Analytical Methods
Table of Contents
http://www.fmmaseattle.com/lab/method.htm
Click on the links below for more information on the following topics
Methods
Preparation of Milk Samples
Fat Content of Raw Milk
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Photo Summary of Ether Extraction
Solids Content of Milk
Protein Content of Milk
Lactose Content of Milk
Technical Supplements
Protein Testing - FAQ's
True Protein and Other Solids;
technical details for keeping pace with federal order reform
Sample Preparation
Shipping and Storage
Milk samples should be drawn in nonabsorbent, leak-proof containers (e.g. plastic vials or bags) and
kept as cold as possible until testing (avoid freezing). Sample containers should be filled to
approximately 75% of capacity, allowing sufficient head-space for thorough mixing. Samples taken for
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USDA verification purposes should be labeled according to MA requirements using bar code labels
and/or a permanent marker.
Samples should be shipped (unless picked up by MA personnel) by overnight express per MA
guidelines. The MA laboratory will supply plastic ice chests, smaller receptacles for packaging, and if
necessary, "blue ice" blocks for shipment. If available, our preference is to ship using wet ice secured
in leak-proof bags. Upon receipt by the MA, samples are checked for temperature and leakage and
immediately refrigerated.
Heating and Mixing
Temper milk samples in a warm water bath to 42+10 C. Intermittent mixing while heating will speed
the tempering process and ensure incorporation of any butterfat that has separated. Immediately prior to
testing, mix samples by repeated gentle inversion until homogeneous (we recommend at least 10x).
Milk subjected to repeated heating and cooling or excessively long heating times will deteriorate and
cause inaccurate test results. Also, milk that is sour or that has been frozen is not suitable for testing.
Temper samples in water bath
Mix by gentle inversion, at
until 42 C/108 F
least 10 times
Immediately run sample
Methods Used for Component Analysis
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
Preface
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
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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
a. 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.
b. Dilution of concentrated sulfuric acid. Always add acid to water, not water to acid. Wear a
face shield or goggles and heavy rubber gloves.
c. 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.
d. 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
a. 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.
b. 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
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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.
c. Petroleum Ether. Extremely flammable. Use effective fume removal device. Avoid static
electricity, flames, and sources of sparks.
d. Ethanol. Flammable. Use effective fume removal device when heating or evaporating.
e. 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
Apparatus
a. 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.
b. 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.
c. 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.
d. 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
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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.
e. 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).
f. 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.
g. 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)
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16
18
20
22
24
rpm
909
848
800
759
724
693
h. Dividers or calipers. For measuring fat column.
i. Water bath for test bottles. Provided with thermometer and device to maintain temperature of
the fat column at 57.5+1oC.
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j.
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.
k. Bottle shaker. Variable speed and matched to the maximum capacity of the centrifuge.
l.
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.
m. 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.
Determination
a. 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.
b. 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.
c. 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
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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
Apparatus
a. 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.
b. 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.
c. 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).
d. 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.
e. 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.
f. Tongs. For handling weighing dishes
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g. Hot plate (steam bath or other heating device). For evaporation of ether at 100oC or less.
h. Corks. High-quality natural cork stoppers for flasks. Soaking corks in water for several hours
will give a better seal.
i.
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.
j.
Water bath for tempering milk samples prior to weighing. Provided with thermometer and
device to maintain milk temperature of 38+1oC.
Reagents
a. Ethyl ether. ACS grade, peroxide free, should leave no residue on evaporation.
b. Petroleum ether. ACS grade, boiling range 30-60oC, should leave no residue on evaporation.
c. Ammonium hydroxide. Concentrated, ACS grade, sp. gr. 0.9.
d. Ethyl alcohol. 95%, no residue on evaporation.
e. Distilled water. Free of oil and mineral residue.
f. Phenolphthalein indicator. 0.5% (weight/volume) in ethanol.
Determination
a. 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.
b. 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.
c. 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.
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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.
Calculation
[(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
=
SR
=
RSDr =
0.015%
0.020%
0.396%
RSDR
r value
R value
=
=
=
0.512%
0.044%
0.056%
Reference: This study to be published in Journal of Association of Official Analytical Chemists.
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http://www.fmmaseattle.com/lab/etherpictures.htm
Ether Extraction
The following photos are provided as a visual supplement to the AOAC-approved Ether extraction method
(which can be found by clicking the back button then clicking on Fat Content of Raw Milk). We hope you
find them useful!
-Seattle Lab Staff
Extraction
Pipetting of 10 mls well-agitated milk at 104 F
Pre-weighing of evaporating dishes that have
into mojonnier flasks.
been heated, cooled, and dessicated.
Addition of ether to flasks containing NH4OH,
Tightly-stoppered flasks containing sample and
EtOH, indicator, and sample. (This is an
Shaker table used to agitate samples following each ether addition.
reagents
explosion-proof fume hood.)
