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Plant Lipids Science, Technology, Nutritional Value and Benefits to Human Health, 2015: 221-238

ISBN: 978-81-308-0557-3 Editors: Grażyna Budryn and Dorota Żyżelewicz

6. Analysis of plant lipids

Marianna Raczyk and Magdalena Rudzińska Institute of Food Technology of Plant Origin, Poznań University of Life

Sciences, Wojska Polskiego 31, 60-624 Poznań, Poland

Abstract. In this chapter selected methods used in plant lipid analyses are discussed. The first step of fat analysis consists in the isolation of lipid components from the plant matrix, thus in the first section the most popular methods of isolation are described. Afterwards, due to the complicated mixture of lipid components their separation is required where different types of chromatography techniques are applied. To detect and quantify them specific detectors are required as part of chromatographic systems, in which mass spectrometry, nuclear magnetic resonance and photometric spectroscopies are often applied. To assess specific chemical characteristic of lipids classical chemical methods using e.g. peroxide, iodine and acid values are very often applied. The last part selected oxidative stability tests such as the Schaal oven test, differential scanning calorimetry and Rancimat (OSI) tests were described. Finally sensory analysis as a quality determination technique was also discussed.

Introduction

Lipids are found in every plant, but their content and composition differ

widely, depending on the type and part of the plant. The main lipids include

Correspondence/Reprint request: Mgr. Marianna Raczyk, IInstitute of Food Technology of Plant Origin, Poznań

University of Life Sciences, Wojska Polskiego 31, 60-624 Poznań, Poland. E-mail: [email protected]

Marianna Raczyk and Magdalena Rudzińska 222

triacylglycerols, diacylglycerols, monoacylglycerols, phospholipids,

glycolipids, sterols, free fatty acids, vitamins soluble in fat, waxes, carotene

and chlorophyll pigments. Storage lipids, such as oil in oilseeds contain the

highest amount of triacylglycerols (95-98%), in the form of fatty acid esters

with glycerol [1]. However, functional lipids in plant cells are composed

mainly of polar lipids such as phospho- and glycolipids with very low

amounts of triacylglycerols. Lipids are insoluble in water, but soluble in

organic solvents such as hexane, ether and chloroform, while polar lipids

require highly polar solvents such as butanol and propanol. The first and the

most important part of lipid analytical procedure consists in the selection of

an appropriate extraction/isolation technique to remove all lipids

representing their composition in the analysed material. A general rule for

the isolation of lipids is to utilize proper solvents such as neutral solvents to

isolate neutral lipids, e.g. tricyglycerides, and polar solvents to extract polar

lipids, for instance phospholipids and glycolipids. Isolation of polar lipids

often requires utilization of elevated temperature and some hydrolysing

agents. Utilization of polar solvents also causes extraction of non-lipid

components such as sugars and in the next step these compounds need to be

separated from lipids, which is usually performed by the application of water

and in the same step separation of the lipid phase from water soluble phase

components. The composition of fatty acids has a significant impact on

physical and biological properties of fat, therefore their assessment is very

often the first step of plant lipid analysis. There are many methods to

determine the composition and quality of fats. The contemporary, precise

and widely used analytical techniques are chromatographic methods.

Currently, also spectrometric methods are used on a large scale in lipid

analysis. The structure and features of analysed compounds have a

significant impact on the method used. Lipids are complex mixtures of many

different types of individual lipid classes, therefore often a single analysing

method would not be able to separate and analyse all compounds present in

fat. Most typically a combination of several techniques has to be applied

[2, 3].

Why are we interested in plant lipid analysis?

To better understand plant lipid metabolism and develop oils with

specific properties, we need to analyse lipids which take part in this process.

The further use of plants, as a source of oils, depends on the quantity and

quality of its plant lipids. Plants, which are good sources of oils, are

oftenpressed to obtain plant oils, but their further application greatly depends

on the required functional properties of the oils. Fatty acid composition has a

Analysis of plant lipids 223

significant influence on nutritional and healthy properties of oils however,

other oil compounds are also of significant importance in defining functional

properties of the oil. Amounts of saturated, unsaturated fats and fat-soluble

substances, such as antioxidants (tocopherols, carotenoids) or prooxidants

(chlorophylls, heavy metal ions), have a high impact on wholesomeness, but

also on oxidative stability and proper application in food processing.

