Babcock AND Ether Extraction Methods for Determination of Fat
Content of Raw Milk
Test Procedures Committee of Market Administrators, February 1988
The following procedures for the Babcock and ether extraction
test methods have been approved by the Market Administrators' Test Procedures
Committee. Both of these chemical methods for determination of fat content
of raw milk are the result of a collaborative study involving 11 laboratories
located in the eastern two-thirds of the United States. Milk samples from
18 dairy farms throughout the United States were tested by each of the
laboratories over a 15-month period.
The goal of the collaborative study was to develop a
butterfat testing and evaluation system that is highly accurate and can be
uniformly applied throughout the dairy industry. These procedures emanated
from this extensive study and, if properly followed, will yield comparable
results between and within laboratories.
Babcock and ether extraction procedures, results, and
statistical analysis from this study were presented to the Association of
Official Analytical Chemists (AOAC) in September 1987. Since then, AOAC
has granted interim official first action approval to both methods, and the
modified Babcock procedure contained herein has been accepted to replace the
prior approved Babcock procedure for raw milk.
The full report is available from the office of any Federal
Milk Market Administrator or from the United States Department of Agriculture,
Agricultural Marketing Service, Dairy Division, P.O. Box 96456, Washington, DC
Handling sulfuric acid. Always wear eye
protection. Avoid contact of acid with skin. If acid is spilled
on skin, wash immediately with large amount of cool water.
Dilution of concentrated sulfuric acid. Always
add acid to water, not water to acid. Wear a face shield or goggles
and heavy rubber gloves.
Centrifugation. Balance the centrifuge by
placing an even number of bottles in positions diametrically opposite from
each other. Make certain that necks of the bottles placed in the
pivot-type head will clear the center when tubes swing to horizontal
position. Do not open the centrifuge cover until the centrifuge stops
Mercury (used for calibrating flask volumes only). Hazardous when in contact with ammonia,
halogens, and alkali. Regard spill on hot surfaces as extremely
hazardous. Vapors are extremely toxic and cumulative. Clean up
promptly. Powdered elemental sulfur sprinkled over spilled mercury can
assist in cleaning up spills. High degree of personal cleanliness is
necessary for persons who use mercury. Handle only in locations where
any spill can be readily and thoroughly cleaned up. A suction device
such as an aspiration flask is often useful for picking up mercury
spills. Mercury waste should be collected and disposed of properly.
Ether Extraction Method
Ammonium Hydroxide. Extremely caustic
liquid. Wear eye, skin, and respiratory protection (or use in a
properly designed hood). Vapors and liquids can burn skin, eyes, and
respiratory tract severely. Ammonia vapors are flammable. Reacts
violently with strong oxidizing agents, halogens, and strong acids. Do
not store in the same cabinet with concentrated acids or halogenated
Diethyl Ether. Store protected from light and
heat. Extremely flammable. Unstable peroxides can form upon long
standing or exposure to sunlight in bottles. Peroxides can react
explosively when in contact with chlorine, ozone, lithium aluminum hydride,
or strong oxidizing agents. Always work with diethyl ether in a fume
hood or appropriate fume removal device that is designed to remove fumes
that are heavier than air. Ether fumes are heavier than air and will
drop to the floor, not rise. Avoid static electricity and any source
of flame or sparks.
Petroleum Ether. Extremely flammable. Use
effective fume removal device. Avoid static electricity, flames, and
sources of sparks.
Ethanol. Flammable. Use effective fume
removal device when heating or evaporating.
Fume Removal During Evaporation of Solvents.
Evaporate flammable solvents on hot plates or heating devices designed for
solvent evaporation. Evaporation of solvents should be done in a hood
designed for removal of fumes from flammable solvents. Exhaust of
fumes to the exterior of the building should be in areas away from other
electrical equipment or source of flames and away from fresh air intake.
Babcock Method for Fat in Raw Milk
Standard Babcock milk-test bottle. 8%, 18g,
milk-test bottle, total height 160-170 mm (6.3-6.7"). Bottom of
bottle is flat, and axis of neck is vertical when bottle stands on level
surface. Quantity of milk for bottle is 18 g.
Bulb. Capacity of bulb to junction with neck
must be > 45 mL. Shape of bulb may be
either cylindrical or conical. If
cylindrical, outside diameter of base must be between 34 and 36 mm; if
conical outside diameter of base must be between 31 and 33 mm, and maximum
diameter between 35 and 37
Neck. Cylindrical uniform diameter from >5
mm below lowest graduation marked to >5 mm above highest
mark. Top of neck is flared to diameter of >10 mm.
