33.2.
35
AOAC Official Method 961.07
Water (Added) in Milk
Thermistor Method
First Action 1961
Final Action 1961
IDF–ISO–AOAC Method
A. Cryoscope
Consists of cooling bath, sample agitator, seeding rod, thermistor
probe (electric resistance thermometer), and Wheatstone bridge and
galvanometer, or taut band meter measuring circuit. Bath may be
cooled by mechanical or electrical means or by insulated ice–salt
mixture. Test sample may be immersed in cooling bath or bath may
be “brought up” to test sample mechanically. Observed fp value is
read from measuring dial, calibrated in millidegrees C (0.001°C;
more correctly, degrees Hortvet or “H”), when galvanometer or
meter is nulled by rotation of dial.
Fill cooling bath at room temperature to proper level each time
instrument is used. Proper coolant level is determined by observing
coolant issuing from bath overflow or by visually checking coolant
level, depending on the make and model of cryoscope. Cooling bath
temperature should be -7° or -8° ± 0.5°C, depending on make of
cryoscope, and temperature is measured by placing thermometer in
empty test sample well.
Locate thermistor probe, both horizontally and vertically, at
midpoint of test sample. Check location visually, using 2.5 mL H2O
in test sample tube.
Amplitude of stirring wire should be great enough to assure
uniform temperature in test sample throughout determination and
may be checked visually, using 2.5 mL H2O in test sample tube to
which small amount of dust, powder, or dye has been added.
(Apparatus and supplies available as Advanced Cryoscope
[current Model 4D3] from Advanced Instruments, Inc., Two
Te c h n o l o g y Wa y, N o r w o o d , M A 0 2 0 6 2 , U S A ; Te l :
+1-800-225-4034; www.aicompanies.com; as Precision Cryoscope
16 Tech Cir, Natick, MA 01760, USA; and as Fiske Cryoscope from
Advanced Instruments, Inc., Fiske Division.)
B. Preparation of Standards
(Use distilled H2O recently boiled and cooled to 20°C for
preparation of standards.)
Prepare following sucrose primary standards or salt secondary
standards. Determine fp values of salt standards as in (b).
(a) -0.408°C (-0.422°H) Standard.—(1) Weigh 7.0000 g NIST
standard sucrose, SRM 17, into 100 mL volumetric flask and dilute
to volume with H2O; or (2) weigh 100 g H2O into 100 mL volumetric
flask and add 0.6889 g reagent grade NaCl (dried to constant weight
just before weighing).
(b) -0.600°C (-0.621°H) Standard.—(1) Weigh 10.0000 g NIST
sucrose into 100 mL volumetric flask and dilute to volume with
H2O; or (2) weigh 100 g H2O into 100 mL volumetric flask and add
1.0207 g reagent grade NaCl.
(Secondary salt standards with fp values equivalent to 7 and 10%
sucrose primary standards may be purchased from cryoscope
manufacturers.)
Mi cro or gan isms at tack su crose af ter lim ited stor age at
re frig er a tor tem per a tures, chang ing fp value of pri mary
standards. Salt (sucrose equivalent) secondary standards, stored
in poly ethylene bottles with screw caps, have long shelf life at
room temperature; use these each time fp value determinations
are made. If it is suspected that salt secondary standards are in
error, check them against freshly pre pared su crose pri mary
standards. It is responsibility of the analyst to be certain that fp
values of salt secondary standards are same as fp values of freshly
prepared sucrose primary standards.
C. Calibration of Cryoscope
(See introduction to 922.07 [see 33.2.34].)
Using calibration controls and fp values of standards, calibrate
cryoscope to obtain correct “span” (0.600 - 0.408 = 0.192°C) and
reference values (-0.600°C and -0.408°C). Follow directions in
man u fac turer’s op er at ing man ual. Cal i bra tion con trols and
procedures vary with make and model of cryoscope but with all
instruments 2 calibration controls (A and B or I and II) are adjusted,
individually or in combination, so that 7 and 10% sucrose primary
standards and/or sucrose equivalent salt secondary standards yield
fp values of -0.408° and -0.600°C, respectively, with 0.192°C span.
D. Determination (Final Action 1974)
[If titratable acidity, 947.05 (see 33.2.06), is >0.18%, results may
underestimate actual amount of added H2O in test portion.]
Apply following technique in exactly same manner for both
standards and test sample to obtain valid milk fp value.