Moreover, previously mentioned factors affect physical properties of oils

(colour, liquidity) and the quality (aroma, taste). The concentration and

quality of plant lipids have an impact in particularly on their application

(edible oils, biofuels and industrial oils) and processing conditions [4].

Lipid extraction

To analyse plant lipid composition and to identify individual

components, first we need to isolate them from the sample matrix. The most

popular way to isolate lipids is extraction, which basically is a separation of

components based on solubility in specific organic solvents. Extraction is

most often used to assess lipid contents in specific products or raw materials,

which is usually followed by gravimetric determination. This assessment is

Water outCondesner

Thimble

Heat source

Solvent

Vapor

Water in

Extraction chamber

Figure 1. The Soxhlet extractor

Figure 1. The soxhlet extractor


often inaccurate due to extraction of components other than lipids, which are

usually present in the sample matrix. The type of solvent or solvent mixture,

mainly polarity of solvents, define which types of lipids are extracted [5].

There are many methods of lipid extraction, choosing one of them is

mainly affected by the characteristics of the tested sample. However, in the

case of plant lipids it is defined by the type of lipids that we want to analyse,

neutral or polar, and accordingly solvents are selected. Utilization of polar

solvents for extraction, usually results in extraction of other components than

lipids, mainly carbohydrates and some protein. In this case to assess lipids

composition or identified compounds it is required to remove those non-lipid

components from the extract. Prior to lipid extraction alkaline, acid or

enzymatic hydrolysis is often used to release the lipid part of complex lipids

however, when the composition and identity of lipid components is the goal

of analysis, extraction of complex lipid components is required without

hydrolysis.

This is particularly important in plant lipids, which are usually combined

with sugars and sometimes with proteins. Prior to extraction hydrolysis is

not required for oilseeds, where lipids are found as storage lipids, not

metabolically involved lipids.

The Soxhlet extraction currently is a standard and reference method

used for food products and raw materials [8]. This method has many

advantages, such as continuous contact between the sample and fresh

solvent, so filtration and other steps are not needed. The main disadvantage

of this method is the required extraction time of 16-24 hours to achieve good

recovery of lipids. This procedure has been modified to improve efficiency

and shorten extraction time by 5-10 times utilizing pressure and elevated

temperature, or modifying the Soxhlet apparatus which makes it possible to

place the sample in boiling solvent (Soxtec Foss-Tecator) [9].

Lipids are found in tissues in a variety of physical forms, but complex

lipids are usually constituents of membranes, where they occur in close

association with such compounds as proteins and polysaccharides, with

which they interact through hydrophobic and van der Waals forces and

perhaps by ionic bonds. Various solvents or solvent combinations have been

suggested as extractants, but most lipid analysts use chloroform-methanol

(2:1 by volume) as suggested by Folch et al. [10]. Endogenous water in the

tissue is a ternary component of the system. The extract is shaken and

equilibrated with one fourth its volume of a saline solution, when the

mixture partitions into two layers, of which the lower is composed of

chloroform-methanol-water at 86:14:1 (by volume) and contains virtually

all the lipids, while the upper phase consists of the same solvents at 3:48:47

(by volume), respectively, and contains much of the non-lipid contaminants.


It is not always recognised how important it is that the proportions of

chloroform, methanol and water in the combined phases should be as close

as possible to the 8:4:3 ratio (by volume), otherwise selective losses of lipids

may occur. If carried out with the correct protocol, this method can give

reliable results [11].

The Bligh-Dyer method is a modified Folch method, which was

applied in the interest of economy and ecology purpose. It is the most often

method used to isolate total lipid content in biological tissues, but is widely

used also in other structures. The advantage of this method is a reducted

amount of solvents used, but also the recovery of total lipids is higher when

compared to the Folch method [12].

