Lab 6.

Determination of Crude Fat

Yundi Liang
ID: 997269081
03/04/2015
yzl5361@psu.edu
Food Science 410, Section 1
Partners: Quinn Mitchell, Brian Wright

Introduction
The term lipid refers to a group of compounds that are sparingly soluble in
water, but show variable solubility in a number of organic solvents (e.g., ethyl ether,
petroleum ether, acetone, ethanol, methanol, benzene). The lipid content of a food
determined by extraction with one solvent may be quite different from the lipid
content as determined with another solvent of different polarity. Fat content is
determined often by solvent extraction methods (e.g., Soxhlet, Goldfish, Mojonnier),
but it also can be determined by non-solvent wet extraction methods (e.g., Babcock,
Gerber), and by instrumental methods that rely on the physical and chemical
properties of lipids (e.g., infrared, density, X-ray absorption). The method of choice
depends on a variety of factors, including the nature of the sample (e.g., dry versus
moist), the purpose of the analysis (e.g., official nutrition labeling or rapid quality
control), and instrumentation available (e.g., Babcock uses simple glassware and
equipment; infrared requires an expensive instrument). (Nielson, 2010)
By using Soxhlet method, fat is extracted, semicontinuously, with an organic
solvent. Solvent is heated and volatilized, then is condensed above the sample.
Solvent drips onto the sample and soaks it to extract the fat. At 1520 min intervals,
the solvent is siphoned to the heating flask, to start the process again. Fat content is
measured by weight loss of sample or weight of fat removed. (Nielson, 2010)
Babcock method is another common way to determine crude fat content.
Sulfuric acid is added to a known amount of milk sample in a Babcock bottle. The
acid digests the protein, generates heat, and releases the fat. Centrifugation and hot
water addition isolate the fat into the graduated neck of the bottle. The Babcock fat
test uses a volumetric measurement to express the percent of fat in milk or meat by
weight. (Nielson, 2010)
The last method is NMR (Nuclear Magnetic Resonance). NMR is the same
technique as Magnetic Resonance Imaging (MRI), which has been widely used in the
medical profession for years to accurately image the human body. In addition, many
industries quantify oils, fats and/or moisture with NMR.Traditionally, NMR has not
been used for wet samples because water protons interfere with the measurement
of fat protons. By combining the microwave drying capability with NMR, the
technology can now be used to accurately measure fat content in almost any type of
food product. A liquid or solid sample is dried to dissipate any hydrogen bound in the
sample as water. Then, the NMR sends a pulse of radio-frequency energy through the
sample, which causes the remaining hydrogen to generate a signal, known as Free
Induction Decay (FID). The intensity of the FID can then be analyzed to determine the
amount of fat protons present in the sample. Since fat protons decay more slowly
than the other constituents do in food (e.g. protein, carbohydrates), they can easily
be directly measured. In addition, NMR measures fat protons throughout the entire
sample and is not affected by surface characteristics (color, ice crystals, sample
changes, etc.) which create problems for some techniques. (CEM, 2011)
The purpose of this lab is to learn three methods of crude fat determination,
Soxtec solvent extraction system, Babcock sulfuric acid assays, and NMR SmartTrac
system.
2

Materials and Methods

The Lab 6. Determination of crude fat experiment was conducted as described in
the laboratory manual (Vanamala, 2015) without any modification.
Results
Table 1. Results of spam lipid analysis by using Soxtec Solvent Extraction System.
Sample #
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Mean
Standard Deviation
%CV

Weight of
lipid (g)
0.9574
0.9372
0.9798
0.9294
0.9088
0.8951
0.8638
0.8794
0.7878
0.8926
0.9281
0.8670
1.1430
0.8674
0.9366
0.9206
0.9375
0.9491

% Crude fat
21.90%
21.30%
26.49%
21.19%
22.11%
22.30%
21.89%
20.91%
19.27%
21.56%
22.27%
20.83%
25.94%
21.62%
22.33%
22.22%
22.33%
23.31%
22.21%
1.690%
7.610%

Table 2. Results of raw milk fat analysis by using Babcock sulfuric acid assays, and by
NMR using the SmartTrac system.
Mean
Standard deviation
%CV
%Erel

Babcock NMR
3.558% 3.794%
0.170%
4.771%
6.807%

0.028%
0.726%
0.148%

Table 3. T-test results of milk fat analysis, calculated by Minitab.