Graduated portion of neck has length >75 mm and is graduated in
whole, half, and tenth percents, respectively, from 0.0 - 8.0%.
Graduations may be etched with black or dark pigment annealed to
graduation, or may be unetched black or dark lines permanently annealed to
the glass. Graduation line widths < 0.2 mm.
Tenth-percent graduations are >3 mm long; half-percent
graduations are >4 mm long and project 1 mm to left; and
whole-percent graduations extend at least half way around neck to the right,
but no more than three-quarters of the way around, and project >2
mm to left of tenth-percent graduations. Each whole-percent graduation
is numbered, with number placed to left of scale. A vertical line may
be etched and annealed with black or dark pigment or may be an unetched
black or dark line permanently annealed to the glass located 1 mm to the
right of the tenth percent graduation marks and extending >1 mm
above the 8% line and >1 mm below the 0% line. The zero line
must be etched, annealed with black or dark pigment and be <0.2 mm
wide. Capacity of neck for each whole percent on scale is 0.200 mL.
Maximum error of total graduation or any part thereof must not exceed 0.008
mL (.04% fat).
bottle must be constructed so as to withstand stress to which it will be
subjected in centrifuge.
Testing. Accuracy of each bottle shall be
determined. Bottle calibration accuracy is determined by placing the
bottle upside down on a Babcock bottle calibration apparatus (modified NAFIS
tester) that is capable of delivering known volumes of mercury into the
Babcock bottle neck. Bottle calibration apparatus delivery is
calibrated and the volumes of mercury contained between the 8 and 4% (0.008
mL), 4 and 0% (0.800 mL), and 8 and 0% (1.600 mL) marks are
determined. Accuracy of any bottle can also be determined by adding
13.5471 g clean, dry mercury at 20 degrees C to a bottle that has been
previously filled to the 0% mark with mercury. This should be equal to
5 +.04% on the scale of an 18 g milk-test bottle.
Pipet. Standard milk pipet conforms to following
< 330 mm
Outside diameter of suction
tube 6-8 mm
Length of suction
Outside diameter of delivery tube
(must fit into bottle
Length of delivery
Distance of grad. mark above
bulb 15-45 mm
Testing the pipet. Place the tip of the pipet against a firm rubber
surface, clamp the pipet in a vertical
fill the pipet to the graduation mark with H2O (at 20oC)
using a buret (Class A -
Acid measure. Device used to measure H2SO4,
should be capable of delivery in the range from 10 to 20 mL and can be set
to consistently deliver the appropriate amount of acid to obtain the desired
milk-acid reaction temperature.
Centrifuge or "tester". Standard
centrifuge, however driven, must be constructed throughout and so mounted as
to be capable, when filled to capacity, of rotating at necessary speed with
minimum vibration and without liability of causing injury or accident.
It must be heated, electrically or otherwise, to temperature of 55 to 60
degrees C during centrifugation. It must be provided with speed
indicator, permanently attached if possible. Proper rate of rotation
may be determined by reference to table below. By "diameter of
wheel" is meant distance between inside bottoms of opposite cups
measured through center of rotation to centrifuge wheel while cups are
Diameter of Wheel (inches) rpm
Dividers or calipers. For measuring fat column.
Water bath for test bottles. Provided with
thermometer and device to maintain temperature of the fat column at 57.5+1oC.
Water bath for tempering milk samples prior to pipeting.
Provided with thermometer, device to maintain temperature of soft water at
60+1oC, and device to deliver water into Babcock bottles.
Bottle shaker. Variable speed and matched to
the maximum capacity of the centrifuge.
Digital thermometer for measurement of milk-acid reaction
temperature. Digital thermometer that reads to the nearest degree
in the range of 100-120oC. Use an acid-resistant probe with a small
diameter (<0.5 mm) to ensure a rapid response time. The length of
the probe should be such that its tip is approximately 1 cm above the bottom
of the bottle when fully inserted.
Reading light. As background when measuring fat
columns. Light should be diffused (soft white color) and provide
illumination from angles above and below level of fat column. A
magnification device must be used to aid reading.
Sample preparation and temperature adjustment. With a
pipet, transfer 17.6+0.05 mL prepared sample at 38oC to milk-test
bottle. Blow out milk in pipet tip about 30 seconds after free outflow
ceases. Adjust milk in test bottles to 21+1oC. Adjust H2SO4
(sp gr 1.82 to 1.83 at 20oC) to 21+1oC. Pipet some additional
milk samples for use as temperature control samples.
