HPO 4 0-47, 0-48
in making up NH2OH.HC1
reagent
Final procedure Final procedure 0-48, 0-49, 0-47
* Values are individual estimations.

Table 2. Effect of different K^HPO^ concentrations on the

estimation of diacetyl
K2HPO4 concentration
H2OH.HC1 reagent Extinction at 530 nm
Twice normal 0-32*, 0-33
Normal 0-36, 0-36
Half normal 0-43, 0-44
None 0-48, 0-47

* Values are individual estimations.

RESULTS AND DISCUSSION

A comparison of an analysis of 5 ml of an aqueous solution of diacetyl containing
20/ig/ml by the Prill & Hammer (1938) and Owades & Jakovac (1963) methods is
shown in Table 1. A 25% increase in extinction with a consequent increase in
sensitivity was found when the reagents recommended by Prill & Hammer (1938)
were used. Dilution of the Owades & Jakovac hydroxylamine reagent with water
instead of K 2 HPO 4 gave results identical to those obtained using the Prill & Hammer
method (Table 1). The major difference in the reagents is the presence of phosphate
in the hydroxylamine reagent used by Owades & Jakovac (1963). Consequently, this
reagent was intensively investigated.
Increasing concentrations of phosphate in the Owades & Jakovac (1963) hydroxyl-
amine reagent decreased colour (dimethylglyoxime) formation while total omission
results in a 25 % increase in extinction (Table 2). Colour formation in the absence of
K 2 HPO 4 (Fig. 1) was complete after 20 min at 75 °C, while in the presence of K 2 HPO 4
the reaction was not complete after 90 min. Omission of phosphate thus allows the
heating time in the formation of dimethylglyoxime from diacetyl to be reduced
from the 60 min recommended by Cogan (1972) to 20 min.
Omission of acetate from the hydroxylamine reagent resulted in a slight increase
in the rate and total amount of dimethylglyoxime formed (Fig. 2). Increasing the
temperature of heating from room temperature to 75 °C resulted in an increase in
the total amounts of dimethylglyoxime formed both in the presence and absence of
acetate. However, at any one temperature, the presence of acetate decreased the
amount of dimethylglyoxime produced. The greatest amount of dimethylglyoxime
formation occurred in the first 5 min at 75 °C and was complete after 20 min. Vir-
tually no increase occurred at any temperature between 20 and 30 min. Thus, it
 Estimation of diacetyl 33
0-5 r-

0-4

5. (H

y o-2

01

20 40 60 80 100
Time, inin

Fig. 1. Effect of phosphate on the rate of formation of dimethylglyoxime. O—O» Absence of

phosphate; • — • , presence of phosphate.

10 20 30
Time. min.
Fig. 2. Effect of time and temperature on the rate of formation of dimethylglyoxime in the
presence (closed symbols and x ) and absence (open symbols and + ) of acetate in the hydroxyl-
amine reagent. O, • , 75 °C; A, A, 60 CC; D, • , 45 °C; x , + , room temperature.

