Engineering Properties of Biological Materials and Food Quality 3(2+1)

Lesson 20. Liquid Food Samples And Extraction Techniques

20.1 Pretreatments:

20.1.1 Removal of extraneous material:

Suspended matter or sediment present in liquids such as beer, wine, juice, or cooking oil is removed by filtration or separated by centrifugation.

Sample reduction:

Liquid samples can be mixed using magnetic stirrers or sonic oscillators.  There are several other factors to consider when reducing a food sample. Food choppers, blenders, and mixers should be constructed of metal alloys that resist corrosion or erosion, and that are inert enough to prevent contamination of the product. Aeration of the product during the blending process should be avoided since this can result in appreciable changes in oxidizable components. It is also important to avoid heating the material during the grinding step since this can accelerate chemical changes in the foodstuff. The surfaces of all mixing equipment should be clean and dry, since changes in sample moisture content can change the chemical and physical nature of the foodstuff. Care should also be taken to prevent the release of volatile constituents during grinding, if this is of concern.

20.2 Extraction methods:

20.2.1 Solid Phase Extraction (SPE)

In SPE a liquid is passed through a solid phase, which selectively retains the analyte. Thereafter, the analyte can be eluted with a strong solvent. In some cases interferences are retained and analytes allowed passing through solid phase unretained. The mechanism of SPE is comparable with LC. The advantages of SPE are that a wide variety of sorbents is available for the selective removal of inorganic, organic, and biological analytes. The good selectivity and efficiency of SPE can be explained by the fact that various modes like reversed-phase (RP), normal-phase (NP), ion-exchange (IE), restricted-access (RAM), Immunoaffinity (IA) and molecular-imprinting (MIP) can be applied. Techniques like SPME and stir bar sorption extraction (SBSE) are relatively new developments approaches to perform extraction procedures without the need for using organic solvents. Furthermore, a number of different sampling formats (e.g., packed syringes and cartridges, disks, pipette tips and 96-well plates) allowing off-line, at-line, on-line and in-line sampling procedures. This means that SPE can be considered to be the most versatile sampling and sample manipulation approach.

20.2.2 Liquid-liquid Extraction (LLE)

Still the most popular technique is LLE. In this case the sample is partitioned between two immiscible phases. The extraction solvent and extraction conditions are chosen in such a way that a maximum difference in solubility is obtained. In order to obtain reliable results one should beware of the formation of emulsions.  Possibilities to break them are heat, addition of salt; change of the KD value by using different solvents or chemicals affecting the equilibrium (such as buffers for pH adjustment, salts for ionic strength, complexing agents, ion-pairing agents, etc.). LLE can be performed manually by using a separatory funnel in case a relatively small number of samples must be analyzed or in an automated way by using packed cartridges or 96-well plates in case larger number of samples must be analyzed.

20.2.3. Dilution

An additional technique is dilution in which the sample is diluted with a solvent compatible with the eluent of the separation system (e.g. LC) to avoid system overloading or to be in linear range of detector. For example, to avoid band broadening the solvent should not be too strong for the LC eluent and should be miscible with LC eluent; “dilute and shoot” is a typical ST method for simple liquid samples such as pharmaceutical formulations.

20.2.4. Evaporation

In evaporation the liquid is removed by gentle heating at atmospheric pressure with flowing air or inert gas or under vacuum. Evaporation should not be performed too quickly and bumping can result in sample losses. Sample losses can also occur on the wall of the container. Evaporation should be done at moderate temperatures, by using an inert gas (e.g. N2) by using a rotary evaporator or an automated system (e.g. Turbovap).

20.2.5. Distillation

In distillation a sample is heated to the boiling point of the solvent, and volatile analytes are concentrated in the vapor phase, condensed, and collected. This approach is mainly used for samples that can easily be volatilized. Problems are that a sample can decompose if heated too high. This means that vacuum distillation can be used for low-vapor-pressure compounds, while steam distillation is rather gentle since maximum temperature is 1000C.

20.2.6. Microdialysis

Microdialysis is a technique in which a semipermeable membrane is placed between two aqueous liquid phases and low-molecular weight molecules transfer from one liquid to the other based on a concentration difference over the membrane. Enrichment techniques such as SPE are required to concentrate the dialysate. Microdialysis is used, for example, for the examination of extracellular chemicals in living plant and animal tissue and in fermentation broths. It has been used on-line with LC. Dialysis with MWCO membranes can also be used for on-line deproteination of samples.

20.2.7. Lyophilization (freeze-drying)

In lyophilization (freeze-drying) an aqueous sample is frozen and water removed by sublimation under vacuum. This technique can be used for nonvolatile organics, the concentration of inorganics and large sample volume can be handled. A potential problem can be the loss of volatile analytes.