AOAC 996.01 is an universally accepted method for nutritional food

labelling, set by the Association of Official Analytical Chemists (AOAC), to

determine total fatty acids in cereal-based products [13]. This procedure has

sufficient accuracy and repeatability and requires application of acid

hydrolysis prior to solvent extraction [14-17].

Most extraction methods mentioned above, are conventional but nowadays

more economic and environmentally friendly methods have been developed,

such as Supercritical Fluid Extraction [18], Pressurized Liquid Extraction,

Focused Microwave-Assisted Soxhlet Extraction [9, 18]. Dynamic

Ultrasound Assisted Extraction [19] and Microwave-Integrated Soxhlet [8,

20].

Microwave-assisted extraction utilizes microwave energy to

disintegrate samples, heat solvents and make extraction efficient and rapid.

According to the research in which this method was used [20], total fats and

oils were qualitatively similar to those obtained by conventional methods.

Fatty acid composition for each extract of tested oils did not show any

significant difference. The method should be consider as valuable, fast and

effective for extraction of fats and oils from food products. Another

technique using microwaves is called the focused microwave-assisted

Soxhlet method [21]. This is a modification of the Soxhlet method, where

microwaves are used in an extraction chamber, as auxiliary energy.

According to the research with the use of this method the best efficiency was

obtained for samples with the moisture content between 20% and 90%

(preferably around 30%). Microwave-assisted extraction was successfully

applied, as an alternative method for oil extraction from olive seeds [21].

Automated hydrolysis and extraction (AHE) is a commercial

procedure which combines acid hydrolysis and solvent extraction in a closed

system. The second part of the analytical process includes extraction of

lipids by the automated Soxhlet method. The advantage of AHE system is

that the whole process is automated and is run in a closed container under


pressure and elevated temperature, making the analysis shorter and more

efficient [16, 22].

Analysis of lipids

Lipids from biological samples, particularly of plant origin, are very

complex mixture samples and to analyse their identity it is required to

separate individual components or at least groups of compounds with

similar properties. For this purpose a variety of chromatographic

techniques are applied, and currently most often high pressure liquid

chromatography (HPLC) is used due to its high efficiency of separation

and high efficient detectors available. Spectrophotometric detectors can be

used in plant lipid analyses, due to the presence of chromophores and a

variety of pigmants. Although most often analysed part of lipid is a fatty

acid composition.

Chromatographic methods

Low-pressure chromatography is used to separate most simple lipids

by column chromatography. One of the effective and widely applied

adsorbents in this method is silicic acid. Substances are selectively eluted by

hexane and diethyl ether mixed at adequate proportions. The precise amount

of diethyl ether must be mixed with hexane to elute every lipid class, which

varies depending on the batch of the adsorbent and should be determined

experimentally. Complex lipids, as a single class, can be obtain with

methanol or chloroform [24].

Thin-layer chromatography (TLC) is a planar adsorption

chromatography, where individual lipids can be separated in multi-

dimension development with solvent. It is one of the oldest, but at the same

time most popular and widely used chromatographic techniques, in which a

thin layer of the stationary phase is placed on glass or aluminium plates. This

method has mainly two applications: purification of specific fractions of

lipids and quantitative analysis, the latter when densitometry with charring

or specific reagents is applied. Many advantages, as for example relatively

inexpensive equipment, easy to set up and operate in laboratories, make TLC

still a useful method. TLC is also widely applied for monitoring reactions

and identification of product formation in organic synthesis [14, 25].

Currently, TLC with pressurized chambers has significantly improved

separation and shorted time of analysis. Also many modification of

adsorbents are available to separate specific groups of lipid components

utilizing some chemical properties [27-29].


Solvent

Time zero

Solvent

After time

Figure 2. Thin-layer chromatography.

The improvement of thin-layer chromatographic methods led to the

development of high performance thin-layer chromatography (HPTLC)

that provides much higher chromatographic resolution compared to TLC.

