WO2010003457A1

WO2010003457A1 - Sample container for use in fat determination

Info

Publication number: WO2010003457A1
Application number: PCT/EP2008/058942
Authority: WO
Inventors: Henrik Thomsen; Christian Born
Original assignee: Foss Analytical AS
Current assignee: Foss Analytical AS
Priority date: 2008-07-09
Filing date: 2008-07-09
Publication date: 2010-01-14
Anticipated expiration: 2011-01-09

Abstract

The invention covers a method of determining fat in a sample, a Soxhlet sample container and a means of filtration (120, 220, 320, 420) for use in a Soxhlet sample container characterised in that said means of filtration is configured to, support hydrolysis by allowing an unaided flow of acid, base and water while retaining fat and to support extraction of fat by allowing an unaided flow of solvent and fat solubilised in solvent.

Description

Sample container for use in fat determination

[0001] The present invention relates to a sample container and a method of determining the fat content of a sample.

[0002] An established conventional laboratory method for determination of the amount of fat (which herein shall be defined as including all fatty acids and all lipids, such as wax, oil and other lipophilic molecules) in a sample of food, feed or foodstuff (hereinafter a sample) is based on variants of the Soxhlet method. The principal steps of a common variant of the method, allowing determination of the total of bound and unbound fat, include placing a well known amount of sample in an appropriate container, subjecting the sample to hydrolysis by boiling acid to release the bound fat, rinsing the sample, drying the sample and finally extracting the fat by a heated solvent such as diethyl ether or petroleum ether and determining the total amount of fat by weighing it. If determination of unbound fat is required separately an additional extraction must be completed prior to hydrolysis, and similarly if only unbound fat is known to be present hydrolysis may be omitted.

[0003] An example of a variant of the method is described in the standard ISO

6492:1999, and other variants are implemented commercially, such as the method marketed by FOSS Analytical under the commercial name Soxtec. For analysis of milk products an alkaline hydrolysis is used due to the special characteristics of milk.

[0004] The present invention relates to the hydrolysis and extraction steps of the Soxhlet method, and to a sample container appropriate for use in this method.

[0005] The sample container is an important element in the practical operation of the hydrolysis and the subsequent extraction. During hydrolysis the container must be able to retain the liberated fat while the hydrolysis acid is allowed to diffuse through a filter into the container; during drying the fat must also be retained, but during extraction the fat of the sample dissolved in solvent must be allowed to pass through the filter, while the non-extractable solids of the sample is retained. In the design of a Soxhlet sample container the combination of the fact that fats float on water due having a lower density and a balance between the required retaining of fats and permeability of aqueous acid and water during hydrolysis and rinsing, a sufficient retaining capacity of fats during drying and an ability to release fats and permeability of fat solubilised in solvent during extraction with solvent is specifically required, and in addition important requirements for the overall method are accuracy of analysis, short total time of analysis and a low amount of labour required.

[0006] The design of a Soxhlet sample container has been handled in several ways over time. In the original method, hydrolysis is performed with boiling acid in a test tube, and the hydrolysed sample is transferred to a paper filter with filter aid, where it is rinsed with water to neutralize the sample. This sample is then dried and transferred to a separate container for extraction. An improvement of this approach is the use of a glass walled sample container with a filter made from a glass frit (herein a glass frit container). The glass frit restrains the flow of polar liquids such as water and acids, as well as fat which has a high viscosity, and therefore during hydrolysis and rinsing with water, the flow has to be aided by vacuum. After rinsing, the glass frit sample container and the hydrolysed sample is dried in an oven or a microwave oven. After the hydrolysed sample has been dried, extraction of the fat with solvent must take place. The free fat will be dissolved in solvent, and due to the low viscosity the mixture will flow freely through the glass frit. To ensure complete and rapid extraction with a minimum consumption of solvent, the solvent is heated and refluxed. After extraction solvent is separated from the fat by evaporation or, in the case of reusing the solvent, distillation, and the remaining fat is weighed to obtain the total amount of fat in the sample. After completion the glass frit must be thoroughly cleaned to avoid that residues from previous samples are blocking the glass frit pores. While the use of a glass frit container is beneficial from the point of view of labour requirements during analysis, the glass frit container is costly and is most often reused, which requires time consuming cleaning.

