Five essentials

of fat extraction
And how they make your process quick
and compliant
 Introduction

Extraction is one of the oldest separation techniques. Records indicate that civilizations going as far as back as the
Babylonians and ancient Egyptians used extraction processes to prepare perfumes and salves. From the 19th
century onwards, increasingly innovative and automated instruments, solvents and materials helped develop an
ever-growing list of extraction applications. In fact, Soxhlet extraction, one of the most widely used extraction
methods was developed in the 19th century, precisely in in 1879, by Franz von Soxhlet1.

Image courtesy of Flickr2

The modern extraction process is part of a larger analysis workflow, which involves a combination of different steps
depending on the sample to be assessed. One of the most common procedures for analysis of solid samples
consists of the following steps:

Homogenization Extraction Clean-up Concentration Analysis

2
The fat extraction principle
The fat extraction principle is illustrated with the figure below using fat extraction from sausage as an example3.

Components soluble
in extraction solvent

Components insoluble
in extraction solvent
Extraction solvent Extraction
substance

Extraction
mixture

Extract

Extration
solvent
Extraction residue Extraction solution

The extraction substance “sausage” is a mixture of meat components and fat enclosed in these
components. The homogenized substance is mixed with a solvent (extraction solvent) . This extraction mixture
is then separated and insoluble components remain as residue . If this procedure is repeated several times, the
proportion of fat in the solvent increases. Pure fat is then obtained by distilling the solvent portion out
of the mixture.

The method described above is used when soluble components are to be separated from insoluble solids.
By using an extraction apparatus, the amount of solvent required and the time of extraction can be reduced to a
fraction.

3
 Factors that influence the extraction process

Importantly, the extraction solvent must always be inert with regards to the extraction substance. Many parameters
influence the recoveries and the speed of the extraction. The most important points to consider when optimizing
the extraction process are listed in the table below.

Factors that influence the recovery rate of the extraction Factors that influence speed of extraction
Solubility of the components to be extracted in the The particle size of the extraction substance
selected extraction solvent (must be of similar polarity)
The thoroughness of the mixing of extraction substance The degree to which extraction substance
with the extraction solvent and extraction solvent are mixed
Size and number of extraction solvent portions Temperature (rule of thumb is that speed
(number of siphonings in the case of Soxhlet or drop of reaction doubles as temperature for
rate of solvent in other methods) every temperature increase of 10 °C)
Nature of sample (enclosed fat, moisture, size
of sample, surface, homogeneity of sample)

One of the best strategies to ensure you are using optimal extraction parameters is to apply standard methods.
These processes contain validated and recognized extraction methods and parameters, which help to increase the
reliability of your extraction workflow.

How to determine the endpoint of the extraction process

During the extraction process, the concentration of soluble components in the extraction substance decreases
steadily until it reaches a point where continuing the extraction is of no further value. This point is called the
practical conclusion3.

Concentration
of soluble
components
in extraction
solutions Pratical conclusion

Number of
solvent portions

The amount of extraction solvent (number of siphonings) required for the extraction to reach the point of practical
conclusion mostly depends on the solubility of the substance being extracted. Although in many cases, determining
the practical conclusion is a matter of experience, there are approaches that can help assess if the point has been
reached. One of the most common ways to determine the end of the extraction is to verify the extraction method
by using reference material with known fat content.

4
1. Why hydrolysis is needed for reliable and compliant fat determination

There are two main types of fat extraction, total and crude fat extraction. Crude fat extraction is a direct extraction
that is limited to determine only free fat. Total fat extraction is used to detect free fat and fat enclosed by other
components of the sample matrix. Total fat extraction is the required method for compliancy with many industry
standard methods.

Total fat extraction differs from crude fat extraction mainly in the addition of a hydrolysis step at the beginning of
the process.

Hydrolysis
Sample (only for
Weighing Extraction Weighing Results
homogenization total fat
extraction)

In general, a large amount of fat is encapsulated in the sample matrix. For example, in baked goods made with
gelatinized starch, fat is often mechanically enclosed by other components, such as carbohydrates to form
glycolipids and protein substances to form lipoproteins. In dairy products like milk, cream and cheese, surface
tension forces cause colloidal components made out of protein to surround the fat droplets. In yeast and eggs,
certain fat components are bound to other components chemically or by adsorption, forming phosphatide-protein
complexes.