Against the traditional, HPTLC plates have significantly smaller size

particles and associated higher surface area, facilitating quicker and clearer

sample separation. A combination of HPTLC with new technologies, such as

the use of pressurized chambers, high resolution cameras, automated sample

applicators and matching software, provides more accurate analysis. These

factors make it an effective analytical technique for quality control [27-30].

Another currently widely used type of chromatographic method is high-

performance liquid chromatography (HPLC), that provides much higher chromatographic resolution compared to TLC. HPLC has been used for accurate separation and quantification of organic substances, including the separation of individual simple lipid classes from the lipid fraction. HPLC has been also used as a standard method to determine tocopherol content in oils [31]. Many different types of column packing materials, eluents and detectors have been used for lipid analysis. The first HPLC methods were elaborated in the 1980s for quantitative analysis of lipids, using mass detectors such as evaporative light-scattering detector (ELSD) and flame ionization detector (FID). The ELSD, depending on the method used, can be applied for analysis of both polar and non-polar lipids. Numerous lipid analyses have used an ELSD and columns with silica gel to separate phospholipid classes in lipid samples. Another very popular and the least expensive detection technique for HPLC is the UV-visible detector, which can be use for lipids that have chromophores (the part of a molecule responsible for its colour), but they have been limited to the analysis of lipid extracts that contain a mixture of saturated and unsaturated molecules. For HPLC analysis, FID was successfully used for several years, but since the sensitivity of ELSD has been improved, the latter method started to

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predominate. The minimum detection limits with modern ELSD is 50-100ng [32, 33]. Another type of detector for lipid analysis which was reported in many researches is an aerosol-based detector, although the lowest limits of detection were found for the nucleation light scattering detector with limits below 1ng. Recently a new, commercially available mass detector is charged aerosol detector (CAD), which was evaluated with normal and reverse phase HPLC systems, widely used for the analysis of molecular species and quantitative analyses of lipid classes [34]. In spite of a huge application of HPLC methods for plant lipid analysis, this technique should be considered as a method that also has some disadvantages such as high costs of equipment and running, large amounts of solvents used during the process, high sensitivity of the column to very high (above 9.0) and low (below 3.0) pH of mobile phases [32-34].

Another widely used separation and identification method is gas

chromatography (GC). In this technique the stationary phase is a

microscopic layer of polymer (gas solid chromatography-GSC) or liquid (gas

liquid chromatography-GLC) on the inert solid support inside a column. The

GC column is a very important part of the system that provides separation,

therefore choosing an adequate one is crucial for the analysis. There are two

types of columns, one of them is a packed column mostly 1.5-10 m long and

2-4 mm wide, polarity of the stationary phase determines separation rate.

The analysis of gas samples is one of the areas where packed columns are

widely used. Another type of column is a capillary with an open cross

section, such columns are usually much longer (10-100 m) and narrower

(0.1-0.5 mm) than packed columns. Among capillary columns, the most

commonly used are fused silica columns: the wall coated open tubular

(WCOT) or porous layer open tubular (PLOT) columns. In the former a

liquid film coated to the deactivated wall of the column is the stationary

phase, but in PLOT it is a solid substance that is coated to the column wall.

Mostly capillary columns are used, because they provide better efficiency

(narrow peaks). The separation power is really high and it facilitates short

time runs, with the use of short columns. Another important part of GC is an

injector, also called an inlet, which provides the introduction of a sample

into a flow of carrier gas. An injector is attached to the column head. There

are three ways of introducing samples in GC: injection with split, splitless

and on-column modes. The following factors have a significant influence on

the separation, polarity of the stationary phase, temperature, flow rate of

carrier gas, length of column and amount of sample injected. Helium has

been applied most often as a carrier gas (mobile phase), but sometimes its

substitutes by hydrogen or nitrogen. Carrier gases are applied to promote

sample transport through the column. Diffusivity provides a measurement


Table 1. Characteristics of carrier gases.

Carrier gas Viscosity at 50 °C

[kg/m s]

Diffusivity

(butane, 100 °C [m2s])

Helium 20.8 5.5 x 10-6

Hydrogen 9.4 6.0 x 10-6

Nitrogen 18.8 1.5 x 10-6

Table 2. Gas chromatography detectors.