[0007] WO 02/33403 A (CAPSULE TECHNOLOGY LTD ) disclose that the Soxhlet method may be carried out using a sample container comprising a filter plug from a porous filtration material, wound in a spiral placed inside a glass or plastic tube and a paper filter thimble - here called a thimble and filter plug container. To obtain the required balance between the required retaining of fats and permeability of aqueous acid and water during hydrolysis and rinsing and a sufficient holding capacity of fats during hydrolysis and drying the filter plug in this design is 15 mm thick, to ensure that all flowing liquid is in contact with the filter plug. This thickness has been obtained by rolling the filtration material into a spiral. During extraction the disclosed method instructs that the container is turned over and that the filter plug is rearranged and a secondary filter - a paper thimble - is used for filtration to separate sample from fat solubilised in solvent. The thimble and filter plug container has the benefit of a low cost, and is therefore suited for single use, but it has the drawbacks that the volume of water retained in the filtration material is high due to the thickness of the filter plug, which requires extra time for drying, and furthermore, that several manual steps are required, both in the production of the container and in the operation of the method.

[0008] An alternative approach is disclosed by US 6479295 B (KOMAREK, A.R. AND KOMAREK R.J.) 01.11.2000 which disclose a method employing a pouch of filter material, which has a pore size of 4 μm, allowing only passage of non-polar liquids, such as solvent and solubilised fat in solvent. Hydrolysis is obtained by using a vapour from a strong acid or base, but the hydrolysis may only be partial.

[0009] The present invention is intended overcome some or all of the problems listed above, and specifically to reduce the amount of manual handling involved in determination of fats in biological materials.

[0010] It has been assumed that combined requirement of (1) a balance between the required retaining of fats and the permeability of aqueous acid and water during hydrolysis and rinsing, (2) a sufficient holding capacity of fats during drying and (3) an ability to release fats and permeability of fat in solvent during extraction with solvent, may only be met by the combined use of two different filter materials such as is the case of the thimble and filter plug container as disclosed in WO02/33403, or by using a filter such as a glass frit, having a high flow resistance to viscous liquids such as fat and requiring the flow of acid, base and water to be aided by vacuum during hydrolysis.

[0011] However the present invention employs the surprising fact that a single thin layer of filter material is beneficial provided that a proper choice of material and a proper design are made. A first aspect of the invention is a means of filtration for use in a Soxhlet sample container, a second aspect is a Soxhlet sample container and a third aspect is a method for determination of fat in a sample.

[0012] The selection of filter materials requires determination of parameters such as the fat retaining capacity and the time of permeation by water and solvent respectively. A test for determination of these parameters is presented below.

[0013] The filter materials appropriate for use in the invention must fulfil multiple criteria. A first criterion is that the materials must be able to withstand the conditions during the Soxhlet process, including hot acid or base during hydrolysis and hot solvent during extraction. Furthermore, during hydrolysis the filter must be permeable to polar liquids such as acid, base and water, and during extraction the filter must be permeable to solvent and solubilised fats. Finally the filter must show sufficient fat retaining capacity to retain substantially all the fat present in the sample analysed. Advantageous materials have been found to be non-woven polymeric materials, such as but not limited to polyethylene (PE), polypropylene (PP), polyester (PET), polyamide (PA), and polyamide-6 (PA6), which possibly may be produced or treated by special methods to enhance certain characteristics of the filter materials, such as increasing the hydrophilicity and the permeability to polar liquids. Suitable treatment methods include chemical treatments such as addition of surfactants, as well as physical methods such as ion bombardments, plasma treatments and ozone treatments. Furthermore the filter materials may also be a woven polymer, a metallic mesh and woven or non woven glass fibres, possibly treated by suitable means to obtain the required characteristics. [0014] To identify appropriate filter materials the following test procedure was developed:

• A test container is made by attaching a disc of filter material 220 in a container with a mechanical fastening means 230 as shown in Fig.2. The internal diameter of the container body 210, and accordingly the diameter of the active filter area was 32 mm.