was decided to omit acetate from the hydroxylamine reagent and not to curtail the
heating period at 75 °C below 20 min.
At this stage of our studies, the buffered hydroxylamine reagent was a 2-5 % (or
0-36 M) solution of hydroxylammonium chloride. However, maximum formation of
dimethylglyoxime occurred when the hydroxylammonium chloride concentration in
the reagent lay between 0-50 and 0-75 M. Concentrations of 0-0-5 M and 0-75-1-50 M
3-2
34 B. WALSH AND T. M. COGAN
decreased the amount formed. The final concentration of NH 2 OH. HC1 in the reagent
recommended by Prill & Hammer is 0-57 M. The effect of using a 0-36 M solution
instead of one of 0-50 M is not great. Nevertheless, it was decided to use a 0-50 M
solution in routine application of the test.
Since dimethylglyoxime formation was similar using the reagents recommended
by Prill & Hammer (1938) and those recommended by Owades & Jakovac (1963)
except that distilled water was used instead of K 2 HPO 4 in the buffered hydroxyl-
amine reagent (Table 1), the implication is that any differences in the remaining
reagents, viz. acetone-phosphate, alkaline sodium-potassium tartrate and FeS0 4
are unimportant. To verify this hypothesis, however, a number of experiments were
carried out. The colour developed when K 2 HPO 4 was omitted from the acetone-
phosphate reagent was less than that obtained in the presence of K 2 HPO 4 . The
potassium-sodium tartrate reagent is c. 3 times more concentrated in the Prill &
Hammer method than in that of Owades & Jakovac. In addition, in the former
method 2-5 ml and in the latter 1-5 ml are used and both reagents crystallize on
storage though not to the same extent. Neither dilution of the Prill & Hammer
reagent up to 1:2 nor using 1-5 or 2-5 ml volumes of either reagent in the test affected
the results (not shown). Decreasing the concentration of sodium-potassium tartrate
1:3 with respect to NH 4 0H, was also without effect (not shown). The alkaline tar-
trate reagent used by Owades & Jakovac, when diluted to 200 ml instead of 150 ml,
resulted in a reagent which did not crystallize on storage and which did not affect
colour development (Table 1) and this is the reagent routinely used in the final form
of the test. In agreement with the findings of Owades & Jakovac (1963), fading of the
developed colour occurred when distilled water was used instead of 33 % K 2 HPO 4
to adjust the final volume to 10 ml. This effect was not reproducible and could be
ignored if the colour were read within 15-30 min of development.

FINAL FORM OF TEST

For the sake of completeness, the detailed procedure now followed in our laboratory
is given:
Reagents
Hydroxylamine. Dissolve 17-5 g Analar grade NH2OH.HC1 (BDH Chemicals Ltd,
Poole, Dorset, England) in water and make up to 500 ml.
Acetone-phosphate. Dissolve 29-0g K 2 HPO 4 or 38g K 2 HPO 4 .3H 2 O in distilled
water, add 40 ml of pure acetone and make up to 200 ml with distilled water. Store
at 3 °C.
Alkaline tartrate. Dissolve 100 g of sodium-potassium tartrate (COONa. (CH0H) 2 .
COOK.4H2O) in distilled water and make up to 200 ml with distilled water. Mix
with concentrated NH 4 0H in the ratio 22:3.
Ferrous sulphate. Dissolve 5 g FeSO4.7H2O in 1 % H2SO4 and make up to 100 ml.
Discard when slight yellow colour appears.
 Estimation of diacetyl 35

Procedure
20 g of culture is steam-distilled using Foamkil (Nutritional Biochemicals Cor-
poration, Cleveland 28, Ohio, U.S.A.) to prevent foaming in the apparatus described
by Cogan (1972), and the first 10 ml of distillate collected.
To 5 ml or other aliquot of the distillate is added 1-5 ml of the hydroxylamine
reagent. The mixture is heated to 75 CC for 20 min in a thermostatically controlled
water-bath and while still warm 0-5 ml of the acetone-phosphate reagent is added,
mixed and cooled to room temperature. 1-5 ml of the alkaline tartrate reagent is
added, mixed and immediately 0-1 ml of the FeSO4 reagent added and mixed. The
volumes are adjusted to 10 ml using 3 3 % K 2 HPO 4 and the extinction read at
530 nm after 15 min. The concentration of diacetyl can be read from a standard
curve obtained by distilling milk samples containing different levels of diacetyl.
Such a curve obeys Beer's Law up to 10/ig/ml.

Grateful appreciation is given to Finbarr Drinan for excellent technical assistance.

REFERENCES
COGAN, T. M. (1972). Journal of Dairy Science 55, 382.
OWADBS, J. L. & JAKOVAC, J. A. (1963). Proceedings of the American Society of Brewing Chemists, p. 22.
PACK, M. Y., SANDINE, W. E., ELLIKER, P. K., DAY, E. A. & LINDSAY, K. C. (1964). Journal of Dairy
Science 47, 981.
PRILL, E. A. & HAMMER, B. W. (1938). Iowa State College Journal of Science 12, 385.

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