Type of Detector Applicable Samples Detection Limit

Mass Spectrometer (MS) any sample 0.25 to 100 pg

Flame Ionization (FID) hydrocarbons 1 pg

Thermal Conductivity (TCD) universal 400 pg/ml

Electron-Capture (ECD) halogenated

hydrocarbons 50 fg

Atomic Emission (AED) element-selective 1 pg

Chemiluminescence (CS) oxidizing reagent Dark current of PMT

Photoionization (PID) vapour and

gaseous compounds 0.002 to 0.02 µg/L

for the diffusion speed of a solute vapour in a given gas and it is different for

each gas (table 1). Diffusion speed is one of the factors that determine the

speed of chromatography [35, 36].

The final, but essential part of the chromatograph is a detector. Different

types of detectors, their applications and detection limits are presented in

table 2. Currently, some of the most common detectors which have been

used with GC are flame ionization detectors (FID), thermal conductivity

detectors (TCD) and electron capture detectors (ECD) or a GC coupled with

a mass spectrometer (MS) [37].

Gas chromatography in the analysis of plant lipids

GC is often used to determine the fatty acid profile of lipids. The

procedure of fatty acid analysis in neutral lipids is as follows: dissolving

dewaxed and degummed oil in alcohol (usually methanol) and titrating by

1M ethanolic potassium standard solution to cover all free fatty acids into

their corresponding fatty acid potassium salts, which then are transformed

into free fatty acids (FFAs) using concentrated sulphuric acid (98%) at 60 °C.


Then, the obtained FFAs are transformed into corresponding fatty acid

methyl esters (FAMEs) using boron trifluoride in (14-15%) alcohol solution

at the same temperature [36, 37].

The fatty acid profile of fat can be obtained by gas chromatography

analysis of the corresponding FAME profiles. Most GC analyses are

performed on the equipment with a spit injector and a flame ionization

detector. Gas chromatography can be set at lower or higher temperatures and

depending on the analytical method, the temperature can be constant or

variable during the whole process. More accurate quantization of fatty acids

has been developed with internal or external standards and a FID response

correction factor the purpose of which is to correct the non-linearity of the

FID response to the mass of fatty acid methyl esters. Theoretical response

correction factors are calculated by a formula considering the detector

response proportional to the relative percentage of carbons in diverse

molecules. The application of this method also requires an optimization of

instrumental conditions [36, 37].

Another application of GC is to check purity of synthesized glycerol

ester (GE), but only a few research groups considered the use of GC-MS for

the analysis of intact GE in edible oils. Other applications where gas

chromatography have been successfully used is to determine the content of

sterols and tocopherols [38].

Short chain, volatile fatty acids are typically analyzed in the free form,

using specialized columns. This group of compounds may be referred to as

free fatty acids (FFAs), volatile fatty acids (VFA), or carboxylic acids. The

analysis of fatty acids in the free form instead of fatty acid methyl esters

results in an easier and quicker sample preparation. In addition, the

formation of undesirable derivatives is eliminated [23, 36].

GC-MS is widely used with other indirect methods for ester analysis, but

so far has not been applied in any direct method. GC-MS is commonly used

because of its advanced sensitivity, selectivity and a high separation power,

but for this reason samples have to be prepared carefully to obtain clear and

easy to interpret results [29, 37].

Mass spectrometry (MS) is a powerful analytical technique that can

provide quantitative and qualitative data, which cannot be obtained by other

methods. Some of the results providedwith the use of this method are

molecular weight, the empirical formula and even the complete structure of

an unknown factor. Using MS, chemical compounds have been ionized to

generate charged molecules or molecule fragments and their mass to charge

ratios are measured. Many examples of applying MS to lipid analysis can be

found in a book by Murphy[39].


The MS principle, in its simplest form, is to bombard organic molecules

with electrons and positively charged ions. By this operation smaller ionized

entities are obtained, which are propelled throught an electrostatic or

magnetic field and are separated according to their mass to charge ratios.