Pouring off of organic phase (clear) being careful
Flasks ready to centrifuge for 30 seconds after
not to pour aqueous phase (pink) into evaporating Evaporation of ether in the fume hood on a heated surface.
addition of petroleum ether and before decanting.
dishes.
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Allow to evaporate until all of the ether is gone
Transfer to 100oC oven for 30 minutes.
Cool dishes in a dessicator to room temperature.
and only fat remains in the dishes.
Clean up
Weighing of dish with fat residue.
Discard residual flask contents down the drain
with running water (or according to local
Fill flasks with HOT soapy water and soak.
regulations).
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Rinse thoroughly and air dry.
Total Solids
Direct Oven Drying Method for Determination of Total Solids Content of
Milk AND (Direct Oven-Ether Extraction) Method for Determination of Solids-Not-Fat
Content of Milk
Preface
The following procedures for determining total solids and solids-not-fat content of milk have been
approved by the Market Administrators' Milk Test Procedures Committee. Both of these component
test methods are the result of collaborative studies which were presented to the Association of Official
Analytical Chemists (AOAC) in May 1988. These methods were granted interim official first action
approval in January 1989.
Ten laboratories analyzed nine pairs of blind duplicate raw milk samples for total solids utilizing the
direct forced air oven method. Solids-not-fat testing was performed by nine laboratories which
analyzed nine pairs of blind duplicate raw milk samples for fat using the Mojonnier ether extraction
method and for total solids using the direct forced air oven method. Solids-not-fat was subsequently
determined by subtracting the percent fat from the percent total solids.
The goal of the collaborative studies was to develop testing procedures for solids and solids-not-fat
determination that would yield highly accurate test results and could be uniformly adopted and applied
throughout the dairy industry. The procedures presented here, when properly followed, will yield
comparable results between and within laboratories.
The full report of the total solids and solids-not-fat testing methods 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.
Direct Forced Air Oven Drying Method for Total Solids in Milk
Principle
Total solids are determined by weighing milk, drying milk, and weighing dried milk residue. The
sample is dried in a forced air oven for 4 hours at 100+1 degree Celsius. Total solids content of milk is
the weight of dried milk residue expressed as percentage of original milk weight.
Apparatus
a. Weighing dishes. Flat bottom dish >5 cm diameter without cover. If disposable aluminum
dishes are used they should be ones manufactured by the "oil free" process to allow more
uniform sample spreading in the dish.
b. Calibration weights. Class S standard calibration weights to verify balance accuracy within
weight ranges to be used for weighing empty dish and dish plus sample.
c. Analytical balance. To read to the nearest 0.0001 g. Accuracy on verification within 0.0002
g. Check periodically and whenever the balance is moved or cleaned. Keep a record
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of balance calibration checks. A digital electronic balance is recommended for fast response
time for weighing warm milk.
d. Water bath. For tempering milk samples prior to weighing, sample temperature should be 38+1
degree Celsius.
e. Desiccator. Room temperature. For cooling weighing dishes after preliminary drying of empty
dishes and after final drying of dish plus sample. Use coarse desiccant (mesh 6-16) that contains
a minimum of fine particles and that changes color when moisture is absorbed. Desiccator
should seal tightly and contain a thermometer.
f. Tongs.
g. Forced air oven. Capable of rapid temperature recovery after oven door is opened and closed
after sample loading and maintaining a temperature of 100 +1 degree Celsius (Blue-M model
490A-2 or equivalent). To evaluate oven temperature recovery: heat empty oven to 100+1oC
and stabilize for 15 minutes, turn off oven and fully open door for 2 minutes, close oven door,
turn oven back on, start timing, and record the time that it takes the oven to reach 99oC. Time
should be < 7 minutes.
Determination
a. Weighing dish preparation. Number the clean weighing dishes and dry for at least 2 hours in a
forced air oven at 100+1 degrees Celsius. Store dishes in dessicator until needed (must be used
within one week or redried). Touch predried dishes only with clean, dry tongs.
b. Sample preparation. Prepare by tempering milk to 38+1oC and mixing. Sample and weigh
milk at 38+1oC.
c. Total solids determination. Remove metal shelf from oven and place next to balance. Record
all weights to four decimal places. Weigh a predried sample dish. Pipet approximately 3 g of
38+1oC milk (nearest 0.1 mg) directly into a preweighed sample dish. Do not chase the
drift. Record the sample weight immediately after the weight stabilizes and just before it starts
its steady drift to lower numbers. Remove dish from balance and place dish on oven shelf for
transport to oven. Check balance zeros between samples.
d. Blanks. Weigh 2 empty, predried dishes.
e. Drying. Preheat the oven to 100+1oC. The hot air exhaust vent should be open to allow
moisture to be removed from the oven. After weighing is completed, turn off the oven and place
shelf plus all dishes (including blanks) into the oven, close the door, turn on the oven, and dry for
4 hours at 100+1oC. Do not open the oven door or place anything else into the oven until the
4-hour drying is completed.
f. Cooling. Remove dishes from the oven and cool to room temperature in desiccator (>30
minutes). Weigh the dish plus dry milk on the same balance as was used for the initial weighings.