To determine the concentration of lipids within the lipoproteins and glycolipids, the bonds that hold lipid and non-
lipid components together must be broken prior to solvent extraction. Hydrolysis is then used to release these
bound lipids into extractable forms. Additionally, during hydrolysis, proteins are hydrolyzed and plant cell walls are
broken down. Physically enclosed fats are also released and made accessible to the solvent during extraction.

Main advantages of hydrolysis:

·· Efficient breakage of matrix structures enclosing the fat fraction of food and feed samples
·· Assures conformity with official regulations for the declaration of total fat content
·· Increased reproducibility thanks to the standardized and exhaustive procedure
·· Ability to process liquid, moist and dry samples without the need to dry or mix samples with sand/sodium sulfate
prior to extraction

Hydrolysis is a necessary method requirement to comply with industry norms and regulations. Reference methods,
such as the ISO 8262-1 Weibull-Berntrop gravimetric method, require acid hydrolysis of fatty acids bound to
glycerides, sterol esters, glycol and phospholipids. Hydrolysis disrupts cell walls and breaks up lipid-protein bonds,
as well as fat emulsions to improve precision, productivity, reproducibility and applicability.

Did you know?

Both manual and semi-automated hydrolysis techniques are possible. Semi-automated hydrolysis is often
preferred because it is faster and safer due to reduced user exposure to hydrochloric acid.

5
 2. How finding the right extraction method improves speed, cost and reproducibility

Three of the most widely used extraction methods for fat determination include Soxhlet extraction, hot extraction
and continuous extraction.

A Soxhlet extraction method is used to determine the content of soluble compounds from dry solid samples.
During the process, the solvent in the flask is heated, the vapor passes the extraction chamber through a side tube
and into the condenser, condenses and the solvent drops into the sample in the extraction chamber until a siphon
point is reached. The extract flows back into the flask. With each extraction cycle, the sample is extracted with
freshly distilled solvent at low temperature.

Classical hot extraction is a process according to the Randall method. The sample is placed in boiling solvent. The
solvent is evaporated, flows to a condenser, condenses and the solvent drops back into the sample. In comparison
to Soxhlet extraction, there is no sample-extract separation.

In the classical Twisselmann extraction, the solvent is placed in a heated flask and the sample is placed in an
extraction thimble in the extraction chamber. In comparison to Soxhlet extraction the vapor does not pass through
a side tube to the condenser, but passes directly into the extraction thimble to the condenser. The vapor condenses
and the solvent drops into the sample.

The extraction methods are similar but offer distinct advantages. For example, Soxhlet is the most robust and
recognized method, but hot extraction offers reduced costs due to lower solvent consumption and faster extraction
processes. Continuous extraction offers high efficiency and accelerated analyte-solvent exchange due to higher
sample temperature than Soxhlet. Continuous and Soxhlet extraction are easier to set up as methods and tend to
produce more reproducible results than hot extraction.

A general overview of the three methods is displayed in the table below5.

A visual representation of how the methods work can also be found in a brief video.

Did you know?

BUCHI is the only extraction solutions provider to offer all three methods in one instrument thanks to an
innovative interchangeable glass assembly design. This feature offers unprecedented flexibility in switching
methods to fit your demands for any particular sample without needing several instruments that would
otherwise overcrowd precious lab space.

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 Soxhlet extraction Hot extraction Continuous extraction
E-500 SOX E-500 HE E-500 ECE

Method and Condenser

apparatus

Condenser

Condenser
Optical Extraction
sensor chamber
with
Magnetic sample
Extraction
valve
chamber
Beaker with
with sample
sample

Beaker Beaker

Method ∙∙ Separation of sample ∙∙ No sample-extract ∙∙ Separation of sample

characteristics and extract separation and extract
∙∙ Freshly distilled solvent ∙∙ Elevated temperature ∙∙ High extraction efficiency
for each extraction cycle of sample leads to ∙∙ Two-fold interaction betwe-
∙∙ Very gentle process at increased exchange en sample and solvent (va-
low temperature ∙∙ High extraction efficiency pors up/ condensate down)