Then, the ions are collected in sequence as the ratio increases. The largest

peak is usually given a value of 100 and the intensities of all other ions are

normalized to this. MS is usually charged with GC or LC. For lipid

applications GC-MS has been used most often, but LC-MS is applied for

structural analysis of lipids that cannot be volatilized by this technique. In

mass spectrometry, electrospray interface (ESI) is also used for lipid

analyses. ESI is a sensitive ionization process which found extensive

applications for structural information of complex phospholipids and

glycolipids. Many successful applications to analysis of non-polar lipids

such as triacyloglycerols with the use of matrix-assisted laser desorption

(MALDI) have been published. Another type of MS is a MSI-mass

spectrometry imaging that provides analysis of spatially localization of

compounds across surfaces in the case of 2D imaging, but volumes can be

analyzed also by 3D, or even 4D imaging. Two new software instruments

implement these new methods for rapid data exploration and visualization of

2D and 3D data sets in full spatial and spectral resolution. The next type of

mass spectrometry which is often used is a tandem mass spectrometry

(MS/MS). This method provides much better sensitivity of detection and

supplies more information about the structure of the molecules [39, 40].

Nuclear magnetic resonance (NMR) spectroscopy is a powerful

technique that is used to obtain quantitative and qualitative information

about the lipid composition of the extracted oil, avoiding invasive

manipulations of the sample. NMR has been applied to identify lipid

structures, and to detect double bonds and their position in fatty acid chains.

The potentiality of this technique in the study of the molecular components

of complex lipid systems has been successfully demonstrated. NMR

spectroscopy has been applied to analyses of the phospholipid profile of

plant extracts. This method has been widely used in conjunction with HPLC

and GC techniques to study the molecular composition of lipids extracted

[41, 42].

Depending on the atom which is used, there are several types of NMR.

Usually the method has been run with 1H,

13C,

15N,

31P. Hydrogen,

1H, is

usually used to analyze organic compounds, but the carbon isotope, 13

C, is

applied for non-polar lipids. 1

H NMR provides high sensitivity, but limited

resolution, whereas 13

C provides a more detailed analysis of various lipids

with higher resolution however, low natural abundance of isotope 13

C makes

this method more expensive [43, 44]. Recently the application of NMR in


the analysis of oils has strongly increased. NMR is a simple, non-destructive

and fast analytical method that enables simultaneous analysis of different

analytes in one sample. Through the comparison with reference spectra, the

determination of many components is possible. An NMR signal is directly

proportional to the amount of the respective nucleus, therefore a molar

concentration can be obtained simply from the NMR spectrum [44]. The

usefulness of the NMR technique in the study of the molecular components

of complex lipid has been presented in many researches [45-47].

Fat values

Despite a wide range of advanced analytical methods used in research

around the world, classical methods are still often applied and a lot of

currently used standard methods are based on them. One of the important

indexes for the determination of vegetable oil quality is acid value (AV),

according to the AOCS Official Method Cd 3d-63 [48], which determines

the amount of free fatty acids contained in oil, and it is used to determine the

degree of lipid hydrolysis. The principle of the method consists in

neutralizing the acid with potassium hydroxide solution versus

phenolphthalein, as an endpoint indicator. Although the AV method has

some disadvantages, such as titration error, mainly in case of strongly

coloured samples, requirements for large solvent and sample amounts

[4, 48].

Peroxide value (PV) is an index of the primary oxidation status of oils

that inform about the concentration of hydroperoxides (primary oxidation

products), which are unstable and easily can decompose into secondary

oxidation products such as ketons and aldehydes. An improved method to

determine PV uses changes in the electrical conductivity values induced by

the reaction of potassium iodide with the hydroperoxides form the sample.

PV can be determined based on the international standard method AOAC

Official Method 965.33 [49].

Iodine value (IV) is a commonly used method to determine the average

degree of lipid unsaturation. The higher IV, the more double bonds in the

fraction there. In general, the analysis consists of the following steps:

weighting lipid, dissolving in suitable organic solvent, adding an iodine

chloride (ICl), which reacts with unsaturated bonds, but the others remain in

the solution. Then, the remaining ICl is assayed by adding excess potassium

iodine, after that the concentration of iodine is determined by titrating with

sodium thiosulfate, in the presence of starch as an indicator. Based on the

result the amount of idodine chloride that reacted with unsaturated bonds can

be calculated [1].