Mean
n
StDev
SE mean
95% CI
Df*
t score
p value

Babcock
3.558%
16
0.1703
0.0426
(3.467%, 3.649%)
15
-5.68
0.000

NMR
3.794%
16
0.02756
0.00689
(3.780%, 3.809%)
15
-0.82
0.427

*Df : Degrees of Freedom

For Babcock method:

Null hypothesis (Ho): The mean of raw milk fat content determined by Babcock
method and the true value of raw milk fat content (3.8%) do not differ
significantly. ( = )

Alternative hypothesis (Ha): The mean of raw milk fat content determined by

Babcock method and the true value of raw milk fat content (3.8%) differ
significantly. ( )
For Babcock method, p = 0.000, which is lower than 0.05. We reject the null
hypothesis, so the mean of raw milk fat content determined by Babcock method and
the true value of raw milk fat content (3.8%) differ significantly.
For NMR method:

Null hypothesis (Ho): The mean of raw milk fat content determined by NMR
method and the true value of raw milk fat content (3.8%) do not differ
significantly. ( = )

Alternative hypothesis (Ha): The mean of raw milk fat content determined by

NMR method and the true value of raw milk fat content (3.8%) differ significantly.
( )
For NMR method, p = 0.427, which is higher than 0.05. We fail to reject the null
hypothesis. So the mean of raw milk fat content determined by NMR method and the
true value of raw milk fat content (3.8%) do not differ significantly.
Sample Calculations
1. Calculation for spam lipid.
Weight of lipid = (Weight of cup + beads + lipid) (Weights of cup + beads)
= 27.9179 g 26.9605 g
= 0.9574 g
% Crude fat =

100%
4

0.9574
4.3721

100% = 21.90%

2. Calculation of statistical parameters.

(Using data of NMR milk fat analysis.)

Mean:
N = 16
=

= 16 (3.79% + 3.82% + 3.81% + 3.81% + 3.80% + 3.78% +

3.83% + 3.84% + 3.80% + 3.76% + 3.74% + 3.79% + 3.77% + 3.82% +

3.76% + 3.79%) = 3.794%

Standard deviation:
N = 16
= 3.794%

( )2

(3.79%3.794%)2 +(3.82%3.794%)2 +(3.81%3.794%)2 +(3.81%3.794%)2

+(3.80%3.794%)2 +(3.78%3.794%)2 +(3.83%3.794%)2 +(3.84%3.794%)2
+(3.80%3.794%)2 +(3.76%3.794%)2 +(3.74%3.794%)2 +(3.79%3.794%)2
+(3.77%3.794%)2 +(3.82%3.794%)2 +(3.76%3.794%)2 +(3.79%3.794%)2
16

0.028%

Coefficient of variation (%CV):

% =

100% =

0.028%
3.794%

100% = 0.7264%

Percent relative error (%Erel)

Absolute error = |(Mean of experimental value) (Expected Value)|
|3.794% 3.8%| = 0.006%
=
Relative error =

0.006%

= 3.794% 100% = 0.148%

3. T-test calculation for one sample t-test.

(Using data of NMR milk fat analysis; actual t-test results I used for discussion are
calculated by Minitab, which is shown in Table 3 above.)

t=

= 3.794% = 3.8% = 0.028% = 16

= 0.82

Discussion
For results of spam lipid analysis by using Soxtec Solvent Extraction System,
values of %Crude fat are relative lower than the expected value, 28.6% (SPAM,
2015). They also have a 7.610% as the percent coefficient of variation, which is
higher than 5%. So Soxtec Solvent Extraction System is not an acceptable and
reproducible technique for lipid analysis. Since we were using this method to do a
crude fat analysis, this Soxhlet method conducted by this Soxtec Solvent Extraction
System might be an acceptable method. The sources of error should be systematic,
which comes from sample preparation step. The used sample might not be
representative enough for a whole can of product due to compositional unevenness
of the product. The different speed of inserting extraction cups might also be a
potential error source of this technique.
For the results of raw milk fat analysis by both methods, the percent coefficients
of variation of both methods are lower than 5%, which means both methods are
acceptable and reproducible. However, comparing the relative error, Babcock
method has a much higher value than NMR does, which is exactly what we expected,
since NMR is the method has the higher accuracy than Babcock method (CEM, 2011).
Potential error source of Babcock method should be systematic, which might come
from sample preparation and results reading. Potential error source of NMR method
should also be systematic, since there is always error within a machine.
For the raw milk fat analysis, we also did a t-test for the results. According to the
results of t-test, values of raw milk fat content came from Babcock method are
significant different from the tabulated value of raw milk (3.8%), while values of raw
milk fat content came from NMR method are not significant different from the
tabulated value of raw milk (3.8%). These results agree with what we expected, since
NMR is the method has the higher accuracy than Babcock method (CEM, 2011).
Babcock method can determine both wet and pre-dried food sample and the
required materials and instruments are normal and simple, which means its a cheap
method. However, it is also has too many error sources. Its defects of milk tests are
curdiness, cloudiness, burnt particles, or dark color, or too light color. If the acid or
the milk be too warm, or the acid too strong, there will be a danger of having charred
fat, or black particles mixed with the fat column. While, on the contrary, if the acid is
too weak, or the milk or acid is too cold, or not enough acid is used, the fat will be
light colored, or curdy. If sufficient speed of the machine has not been maintained, or
it has not been whirled for sufficient length of time, the tests are liable to be cloudy.
This is especially true if the samples are not kept warm enough while being whirled.
NMR method is faster and more accurate, but NMR machine is an expensive
instrument.