Extraction time
(incl. extraction, rinse 1.5 h 40 min 1h
and dry)

Solvent
consumption
(per position)

100 mL 50 mL 70 mL
Sample
temperature

Compliance /
Reference
methods ISO / AOAC / EPA ISO / AOAC / EPA ISO / LFGB

Method
programming
Simple method selection Method development required Simple method selection

Reproducibility
(RSD)
Excellent Good Very good

7
 Fat determination by extraction

3. How the right instrument set-up can improve the efficiency of your extraction

There are numerous instruments available for performing extraction for fat determination purposes. The type of
instrument and extraction method you choose largely depends on your sample type, solvent use and the need of
compliance that your process requires among other factors.

Certainly, having the right instrument at hand can make all the difference in achieving cost-efficient and effective
results that satisfy your individual needs.

Use the decision tree below to easily find the instrument that best fits your sample and application.

Fat Fat (gravimetric)

Extraction either total fat or crude fat

Do you choose a solvent from the list? Does your food

No • Petroleum ether Yes sample require a Yes
• Diethyl ether hydrolysis prior
Manual
• n-Hexane to extraction?
• Chloroform
No
semiautomated
Talk to expert: HydrolEx H-506
[email protected]
or visit the decision tree:
www.buchi.com/extraction-decision-tree

Sample type

Non-processed food
• Raw Material Processed Fat > 5% mostly free fat
• Natural Products Food sample Fat ≈ extractable matter

Low RSD («1%)

Fat < 5%
Fat < extractable matter

Extraction SOX: Soxhlet

Extraction HE: Hot Extraction
Extraction ECE: Economic Continous Extraction (Twisselmann)

8
 See standard and regulations

Application Soxhlet Extraction Hot Extraction Extraction ECE

Chocolate AOAC 963.15 LFGB §64
AOAC 920.75
ISO 23275-1:2006
Dairy ISO 8262-1 LFGB §64
Bakery, cereal, nut AOAC 945.16 ISO 11085:2008 LFGB §64
AOAC 948.22 AOAC 2003.05
Meat ISO 1443:1973 AOAC 991.36 LFGB §64
ISO 1444:1996
Oilseed ISO 734-1 ISO 734-2 ISO 659
ISO 734-1
ISO 734-2

Sample type
No Non-processed food Bulky residue after
Processed • Raw Material hydrolysis
food • Natural Products and/or fat < 5%?

Extraction ECE
• Costs are key Yes
Yes
See standard • Solvent consumption (70 mL)
and regulations • Convenience is
important

Yes

Extraction HE
No • Speed is important No
Compliant to
• Very low solvent
any method?
consumption (50 mL)
• Correspondance to
third party automated
extraction systems

Extraction SOX
Yes Yes
• High reproducibility (RSD «1%))
• High analytical safety
• Very gentle process
at low temperature

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 4. How competent troubleshooting generates reliable results

Quickly resolving any issues arising during the extraction process can help minimize downtimes and increase the
reproducibility of the final data. Below are a few common pitfalls in fat extraction and suggestions on how to
resolve any issues that threaten the integrity of the extraction process.

Issue to be resolved Possible cause

Drying of extract was not sufficient

Oxidation of fat due to excessively high temperature during drying step

Fat content is too high

Celite® was washed out during extraction

Impurities in solvent

Loss of sample during hydrolysis step

Fat content is too low Incomplete extraction

Presence of water

Differences in beaker temperature

Sample weight is too small

Inhomogeneous sample
Variation is too large
Low fat content

Incorrect parameter set-up

Insufficient drying of sample after hydrolysis

Solvent evaporation is too fast.

Accumulation of solvent
Incorrect amount of sand on top of Celite® layer
on top of the sample
Compacted Celite® layers

Frit is blocked

10
 Did you know?

If you find yourself facing a tough challenge and you are stuck for answers, you are welcome
to contact the BUCHI extraction specialists. With over 30 years of experience in fat
extraction, we’d be happy to assist you in moving your project forward and achieving the
best possible recoveries from your sample. Please get in touch via e-mail.