• The mass of the test container with a dry filter is recorded as m_dry.

• The time required for 20 ml of water at 22⁰C to pass the test container with the dry filter, is recorded as the time of permeation of water.

• The filter is dried

• The time required for 20 ml of petroleum ether at 22⁰C to pass the test container with the dry filter, is recorded as the time of permeation of solvent.

• The filter is dried

• The test container is filled with 5 ml of rape seed oil, and is held above a surface to allow the filter to drip until no drops are observed for 60 seconds.

• The mass of the test container with oil-wetted filter is recorded as m_ol|.

• The fat retaining capacity of the filter is calculated as m_cap.= m_oιrm_dry. [0015] Fig.1 shows a Soxhlet sample container with a permanently attached filter,

Fig.2 shows a Soxhlet sample container with a filter fixed by a screw thread union, Fig.3 shows a Soxhlet sample container with two layers of filter material and Fig.4 shows a Soxhlet sample container with three layers of filter material. [0016] A preferred exemplary embodiment is shown in Fig.1 , where a Soxhlet sample container appropriate for single use is made from a filter 120 made from a disc of filter material permanently attached to a cylindrical container body 110, by melting the two parts together by a heating source, such as a contact heating element, infrared heating, ultrasonic heating, lasers or friction. The filter material 120 is preferably a non-woven mixture of polyethylene and polypropylene, which in the same configuration, supports hydrolysis by allowing an unaided flow of acid, base and water while retaining fat and supports extraction of fat by allowing an unaided flow of solvent and solubilised fat in solvent.

[0017] A porous filter with an ability to absorb and thus retain fat may often also retain water and may therefore require a longer time for rinsing away acid and drying and therefore it is beneficial if the porous filter is having a thickness of less than 5 mm, preferably less than 2 mm, and more preferably less than 1 mm.

[0018] An alternative production method based on attaching the filter material 120 to the container body 110 by means of glue will have the benefit of avoiding the need of a heat source in production of the sample container.

[0019] In another exemplary embodiment shown in Fig.2 a sheet of filter material 220 is attached to a container body 210 by a mechanical fastening means 230, such as a union fastened by a screw thread, a bayonet coupling or spring loaded coupling which seals the sheet of filter material 220 against the container body 230. The use of a mechanical fastening means 230 has the advantage of supporting a re-use of the container body 210, while allowing the filter material 220 to be discarded.

[0020] An appropriate Soxhlet sample container may also be produced by insert moulding or by production from a pouch of filter material, where the container walls are made impermeable by an appropriate treatment such as covering the walls with an appropriate material or sintering by heat.

[0021] In the third and fourth embodiment below, the means of filtration comprises multiple layers of filter materials with different functionality but the dominant fat retaining ability is due to a single material, which is preferably in a thin layer, for the reason discussed above, and which for simplicity is called the fat retaining filter layer.

[0022] In Fig.3 is shown a third embodiment of the invention, having the benefit that the risk of the sample blocking the flow is reduced, by removing the sample 350 from immediate contact with the fat absorbing filter 320. The Soxhlet sample container according to this embodiment of the invention comprises a container body 310 and two layers of filter material 320, 330. A thin fat retaining filter material layer 320 is attached to the bottom of the container body 310, and a thicker coarse filter 330 is placed above this, separating the sample 350 from the fat retaining filter material 320. [0023] In Fig.4 a fourth embodiment of the invention is shown, wherein the means of filtration comprises 3 layers of filter materials, with potential production and handling benefits. In this embodiment a coarse grid 440 at the bottom of the container body 410, has the main function of supporting a thin plug of fat retaining filter material layer 420. The optional coarse filter 430 has the same function of separating the fat absorbing filter material 420 from the sample 450. This embodiment has the benefit during production that the container body 410 and the coarse grid 440 may be moulded from one material, and that a thin plug of the fat retaining filter material 420 may be placed without requiring production equipment, for instance in the case where it is desired that the body of the Soxhlet sample container is reused.

[0024] The use of multiple layers of filter material will also give the benefit of a better optimisation of the balance between the desired capabilities of the means of filtration, by combining capabilities of different materials.

