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Extraction Application
B.1.1.a. HYDROTHERM Total Fat in (Whipped) Cream
C. Gerhardt GmbH & Co. KG
Cäsariusstraße 97
53639 Königswinter, Germany
@ www.gerhardt.de
© 2020
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B.1.1.a. HYDROTHERM Total Fat in (Whipped) Cream
Application_B.1.1.a. HT HYDROTHERM Total Fat in Whipped Cream_09.2020_english.docx
23.08.2020 • Rev.: A • Page 2 of 8
1 Principle and Introduction
The sample is heated up with hydrochloric acid to digest protein and free bound lipids.
The digestion solution is filtered. After the drying process, the fat remaining in the filter is
extracted with petroleum ether. The solvent is distilled and the dried residue is weighed.
The fat content is calculated from the difference between initial sample weight and the
extract.
2 Methods
This application note is meant to be a guideline for the operation of your C. Gerhardt
analysis system and has to be adapted to your sample matrix and the local peculiarities
in your laboratory.
This document is based on the following official methods:
• Amtliche Sammlung von Untersuchungsverfahren nach § 64 LFGB (vormals
§ 35 LMBG), L 01.00- 20 Bestimmung des Fettgehaltes von Milch und
Milchprodukten, Verfahren nach Weibull, Mai 1988
• AOAC 989.04: Fat in raw milk
• AOAC 989.05: Fat in milk
3 Chemicals and material
Quality p. a.
• Water: demineralized or distilled
• Hydrochloric acid HCl, 15 %
• Petroleum ether, boiling range 40 to 60 °C
• pH indicator paper
• Silica order number Nr. 14-0022, 25 g absorbent for liquid
samples, alt. Silica Supelco 18514 (Sigma- Aldrich)
• Disposable syringe, 5 ml
• Folded filter FF240, order number 1004092
• Extraction thimbles 33 x 80 mm, order number 13-0054
• Extraction thimbles 33 x 94 mm, order number 13-0057
• Cotton wool, chemically pure and defatted
4 Instruments
• Analytical balance, precision 0.1 mg
• Water bath
• Desiccator with drying agent, e.g. silica gel blue
• HYDROTHERM HT 6 hydrolysis system
• SOXTHERM extraction system micro/macro with control unit MULTISTAT order
number 13-0011 or SOXTHERM-Manager order number 13-0012
• Drying oven, heated electrically, with natural aeration and automatic temperature
control
5 Procedure
5.1. Sample preparation cream
Fill 3-5 boiling stones into each extraction beaker and leave the beakers in the drying
oven at 100 ± 2 °C for 1 hour. After that, allow the beakers to cool down to room
temperature in the desiccator and weigh them with an accuracy of 1 mg.
Store the sample closed at 2 - 6 ° C to prevent any spoilage or alteration of its
composition. Before weighing, stir the sample thoroughly with the help of a spoon to
achieve an even fat distribution. Place the sample in a beaker glass and warm it in a 40 °
C water bath to approx. 38 ° C. Keep the sample at this temperature for a few minutes
and carefully stir from time to time. Allow cooling down to room temperature and weigh
with samples using a disposable syringe.
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B.1.1.a. HYDROTHERM Total Fat in (Whipped) Cream
Application_B.1.1.a. HT HYDROTHERM Total Fat in Whipped Cream_09.2020_english.docx
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5.2. Hydrolysis
Choose the sample weight in relation to the expected fat content. After the analysis, the
resulting weight of the extracted fat should be 1.0 g. Weigh 2.5-3.0 g of the sample with
an accuracy of ± 0.1 mg by differential weighing (disposable syringe) directly into the
hydrolysis beakers. Add 0,2g +/- 0,05 Silica (Kieselgur) to ensure the fat will be
quantitively transferred from the extraction beaker into the filter during filtration and
moreover to achieve an even fat distribution in the filter. Without adding Kieselgur,
approximately 0.2 % less fat will be determined.
Put HYDROTHERM into operation following the instructions in the operation manual.
Then place the beakers with the sample into HYDROTHERM and close the lid.
Place dry folded filters into the respective positions. Close the instrument and start the
program. Hydrochloric acid is added then automatically and brought to boil. It is kept
boiling for approximately 1 h at a reduced heating power. After the hydrolysis is
completed, the digestion solution is diluted with hot water (1.5 times the amount of HCl)
and immediately filtrated through a moisturized folded filter. Digestion vessel, condenser
and filtration area are rinsed several times with hot water. Finally, the paper filter is
washed neutral. HYDROTHERM performs all steps of the process automatically.