Corrective measure

Dry to a constant weight

Reduce the duration of the drying step, use the SmartDrying function

Dry the extract at lower temperatures and reduced pressure in a vacuum oven

Loose the pulp carefully before drying the hydrolyzed sample

Use new solvent or freshly distilled solvent

Wash the hydrolysis vessels with several aliquots of water so that the sample is transferred quantitatively
into the glass sample tube
Adjust the temperature of the water (40-60°C). Water that is too hot can result in loss of fat,
water that is too cold cannot dissolve remaining sample sufficiently for a complete sample transfer
Set the level sensor to the top end of the sample to assure complete immersion in solvent

Choose appropriate extraction times and cycles

Avoid accumulation of solvent on top of the sample

Water must be thoroughly removed, as it repels lipophilic solvents and hinders the extraction process.
Careful dry the sample before extraction.
Make sure to use the same settings for drying (time and temperature) and cooling (time) of the beaker
when weighing prior to and following the extraction
If sample is very inhomogeneous, increase sample weight

Homogenize your sample by using a powerful blender, such as Mixer B-400 or by using mortar and pestle

The sample weight can be increased up to 10 g

Setting the correct number of cycles is essential for Soxhlet extraction, whereas setting extraction time
matters most for hot and continuous extraction
Ensure the glass sample tube with the sample is dried sufficiently in microwave or drying oven
Ensure that the right solvent from the library is selected and adjust heating level setting depending
on altitude and ambient temperature.
Use enough sand on top of the Celite® layer. Amounts that are too low can result in solvent accumulation.

Mix the Celite® layers carefully and thoroughly using a spatula prior to drying
Rinse frit thoroughly to remove any remaining sand and Celite® prior to cleaning in dishwasher.
If the frit cannot be unblocked, the glass sample tube must be replaced

11
 5. How to find the right extraction parameters to make fat determination easier and
faster

Fat determination is one of the most widely used analysis in the dairy industry. Parameters established in literature
or for a certain instrument are a good starting point for initial method development. Here, simple and reliable
procedure for fat determination in milk powder and yogurt using continuous extraction, hot extraction and Soxhlet
are presented.

Method

Equipment: HydrolEx H-506, FatExtractor E-500 Soxhlet

Sample: milk powder LUV No. 17-4b with a certified fat content of 24.27 g/100 g (+/-0.542 g/100 g),
Yoghurt muva-jo-1422 with a certified fat content of 3.76 g/100 g (+/-0.13 g/100 g).

Process: the sample was mixed with quartz sand in a glass sample tube and Celite®545 was added on top. The
samples were weighted and hydrolyzed with hydrochloric acid on the H-506 for 30 min. the hydrolysate was
transferred and the vessels washed with warm water until a neutral pH was reached. The glass sample tubes were
dried and cooled down, then another layer of quartz sand was added to the sample tube. The extraction was
performed using the E-500 with the parameters specified in Table 1.

Table 1. Parameters for extraction with fat extractor E-500 ECE, HE and SOX

Method Continuous Extraction Hot Extraction Soxhlet

Parameters
Solvent Petroleum ether / Hexane / Petroleum ether / Hexane / Petroleum ether / Hexane /
Diethyl ether / Chloroform Diethyl ether / Chloroform Diethyl ether / Chloroform
Extraction step 60 min 5 min 20 cycles
(heating level 5-8)* (heating level 4-8)* (heating level 5-9)*
Rinse step 30 min 5 min
(heating level 5-8)* (heating level 5-9)*
Drain 3
Drying step 6–12 min 3 min 10-13 min
(heating level 4-7)* (heating level 3-5)* (heating level 5-7)*
Solvent volume 70 mL 50 mL 100 mL

* Heating level proposed by the system depending on the selected solvent

The samples were extracted in triplicate. The extracts were dried to a constant weight the total fat content was
determined gravimetrically.

12
Results

The results are summarized in Table 2. The data corresponded well to certified values of the reference materials.
The results show low relative standard deviations (rsd).