Previously mentioned methods such as AV, PV and IV, in spite of many disadvantages such as titration error, large amounts of solvents and sample used, play an important role in quality control and identification of lipids.

Oxidation stability of lipids

Lipid oxidation is one of the main reasons of quality deterioration of oils

and other fats and in the advanced stages it can lead to reduction of their

shelf life. Moreover, changes in oils caused by oxidative processes have a

significant influence on sensory and health characteristics of oils. Therefore,

the oxidation stability and the level of oxidation processes should be

determined.

Oxidative stability testing methods are based on quantity measurement of oxidation products or determination of antioxidant activity. Most oxidative stability testing methods focus on monitoring of the oxidation products. The change in the concentration of oxidation products can be analysed by many methods such as gas chromatography, liquid chromatograph, differential scanning calorimetry, the Rancimat test or the Schaal oven test. Some selected methods are described. The Schaal oven test is one of the oldest methods, where a peroxide value is determined daily in oil samples. Samples are kept in open glass and stored at an constant temperature, usually at 60°C. At the end induction time is calculated based on the plotted curve of PV against time [2]. The modification of the Shaal oven test is a reference method recommended by the AOCS and it is called the Oven Storage Test for Accelerated Aging of Oils. In this method oil oxidation level is analysed after storage period using more than one method, preferably one each for primary and secondary oxidation products [60]. Differential scanning calorimetry (DSC) is a method that is used to evaluate oxidation stability of oils. The same technique, but applying high pressure is called pressure differential scanning calorimetry (PDSC). In this method, the time (oxidation induction time-OTI), in which a rapid exothermic reaction takes place under certain conditions (temperature, pressure, oxygenation), is measured. In this technique high pressure decreases sample volatility by elevating its boiling point and it increases the concentration of the reacting gases. PDSC could potentially be used to study oxidative stability of plant lipids as the reaction of oil oxidation is exothermic, but literature reports on the measurement of the oxidative stability of oils by this technique are few. The technique has many advantages such as small sample required, short time and easy performance. In PDSC the experiment is carried out at lower temperatures and the heat of transition is more precisely defined [51-54].


The Rancimat test is a popular method for determining oil stability.

This technique was developed by Hadom and Zürcher, it consists in the

conductometric determination of volatile oxidation products. This method

has many advantages, such as easy procedure, relatively low cost and short

time of analyses [55]. The test is performed with the use of a Rancimat

apparatus (Figure 3) and it is an official method for determining oil stability

[56]. This test is based on automatic determination of time, known as the

oxidative stability index (OSI), before the maximum rate change of

oxidation, by measuring the increase in conductivity of deionised water,

caused by air that bubbled through a heated oil sample, that carries the

resulting volatile products into a separate chamber with deionised water

(Matthaus 1996). However, the technique has some limitations e.g. drastic

conditions (high temperature and a continuous supply of oxygen) [57].

Sensory analysis is a non-instrumental method of evaluating oil quality.

This method has a significant place in oil analysis but requires a trained

panel, it is time consuming and is rarely objective. Sensory analysis requires

a panel that is a group of trained human assessors. Procedures are strictly

specified, all tested products have to be specially prepared and need to be

monitored during the test. The method makes it possible to make inferences

and gain insight into tested products by applying statistical techniques. This

method is useful, because of irreplaceable sensitivity of the human senses.

Figure 3. A simple scheme of the Rancimat test.


Sensory analysis is a successfully used method, in spite of some

disadvantages such as high costs. Strict procedures and guidelines provide

repeatable and reproducible results. Sensory quality plays an important role

in plant lipid analysis, because it directly reflects the potential consumer

interest, therefore despite the difficulties it should be carried out as the last

step of testing of food products [58, 59].

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6. Analysis of plant lipids - BRIC

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