20.2.8.Water sampling

In the case of water sampling it is important that collection containers are pretreated before the sample can be collected. In principle, only polyethylene or PTFE containers should be used and they should be washed and stored in 10% of HNO3 for 2 days and rinsed with double distilled deionised water. Following collection, acidification of the sample (normally with 2 mL of 10% HNO3 or 5 M HCl) will reduce or eliminate trace element adsorption and hydrolysis.

 Depending on the type of water precautions must be taken. For the collection of tap water, the first water running from the tap must be avoided because there will be a high concentration of trace elements from the pipes, soldering and welds. Normally, sampling is performed by running the tap for 5 – 30 min, before the actual sample is taken.

Most water samples require filtration immediately after collection to remove bacteria, algae and particulate matter. In most cases 0.5 mm membrane filters are used. Stabilizing agents like nitric, hydrochloric and sulphuric acids are frequently added to lower the pH to about 1 – 3.5. Before storage all sample containers should be completely full, because the presence of air may chemically or biologically alter the sample. Water samples shouldbestored in the dark, either by refrigeration (40C) or by deep-freezing (-200C).

The most important problem during the sampling of surface water is that, in principle, no samples may be taken from a stagnant water source because in those cases contaminants from valves, connectors, pipes, lubricants, etc. can be dissolved in the water. The system must therefore always be flushed for a while before taking the sample. The material of the container, used for storage, normally is not critical. However, the container must be carefully closed using aluminum or PTFE cap to avoid that pollutants fro the cap will pollute the sample. It is important that the sampling is performed at the same temperature as the surroundings. Using pressurized systems the sample must be done at flow rates of 500 mL/min or higher.

20.2.9 Microextraction techniques

Two equilibrium-based microextraction techniques serve as alternatives to classical solid-phase extraction: solid-phase microextraction (SPME) and stir-bar sorptive extraction (SBSE). Stir bar sorptive extraction is a similar equilibrium technique that requires submersion of a stir bar (that is encapsulated in a glass jacket and coated with a solid-phase) into the liquid sample. In this case, the solid-phase is usually a relatively high amount (25-125 ,ul) of polydimethylsiloxane (PDMS) polymer. The stir bar is then thermally desorbed on-line in the heated injector of a gas chromatograph. The advantage to utilizing SBSE for sampling liquid samples or extracts that are amenable to the PDMS solid-phase technique is that a 500-fold increase in enrichment, and therefore sensitivity, can be achieved compared with a 100 pm PDMS SPME fiber.

20.2.10. Membrane techniques

Membrane extraction methodologies encompass both the non-porous techniques of supported liquid membrane extraction (SLM), microporous membrane liquid-liquid extraction (MMLLE), polymeric membrane extraction (PME), and membrane-extraction with a sorbent interface (MESI), in addition to the porous membrane technique of dialysis. Variations of the latter are microdialysis and electrodialysis. Unlike the non-porous membrane methodologies, the porosity-based techniques are not characterized by analyte enrichment. There is no discrimination between small-sized molecules that are similar in size to the analyte, and only partial sample clean-up is achieved by membrane separation of lower molecular weight species from higher molecular weight matrix components. A dialysis clean-up step is therefore often combined with a subsequent enrichment technique, for example on an automated trace enrichment of dialysates system, also known as ASTED.

20.2.11. Microwave-assisted extraction

Microwave-assisted extraction (MAE) is one of several techniques that have

been developed in response to the increased demand for techniques that have a

shortened extraction time and reduced solvent consumption. One of the primary benefits of MAE is the ability to directly heat the sample with the application of microwaves. This type of heating is fast and temperature gradients are kept to a minimum. A drawback to the technique is the requirement for an extraction solvent that is able to absorb microwaves. In addition, a subsequent clean-up step is usually required once the microwave vessel has cooled sufficiently for handling.

Microwave techniques have been applied to biological and food samples quite extensively.

20.2.12. Pressurized liquid extraction

Pressurized liquid extraction (PLE) methods frequently utilize the Accelerated Solvent Extraction (ASE) system, or any other system that performs static or dynamic solvent extractions at elevated temperatures and pressures. The advantage to performing extractions under pressurized conditions is that the upper extraction temperature is not limited by the boiling point of the solvent, as is the case with the traditional Soxhlet system. A flow-through system such as the ASE is also particularly beneficial in food analysis. Static extractions are performed inside steel extraction vessels that have ample capacity for food samples, from 11-100 ml. The static extraction period is followed by elution of the extraction solvent into a collection vial.

20.2.13. Supercritical fluid extraction

Super critical fluid such as super critical carbon dioxide (SC- CO₂), super critical water are used for extraction as the solubility of materials at supercritical stage increases. SC-CO₂ continues to be the fluid of choice, since its critical parameters (31.1”C, 72.8 bar) are easily achieved with high pressure instrumentation. Further, it is non-toxic and easy to obtain. Some of the SF-based methodologies utilize suitable modifiers to enhance analyte recovery.