Check cooling bath level, cooling bath temperature, stirring
efficiency, and probe position in test portion tube as in A.
Check reference fp values and “span,” using salt secondary
standards or sucrose primary standards. If “span” is other than
0.192°C, recalibrate cryoscope as in C. If “span” is correct but
reference fp values differ from known values of standards, it is not
necessary to recalibrate cryoscope; simple arithmetic correction
will give correct observed fp value of test portion.
Using clean, dry syringe or pipet, measure 2–3 mL test portion
and transfer to clean, dry test portion tube supplied by manufacturer.
Set measuring dial to expected fp value. Place test portion tube in
cooling bath test portion well or in operating head and lower
operating head to position test portion in cryoscope cooling bath.
Cool test portion if not already being cooled above. Proper
cooling is indicated by rapid and uniform (steady) movement of
light spot or needle from right to left over scale of galvanometer or
meter.
If cryoscope raises cooling bath to test portion level, begin slow
cooling at -1.5° to -2.0°C, depending on extent of supercooling
desired. If cryoscope immerses test portion in cooling bath, do not
isolate test portion in air above cooling bath.
Seed test portion at -2.0° or -3.0°C, depending on make of
cryoscope. Galvanometer spot or meter needle will jump to right as
temperature of supercooled test portion rises toward fp. Extent of
supercooling (seeding point) must be same for sucrose primary
standards or salt secondary standards as for milk test portions.
Adjust galvanometer spot or meter needle to 0 if necessary.
Switch galvanometer or meter to high sensitivity position. With
temperature dial, keep galvanometer or meter nulled (reading 0).
Galvanometer spot or meter needle will cease to move to right,
remain steady at 0, and finally begin to move to left. Read fp value
from measuring dial to nearest millidegree, while spot or needle is
steady just before movement to left begins. Do not read fp value
from measuring dial at some predetermined time after seeding test
portion; always wait for movement of galvanometer spot or meter
needle to left before recording fp value. Spot or needle will become
2006 AOAC INTERNATIONAL
ã 2005 AOAC INTERNATIONAL
steady and begin to move to left sooner with standards than with
milk test portions. Check 0 point of galvanometer or meter.
E. Interpretation
If fp is -0.508°C (-0.525°H) or below, milk may be presumed to
be H2O-free or may be confirmed as H2O-free by tests specified
be low. If fp is above -0.508°C, milk will be des ig nated
“presumptive added H2O” and will be confirmed as “added H2O” or
“H2O-free” by tests specified below. Evaluate extreme daily
fluctuations in fp of herd, pooled herd, or processed milk for
presence of added H2O.
To confirm herd milk as “added H2O” or “H2O-free,” determine
fp of authentic laboratory sample of herd milk obtained £ 72 h after
laboratory sample to be “confirmed.” Authentic laboratory sample
is sample of milk from 1 complete, supervised herd milking (either
AM or PM but beginning not <11 or >13 h after beginning of
previous milking) obtained from bulk tank after entire herd has been
milked through approved, properly sanitized, and thoroughly
drained milking system into empty bulk tank but before rinsing or
washing of system has begun. Compare fp of authentic laboratory
ã 2005 AOAC INTERNATIONAL
sample and laboratory sample to be confirmed. If fps differ by
£0.010°C, laboratory sample is confirmed as H2O-free.
To confirm pooled herd milk as “added H2O” or “H2O-free,”
determine fp of authentic laboratory samples of all herd milks
composing pooled herd milk, calculate weighted average fp for the
authentic laboratory samples of herd milk, and compare values as
above.
To confirm processed milk as “added H2O” or “H2O-free,”
determine fp of test samples of all pooled herd milk received by
processing plant, calculate weighted average fp for milk received,
and compare with fp value to be confirmed. If fps differ by
£0.010°C, processed test sample is confirmed as H2O-free during
processing. If ³1 test sample of pooled herd milk is “presumptive
added H2O,” proceed as above for pooled herd milk. Fp for
pasteurized-homogenized milk should be same as that of pooled
herd milk unless processing includes vacuum pasteurization, which
raises the freezing point approximately 0.005°C.
References: JAOAC 44, 438(1961); 51, 816(1968);
53, 539(1970); 55, 410, 504(1972).
ISO 5764:2002.
2006 AOAC INTERNATIONAL