Primary Method Feature: Extraction (Part 1: Extraction Foundations)

Another blog not about NIR?

Near-infrared (NIR) spectroscopy is a secondary method. That means that NIR doesn’t measure things like fat, protein, moisture, ash, %-polymerization or anything else directly. Instead, we use an acceptable primary method to train our NIR to make those measurements. More details on NIR calibration can be found in this earlier blog post.

If you follow the blog, you may be feeling a little déjà vu. We just wrapped up a 2-part series about the primary method responsible for the protein data we use to train NIR with: Kjeldahl. The first post provided an overview of Kjeldahl foundations, then we went for blood in a Kjeldahl vs NIR head-to-head match-up.

Now it’s time to chew on a more calorically dense topic: fat. For that, we rely on a separation technique that came in vogue when ancient civilizations used it to make perfume and salves. Time brought further innovation, and with it, growing applications. In this blog, we will focus on the principles that apply to the fat extraction.

Basic extraction workflow

The modern extraction process is part of a larger analysis workflow; while the workflow itself with vary depending on the sample being analyzed, a general workflow looks like this:

Since fat is typically enclosed within other components of a complex sample matrix, sample prep for extraction begins with homogenization. Commercial mixers like the BUCHI Mixer B-400 makes the fat content is more accessible to solvents used downstream in the extraction; it also plays an important role ensuring method accuracy and repeatability.

The extraction process itself involves mixing the homogenized sample with a solvent. Components soluble in the extraction solvent are isolated from the sample mixture, while insoluble components remain in the extraction residue. The process is repeated several times to improve the extraction recovery (i.e., the amount of fat extracted from the sample). 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.

Hydrolysis as part of the extraction workflow

There are two main types of fat extraction: total fat 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 in the sample matrix. Total fat extraction is compliant with many industry standard methods.

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

In general, a large amount of fat is encapsulated in the sample matrix. For example, in baked goods made from gelatinized starch, fat is often mechanically enclosed by other components like carbohydrates (forming glycolipids) and protein substances (forming lipoproteins). In dairy products like milk, cream and cheese, surface tension forces cause colloidal components made out of protein to surround fat droplets. In yeast and eggs, certain fat components are bound to other components chemically or by absorption, 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 extraction. Hydrolysis is then used to release these bound lipids into extractable forms. Similarly, plant cell walls are broken down and physically enclosed fats are released and made accessible to the solvent during hydrolysis.

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 method
  • 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 requirement for compliance with industry norms and regulations. This short video gives an overview of the semi-autonated BUCHI HydrolEx H-506 hydrolysis unit.

Factors influencing extraction

Many parameters influence the recoveries and speed of extraction. The most important ones to consider when optimizing the extraction process are summarized here in terms of their impact on recovery rate or the speed of extraction.

Factors influencing the recovery rate

  • Solubility of the components in the extraction solvent (i.e. they must have similar polarity)
  • Thoroughness of mixing of extraction substances with solvent
  • Size and number of extraction solvent portions (i.e. number of siphonings in the case of Soxhlet, or drop rate of solvent in other methods)
  • Nature of the sample (e.g. enclosed fat, moisture, size of sample, surface, degree of homogeneity)

Factors influencing the speed of extraction

  • Particle size of the extraction substances
  • The degree of mixing of extraction substance and extraction solvent
  • Temperature; speed of reaction typically doubles with every 10 deg. C

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

End point determination

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 conclusion.

The amount of extraction solvent (i.e. number of siphonings) required for the extraction to reach the point of practical conclusion depends mostly on the solubility of the substance being extracted. While experience is often relied on for end point determination, a common approach is to verify the extraction method by using reference material with known fat content.

Types of Extractions

Three of the most widely used extraction methods for fat determination include Soxhlet extraction (SOX), hot extraction (HE), and continuous extraction (ECE).

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. Condensed 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 and flows to a condenser. Condensed solvent drops back into the sample. Unlike Soxhlet extraction, there is no sample-extract separation.

In the classical Twisselman (Continuous) extraction, the solvent is placed in a heated flask and the sample is placed in an extraction thimble in the extraction chamber. The vapor passed directly into the extraction thimble to the condenser. The vapor condenses and the solvent drops into the sample.

These extraction methods are similar, but offer unique 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 relative to Soxhlet. Continuous and Soxhlet extraction are easier to set up as methods and tend to have better reproducibility than hot extraction.

The illustration below gives a comparison of the methods in terms of the apparatus used, method characteristics, extraction time, solvent consumption, sample temperature, reference methods, method programming and reproducibility.

Excerpt from 5 Essentials of Fat Extraction booklet.

Sample characteristics are clearly another factor to be weighed in the selection of an extraction method. Here, the standard methods offer clear direction. However, the 5 Essentials of Fat Extraction booklet offers a decision tree that takes into account things like solvent type, sample type (e.g. non-processed food, natural products or raw materials vs. processed food samples), expected fat content, whether a bulky residue is seen after hydrolysis, and compliance.

BUCHI Extraction Solutions

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 multiple instruments.

See a short video to get an overview of the fast, flexible, and compliant BUCHI FatExtractor E-500:

Interested in Learning More?

The content used in this blog post was extracted (pun intended, of course) from the illustrated booklet, 5 Essentials of Fat Extraction.

Download the complete booklet from BUCHI for expert insight on key topics, including:

  1. Why hydrolysis is needed for reliable and compliant fat determination
  2. How finding the right extraction method improves speed, cost, and reproducibility,
  3. How the right instrument set-up can improve the efficiency of your extraction,
  4. How competent troubleshooting generates reliable results, and
  5. How to find the right extraction parameters to make fat determination easier and faster.

Or, contact an expert at BUCHI to discuss your extraction application.

More resources:

BUCHI Labortechnik AG (2017). Guidebook to Proximate Analysis

Application note 348/2019 Fat determination in dairy products (by Weibull-Stoldt Method), BUCHI Labortechnik AG.

Application note 349/2019 Fat determination in dairy products (by Economic Continuous Extraction), BUCHI Labortechnik AG.

Application note 250/2019 Fat determination in dairy products (by Hot Extraction), BUCHI Labortechnik AG.

Application note 388/2020 Crude fat determination in hemp seeds, BUCHI Labortechnik AG.

Application note 392/2020 Determination of oil in fish meal according to AOCS Ba 3-38, BUCHI Labortechnik AG.

Application note 389/2020 Determination of oil in seed meals according to AOCS Ba 3-38, BUCHI Labortechnik AG.

Application note 387/2020 Determination of total fat content in plant-based meat substitutes, BUCHI Labortechnik AG.

Application note 396/2020 Oil in Soybeans according to AOCS Ac 3-44, BUCHI Labortechnik AG.

Application note 393/2020 Oil in Soybeans according to AOCS Am 2-93, BUCHI Labortechnik AG.

Application note 390/2020 Oil determination in peanuts by Twisselman extraction, BUCHI Labortechnik AG.

Browse the entire BUCHI Application Note library using the BUCHI Application Finder

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