Application Note

Determination of Fat in Manually Hydrolyzed Food and Feed Samples using the Extraction Unit E-816 SOX

018/2009

Extraction Unit E-816 SOX

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SHORT NOTE Determination of Fat in Manually Hydrolyzed Food and Feed

Samples using the Extraction Unit E-816 SOX

The determination of fat in food and feed is a routine procedure in quality assurance and labelling. A simple and fast procedure for the total fat determination in various certified food and feed samples is introduced. The samples are hydrolyzed manually, followed by a Soxhlet extraction with the Extraction Unit E-816 SOX. The total fat content is determined gravimetrically after the extract is dried to a constant weight. The fat contents correspond with the certified values of the reference materials.

Introduction

Total fat determination is one of the key analyses performed in the food and feed industry. Complex matrices typically require acidic hydrolysis prior to the solvent extraction. An easy and reliable procedure to determine the total fat content in various food and feed samples is presented below. In this procedure, a manual hydrolysis is performed prior to the Soxhlet extraction.

Experimental

Instrumentation: Mixer B-400, Extraction Unit E-816 SOX, heater, drying oven Samples: certified reference materials: boiled sausage, canned pet (cat) food, milk powder, shortbread, and chocolate

Figure 1: boiled sausage (1), shortbread (2), chocolate (3), milk powder (4),

and canned pet food (5)

The homogenized samples were weighed in a beaker and manually hydrolyzed for 30 min on a heater using 4 mol/l HCl. The solution was diluted, filtered, and washed to a neutral pH with boiling water. The filter was placed into a paper thimble and dried in a drying oven. The Soxhlet extraction was carried out with the Extraction Unit E-816 SOX using the parameters shown in Table 1. The samples were extracted in triplicate. The extracts were dried in a drying oven (102°C) to a constant weight and the total fat content was calculated. Table 1: Soxhlet Extraction parameters (Extraction Unit E-816 SOX)

Solvent Petroleum ether

Extraction 120 min (Heater 100 %)

Rinse 5 min (Heater 100 %)

Drying 25 min (Heater 100 %)

Solvent volume 120 ml

Figure 2: Heater with sample in triplicate and filtration rack

Results

The determined fat contents are presented in Table 2. The results correspond with the values of the certified reference materials and show low standard deviations. Table 2: Determined fat contents in food and feed samples, fat in g/100 g, (standard deviation for the experimental results in brackets), n = 3

Sample Certified value Experimental results

Boiled sausage 23.93 +/- 0.09 23.98 (0.14)

Canned pet (cat) food

4.46 +/- 0.17 4.46 (0.03)

Milk powder 26.24 +/- 0.39 26.26 (0.05)

Chocolate 31.64 +/- 0.50 31.90 (0.04)

Shortbread 12.11 +/- 0.28 12.20 (0.10)

Conclusion

The determination of the total fat contents in manually hydrolyzed food and feed samples by Soxhlet extraction provides reproducible results which correspond with the certified values of the reference materials. The determinations are in line with the standard deviations reported in the certificates.

References

Extraction Unit E-816 SOX operation manual

For more details see Application Note 018/2009

018/2009

Extraction Unit E-816 SOX

(1) (2)

(3)

(4) (5)

Application Note 018/2009 Version A, Copyright © 2009 Büchi Labortechnik AG 3/7

1 Introduction

A simple procedure for total fat determination is introduced. The samples are hydrolyzed manually prior to a Soxhlet extraction with the Extraction Unit E-816 SOX. The total fat content is determined gravimetrically after the extract is dried to a constant weight.

2 Equipment

− Extraction Unit E-816 Sox − Mixer B-400 − Analytical balance (accuracy +/- 0.1 mg) − Drying oven / Vacuum drying oven − Heater − Beaker, 250 ml − Digestion tube − Glass funnel − Watch glass − Expansion element (Büchi order no. 051957)

3 Chemicals and Materials

− Hydrochloric acid 4 mol/L, 4 L HCl 32 % (Hänseler, 20-2000-5) are filled to 10 L with deionized water