Table 2. Determined fat content in dairy products, fat in g/100 g (relative standard deviation in brackets), n=3

Continuous Extraction Hot Extraction Soxhlet

Solvent Milk powder Yogurt Milk powder Yogurt Milk powder Yogurt
Petroleum ether 24.72 (1.06) 3.68 (0.94) 24.26 (0.33) 3.66 (0.33) 24.35 (0.26) 3.80 (1.07)
Hexane 24.43 (0.03) 3.70 (0.72) 24.27 (0.24) 3.64 (1.42) 24.45 (0.28) 3.70 (0.75)
Diethyl ether 24.36 (0.44) 3.74 (0.56) 24.39 (0.35) 3.77 (0.55) 24.50 (0.22) 3.75 (0.26)
Chloroform 24.52 (0.25) 3.75 (1.31) 24.68 (0.28) 3.73 (0.36) 24.70 (0.41) 3.81 (1.29)

Conclusion

The determination of fat in different dairy products using the HydrolEx H-506 and the FatExtractor E-500 provides
reliable and reproducible results. These results correspond well to the labelled values, with low relative standard
deviations.

Did you know?

Application notes for fat extraction of all types of samples ranging from dairy products to
chocolate could be found on the BUCHI Application Finder. Follow the QR code to see
what parameters could be ideal for your own fat extraction process.

13
 Outlook – what other methods exist for fat
analysis?

Besides solvent extraction, which remains the standard in fat determination, other methods are available for fat
analysis. The table below compares fat extraction to another common method for fat determination, NIR analysis.

Area of application Fat «Extraction» ProxiMateTM «NIR»

R&D +++ +
Production + +++
Goods inspection + +++
Quality control / labeling +++ ++
Characterictics
Range of applications + +++
Variation in sample types +++ ++
Automated throughput ++ +
Speed of analysis + +++
Compliance 1)
+++ +
Detection of adulterants + +++
Unattended operation ++ +
No contact with chemicals + +++
Ingress protection rating + (IP 20)
+++ (IP 65)
Low initial costs +++ +
Low running costs ++ +++
Eco-friendly ++ +++
Technical Data
Throughput in 9 h 3)
~ 36 samples 400+ samples
Analysis time ~ 90 min/6 samples ~ 15 s/sample
Max. sample amount 10 g 395 cm
Limit of detection (LOD) 0.1% 0.1%
1)
With respect to application regulations such as AOAC, ISO, DIN etc. + applicable
2)
Initial costs of the Kjeldahl products are very much depending on the level of automation ++ more applicable
3)
Depending on sample composition, packaging material. No shift work assumed. +++ most applicable

Did you know?

BUCHI’s FatExtractor E-500 offers reduced time-to-result and unprecedent sample throughput per day.
The Soxhlet process as performed by the FatExtractor E-500 is the reference method for NIR calibrations.

14
References Further reading

1. Soxhlet, F. (1879). “Die gewichtsanalytische 1. Application note 348/2019 Fat determination

Bestimmung des Milchfettes”. Dingler’s in dairy products, BUCHI Labortechnik AG.
Polytechnisches Journal 232: 461–465. Available for download:
https://www.buchi.com/sites/default/files/
2. inpastel. “egyptians wearing perfume cones”. AN_348_2019_Fat_determination_in_dairy_
(06 Sep 2009). Online image. Flickr. 08 July 2019. products.pdf
https://www.flickr.com/photos/g-dzilla/3899408057/
2. Application note 349/2019 Fat determination
3. BUCHI Labortechnik AG (2013). Basic Training in dairy products, BUCHI Labortechnik AG.
Primer – Extraction: available upon request. Available for download:
https://www.buchi.com/sites/default/files/
4. ISO 8262:2005, milk products and milk AN_349_2019_Fat_determination_in_dairy_
based foods – determination of fat content products.pdf
by the Weibull-Berntrop gravimetric method
(Reference Method). Retrieved from: 3. Application note 350/2019 Fat determination
https://www.iso.org/obp/ui/#iso:std:iso:8262:-3:ed- in dairy products, BUCHI Labortechnik AG.
2:v1:en Available for download:
https://www.buchi.com/sites/default/files/
5. BUCHI Labortechnik. AG Classical Extraction AN_350_2019_Fat_determination_in_dairy_
Methods: available upon request. products.pdf

6. BUCHI Labortechnik. AG (2017). Guidebook to

Proximate Analysis:
https://www.buchi.com/en/guidebook-to-
proximate-analysis-by-buchi

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