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Fat Determination

Babcock AND Ether Extraction Methods for Determination of Fat Content of Raw Milk

Prepared By
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 20090-6456.

Safety Precautions

Babcock Method

  1. 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.

  2. Dilution of concentrated sulfuric acid.  Always add acid to water, not water to acid.  Wear a face shield or goggles and heavy rubber gloves.

  3. 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 completely.

  4. 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

  1. 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 solvents.

  2. 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.

  3. Petroleum Ether.  Extremely flammable.  Use effective fume removal device.  Avoid static electricity, flames, and sources of sparks.

  4. Ethanol.  Flammable.  Use effective fume removal device when heating or evaporating.

  5. 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


  1. 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.

  2. 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 mm.

  3. 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).

          Each bottle must be constructed so as to withstand stress to which it will be subjected in centrifuge.

  1. 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.

  2. Pipet.  Standard milk pipet conforms to following specifications:
        Total length                                       < 330 mm
        Outside diameter of suction tube        6-8 mm
        Length of suction tube                        130 mm
        Outside diameter of delivery tube
            (must fit into bottle (a))                    4.5-5.0 mm
        Length of delivery tube                        100-120 mm
        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
            position, and fill the pipet to the graduation mark with H2O (at 20oC) using a buret (Class A -
            graduations < 0.05 mL).

  1. 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.

  2. 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 horizontally extended.

                                        Diameter of Wheel (inches)        rpm
                                                    14                                  909
                                                    16                                  848
                                                    18                                  800
                                                    20                                  759
                                                    22                                  724
                                                    24                                  693

  1. Dividers or calipers.  For measuring fat column.

  2. Water bath for test bottles.  Provided with thermometer and device to maintain temperature of the fat column at 57.5+1oC.

  3. 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.

  4. Bottle shaker.  Variable speed and matched to the maximum capacity of the centrifuge.

  5. 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.

  6. 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.


  1. 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.

  2. 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 acid.

  3. 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% fat
                At 3.6% fat
                    Sr      =   0.029%                RSDR   =      1.014%
                    SR*   =   0.037%                r value   =      0.081%
                    RSDr =   0.742%                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 J.AOAC.

Ether Extraction Method for Fat in Raw Milk


  1. 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.

  2. 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.

  3. 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).

  4. 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.

  5. 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.

  6. Tongs.  For handling weighing dishes

  7. Hot plate (steam bath or other heating device).  For evaporation of ether at 100oC or less. 

  8. Corks.  High-quality natural cork stoppers for flasks.  Soaking corks in water for several hours will give a better seal.

  9. 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.

  10. Water bath for tempering milk samples prior to weighing.  Provided with thermometer and device to maintain milk temperature of 38+1oC.


  1. Ethyl ether.  ACS grade, peroxide free, should leave no residue on evaporation.

  2. Petroleum ether.  ACS grade, boiling range 30-60oC, should leave no residue on evaporation.

  3. Ammonium hydroxide.  Concentrated, ACS grade, sp. gr. 0.9.

  4. Ethyl alcohol.  95%, no residue on evaporation.

  5. Distilled water.  Free of oil and mineral residue.

  6. Phenolphthalein indicator.  0.5% (weight/volume) in ethanol.


  1. 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.

  2. 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 matter.

  3. 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 first extraction.
    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 100
                                                    (wt of milk)

Maximum recommended difference between duplicates <0.03% fat

        At 3.6% fat:

                Sr      =     0.015%                    RSDR      =     0.512%
                SR     =    0.020%                     r value      =    0.044%
                RSDr =    0.396%                     R value     =    0.056%   

Reference:  This study to be published in Journal of Association of Official Analytical Chemists.

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