− Petroleum ether 40-60 °C, analytical grade, Scharlau, EGT (ET 0093) − Boiling stones, Merck, 1.07913.0100 − Folded filters, Schleicher & Schuell, 595 ½, diameter 150 mm − Paper thimbles, 33 x 94, Macherey-Nagel, MN 645 − pH paper, Merck, 1.09535.0001

4 Samples

Boiled sausage, certified reference material LVU 2007 (www.lvus.de), specified fat content: 23.93 +/- 0.09 g/100g

Canned pet (cat) food, certified reference material LGC 7176 (http://lgcstandards.com/home/home_de.aspx), specified fat content: 4.46 +/- 0.17 g/100g

Milk powder (spray dried), certified reference material LVU 04a-08 (www.lvus.de), specified fat content: 26.24 +/- 0.39 g/100g

Chocolate, certified reference material LVU 13-08 (www.lvus.de), specified fat content: 31.64 +/- 0.50 g/100g

Shortbread biscuits, certified reference material LVU 11-09 (www.lvus.de), specified fat content: 12.11 +/- 0.28 g/100g

5 Procedure

The fat determination according to Weibull-Stoldt includes the following steps: 1. Sample homogenization 2. Manual hydrolysis of the sample with 4 M hydrochloric acid to break up the

matrix and filtration of the hydrolysis solution to separate the fat 3. Drying of the filtration residue 4. Soxhlet extraction of the dried residue 5. Drying and weighing of the extract

Application Note 018/2009 Version A, Copyright © 2009 Büchi Labortechnik AG 4/7

6. Calculation of the fat content

5.1 Sample homogenization − Homogenize the boiled sausage and the canned pet food using the mixer B-400,

once for 2 s to obtain visual homogeneity − Use the milk powder without further homogenization − Melt the chocolate in a drying oven at a temperature of 45 °C for 60 min and stir

well for 1 min using a spatula − Break the shortbread into small pieces and homogenize it using the mixer B-

400, once for 2 s to obtain visual homogeneity

5.2 Manual hydrolysis − Perform the hydrolysis in a fume hood − Weigh in the homogeneous sample into a 250 ml beaker. Note the accurate

weight of the sample and add some boiling stones − Add 100 ml of hydrochloric acid (4 M) − Place the beaker on the heater; cover it with a watch glass and start heating.

Watch the solution until stable boiling − After boiling occurs, the sample is hydrolyzed for 30 min − Prepare a rack with digestion tubes, glass funnels and folded filters. Wet the

filters with hot water − After the hydrolysis time has been finished, rinse the watch glass with boiled

water into the beaker. Dilute the solution with 100 ml of boiling deionized water − Filtrate the solution through the filter and wash the beaker with boiling water until

the sample is transferred completely − Wash each filter with a total of at least 250 ml of boiling water until the filtrate is

neutral. Check the pH with pH paper

5.3 Drying of the filtered samples − Place a paper thimble into a paper thimble holder − Place the folded filter with the filtration residue into the paper thimble − Dry the sample in a drying oven for 45 min at 102 °C − Allow the sample to cool down to ambient temperatures in a desiccator

5.4 Soxhlet extraction of the fat Preparation of the beakers − Always us dry and clean beakers for Soxhlet extraction − Add 2-3 boiling stones to each beaker and dry them for at least 30 min at 102 °C − Let them cool down to ambient temperature in a desiccator for at least 1 h − Record the exact weight prior to extraction

Soxhlet extraction − Place the expansion element (feet upside down) into the extraction chamber that

the space between two feet points to the solvent’s outlet of the extraction chamber

− Put the paper thimble containing the sample filter into the extraction chamber and adjust the level sensor. The height of the sample in the filter can not be seen why the position of the level sensor has to be estimated, approximately 1-2 centimetre beneath the top of the expansion element (see Figure 1)

− Close the safety shield, lower the rack and add the solvent carefully through the condenser

Application Note 018/2009 Version A, Copyright © 2009 Büchi Labortechnik AG 5/7

Figure 1: Extraction chamber with paper thimble and expansion element, position of the level sensor

− Carry out the extraction using the parameters of table 1. The extractions of the samples were done in triplicate

Table 1: Parameters for Soxhlet extraction with Extraction Unit E-816 SOX

Parameter Value

Solvent Petroleum ether

Extraction step 120 min (Heater 100 %)

