L

Liquid Extraction: Bligh and Dyer an efficient and mild procedure to study lipid
decomposition in frozen fish (Bligh and Dyer
Annika S€
undermann, Lars F. Eggers and Dominik 1959). The basic principle for the extraction is
Schwudke based on Folch’s method (Folch-Pi et al. 1957)
Research Center Borstel, Borstel, Germany using less amounts of chloroform and methanol
for the primary extraction step. A biological sam-
ple (e.g., tissue, blood, plasma) is homogenized in
Abbreviations a monophasic chloroform/methanol/water mix-
ture. Afterward a phase separation is induced by
ESI-MS/MS Electrospray ionization tandem addition of chloroform and water. The same phy-
mass spectrometry sicochemical principles for the distribution of
GC Gas chromatography chemical components between two nonmiscible
GC-MS Gas chromatography mass solvents apply concerning Nernst’s partition law
spectrometry and diffusion as discussed in Folch’s method
HCl Hydrochloric acid (Liquid Extraction: Folch). Although the method
HPLC High-performance liquid has been introduced for lipid extraction of fish
chromatography muscle, it found many applications in biological
LC-MS Liquid chromatography mass and biomedical research. In 1999, Smedes and
spectrometry Askland revisited the composition of the ternary
solvent mixture chloroform/methanol/water of the
Definition original Bligh and Dyer recipe using GC (solvent
compositions) and HPLC (lipid patterns). They
The lipid extraction method by Bligh and Dyer is reported that the obtained lipid yield is strongly
one of the standard procedures to isolate total lipid associated to the methanol content and could be
fractions from biological matrices based on a sol- improved with a higher proportion of methanol
vent system consisting of chloroform/methanol/ (Smedes and Askland 1999).
water 2:2:1.8 (v/v/v). Iverson et al. showed limitations of the original
Bligh and Dyer recipe for animal tissues of high
lipid content, especially neutral lipids (Iverson
Background et al. 2001). The extraction yields for samples
with lipid contents lower than 2 % were compa-
The extraction method by E.G. Bligh and rable to Folch, whereas for high-fat tissues recov-
W.J. Dyer was introduced in 1959 in search of ery was significantly less. This can be attributed to
# Springer Science+Business Media Dordrecht 2016
M. Wenk (ed.), Encyclopedia of Lipidomics,
DOI 10.1007/978-94-007-7864-1_88-1
2 Liquid Extraction: Bligh and Dyer
a lower solubility of neutral lipids in the Bligh and
Dyer solvent system indicating that for different
sample types, solvent/sample ratios and/or num-
ber of re-extractions steps should be optimized.
Such customization of the original protocol can be
key to the success of a study and should be well
documented. This is often not set into practice,
which makes it difficult to reproduce lipidomics
experiments.
Procedure
Workflow according to the original protocol
(Bligh and Dyer 1959): Homogenized tissue sam-
ple is mixed with 1 volume of chloroform and
2 volumes of methanol. After thoroughly
vortexing, 1 volume of chloroform is added to
the homogenate followed by another mixing
Liquid Extraction: Bligh and Dyer, Fig. 1 Workflow for performing lipid extraction according to Bligh and Dyer
step. Afterwards, 1 volume of distilled water is
added and subsequently the suspension is stirred
for an additional incubation period. The resulting
suspension consists of non-extractable residues in
a chloroform/methanol/water mixture with volu-
metric ratios 2:2:1.8 (v/v/v). This suspension is
subsequently filtered through a medium flow filter
paper. After a short incubation period, the filtrate
is completely phase separated and the upper aque-
ous layer can be removed. Bligh and Dyer stated
that an additional extraction step using chloro-
form should be performed to give better yields
for nonpolar lipids like triacylglycerols. Nowa-
days, the filtering step is often omitted in
lipidomics applications and phase separation is
supported by centrifugation for a few minutes
(Gardner et al. 1985; Booij and van den Berg
1994) (Fig. 1).
Liquid Extraction: Bligh and Dyer 3

a 1500

1250 *
citations per year

1000

750

500

250

0
1985
1986
1987
1988
1989
1990
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
2014
2015
* value for 2015 extracted November 30, 2015

OTHER MICROBIOLOGY AGRICULTURE

b MARINE FRESHWATER BIOLOGY BIOPHYSICS

CELL BIOLOGY CHEMISTRY FOOD SCIENCE TECHNOLOGY

BIOCHEMISTRY MOLECULAR BIOLOGY NUTRITION DIETETICS

100

80
% of citiations

60

40

20

0
1985
1986
1987
1988
1989
1990
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
2014
2015

Liquid Extraction: Bligh and Dyer, Fig. 2 (a) Number (b) Distribution of citations per research area according to
of citations per year for Bligh and Dyer extraction (Data categorization of Web of Science and year in percent (Data
was extracted from Web of Science, Thomson Reuters). was extracted November 30, 2015)
4 Liquid Extraction: Bligh and Dyer
Applications
With 32,131 citations the original method has
become one of the most influential articles in
scientific literature of all time (Van Noorden
et al. 2014) reflecting its outstanding importance
for studying lipid biochemistry (Fig. 2). It is
widely used as extraction method for studying
total lipid composition using GC (Smedes and
Askland 1999; Jensen 2008), GC-MS (Long
et al. 2015) and LC-MS (Retra et al. 2008; Reis
et al. 2013; Pellegrino et al. 2014). The majority of
studies are performed in Biochemistry and Molec-
ular Biology or Biophysics. Increased interest in
the Bligh and Dyer protocol from the mid-2000s
onward can be attributed to biomedical applica-
tions utilizing high-throughput lipidomics plat-
forms based on ESI-MS/MS (Leidl et al. 2008;
Liebisch et al. 2004).
Many modifications to the original protocol
have been made since the introduction of the
protocol in 1959 by customizing the volumetric
ratio of sample to extraction solvent and/or com-
position of the chloroform/methanol/water sys-
tem. An appropriate way to enhance recovery of
acidic phospholipids and/or fatty acids can be
achieved by acidification of the extraction solvent
mixture using HCl (Retra et al. 2008) (Jensen
2008) and acetic acid for oxidized fatty acids
(Liu et al. 2013).
References
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tion and purification. Can J Biochem Physiol.
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Booij K, van den Berg C. Comparison of techniques for the
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1994;53(1):71–6.
Folch-Pi J, Lees M, Stanley GHS. A simple method for the
isolation and purification of total lipides from animal
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Gardner WS, Frez WA, Cichocki EA, Parrish
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