Measurement of milk-acid reaction temperature and
determination of amount of sulfuric acid to use. Prior to testing a
group of samples, determine the correct amount H2SO4
to be used by measuring milk-acid reaction temperature. Start by
adding 17.5 mL of 21+1 degree H2SO4
to a bottle containing 18 g of milk of the same temperature. Add the
17.5 mL of acid in one delivery that washes all traces of milk into bulb and
cleanly layers the acid under the milk. Fully insert the
digital-thermometer probe down the bottle neck, immediately shake by hand
rotation until all traces of curd disappear. The peak reaction
temperature should be 108+2oC. Adjust the amount of acid added until
the reaction temperature us within this range and the color of the fat
columns is a translucent golden-yellow to amber. The amount of acid
required may be different for different technicians and different batches of
Testing milk samples. Add the appropriate amount of H2SO4
(as previously determined in (b)) by delivering the acid in one addition
that washes all traces of milk into the bulb and cleanly layers the acid
under the milk. Immediately shake by hand rotation until all traces of
curd disappear. Place the bottle in a Babcock bottle shaker set at
medium speed. Continue to add acid to all samples to be tested.
After acid has been added to all samples, shake the full set 1 additional
minute. The temperature of milk plus acid in the first bottle should not be
less than 60oC at time bottles are transferred to the centrifuge.
Place bottles in heated centrifuge, counterbalance, and after proper speed
is reached, centrifuge 5 minutes. Add soft H2O
at 60+1oC until bulb of bottle is filled. Centrifuge 2
minutes. Add soft H2O at 60+1oC until fat
column top approaches the 7% mark of the bottle calibration.
Centrifuge 1 minute longer at about 60oC. Transfer bottle to warm H2O
bath kept at 57.5+1oC, immerse it to level slightly above the top of
fat in column, and leave until column is in equilibrium and lower fat
surface assumes final convex form (>5 minutes). Remove one bottle
from bath, wipe it, and with aid of reading light and magnification use
dividers or calipers to quickly measure fat column (before it begins to cool
and contract). Place caliper points in the vertical line on the neck
of the bottle with one point at the lowest surface of the lower meniscus and
the other point at the top of the upper meniscus. Without changing the
distance between the tow points on the calipers, move the calipers down the
bottle neck until the lower point rests in the etched horizontal graduation
mark at 0%. Place the upper point of the calipers against the bottle
graduation and read the test in % by weight to the nearest 0.05%.
Repeat for each bottle.
Fat column at time of measurement should be translucent,
golden-yellow or amber, and free of visible suspended particles.
Reject all tests in which fat column is milky or shows presence of curd or
charred matter, or in which meniscus is indistinct or distorted; repeat
test, adjusting the volume of H2SO4
added to obtain proper color and milk-acid reaction temperature.
Maximum recommended difference between duplicates 0.1%
r value = 0.081%
R value*= 0.104%
*using regression equations for milk containing 3.6% fat.
SR = (0.0080 x 3.6) + 0.0800
R value = (0.0227 x 3.6) + 0.0226
References: Journal of the Association of Official Analytical
Chemists 8, 4(1924); 8, 471(1925); 56, 949(1975); this study to be published in
Ether Extraction Method for Fat in Raw Milk
Flask. A Mojonnier style ether extraction flask
with a volume of 22+1 mL in the lower bulb plus neck at the bottom of
the flask. The flask should have a smooth and round opening at the top
that will seal when closed with a cork.
Weighing dishes. Metal (8.5 to 9.5 cm diameter and
4.5 to 5.5 cm tall) or 70mm x 50mm glass crystallizing dishes or 250 mL glass beakers.
Calibration weights. Class S standard calibration
weights to verify balance accuracy within weight ranges to be used for
weighing flask (empty flask and flask plus sample) and weighing dish (empty
dish and dish plus fat).
Analytical balance. Readability to nearest
0.0001 g. Accuracy on verification within 0.0002 g, checked
periodically and whenever the balance is moved or cleaned. A record
should be kept of balance-calibration checks.
Dessicator. Room temperature for cooling weighing
dishes after preliminary and final drying. Should contain coarse
desiccant (mesh size 6-16) that contains a minimum of fine particles and
that changes color when moisture is adsorbed.
Tongs. For handling weighing dishes
Hot plate (steam bath or other heating device).
For evaporation of ether at 100oC or less.
Corks. High-quality natural cork stoppers for
flasks. Soaking corks in water for several hours will give a better
Vacuum or forced air oven. Vacuum oven capable
of maintaining a temperature of 70-75oC at greater than 50.8 cm (20 inches)
of vacuum for at least 7 minutes or a forced air oven maintained at a
temperature of 100+1oC.
Water bath for tempering milk samples prior to weighing.