Calculation
Always use blank values in the calculation. If blank values are negative, be sure to add the blank
value instead of subtracting it. The cause of negative blanks should be determined and
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corrected. Negative blanks are usually caused by the pans not being completely dry before use in the
analysis.
[(weight of dish + dry milk) - (wt of dish)] - (mean blank)
Percent TS = ------------------------------------------------------------ x 100
[(wt of dish + milk) - (wt of dish)]
Maximum recommended difference between duplicates 0.05% total solids
Sr = 0.019
SR = 0.042
RSDr = 0.149%
RSDR = 0.327%
r value = 0.054
R value = 0.118
Reference: J.L Clark, et al., 1989. Journal of Association of Official Analytical Chemists, 72:
712-718.
(Direct Oven - Ether Extraction) Method for Solids-Not-Fat in Milk
Principle
Solids-not-fat is determined by conducting total solids and fat analyses. Percent fat is subtracted
from percent total solids to obtain percent solids-not-fat.
Determination
a. Conduct total solids analysis of milk by the direct forced air oven drying method.
b. Conduct fat analysis of milk by the modified Mojonnier ether extraction method.
Calculation
Percent solids-not-fat = Percent total solids - Percent fat
Maximum recommended difference between duplicates 0.06% total solids
Sr = 0.019
SR = 0.041
RSDr = 0.218%
RSDR = 0.466%
r value = 0.055
R value = 0.117
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Protein Determination
Nitrogen (Total) in Milk - Kjeldahl Methods
Protein Nitrogen Content of Milk - Kjeldahl Method (Direct Method)
NonProtein Nitrogen in Whole Milk - Kjeldahl Method
Protein Nitrogen Content of Milk - Kjeldahl Method (Indirect Method)
Prepared by
Test Procedures Committee of Market Administrators, June 1991
Preface
The following procedures for determining the protein content of milk have been approved by the
Market Administrators' Milk Test Procedures Committee.
These procedures involve the following Kjeldahl methods:
1. Nitrogen (Total)
2. Protein Nitrogen
3. Nonprotein Nitrogen
4. Protein Nitrogen (Indirect Method)
These methods resulted from collaborative studies which were presented to the Association of
Official Analytical Chemists (AOAC) in September 1989 and were published in "Changes in Official
Methods of Analysis", 1991 journal.
Nine laboratories analyzed nine pairs of blind duplicate raw milk samples for percent protein by these
prescribed methods. The goals of these collaborative studies were to develop testing procedures for
protein determinations that would yield highly accurate results and ones which could be uniformly
adapted and applied throughout the dairy industry.
These procedures when properly followed will yield comparable results between and within
laboratories.
The full report for each of these protein determination procedures by the Kjeldahl method is available
from the office of any Federal Milk Market Administrator or from the USDA, Ag. Mkt. Service, Dairy
Division, P.O. Box 96456, Washington, DC 20090-6456.
Safety Precautions
Sulfuric acid. Sulfuric acid can cause severe burns. Wear eye protection and acid resistant
15
gloves. If acid is spilled on skin, wash immediately with large amounts of cool water. NEVER
NEUTRALIZE ACID ON SKIN WITH BAKING SODA.
Sodium Hydroxide. Alkalies can cause severe burns. Violent boiling can occur when sodium
hydroxide is added to the digest (especially if there is too much residual acid). Wear eye protection and
heavy rubber gloves when working with sodium hydroxide. If sodium hydroxide is spilled on skin,
wash immediately with large amounts of cool water.
Trichloroacetic acid (TCA). TCA is very corrosive and can cause burns. Wear eye protection and
plastic gloves when working with TCA. If TCA is spilled on the skin, wash immediately with large
amounts of cool water. NEVER pipet TCA solutions by mouth.
0.1N and .01N Hydrochloric acid (HCL). HCL solutions can cause burns. Wear eye
protection. Wearing acid resistant gloves when handling HCL is recommended but may not be practical
during titration. If HCL is spilled on the skin, wash immediately with large amounts of cool water.
Digestion. Check the bottom of the Kjeldahl flasks for star cracks prior to adding the sample and
reagents. Discard cracked flasks. Acid fumes are generated during digestion. Make sure the digestion
apparatus (traditional or block) effectively removes fumes. Wear eye protection and heavy rubber
gloves when handling Kjeldahl flasks.
Distillation. Distillation involves the heating of an acid digest plus sodium hydroxide with the
release of ammonia gas. Care should be taken under conditions such as these. Always wear eye
protection and heavy rubber gloves. Most Kjeldahl distillation units have a protective glass (or plastic)
barrier that is used as protection for the operator during distillation. If your unit has this, use it!
Nitrogen (Total) in Milk Kjeldahl Methods
Principle
Milk is digested in H2SO4, using CuSO4 . 5H20 as catalyst with K2SO4 as boiling point elevator, to
release nitrogen from protein and retain nitrogen as ammonium salt. Concentrated NaOH is added to
release NH3, which is distilled, collected in H3BO3 solution, and titrated.
