Indian J. Dairy Sciellce, !l.I, ,1, 198:!

A SIMPLE COLORIMETRIC METHOD FOR THE

DETERMINATiON
OF UREA IN MILK
~I.K. BI1:\V:\I)"\S . \ ~.

\".S. RA,IP CT and \' .C. G.\ ;\GU .[

Na/iOlwl Dairy ne .~('lfl"ch Instilt/ te. /{ artwl-J.12()O!

l K THOI)L"CT[O N

In rccent Yl'a rs, sc\'e ral Ic j.urls ha \'c pro

\'ided condncing e\'idence ,thai the lel'c l oj
urea in cow milk plays a sigll ilicanl rule in
regula ting its h C1l 1 s tability i ~luir and Sweet~u ... 1976,
19 77 , 1\178 ; lIull el al.. 1978;
'fuir et al., Hli 9 ; F"x et (Ii., 1980 ) . it ha ~
"t'cn sugges ted thai the I"HlItine
IHca 'iUrl'Illenl of urea in eu\\' mil k du ri ng its u sc in
Ihe manufacture of some da iry products,
~ uch as ste rilized milk, would help solving
problems arising as a rcsu lt of "aria tion s in
the heat stabilit y of milk Dluir and Sw ee t,ur, 1978}. Hence, estimalion of \lrca in
milk becomes a necessa ry step . The existing
method for urea estimation in milk in\' (J"'cs
the con version of urea to a mm onia
b~'
IIr~'lI. !\e and ,vhic h is su bsequently c1c te rminld colorime tric ally (Muir and Sweetsu r.
HliG) . .-\!though the method is preci se and
'l' n~iti\'('. it requ ires thl' usc of SpCd AI pu rity
IIrl'A SI,.' whi ch is difficult to prnc ure in Inany
dairy plants anci labo ratories in our ('ounIry. ~for(>o\'er , conlamina tion by "Ill ali
III!Iuun l of atmospheric ammonia wou ld
":In' misleading results . Thi s prompted us
to ~Ie\'clop an alte rnate method , equally
'(n .~ ili\c and preci se for es tim ation of urea.
rht, present paper describes such a colori IIwtric method fo r ure a (>s tim ation in mil k ,
"Meet on the m odifica tion o f the mic roanaly'I, or urea in bloocl ('Vootton. 19"1).

MATERI ALS A;./J)

~IETJI()nS

The indh'idll al milk sal1lple ~ w en! eoliec"'d (rom cows and hllifaloes mainta ined a l
lilt' IO$titutc herd. Urease wns purchn ,> cd

from Si;:lua Chem ical Co .. C .S ..-\ . .\\1 o the r

!"eu ~l: nls used in thi s .~ lud 'y wel"e procure,'
loea Jl y and Wl're of allldylical ~I"adc.
~IETlIflI)S

a) n e(l!J '/! I .~:

i)

Acid

the prcparati'm o f acid I"eal,:"l'n l , '1 .;; Ill" of ferri c

c hlori de was (\i s~ olye(j in -15 ml dis
tilled water to which MOul of phosphoric acid soluli(Jn w as added . The
"olullte
Itf the mix tu re wa s then
made up to '1;")0 ml with g :"J H 2SO"
n'u:/t'li f

.. ' F or

ii ) JUre d cO /O ur r l!OY Cllt - T he mixed

colour re a;:;-('nt WII S prellared by
mi xing up 16.7;) ml each of di acetyl
111onoximc (0.1'1:,%) a nd thiosemi
carbazide (0.313 % ). The mlumc of
the mi xture was then made up to
'2i)O 011 with di s1illed water.
h) Prepara tion of milk xe/"um :

To 3 ml t)f cow or hu ifal n milk 3 ml (If

10 % tridlluroacctic acid was added. The
pret: ipitalcd protein was /Htcred through
" ' h a tman :\Iu. 1 filt e r paper. An a liquot of
0.'1 llIi of the li!tr~t c was diluted to 5 011
w ith d istilled \\"ukr and 1.0 1111 of the dilut
1'(1 filtrat e W tl!<: used for urea estimation.
(" ) RstimntifJlI of l/rea:

T wo Illl ,)r standard urea solutions ('on

tai ning- "! .il to 20 r'g 'If ure a wcre usI"1
fol' p rep aring til(' .~ Iancl a rd cUl'ye . For milk

/Jelerlllill ({ lirin 01 ["rea

sa lllp k ... , \0 1.0 Ill i of di luh'd fi ll ratc frlllll

milk a .. in (h). 1.0 Ill \ (If w a tel" Wl\ :; :Hhkd.
Two IllL of Ildxerl fi eid fla genl foLlo wl'd hy
2 .0 Ill! of mixe d (olour reage nt wer,' added
to eac h t uhc , T h e tuhlS we re ke p t ill h(lll ing water bath for :W min , coo led tn ["/JO lll
il'mpe raturt' and the de\c1oJlt'd p in k \'olour

was read at

r) ~o

iii milk

by this m e thod it w a s m:cc :'> Nl ry 10 l.n:; pa r

the mill; fiLtrate ( rec fram 111"('8 . This w~
ad lil'\cd hy pn: pal'i nt-: a un'll-dcjJ l('\e(\li'iH
Sll llljllc by incu !J~ t ing o,:! 1111 of d ilu ted mil
(I :~ w ith 0. 1 11I1 of
u rcahl.'
solution (I
unit s/11l1. in Jlh [)~pha lc huffer, 0,0;);\1, pi

nm .

RES CLTS ..... ~D DIS CCSSIO.\""

~ 0 Ii

As the first (' x c!"d ~(' , a st undard

t:urn;
llsinH pure ... o lu tiull :.f lI rI'fI !:! .:. to '.!O
)
was prepared. F ro rll Fi g I it i.~ appare nt
thll t un:a conce ntratiu ll and the d Cn':oJll'd
colour 1"0110\\ a lineal" Icla tiu u , hi IJ. IIwl"pby
obeyi ng the Bee rs La w, Th is c un'c wa .~ then
utili zed for t he e sti mation of lIrea in mi lk
sa mples.
Prec{.~i(m

and ,~ell.\'iti{1ily

'4

"<o-,~-..,"--".".--,"",.--r,,,~.--T,,,""--c,,,.<,--,t."""~

thr me/ hot! :

To lind uu t w IH' tlw r t hc nul u raL so lu ble

<:ompuncn t s o lhe r than urca uf lUiJl,; art'
res ponsible for tilt, d en'lopcr! pi nk c(,!our

U~E/I.

(~gJ

Fig. 1

Standard curve for urea showing the lincar

relationship with concentration

T ..... B LE 1

Rel'OH' ry I, f a dded u n u ill c o w and

Sa mpk,

rni tiaL

u rea

j'\ t> .

- -_.

.,

"

4
"

__

!(. \.e\
il1!;r/IOOnd )
..

--- - ') -

- 'I

- I , I _

;.I:.!. W
:\ j . l'!
I".! ;11;
t ";", i~
1 ~ . H:1

hu i ~ a l t,

Added

u rea
le \ cI
i lll'0 "'' IOOIllL)

1:),00
BO.OO
-1.-).00

fIO.OO
7'-.,00
!jO.OO

milk
Final

Re.cover.""

e... ti ma tcd

- - - ---

urea lew!
1111 :.:-/ tOOml }

.t :! .70
O:::! .:)H
~:! .:) I
i'.! A:!
\1:2.l\l
tOH .O:!

(%)
rlfl.S(j
tOO.40

IOO .:W
100.10
99.81
SlI1.10

JI.ll. BlulPwlason ct al.

(Lv) at 37C for lO min. The trichloruacetic

aCid Jiltmte was ubtained frum this mea<it!pictcd milk and subjeeled tu colour de\-elopmcnt.
It was foun!1 that .~uch filtrate
aJti;r: treatment with the reagl'nts did not gin)
any absorbance at ;)20 nill. Thus the interf(.'tcnce of milk eOlllpOlicnts other than urea
in this method was I'lIled out. This fmther
strengthens the method as the dc\-eloped
t;'olour was due to urea only_
Accuracy vf lhe ml'lhfJr/,'