Provided with thermometer and device to maintain milk temperature of 38+1oC.
Ethyl ether. ACS grade, peroxide free, should
leave no residue on evaporation.
Petroleum ether. ACS grade, boiling range 30-60oC, should leave no residue on evaporation.
Ammonium hydroxide. Concentrated, ACS grade, sp.
Ethyl alcohol. 95%, no residue on evaporation.
Distilled water. Free of oil and mineral
Phenolphthalein indicator. 0.5% (weight/volume)
Sample preparation. Prepare by tempering milk
to 38oC and weigh about 10 g of milk (to the nearest 0.0001 g) directly into
a clean and dry sample flask.
Weighing dish preparation. Number the clean
weighing dishes and predry under the same conditions as those that
will be used for the final drying after the fat extraction. Be sure
that all surfaces where weighing dishes will be placed (ie., hot plate,
desiccator, ect.) are clean and free of particles. At the end of
oven drying, place the pans in a room temperature desiccator and cool to room
temperature. Weigh the dishes to the nearest 0.0001 g and record
weights. (This should be done on the same day as fat
extraction.) Check balance zero after weighing each pan. Once
pans have been weighed, protect them from contamination with extraneous
Fat extraction. At this step, remove the dry cork
stopper used during the sample weighing process and replace with a wet cork
stopper which has been previously soaked in water for several hours.
Add 1.5 mL NH4OH to each flask and mix
thoroughly. The NH4OH neutralizes any acid
present and dissolves casein. Add 3 drops of phenolphthalein
indicator. The indicator will help sharpen the visual appearance of
the interface between the ether and aqueous layers during the
extraction. Add 10 mL ethyl alcohol, stopper with cork, and shake for 15
seconds. For the first extraction, add 25 mL ethyl ether, stopper with cork,
and shake flask very vigorously 1 minute. Release pressure by
loosening stopper. Add 25 mL petroleum ether, stopper with cork, and
repeat vigorous shaking for 1 minute. Centrifuge the flasks at about
600 rpm for at least 30 seconds to obtain a clean separation of aqueous
(bright pink) and ether phases. Decant ether solution into suitable
weighing dish prepared as (b). When decanting the ether solution into
the dishes, be careful not to pour over any suspended solids or aqueous
phase into the weighing dish. Ether can be evaporated at <100oC
from the dishes while conducting the second extraction.
For the second extraction, add 5 mL of ethyl alcohol, stopper with cork, and
shake vigorously for 15 seconds. Next, add 15 mL ethyl ether, stopper
with cork, and shake flask vigorously 1 minute. Add 15 mL petroleum
ether, stopper with cork, and shake flask vigorously for 1 minute.
Centrifuge the flasks at about 600 rpm for at least 30 seconds to obtain a
clean separation of aqueous (bright pink) and ether phases. If the
interface is below the neck of the flask, add distilled water to bring the
level about halfway up the neck. Addition of distilled water should be
done slowly and allowed to run down the inside surface of the flask so that
there will be minimal disturbance of the separation. Decant either
solution for the second extraction into the same weighing dish as for the
For the third extraction, omit the addition of ethyl alcohol and repeat the
procedure used for the second extraction.
Evaporate solvents completely on hot plate at <100oC (avoid
splattering). Dry the extracted fat plus weighing dish to constant
weight in a forced air oven at 100+1oC (30 minutes or more) or vacuum
oven at 70-75oC at greater than 50.8 cm (20 inches) of vacuum for at
least 7 minutes. Remove weighing dishes from oven and place in a
desiccator to cool to room temperature. Record the weight of each
weighing dish plus fat to the nearest 0.0001 g.
A pair of reagent blanks should be run each day that
tests are conducted. To run a reagent blank, replace the milk samples
with 10 mL of distilled water and run the test as normal. Record the
weight of any dry residue collected and use it in the calculation.
Reagent blank should be less then 0.0020 g of residue. If the reagent
blanks for a set of samples are negative, use the negative number in the
calculation. (Caution: Subtraction of a negative number means that you
add it to the difference in the weight of pan plus fat and the weight of the
empty pan.) A negative blank usually indicates that your pans were not
completely dry when you started or that your balance calibration shifted
between the weighing of the empty pans plus fat. Cause of negative
blanks should be identified and corrected.
[(wt pan + fat) - (wt pan)] - (avg. wt blank residue)
Percent fat = -------------------------------------------------------------- x
(wt of milk)
Maximum recommended difference between duplicates
r value = 0.044%
R value = 0.056%
Reference: This study to be published in Journal of
Association of Official Analytical Chemists.
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