The fatty acid (FA) composition of photosynthetic tissue differs from that in other plant or animal tissues. In leaves, the lipid fraction constitutes less than 10% of the dry weight and is mostly located in the chloroplasts. An extraction solvent should dissolve polar lipids readily, but should also overcome interactions between the lipids and the tissue matrix. A mixture of chloroform/methanol (C/M) is commonly used. However, less toxic alternative methods such as hexane/isopropanol (H/I) and ethanol (E) have been suggested. In this preliminary study we compared the effectiveness of these three methods which are used as standard extraction protocols for FA analysis of plant material at three different European Universities. C/M extraction gave the highest total FA content and H/I the lowest, suggesting that C/M is indeed the best general-purpose lipid extraction solvent. Significant differences were also observed for FA composition including the ratio of saturated to unsaturated FA indicating selectivity of the various solvents in extracting different individual FA. Further and more detailed investigations are required to confirm this hypothesi

Arvidsson, K.

Elgersma, A.

Fievez, V.

Gustavsson, A.M.

Lourenço, M.

Witkowska, I.M.

English

Wageningen Yield 

190 
Comparison of methods for determining the fatty acid composition of 
photosynthetic tissues 
Izabela Magdalena Witkowska1, Marta Lourenço2, Veerle Fievez2, Katarina Arvidsson3, Anne-
Maj Gustavsson3, and Anjo Elgersma1  
1Wageningen University, Crop and Weed Ecology, Haarweg 333, Wageningen, 6709 RZ, The 
Netherlands 
2Ghent University, Laboratory for Animal Nutrition and Animal Product Quality, 
Proefhoevestraat 10, 9090 Melle, Belgium 
3Swedish University of Agricultural Science, Dept. of Agricultural Research for Northern 
Sweden, S-904 03 Umeå, Sweden 
To whom correspondence should be addressed (email  Anjo.Elgersma@wur.nl)  
 
Abstract 
The fatty acid (FA) composition of photosynthetic tissue differs from that in other plant or 
animal tissues. In leaves, the lipid fraction constitutes less than 10% of the dry weight and is 
mostly located in the chloroplasts. An extraction solvent should dissolve polar lipids readily, but 
should also overcome interactions between the lipids and the tissue matrix. A mixture of 
chloroform/methanol (C/M) is commonly used. However, less toxic alternative methods such as 
hexane/isopropanol (H/I) and ethanol (E) have been suggested. In this preliminary study we 
compared the effectiveness of these three methods which are used as standard extraction 
protocols for FA analysis of plant material at three different European Universities. C/M 
extraction gave the highest total FA content and H/I the lowest, suggesting that C/M is indeed 
the best general-purpose lipid extraction solvent. Significant differences were also observed for 
FA composition including the ratio of saturated to unsaturated FA indicating selectivity of the 
various solvents in extracting different individual FA. Further and more detailed investigations 
are required to confirm this hypothesis. 
 