Table 1: Recommended parameters and settings: HYDROTHERM Best Practice
Parameter Setting Scale unit Note
Filling levels
HCl amount 100 ml
H2O amount / dilution
50 ml
Heating / cooling phases
Heat-up phase 12 min
Boiling phase 1 20 min Power 50%
Boiling phase 2 30 min Power 70%
Cooling down phase - duration 15 min
Filter moisture
Number of moisture cycles 3
Moisture amount per cycle 30 ml
Filter phase
Filter wait time 5 s
Rinsing cycles 20
Pipe opening time 200 ms
Sample rinse time 10 s
Sample shower - amount 30 ml
Condenser shower - amount 30 ml
Filter shower - amount 50 ml
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B.1.1.a. HYDROTHERM Total Fat in (Whipped) Cream
Application_B.1.1.a. HT HYDROTHERM Total Fat in Whipped Cream_09.2020_english.docx
23.08.2020 • Rev.: A • Page 4 of 8
33 x 94 mm /13-0057
Parameter
Sample must be immersed
completely
the thimble
After B the extraction beaker
extraction agent.
After the program is completed, place the wet filters on a watch glass and leave them to
dry at room temperature overnight. Alternatively dry the filters approximately 1.5 h at
103 ± 2°C in a drying oven. A higher temperature could lead to a loss of easy volatile
fatty acids. Extract the folded filters immediately after drying or store them in a desiccator
before the extraction. As moisture could eventually affect the result, add sodium sulphate
(approx. a half tea spoon) to the sample into the extraction thimble.
The values given in the attachment can be used as orientation for the sample weight.
5.2. Extraction
After cooling, place the filter in the extraction thimble and cover him with cotton wool. In
case any fat traces remain on the watch glass, moisten cotton wool with the extraction
solvent to absorb the fat and place the wet cotton wool inside the thimble.
Add 150 ml solvent (SOXTHERM macro) or 100 ml solvent (SOXTHERM micro) and
extract samples with the following program:
Table 2: Instrument configuration SOXTHERM
Parameter Note / Order number
Solvent Petroleum ether
Boiling point/range 40-60 °C
Solvent amount 100 ml SOXTHERM micro
150 ml SOXTHERM macro
Sealing type Viton / 1000578
Extraction thimbles
Thimble holders SHK2 /13-0062
Boiling stones 13-0047
Compressor / Connection compressed air 13-0010, minimum 4.5 bar
Water connection / Chiller minimum 0.5 bar
33 x 80 mm /13-0054
Table 3: SOXTHERM Program
Program step
T-classification 200 °C
Extraction temperature 150 °C
Reduction interval 4 min.
Reduction pulse 2 s
Note
Hot extraction 30 min
Evaporation A
Rinsing time 60 min.
Evaporation B
Evaporation C 4 min.
4-5 x interval
3-4 x Intervall
After phase A the solvent level
should be at least 10 mm below
should be widely free of
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B.1.1.a. HYDROTHERM Total Fat in (Whipped) Cream
Application_B.1.1.a. HT HYDROTHERM Total Fat in Whipped Cream_09.2020_english.docx
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After the extraction program is finished, leave extraction beakers in the drying oven for
30 minutes at a temperature of 103 ± 2 °C. After that place the extraction beakers in the
desiccator, allow cooling down to room temperature and weigh them out with an
accuracy of 1 mg.
In order to check the weight consistency, dry the samples for another 30 minutes and
weigh again after cooling down. Repeat this procedure until the difference between two
successive weighings does not exceed 1 mg of the final extract weight. Should the
weight increase, take the previous lower value.
Extraction, drying and weighing should be performed consecutively, without interruption.
6 Evaluation:
6.1. Calculation
The total fat content w in g/100 g (corresponds to %) of the sample is calculated using
the following equation:
m1: mass of the empty extraction beaker with boiling stones [g]
(
2 − 1) ∗ 100
=
m2: mass of the extraction beaker with fat after drying [g]
mo: initial sample weight [g]
The result is rounded to one decimal place.
6.2. Repeatability
r = 0,20 g/100 g (requirement acc. to Röse-Gottlieb-Method)
7 Exemplary results
The following table shows exemplary results for commercially available long-shelf-life
cream. The expected fat content is 30 %. The last line displays the average value and
the standard deviation (value in brackets). The standard deviation of 0.07 is significantly
below the standard requirement of 0.20g/100g.