Rinse step 5 min (Heater 100 %)

Drying step 25 min (Heater 100 %)

Solvent volume 120 ml

5.5 Drying and weighing of the extract − Dry the beaker containing the extract in a drying oven at 102°C to constant

weight − Allow the beaker to cool down to ambient temperature for at least 1 h in a

desiccator and record the weight. Make sure, that the cooling time of the beaker in the desiccator is the same before and after extraction. Differences in beakers’ temperature falsify the result

5.6 Calculation of the fat content − Calculate the results as percentage of fat using equation (1)

%100)

•=

m

m -(m%Fat

Sample

beakertotal (1)

%Fat : percentage of fat in the sample mtotal : beaker weight + extract [g] mbeaker : beaker weight [g] mSample : sample weight [g]

(1) (2)

Expansion element

Level sensor

Space (1) between two feet points to the solvent’s outlet (2)

Application Note 018/2009 Version A, Copyright © 2009 Büchi Labortechnik AG 6/7

6 Results

The results of the fat determination in the different samples are presented in tables 2 - 6

Table 2: Results of fat extraction of boiled sausage, specified fat content: 23.93 +/- 0.09 g/100g

msample mbeaker mtotal % Fat

Sample 1 3.5111 102.9907 103.8307 23.92

Sample 2 2.5099 98.2592 98.8585 23.88

Sample 3 2.2731 100.7712 101.3199 24.14

Mean value 23.98

sd 0.14

rsd [%] 0.58

Table 3: Results of fat extraction of canned pet (cat) food, specified fat content: 4.46 +/- 0.17 g/100g

msample mbeaker mtotal % Fat

Sample 1 9.6996 101.7613 102.1908 4.43

Sample 2 10.1336 101.1527 101.6068 4.48

Sample 3 9.7535 101.3530 101.7879 4.46

Mean value 4.46

sd 0.03

rsd [%] 0.60

Table 4: Results of fat extraction of milk powder, specified fat content: 26.24 +/- 0.39 g/100g

msample mbeaker mtotal % Fat

Sample 1 3.3568 101.3646 102.2471 26.29

Sample 2 2.6442 101.1600 101.8528 26.20

Sample 3 2.5588 98.2229 98.8956 26.29

Mean value 26.26

sd 0.05

rsd [%] 0.20

Table 5: Results of fat extraction of chocolate, specified fat content: 31.64 +/- 0.50 g/100g

msample mbeaker mtotal % Fat

Sample 1 3.1909 98.2440 99.2614 31.88

Sample 2 2.6063 98.1956 99.0263 31.87

Sample 3 3.0729 102.0729 103.0545 31.94

Mean value 31.90

sd 0.04

rsd [%] 0.12

Application Note 018/2009 Version A, Copyright © 2009 Büchi Labortechnik AG 7/7

Table 6: Results of fat extraction of shortbread biscuits, specified fat content: 12.11 +/- 0.28 g/100g

msample mbeaker mtotal % Fat

Sample 1 4.8700 98.2815 98.8780 12.25

Sample 2 4.9482 103.0097 103.6166 12.27

Sample 3 4.9052 99.2468 99.8398 12.09

Mean value 12.20

sd 0.10

rsd [%] 0.80

The determined fat contents of the certified reference materials are in accordance with the specified values. The relative standard deviations (rsd) are very low, being < 1% for all samples.

7 Comparison to Standard Methods

The parameters used for the hydrolysis and the extraction were following the standard method § 64 LFGB L 01.00-20 (Bestimmung des Fettgehaltes von Milch und Milchprodukten).

8 Conclusion

The determinations of total fat by manual hydrolysis prior to Soxhlet extraction with the Extraction Unit E-816 SOX provided reproducible results which correspond with the certified values of the reference materials.

9 References

Operation manual Extraction Unit E-816 SOX

Operation manual Mixer B-400

§ 64 LFGB L 01.00-20 (Bestimmung des Fettgehaltes von Milch und Milchprodukten).

AOAC 963.15

BÜCHI Labortechnik AG CH-9230 Flawil 1/Switzerland T +41 71 394 63 63 F +41 71 394 65 65 www.buchi.com Quality in your hands