Traditional Method
Apparatus
a. Digestion flasks. Kjeldahl. Hard, moderately thick, well annealed glass. Total capacity is ca
500 or 800 mL.
b. Distillation flasks. Same as Kjeldahl flask as in (a), fitted with rubber stopper through which
passes lower end of efficient connecting bulb or trap to prevent mechanical carryover of NaOH
during distillation. Connect upper end of bulb to condenser tube with rubber tubing. Use
graduated 500 mL Erlenmeyer titration flask to collect distillate. Trap outlet of condenser in
manner to ensure complete absorption of NH3 distilled into boric acid solution.
c. Digestion/distillation system. Traditional apparatus with adjustable controls for individual
flasks.
d. Titration buret. 50 mL. Class A or equivalent.
16
Reagents
a. Sulfuric acid. 95-98% H2SO4. Nitrogen free.
b. Copper catalyst solution. CuSO4 . 5H2O. Nitrogen free. Prepare solution 0.05 g/mL H2O.
c.
d.
e.
f.
Potassium sulfate. K2SO4. Nitrogen free.
Sodium hydroxide solution. 50% w/w nitrate-free NaOH.
Boiling chips. Mesh size 10 suggested. High purity, amphoteric alundum granules, plain.
Methyl red/bromocresol green indicator solution. Dissolve 0.2 g methyl red and dilute to 100
mL in 95% ethanol. Dissolve 1.0 g bromocresol green and dilute to 500 mL in 95%
ethanol. Mix 1 part methyl red solution with 5 parts bromocresol green solution (combine all of
both solutions).
g. Boric acid solution. 4%, with indicator. Dissolve 40 g H3BO3 and dilute to 1 L in water and
add 3 mL methyl red/bromocresol green indicator solution, (f). Solution will be light orange
color.
h. Hydrochloric acid standard solution. 0.1000N. Prepare as in Association of Official Analytical
Chemists method number 936.15 or use pre-made solution of certified specification range
0.0995-0.1005N and use 0.1000N for calculation.
i. Ammonium sulfate. 99.9% (NH4)2SO4.
j. Tryptophan or lysine hydrochloride. 99% C11H12N2O2 or C6H15ClN2O2.
k. Sucrose. Nitrogen free.
Sample Preparation
Add 15 g K2SO4, 1 mL CuSO4 . 5H2O catalyst solution and 8-10 boiling chips to digestion
flask. Warm milk to 38+10C and mix thoroughly. Weigh warm sample (5+0.1 mL) and immediately
place in digestion flask. (Note: Weights mist be recorded to nearest 0.0001 g.) Add 25 mL H2SO4,
rinsing any milk on neck of flask down into bulb. Flask may be stoppered and held for digestion at later
time. Digest and distill a blank (all reagents and no sample) each day.
Determination
a. Digestion burner setting. Conduct digestion over heating device that can be adjusted to bring
250 mL H2O at 250 to rolling boil in ca 5-6 min. To determine maximum heater setting to be
used during digestion, preheat 10 minutes (gas) or 30 minutes (electric) at burner setting to be
evaluated. Add 3 or 4 boiling chips to 250 mL water at 250C and place flask on preheated
burner. Determine heater setting that brings water from 250 to rolling boil in 5-6 minutes on
each burner. This is maximum burner setting to be used during digestion.
b. Digestion. Place flask in inclined position with fume ejection system on. Start with setting low
enough so that sample does not foam up into neck of Kjeldahl flask. Digest at least 20 minutes
or until white fumes appear in flask. Next, increase burner setting half way to maximum setting
determined in (a) and heat for 15 minutes. Next, increase heat to maximum setting determined
in (a). When digest clears (clear with light blue-green color), continue to boil 1-1.5 hr at
maximum setting (total time ca 1.8-2.25 hr).
To determine specific boil time needed for analysis condition in your laboratory, select a high
17
protein, high fat milk sample and determine protein content using different boil times (1-1.5 hr)
after clearing. mean protein test increases with increasing (0-1.5 hr) boil time, becomes constant,
and then decreases when boil time is too long. Select boil time that yields maximum protein test.
At end of digestion, digest should be clear and free of undigested material. Cool acid digest
to room temperature (ca 25 min.). Cooled digest should be liquid or liquid with few small
crystals. (Large amount of crystallization before addition of water indicates too little residual
H2SO4 at end of digestion and can result in low test values.) After digest is cooled to room
temperature, add 300 mL H2O to flask and swirl to mix (for 800 mL flasks add 400 mL
H2O). When room temperature water is added some crystals may form and then go into solution;
this is normal. Let mixture cool to room temperature before distillation. Flasks can be
stoppered for distillation at a later time.