[0025] According to a second aspect of the present invention, there is provided a method of testing a fat content of an item, comprising one or more of the following steps

(a) Grinding the sample and placing it in the Soxhlet sample container

(b) Weighing the sample

(c) Drying the sample

(d) Weighing the dry sample

(e) Solvent extraction of (free) fat

(f) Weighing the sample

(g) Using a hydrolysis liquid such as acid or base to release bound fat and removing the liquid and soluble components from the container and, optionally, re-weighing

(h) Washing in water, removing the hydrolysis liquid and other water soluble components from the container and, optionally re-weighing the container ;

(i) Drying the container to remove moisture and weighing the container ; (j) Solvent extraction of fat from the container with the relative configuration of the sample and the means of filtration of the Soxhlet sample container being the same as in (g) (k) Evaporation of solvent (I) Weighing of extracted fat In the method description above steps (b) to (e) are included for full compatibility with the ISO method and to allow the separate determination of unbound and bound fat, and may be excluded, especially for a known sample type. Especially during step (h) the use of cold water may be beneficial to avoid mixing of fats and water, and thus leakage of fats through the means of filtration.

Claims

1. A means of filtration (120, 220, 320, 420) for use in a Soxhlet sample container characterised in that said means of filtration is configured to support hydrolysis by allowing an unaided flow of acid, base and water while retaining fat and to support extraction of fat by allowing an unaided flow of solvent and fat solubilised in solvent.

2. A means of filtration according to claim 1 comprising one or more layers of filter materials (120, 220, 320, 330, 420, 430, 440) of which at least one (120, 220, 320, 420) is fat retaining wherein the dominating fat retaining filter material layer is having a thickness less than 5 mm, preferably less than 2 mm and more preferably less than 1 mm.

3. A means of filtration , according to claim 1 or 2 having a time of water permeation which is less than 60 seconds and a time of solvent permeation which is less than 60 seconds, in accordance with the test procedure of the description.

4. A means of filtration , according to claim 1 to 3 having a fat retaining capacity of at least 0.1 g, in accordance with the test procedure of the description.

5. A Soxhlet sample container comprising a means of filtration according to any claim above further comprising a container body (110, 210, 310, 410) being impermeable to liquids.

6. A Soxhlet sample container according to any claim above obtained by attachment of at least one layer of filter material (120, 220, 320, 440) and the container body (110, 210, 310, 410).

7. A Soxhlet sample container according to claim 6 wherein the attached layer of filter material (120, 320, 440) and the container body (110, 310, 410) are permanently attached, preferably by thermal fusing or gluing.

8. A Soxhlet sample container according to claim 6 further comprising a mechanical fastening means (230) configurable to attach said filter (220) and the container body (210).

9. A Soxhlet sample container according to any of the claims 5 to 8, where the container body (110, 210, 310, 410) is substantially inflexible.

10. A Soxhlet sample container according to any of the claims 5 to 9, wherein the filter material dominating the fat retainment (120, 220, 320, 420) is a non-woven polymer.

11. A Soxhlet sample container according to any of the claims 5 to 10, wherein the filter material dominating the fat retainment (120, 220, 320, 420) is made from one or more of the following materials; polyethylene, polypropylene, polyester, polyamide and polyamide-6.

12. Use of a Soxhlet sample container in the determination of the fat content of biological materials, characterised in that the Soxhlet sample container is one according to any of the claims 5 to 11 or one employing a means of filtration according to any of the claims 1 to 4.

13. A method of determination of fat, employing a Soxhlet sample container according to claims 5 to 11 or one comprising a means of filtration according to claims 1 to 4, and comprising at least steps (g) and (j) of the following steps:

(a) Grinding the sample and placing it in the Soxhlet sample container

(b) Weighing the sample

(c) Drying the sample

(d) Weighing the dry sample

(e) Solvent extraction of (free) fat

(f) Weighing the sample

(i) Drying the container to remove moisture and weighing the container ;

(j) Solvent extraction of fat from the container with the relative configuration of the sample and the means of filtration of the Soxhlet sample container being the same as in (f)

(k) Evaporation of solvent

(I) Weighing of extracted fat