To a,'>sess the acellral'Y (Jf thc methud, [~

('overy experiment was eonducted
using
known quantity of uJ'{~a added to c(O\y and
buffalo milk samples. The levels of urea
wete then estimated in these samples. Tabk 1
shows the values of urea obtained by this
method, indicating an exel'llcnt reCoyery"
Hence it could be concluded that this simple
melhod for estimation of urea in milk is an
accurate one.
Urea levels ill cow awl buffalf) milk,'

The range alongwith the an'rage yaiues

foi' urea lcwls in 20 samples each of cow
andbuH'alo milk are shown in Table 2. In
general the urea !e\-eI in buJl'alo lllilk 117

2!i;)

mg/lOO ml) was lower than in (;OW Jllilk (;H

mg/lOO IllI).
I-fence, the possibility of the
lowe! heat ,;tahility in hulralo milk due to
the lower h~\-e1 of innate urea cannot be
ruled out due to corr('\atiol1 /wt\\"ccn heal
stability and un~a le\'cl as obser\"(;d in ca_~e
of cow mitk {\luir alHI S\n'ehur, l!J7S).
These results [lroyide ((!O\"ilwing eddenCl'
on tbe lise oj' tbis to!oriUlctri(- mcth"d for
lhl' estimation of ur!~a in milk as it satislie.-;
alt the requiretlll'llh oj' such procedure. Re("ently, in another iUH'stigatioll IGanguii
and Sharma, 1!J~()) it was ubse!"\-ed that the
addl'd urea lin ,{)Iit! ((Irml in !lIilk cannot
he quantilatinty dc\el"lllimd
tjlher yolumetric-ally 01' gradtllctri<'ally although it is
supposed 10 giye an e!lui\alenl increase in
either Si\"F or total solids. The reetJ\-ery in
Yulumetric method using
Ri("hrnond'~ furmula is aiJout iO% where[l~ grad metric-ally
it is eYC'n poor [-W%l, Thi,; further demand'>
the u,>e of a nnn(\cslru('ti\'e method for urea
C'~tilllatiun in mille The pr(-'sent colorimetric
method oyercumes sueh dJ'a\yh[lci-;s_ \Vork
is 110\\' in prngr('~s on thl' applicability of
the tllethod for urea estimation in milk
from other species, ill diff('rl'nt milk products and to {'valUate the illlpact of fcedurea un the milk-urea len:\.

TABLE 2

Urea it'n'l in cow and bulfalo milk

Range
.-\.\crage
-- (mgj 100m I) -

Spcdes*

1H. 69--11(:-\;')
12,02-2!l.6-1

Bll/Jalo

'Xo. of samples annly~('d in ,:adl (usc was

20:.
'-'\lcan

S.D_

SC\I\I.-\HY
A simple eol()J"imctl'ic
llH'th()(1
for the
estimation of urea in milk has been l1e,-clop('(!. The urea-depleted milk did not respond to the ('o\out" dl'n::lo[lml'n!. thus showing the mclhod as preelsc' and sensitive.
The re('{)\"cry of knowil amount of a!lderl
urea In Illilk by this melhod
was nearly
lOon;, _slIgw'sting good al'(ura("~. Estimation
of urea in cow and bulfaln milk by Ihis
method led to the conclusion that urea level
in huHalo milk is ("onsideJ"ahly luwer than
ill cow milk.

Dl'lf'rmina.lioll

:!()()

or

"f("([

ill milk

REFERENCES
Fox, P.F., Nash. :M.G., Horan, T.:: .. Obrien, J.
and Morrissey, P.A. (1980)J Dairll Res., 47,
199.

Ganguli, N.C. and

ration.

Sharm~,

B.M. (198.!) In prepa-

D.D. and Sweetsur, A."\\'.l\1.

Holt, C.D.. Muir.
(1978) J. Dairy Nes, 45, 183.

Muir, D.D. and Sweelsur, A.W.M. (1877) J. Dairy

Res .. 43, 495.

Muir, D.D. and Sweetsur, A.W.M. (97.) J. Dair-u

Rf's., 44, 249.

Muir, D.D. and Swe-etsur, A.W.M. (l9'B) J. Da:irll

Res., 4.>, 37.
Muir, D.D" Sweetsur, A.W.M. and Holt, C. (1979)
J. Dairy Res., 46, 381.
Wootton. I.D.P. (l9';"4)
Microanalysis in Medical Biochemistry,
Vth Ed.
p. ';".i,