c. Distillation. Turn on condenser water. Add 50 mL H3BO3 solution with indicator to graduated
500 mL Erlenmeyer titration flask and place flask under condenser tip so that tip is well below
H3BO3 solution surface. To room temperature diluted digest, carefully add 75 mL 50% NaOH
down sidewall of Kjeldahl flask with no agitation. NaOH forms clear layer under the diluted
digest. Immediately connect flask to distillation bulb on condenser. Vigorously swirl flask to
mix contents thoroughly; heat until all NH3 has been distilled (>150 mL distillate; >200 mL total
volume). Do not leave distillation unattended. Flasks (500 mL) may bump at this point (ca 150
mL distillate; 200 mL total volume). Lower receiving flask and let liquid drain from condenser
tip. Turn off distillation heater. Titrate H3BO3 receiving solution with standard 0.1000N HCL
solution to first trace of pink. Lighted stir plate may aid visualization of end point. Record mL
HCL to at least nearest 0.05 mL.
Nitrogen Recovery Verification
Run nitrogen recoveries to check accuracy of procedure and equipment.
a. Nitrogen loss. Use 0.12 g ammonium sulfate and 0.85 g sucrose per flask. Add all other
reagents as stated in Sample Preparation. Digest and distill under same conditions as for a
milk sample. Recoveries shall be at least 99%.
b. Digestion efficiency. Use 0.16 g lysine hydrochloride or 0.18 g tryptophan, with 0.67 g sucrose
per flask. Add all other reagents as stated in Sample Preparation. Digest and distill under
same conditions as for milk sample. Recoveries shall be at least 98%.
Calculations
Calculate results as follows:
1.4007 x (mL HCL, sample - mL HCL, blank ) x normality HCL
Nitrogen, % = -------------------------------------------------------------------g sample
18
Multiply percent nitrogen by factor 6.38, to calculate percent "protein." this is "protein" on a total
nitrogen basis.
Maximum recommended difference between duplicates is 0.03% "protein."
Repeatability and Reproducibility Values
For method performance parameters obtained in collaborative study of this method Sr = 0.014, SR =
0.017, RSDr = 0.385%, RSDR = 0.504%, r value = 0.038 and R value = 0.049.
Reference: D.M. Barbano, J.L. Clark, C.E. Dunham, and J.R. Fleming. 1990.
Kjeldahl Method for Determination of Total Nitrogen Content of Milk:
Collaborative Study. Journal of Association of Official Analytical Chemists
73: 849-859.
Block Digestor / Steam Distillation Method
Apparatus
a. Digestion block. Aluminum alloy block or equivalent apparatus, with adjustable temperature
control and device for measuring block temperature.
b. Digestion block tubes. 250 mL capacity.
c. Distillation unit. For steam distillation. To accept 250 mL digestion tubes and 500 mL titration
flasks.
d. Titration buret. 50 mL. Class A or equivalent
Reagents
See Reagents (a)-(k) for the Traditional Method.
Note: 40% w/w NaOH may be used instead of 50% w/w. Boiling chips should not be used if
equipment manufacturer does not recommend such use.
Sample Preparation
Add 12 g K2SO4 and 1 mL CuSO4 . 5H2O catalyst solution to digestion tube. Warm milk to 38+1oC
and mix thoroughly. Weigh warm sample (5 + 0.1 mL) and immediately place in digestion tube. (Note:
weights must be recorded to nearest 0.0001 g.) Add 20 mL H2SO4. Tube may be stoppered and held
for digestion at later time. Digest and distill a blank (all reagents and no sample) each day.
Determination
a. Digestion. Set block at al low initial temperature to control foaming (ca 180-230oC). Place
tubes with aspirator connected in block digestor; suction should be just enough to remove
fumes. Digest 30 minutes or until white fumes develop. Increase temperature to 410-430oC
and digest until clear. It may be necessary to increase temperature gradually over ca 20 minutes
to control foaming. Do not let foam in tube rise higher than ca 4-5 cm below surface of fume
collection device inserted into top of tube. After digest clears (clear with light blue-green color),
continue to boil (H2SO4 must be boiling) for at least 1 hr, total digestion time ca 1.75-2.5 hr.
19
To determine specific length of boil time needed for analysis conditions in your laboratory,
select high protein, high fat milk sample and determine protein content using different boil times
(1-1.5 hr) after clearing. Mean protein test increases with increasing (0-1.5 hr) boil time,
becomes constant, and then decreases when boil time is too long. Select boil time that yields
maximum protein test. (Note: before removing hot tubes from block, make sure there is no
condensate layer in aspirator manifold. If there is a liquid layer, increase aspiration to remove
liquid.)