Introduction 
Classically, determination of FA composition is done in three steps: first, lipids from the sample 
are extracted with a solvent; second, the isolated lipids are (trans)esterified to form fatty acid 
methyl esters (FAMEs); and in the last step, FAMEs are quantified by gas chromatography (GC). 
Various methods are used for these three steps; however, not all of them are suitable for plant 
material, as the lipid composition of photosynthetic tissue differs significantly from that of other 
sources. This lipid fraction constitutes less than 10% of the dry weight of leaves and mainly 
consists of complex polar and highly unsaturated membrane lipids. Consequently, a chosen 
solvent should not only be suitable to dissolve polar lipids readily, but should also overcome the 
interactions between the lipids and the tissue matrix.  
The extractability of lipids from a given tissue is variable and depends both on the nature 
of the tissue and of the lipids (Christie, 2003). For instance, simple lipids or triacylglycerols are 
extracted relatively easily, while galactolipids or phospholipids are more difficult to extract since 
they are constituents of membranes. Furthermore, lipids lacking polar groups are very soluble in 
hydrocarbons such as hexane, toluene and cyclohexane, or in moderately polar solvents, such as 
chloroform. Lipids with polar groups are only slightly soluble in these solvents. The polar lipids 
are extractable from plant tissue only in highly polar solvents such as ethanol or methanol, which 
are able to counterbalance the hydrogen bonds and ionic forces between these membrane-
associated lipids and proteins. A combination of moderately polar chloroform and polar 
methanol (2/1, v/v), C/M (Folch et al., 1957) has proven to extract both polar and non-polar 
lipids  from various kinds of tissues more fully than most other solvents (Christie, 2003). 
However, since both components are harmful to the analysts and environment other methods 
have been applied over the years to find a solvent system with low toxicity, e.g. Hara and Radin 
191 
(1976) and Elgersma et al. (2003). Consequently, various methods are used for the extraction of 
FA from herbage tissues; however, there is no assurance that results obtained with these different 
methods are comparable with each other. Therefore, in this preliminary study we compared two 
extraction methods: hexane/isopropanol, H/I (Nourooz-Zadeh and Appelquist, 1988) and 
ethanol, E (Elgersma et al., 2003) for their efficiency in the extraction of lipids from 
photosynthetic tissue against the “reference” method, C/M (Folch et al., 1957).  
 
Material and Methods 
Plant material was collected from an experimental field at Wageningen University, the 
Netherlands. Fresh herbage (± 200g) was sampled immediately, collected in plastic bags in dry 
ice (-80C) and moved to the lab. Three perennial ryegrass samples with a different leaf blade 
proportion were chosen. In addition, a grass silage sample was taken from an experimental farm 
of Wageningen University. Each sample was divided into four sub-samples, which were kept 
frozen. A paper cutter was used to cut the sub-samples into 5-10 mm pieces, that were 
representatively divided into three centrifuge tubes, as individual replicates for each method. 
Tubes were transported frozen (-80°C) and analyzed on the same day (11 days after sampling) in 
each lab with their standard method: chloroform/methanol (2/1, v/v) (C/M, Folch et al., 1957), 
hexane/isopropanol (H/I, Nourooz-Zadeh and Appelquist, 1988) and ethanol (E, Elgersma, et al., 
2003). The (trans)esterifications were done as described by: Raes et al. (2001), Sukhija and 
Palmquist, (1988), and Badings and de Jong (1983), for C/M, H/I and E, respectively. The GC 
conditions were similar for all methods, except differences in the length of the column, which 
was 100 m for C/M and H/I (CP-Sil-88 for FAME, Chrompack, The Netherlands), and 30 m for 
E (ZB-Wax, Zebron, Phenomenex, USA). 
  
Statistical analysis 
Data were processed using the SPSS statistical package (SPSS for Windows, Rel. 11.0.1, 2001. 
SPSS inc., Chicago, USA). Grass (two-way mixed model Anova) and silage (one-way Anova) 
samples were analyzed separately. Comparison of means for the individual treatments was done 
at the 5% significance level based on the F-test of the analysis of variance. 
 
Data summary 
The assessment of extraction solvent and methylation procedure on grass FA results was based 
on the seven major grass FAs (C14:0, C16:0; C16:1, C18:0; C18:1; C18:2 and C18:3), called 
BIG7. The profiles of these seven individual FAs were re-calculated relative to the total of BIG7 
(93% (C/M and H/I) to 95% (E) as proportion of total FAs). Concentrations of total BIG 7 and 
individual FAs differed significantly between methods. Data for concentrations of three major 
FA (C16:0, C18:2 and C18:3 are presented in Figure 1. For grass samples, the highest 
concentrations were reported with the C/M method and the lowest with H/I, which gave only 8% 
of C/M value for BIG7. 
192 
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
1 (C/M) 2 (C/M) 3 (C/M) 4 (C/M) 1 (H/I) 2 (H/I) 3 (H/I) 4 (H/I) 1 (E) 2 (E) 3 (E) 4 (E)
Sample number and Method
FA
 c
on
ce
nt
ra
tio
ns
 (g
 k
g-
1  
D
M
)
C16:0 C18:2 C18:3
 