Sample
Sample Weight [g]
Fat content [%]
Cream 3890 2.7253 30.11
Cream 3890 2.8047 29.95
Cream 3890 2.5701 30.14
Cream 3890 2.4266 30.08
Cream 3890 2.5568 29.96
Cream 3890 3.1350 30.03
Average and sd 30.05 (0.07)
The folded filters were dried for 1.5 h at 100 °C or at room temperature overnight.
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B.1.1.a. HYDROTHERM Total Fat in (Whipped) Cream
Application_B.1.1.a. HT HYDROTHERM Total Fat in Whipped Cream_09.2020_english.docx
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→recovery too low
hotplate
No boiling bubbles ascending at the end of
boiling →recovery too low
The screw connect (screw cap GL 18) is
hydrolysis beaker plane on the hotplate
low
Fat is remaining in the hydrolysis beaker→
Increase rinsing cycles; add
beaker to get it clean after filtration
necessary to set it again to 15 minutes.
Fat is lost to the waste tank → recovery is too
During the filtration the whole fat isn´t
double folded filter
Clean hydrolysis beaker before starting
clean before starting hydrolysis
the usual drying time
oxygenized and mass increases
Drying time too long, fat is oxygenized and
mass increases
weighing and cooling off process
Solvent is used several times; it has taken up
fat which is detected, too.
Parts of the thimble or sample residue are left
Check whether the fat film is clear
spills over
8 Troubleshooting Hydrolysis
Cause Remedy
Sample with hydrochloric acid is not boiling
well, sample won´t be hydrolyzed completely
the hydrolysis beaker overflow, no continuous
Due to delays in boiling fat is sticking to the lid
which won´t be transferred → recovery too
Recovery is too low during the fat extraction
Fat is burnt in the hydrolysis beaker Reduce boiling power. If the cool down
low during the fat extraction
Hydrolysis beaker is dirty → recovery too high
Increase boiling power; be sure that the
hydrolysis beaker is well placed at the
not tight; screw it tight, place the
Reduce boiling power and/or sample
weight
0,2g +/- 0,05 of Silica to the hydrolysis
duration was reduced it´s absolutely
collected completely in the folded filter;
reduce sample weight and/or take a
hydrolysis; control the beaker if it is
9 Troubleshooting Extraction
9.1 Results too high
Cause Remedy
Water in the sample, water drops swim on the
fat surface, fat can’t be concentrated within
Too high temperature in the drying oven, fat is
Extraction beaker is dirty
in the extraction beaker
Filter or sample must be dry when they
are put in
Check temperature of the drying oven
Observe needed drying time
Work as clean as possible in drying,
Check blank value of the solvent
Check whether the thimble is porous
Check whether the grinding of the
sample is too fine. Use a second filter
Thimble is too impermeable, sample
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B.1.1.a. HYDROTHERM Total Fat in (Whipped) Cream
Application_B.1.1.a. HT HYDROTHERM Total Fat in Whipped Cream_09.2020_english.docx
23.08.2020 • Rev.: A • Page 7 of 8
Incomplete extraction
Extract thimble again
Change thimble
surface e.g. by using sodium sulphate
Wrong or missing boiling stones, glass
extraction temperature
Check for leakages in the apparatus at
O-rings and connectors
Delayed boiling during extraction, sample
residues may stick to the teflon connector
Delayed boiling because of usage of glass
beads
Filtration problems, paper filter was not evenly
washing solution
9.2 Results too low
Cause Remedy
Incomplete extraction
Incomplete extraction
Cooling off time too short Follow correct weighing procedure
Boiling delay during concentration process, fat
splashes can be found in the thimble
Flowrate cannot be achieved because of
too low permeability of the thimble.
Sample sticks together, enlarge the
beads cannot be used.
Heating power too high, reduce the
9.3 Varying Results
Cause Remedy
Uneven extraction due to solvent losses
Use boiling stones
Use standard boiling stones
wetted, fat losses because of fat running
through. Can be seen from fat residues in the
Make sure filter in HYDROTHERM is
wetted evenly
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B.1.1.a. HYDROTHERM Total Fat in (Whipped) Cream
Application_B.1.1.a. HT HYDROTHERM Total Fat in Whipped Cream_09.2020_english.docx
23.08.2020 • Rev.: A • Page 8 of 8
COMPREHENSIVE APPLICATION DATA BASE
C. Gerhardt offers a wide range of application notes for many methods and procedures.
Please contact our application lab team via [email protected]
information on:
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Crude fibre, ADF and NDF in feed
•
Fat in food and feed
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Alcohol determination
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Trace metal in soil and sludge
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COD determination in water
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Total nitrogen determination in water, soil and plants
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