At the end of digestion, digest should be clear and free of undigested material. Cool digest to
room temperature (ca 25 min). Cooled digest should be liquid or liquid with a few small
crystals at bottom of tube. (Excessive crystallization indicates too little residual H2SO4 at end of
digestion and may cause low results. To reduce acid loss during digestion, reduce fume
aspiration rate.) After digest has cooled to room temperature, add 85 mL H2O (blanks may
require 100 mL) to each tube, swirl to mix, and let cool to room temperature. When room
temperature water is added some crystals may form and then go into solution; this is
normal. Tubes can be stoppered for distillation at a later time.
b. Distillation. Place 50% (or 40%) NaOH in alkali tank of distillation unit. Adjust volume
dispensed to 55 mL (65 mL for 40% NaOH). Attach digestion tube containing diluted digest to
distillation unit. Place graduated 500 mL Erlenmeyer titration flask containing 50 mL H3BO3
solution with indicator on receiving platform, with tube from condenser extending below surface
of H3BO3 solution. Steam-distill until >150 mL distillate is collected (> 200 mL total
volume). Remove receiving flask. Titrate H3BO3 receiving solution with standard 0.1000N
HCL to first trace of pink. Lighted stir plate may aid visualization of end point. Record mL
HCL to at least nearest 0.05 mL.
Nitrogen Recovery Verification
Run nitrogen recoveries to check accuracy of procedure and equipment.
a. Nitrogen loss. Use 0.12 g ammonium sulfate and 0.85 g sucrose per flask. Add all other
reagents as stated in Sample Preparation. Digest and distill under same conditions as for a
milk sample. Recoveries shall be at least 99%.
b. Digestion efficiency. Use 0.16 g lysine hydrochloride or 0.18 g tryptophan, with 0.67 g sucrose
per flask. Add all other reagents as stated in Sample Preparation. Digest and distill under
same conditions as for a milk sample. Recoveries shall be at least 98%.
Calculations
See Calculations from Traditional Method.
Repeatability and Reproducibility Values
See Traditional Method. Values are the same.
20
Reference: D.M. Barbano, J.L. Clark, C.E. Dunham, and J.R Fleming. 1990. Kjeldahl Method for
Determination of Total Nitrogen Content of Milk: Collaborative Study. Journal of Association of
Official Analytical Chemists 73:849-859.
Protein Nitrogen Content of Milk Kjeldahl Method (Direct Method)
Principle
Protein is precipitated from milk by trichloroacetic acid (TCA) solution. Precipitation must be done
in Kjeldahl flask or tube. Final concentration of TCA in mixture is ca 12%. The 12% TCA solution,
which contains nonprotein nitrogen components of a sample, is separated from protein precipitate by
filtration. Nitrogen content of protein precipitate is determined as in method titled Nitrogen Total in
Milk - Kjeldahl Methods.
Apparatus
See method titled Nitrogen (Total) in Milk - Kjeldahl Methods for both traditional and block
digestor systems.
Reagents
See method titled Nitrogen (Total) in Milk - Kjeldahl Methods for both traditional and block
digestor systems, and in addition:
(a) Trichloroacetic acid solution. 15% w/v, analytical grade CCl3COOH. (Caution: see safety note on
trichloroacetic acid.) TCA is a soft, white, deliquescent crystal, which should be stored in a container
protected from light and moisture.
Preparation of Sample
Warm milk to 38+1oC and mix thoroughly. Immediately place weighed sample (5 + 0.1 mL) in
Kjeldahl digestion flask. Record all weights to nearest 0.0001 g. Add 5 + 1 mL H2O rinsing any milk
on the neck of the flask into the bulb. Add 40 + 0.5 mL 15% TCA solution to flask. Swirl
mixture. Let precipitate settle (ca 5 minutes). Pour mixture from Kjeldahl flask through filter paper
(Whatman No. 1, 15 cm, N-free; or equivalent) and collect filtrate. (Some protein precipitate will
remain in Kjeldahl flask and some will be collected on paper. It is not necessary to remove precipitate
from flask.)
Immediately after pouring mixture (do not let precipitate dry on neck of Kjeldahl flask), use pump
dispenser to add 10+0.5 mL 15% TCA to Kjeldahl flask and rinse any precipitate on neck of flask down
into bulb. Swirl to mix. Pour mixture from Kjeldahl flask through same filter paper, and add filtrate to
that previously collected. Immediately rinse neck of Kjeldahl flask with another 10+0.5 mL rinse of
TCA solution. Swirl to mix and pour mixture from flask through same filter paper used
earlier. Collect entire filtrate. Filtrate should be clear and free of particulate matter. At this
point. filtrate is no longer needed and may be discarded in the appropriate manner.
Wearing TCA-resistant gloves, pick up filter paper; take care not to lose any precipitate. Pinch paper
at top and twist sides and bottom to form oblong shape. If any precipitate remains on either inner or
outer lip of Kjeldahl flask, wipe with filter paper so precipitate adheres to paper. Drop filter paper in
Kjeldahl flask. Add boiling chips, K2SO4, CuSO4 . 5H2O catalyst solution, and H2SO4 as in method
21
titled Nitrogen (Total) in Milk - Kjeldahl Methods. Flask may be stoppered and held for digestion at
a later time. Digest and distill a blank (filter paper) each day that samples are analyzed. Keep record
of blank values. If blank values change, identify cause.
Determination
Proceed as in Method titled Nitrogen (Total) in Milk - Kjeldahl Methods.