Figure 1. Effect of three extraction methods (C/M, E, H/I) on estimates of the concentrations of three major FAs in 
four grass samples (1-4) 
 
Table 1. Effect of three extraction methods (C/M, E, H/I) on estimates of FA profiles and of their total FA 
concentration (BIG7) in four grass samples (1-4) 
  (%) (g kg-1 DM) 
Method Sample C14:0 C16:0 C16:1 C18:0 18:1 18:2 18:3 BIG7 
  FRESH GRASS SAMPLES (n=4) 
1 3.46 15.52 1.68 3.24 5.76 11.16 59.18 55.31 
2 4.17 13.23 2.09 2.00 3.24 11.92 63.35 48.12 
C/M 
3 3.56 11.58 2.59 1.36 1.93 10.54 68.43 55.11 
1 0.83 18.36 0.74 5.21 2.68 10.96 61.22 3.75 
2 0.77 17.38 0.50 5.30 2.74 10.91 62.39 3.90 
H/I 
3 0.56 14.67 0.16 4.53 1.97 8.55 69.56 4.38 
1 0.26 12.85 0.20 0.96 1.85 12.69 71.20 32.53 
2 0.26 13.12 0.29 1.10 2.44 13.04 69.76 29.78 
E 
3 0.19 11.99 0.10 0.89 1.63 11.18 74.10 35.24 
         
Method (M) Sign *** ** ** *** NS ** ** *** 
Sample (S) Sign NS NS NS NS NS ** * NS 
 s.e.d. 0.145 0.698 0.092 0.355 0.547 0.403 1.508 1.098 
M * S Sign NS NS *** NS NS NS NS NS 
  SILAGE SAMPLES (n=4) 
C/M 4 9.97 16.39 5.78 1.52 2.80 16.53 47.02 23.32 
H/I 4 1.20 21.27 0.70 2.02 3.06 19.49 52.27 10.95 
E 4 0.81 15.19 0.24 1.36 2.55 21.56 58.21 8.08 
          
Method (M) Sign *** *** *** *** *** *** *** *** 
 s.e.d. 0.398 0.210 0.193 0.129 0.335 0.266 0.729 0.387 
Asterisks indicate significant effects: *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant.  
s.e.d. standard error of difference. 
Differences in estimates of the proportions of individual FAs were also significant 
between methods (Tab. 1). It was distinctive that with H/I the highest profiles of C16:0 and 
C18:0 were reported, with C/M the highest profiles of C14:0 and C16:1, and with E the highest 
profiles of C18:3. Moreover, the ratio between saturated and unsaturated FAs was significantly 
different for the investigated methods, with H/I the estimated ratio was highest (0.33, 0.31, 0.25 
and 0.32 for sample 1, 2, 3 and 4, respectively) and with E lowest (0.16, 0.17, 0.15 and 0.21 for 
sample 1, 2, 3 and 4, respectively).  
 