Questions
1. Why is temperature control critical for the Babcock method?
If the acid or the milk be too warm, there will be a danger of having charred fat,
or black particles mixed with the fat column. While, on the contrary, if the milk
or acid is too cold, the fat will be light colored, or curdy. If sufficient speed of
the machine has not been maintained, or it has not been whirled for sufficient
length of time, the tests are liable to be cloudy. This is especially true if the
samples are not kept warm enough while being whirled.
2. What are possible causes of charred particles in the column of the Babcock
bottle? Why is it a problem for this measurement technique?
If the milk or the acid is too warm, charred particles may appear in the column.
Given we are using Babcock assay to measure fat content, appearance of
charred particles will decrease the result because those particles are burnt fat.
Appendix
Table 4. Raw data collected for spam fat analysis.

Sample #
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18

Weight of
sample
(g)
4.3721
4.3997
3.6988
4.3857
4.1111
4.0135
3.9467
4.2062
4.0874
4.1404
4.1678
4.1626
4.4060
4.0122
4.1947
4.1436
4.1987
4.0721

Weight of
Cup+beads
(g)
26.9605
27.0842
26.8284
26.7831
26.7064
27.1571
26.6824
26.1153
27.3611
26.9728
26.4069
26.9040
26.8100
26.4451
26.0879
27.2990
26.9544
26.8299

Weight of
Cup+beads+lipid
(g)
27.9179
28.0214
27.8082
27.7125
27.6152
28.0522
27.5462
26.9947
28.1489
27.8654
27.3350
27.7710
27.9530
27.3125
27.0245
28.2196
27.8919
27.7790

Table 5. Raw data collected for raw milk fat analysis by using Babcock sulfuric acid
assays.
Sample #

Rep 1

(%fat)

Rep 2 (%fat)
7

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

3.50%
3.80%
4.00%
3.30%
3.40%
3.60%
3.70%
3.60%
3.50%
3.50%
3.40%
3.20%
3.70%
3.60%
3.50%
3.50%

3.50%
3.85%
3.70%
3.60%
3.30%
3.50%
3.60%
3.50%
3.80%
3.50%
3.60%
3.20%
3.90%
3.50%
3.50%
3.50%

Table 6. Raw data collected for raw milk fat analysis by NMR using the SmartTrac
system.
Sample #
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

% total
solids
12.27%
12.34%
12.45%
12.37%
12.34%
12.31%
12.35%
12.36%
12.33%
12.32%
12.32%
12.19%
12.36%
12.38%
12.28%
12.56%

% fat
3.79%
3.82%
3.81%
3.81%
3.80%
3.78%
3.83%
3.84%
3.80%
3.76%
3.74%
3.79%
3.77%
3.82%
3.76%
3.79%

References
Caprette, D. (1997, May 8). Selected Critical Values of the t-Distribution. Retrieved
March 4, 2015, from http://www.ruf.rice.edu/~bioslabs/tools/stats/ttable.html
Nielsen, S. (2010). Standard Solutions and Titratable Acidity. In Food analysis
laboratory manual (2nd ed., pp. 97-101). New York: Springer.
SMART Trac II - Moisture & Fat Analysis Technology - CEM Corporation. (2011,
January 1). Retrieved March 4, 2015, from
http://www.cem.com/smart-trac-2-technology.html
SPAM Classic. (2015, January 1). Retrieved March 4, 2015, from
http://www.spam.com/varieties/spam-classic
Vanamala, J. (2015). Lab 6. Determination of Crude Fat. In Food Science 410 Lab
Manual. State College.