Calculation
Calculate protein nitrogen in milk as in method titled Nitrogen (Total) in Milk - Kjeldahl Methods.
Repeatability and Reproducibility
For method performance parameters obtained in collaborative study of this method, Sr = 0.008, SR =
0.021, RSDr = 0.285%, RSDR = 0.702%, r value = 0.024 and R value = 0.059.
Reference: D.M. Barbano, J.M Lynch, and J.R. Fleming. 1991. Direct and Indirect Determination of
True Protein Content of Milk by Kjeldahl Analysis: Collaborative Study. Journal of Association of
Official Analytical Chemists. 74:281-288.
Nonprotein Nitrogen in Whole Milk Kjeldahl Method
Principle
Protein is precipitated from milk by addition of trichloroacetic acid (TCA) solution. Final
concentration of TCA in the mixture is about 12%. Precipitated milk protein is removed by
filtration. Filtrate contains nonprotein nitrogen components of milk. Nitrogen content of filtrate is
determined as in method titled Nitrogen (Total) in Milk - Kjeldahl Methods.
Apparatus
See method titled Nitrogen (Total) in Milk - Kjeldahl Methods.
Reagents
See method titled Nitrogen (Total) in Milk - Kjeldahl Methods, and in addition:
(a) Trichloroacetic acid solution. 15% w/v, analytical grade CCl3COOH. (Caution: See safety note
on trichloroacetic acid.) TCA is a soft, white, deliquescent crystal, which should be stored in a
container protected from light and moisture.
(b) Hydrochloric acid standard solution. 0.0100N HCL. Prepare as in Association of Official
Analytical chemists method number 936.15. Alternatively, use pre-made solution of certified
specification range 0.0101-0.0099N and use 0.010 for calculation.
Preparation of Sample
Warm milk to 38+1oC and mix thoroughly. Immediately pipet milk (10+1 mL) into preweighed 125
mL Erlenmeyer flasks and weigh. Record all weights to nearest 0.0001 g. Add 40 + 0.5 mL 15% TCA
solution to flask. Weigh flask and contents, swirl to mix. Let precipitate settle (ca 5 min). Filter
(Whatman No. 1 paper, 15 cm, N-free; or equivalent) and collect entire filtrate. Filtrate should be clear
and free of particulate matter; if it is not, repeat sample preparation. Swirl filtrate to mix. Pipet 20 +
22
0.2 mL filtrate into a 50 mL beaker and weigh. Pour filtrate from beaker into Kjeldahl digestion flask
that contains boiling chips, Cu2SO4, and CuSO4 . 5H2O catalyst solution as in method titled Nitrogen
(Total) in Milk - Kjeldahl Methods. Immediately reweigh empty beaker. Add H2SO4 as in method
titled Nitrogen (Total) in Milk - Kjeldahl Methods. Flask may be stoppered and held for digestion at
a later time. Digest and distill a blank solution 916 + 0.5 mL 15% TCA and no sample) each day that
samples are analyzed. Keep record of blank values. If a blank value changes, identify the cause.
Determination
Proceed as in method titled Nitrogen (Total) in Milk - Kjeldahl Methods, substituting 0.0100N
HCL solution for 0.1000N HCL solution as a titrant.
Calculation
Calculate as follows:
1.4007 x (Vs - Vb) x N
Nitrogen % = ---------------------------------(Wf x Wm)/[Wt - (Wm x 0.065)]
Where:
Vs
Vb
N
Wf
= mL titrant used for sample
= mL titrant used for blank
= normality of HCL solution
= weight, g, of 20 mL filtrate
Wm = weight, g, of milk
Wt = weight, g, of milk plus 40 mL 15% TCA solution
Note: Factor 0.065 in denominator assumes that milk contains about 3.5% fat and 3.0% true protein (i.e.,
0.035 + 0.030). Factor may need to be adjusted if liquid diary products of different composition are
analyzed (i.e., concentrated or fractionated skim or whole milk products, etc.).
"protein equivalent, " % = nitrogen x 6.38
which is nonprotein nitrogen expressed as protein equivalent.
Repeatability and Reproducibility
For method performance parameters obtained in collaborative study of this method, Sr = 0.0006, SR =
0.012, RSD r = 2.817%, RSDR = 5.707%, r value = 0.016, and R value = 0.033.
Reference: D.M> Barbano, J.M. Lynch, and J.R. Fleming. 1991. Direct and Indirect Determination of
True Protein Content of Milk by Kjeldahl Analysis: Collaborative Study. Journal of Association of
Official Analytical Chemists 74:281-288.
Protein Nitrogen Content of Milk Kjeldahl Method (Indirect Method)
23
Principle
Total nitrogen and nonprotein nitrogen contents of milk sample are determined
separately. Difference between results of these 2 determinations is protein nitrogen content of milk.
Determination
(a) Total nitrogen. Determine as in method titled Nitrogen (Total) in Milk - Kjeldahl Methods.