193 
Discussion  
In this preliminary research, no distinctive evaluation has been made of the extraction and 
esterification procedure and GC conditions. Consequently, differences in estimated values 
between methods could not be related to extraction or (trans)esterification separately, only by the 
overall procedure. This discussion is limited to the first two steps of the procedure, extraction 
and (trans)esterification, since the methods and equipment used for GC analysis of FAMEs were 
similar for the three methods.  
Polar ethanol was presented as a successful solvent for lipids from the liver (Lucas and 
Ridout, 1970), cyanobacterium (Mendes et al., 2005 and 2006) and fresh grass and silage 
(Elgersma et al., 2003). In the present comparison, in agreement with Mendes et al. (2005 and 
2006), E yielded 60 to 70% lower concentrations of total and individual FAs than C/M. Although 
H/I was presented previously to be a good solvent for the lipids from the brain tissue (Hara and 
Radin, 1976) and milk powder products (Nourooz-Zadeh and Appelquist, 1988), it seems to be 
unsuitable for fresh plant tissue with complex and polar membrane-associated lipids as only 8% 
of the lipids were estimated compared to C/M. This conclusion is consistent with the fact that 
hexane is a good solvent for lipids that do not contain markedly polar groups, for example 
triglycerides or cholesterol esters (Christie, 2003) and that isopropanol is used mainly to 
deactivate enzymes (Hawke, 1973). Overall, the mixture of chloroform/methanol once more 
proved to be a more general solvent, as it extracted lipids more readily than any other 
(combinations of) solvents. Furthermore, considering the significant differences in the FAs 
profiles and ratio of saturated to unsaturated FAs between these three methods, it is apparent that 
the used solvents showed selectivity in extracting different lipid classes. E appeared to be a good 
solvent for membrane-associated lipids, especially galactolipids containing high proportion of 
C18:3, whereas H/I does not have these properties and consequently extracted fewer lipids from 
the photosynthetic tissue.  
Moreover, the (trans)esterification step seems to be especially crucial for explaining the 
differences in the concentration of total and individual FAs for the silage sample. Elgersma et al. 
(2003) found that the level of free fatty acids (FFA) in silage samples varied from 27 to 73% of 
total FAs. Acid-based methylations, used with the C/M and H/I methods, esterify FFAs or 
transesterify FAs linked by ester bonds (EFA) to glycerol or cholesterol (Christie, 2003). In 
contrast, alkaline-based methylation, used after E extraction, only transesterifies EFAs but does 
not esterify FFAs. So the total concentrations of FAs in silage samples analyzed with C/M and 
H/I were equal to FAMEs of FFAs plus EFAs, whereas in samples analyzed with E results 
represented only FAMEs of EFAs. Consequently, the differences in the total concentration of 
FAs especially between C/M and E might be explained not only by the extraction step, but also 
by the (trans)esterification step. On the other hand, since the proportion of FFAs in fresh samples 
is usually low, from 1 to 2% (Elgersma, et al. 2003; personal communication M. Lourenço), the 
differences in the total concentration of FA in fresh grass samples cannot be explained by the 
methylation step, and thus are mainly a result of the extraction. 
 
Conclusions 
Three methods showed significant differences in concentration and proportion of FAs in fresh 
grass and silage. These were mainly related to the selectivity of the used solvents in extracting 
different lipid classes. The mixture of chloroform/methanol (2/1, v/v) proved to be the most 
general and efficient solvent in extracting lipids. On the other hand, hexane/isopropanol appeared 
to be unsuitable for the extraction of membrane-bound polar lipids.  
 
Acknowledgements 
We thank Mr. F. Bakker for help with the field work, Ms A.C. Wever for analytical help and Mr. 
194 
A. Otten for statistical advice. I.M. Witkowska and A.C. Wever thank the Grassland Science 
Foundation SGW for financial support. 
 
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Comparison of the fatty acid composition of fresh and ensiled perennial ryegrass (Lolium perenne L.), affected by cultivar and regrowth interval, Anim Feed Sci Technol 108: 191-205 Folch

Fatty livers and lipotropic phenomena. Prog Chem Fats and Other Lipids 10: 1-150

Glass capillary gas chromatography of fatty acid methyl esters. A study of conditions for the quantitative analysis of short- and long-chain fatty acids in lipids,

Supercritical CO2 extraction of -linolenic acid and other lipids from the cyanobacterium Arthospira (Sprirulina)maxima: Comparison with organic solvent extraction.

Comparison of methods for determining the fatty acid composition of photosynthetic tissues

Comparison of methods for determining the fatty acid composition of photosynthetic tissues

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