(b) Nonprotein nitrogen. Determine as in method titled Nitrogen (Total) in Milk - Kjeldahl Methods.
Calculation
Subtract nonprotein nitrogen content from nitrogen content of milk sample and multiply result by
6.38.
Repeatability and Reproducibility
For method performance parameters obtained in collaborative study of this method, Sr = 0.014, SR =
0.031, RSDr = 0.483%, RSDR = 1.051%, r value = 0.040, and R value = 0.088.
Reference: D.M Barbano, J.M. Lynch, and J.R. Fleming. 1991. Direct and Indirect Determination of
True Protein Content of Milk by Kjeldahl Analysis: Collaborative Study. Journal of Association of
Official Analytical Chemists 74:281-288.
Fact Sheet - Milk Protein Testing - FAQ'S
Changing from Crude Protein to True Protein
David M. Barbano and Joanna M. Lynch
Cornell University, Ithaca NY.
What is the difference between crude protein and true protein?
Crude protein, sometimes called total protein, is estimated from measuring the total nitrogen content of
milk. Nitrogen is multiplied by 6.38 to express the results on a protein equivalent basis. The total
amount of nitrogen in milk, however, comes from both protein and non-protein sources. True protein
reflects only the nitrogen associated with protein and does not include the nitrogen from non-protein
sources.
What is non-protein nitrogen?
This is a normal part of milk. The non-protein nitrogen (NPN) fraction is composed of urea and other
low molecular weight nitrogen containing compounds such as creatine and creatinine. About 50% of
the NPN in milk is urea, and variation in NPN is attributed primarily to variation in urea
content. Non-protein nitrogen has little nutritional value and does not contribute to cheese
24
yield. Therefore, it does not have the same economic value as "true" milk protein to either the
processor or the consumer.
How much of the crude protein is NPN?
The amount of NPN in milk varies naturally, just like any other milk component. On average, NPN
represents approximately 6% of the total nitrogen. On absolute basis, NPN accounts for about 0.19% of
the "protein" in a crude protein value, but may range at the extremes between 0.12-0.25%.
How are crude protein and true protein measured?
Kjeldahl nitrogen analysis forms the basis for the reference tests for both crude and true protein. In both
cases, nitrogen is multiplied by 6.38 to express the results on a protein equivalent basis. Milk infrared
analyzers are the most common testing instruments used for determination of protein for payment
testing. They are calibrated using results from Kjeldahl reference testing. These instruments detect a
signal generated from the protein molecules. In simple terms, the machines "see" protein but cannot see
NPN substances.
Why change the basis for measurement of protein concentration in milk from crude protein to
true protein?
In the past, most electronic milk testing equipment was calibrated on a crude protein basis. This created
problems because, although the NPN varied, the machine could not measure this variation. By
calibrating on crude protein, a certain amount of error was inevitable when the machine attempted to
predict something it could not measure. The direction and magnitude of these errors are not easily
predicted, as NPN is not well correlated with either crude or true protein level. These errors are
eliminated when true protein is used as the basis for calibration because the electronic testing
instruments can directly detect the protein signal.
Are there differences in NPN between farms? Between breeds?
Milk NPN levels are influenced primarily by farm management and feeding practices. Feeding
practices account for much of the variation in NPN observed between farms, regions and seasons. Any
differences in NPN between breeds will be small compared to the effects of diet.
Will expressing protein as true protein rather than crude protein decrease my protein test?
On an absolute basis, yes.
Will the lower protein decrease the milk price?
No. The value of protein will be increased to compensate for the decrease in protein. The change in
test level in the Federal Milk Markets will be revenue neutral.
How do I compare my true protein to my previous crude protein records?
Add 0.19% to the true protein values to get an approximate estimate of crude protein.
You say that NPN levels can vary. So how is adding a constant correction of 0.19% to estimate
crude protein from true protein accurate?
Estimates of crude protein based on electronic milk testing have never been accurate with respect to the
25
actual amount of NPN in milk, since this is not a component that the machine can measure. Adding a
constant factor contributes no greater error than previously occurred when instruments were calibrated
on a crude protein basis.
How will changing from crude protein to true protein influence genetic selection for protein
production?
Using true protein will reduce the amount of random error in milk protein production data and improve
the data quality for genetic selection. This will be an advantage for genetic selection for improved
protein production in all breeds within the US. The actual value of protein production can be adjusted
to a crude protein basis by adding 0.19% to the true protein test to make data comparable to historic data
and data from other countries that still express milk protein on a crude protein basis.
Will this change in payment testing affect nutritional labeling?
No. Crude protein is the basis for nutritional labeling on an international basis.
Do any other countries express milk protein content for payment testing on a true protein basis?
Yes. France and Australia.
Please summarize the advantages of using true protein instead of true protein.
Using true protein instead of crude protein will better reflect the economic value of milk
protein. Additionally, it will improve the accuracy of payment testing for protein by eliminating
sources of random error. This will result in more equitable and accurate protein tests, and improve the
quality of data used for genetic selection and farm management.
26