revisiting the development of the bligh

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Revisiting the Development of the Bligh

and Dyer Total Lipid DeterminationMethodFOPPE SMEDES* and TORSTEN K. ASKLAND

Ministry of Transport, Public Works and Water Management, National Institute for Coastal and Marine Management/

RIKZ, P.O. Box 207, 9750 AE Haren, The Netherlands

The experiments leading to the development of the most

well-known method for total lipid determination in marinebiological tissues (Bligh, E. G. and Dyer, W. J. (1959)

Can. J. Biochem. Physiol. 37, 911±917) were repeated in

order to discover the secrets of its success. Along with

measuring the phase volumes of the water/methanol/

chloroform mixtures investigated by Bligh and Dyer, the

phase compositions were determined by gas chromato-

graph (GC). An examination showed that, although Bligh

and Dyer applied largely di�erent solvent ratios, the

composition of both phases varied only within a limited

range resulting in an incomplete investigation of the ef-

fects of changing this factor. Using Bligh and DyerÕs

solvent mixtures the recovered fraction of organic phase

was found to be the key factor determining the extracted

lipid yield. On its turn the recovered fraction was posi-

tively correlated to the size of the organic phase and its

methanol content. Additional experiments applying new

extraction points with higher methanol contents revealed

an increase of extracted lipid. This increasing yield was

mainly due to a better extraction of the phospholipids as

could be deducted from lipid patterns recorded by normal

phase high performance liquid chromatography (HPLC)

using an evaporative mass detector. Ó 1999 Elsevier

Science Ltd. All rights reserved.

For studies of pollution levels in the marine environ-ment the lipid content of biological material is a crucial

parameter to interpret data on organic contaminants

(Schneider, 1982; Delbeke et al ., 1995). Lipid is a natural

mixture of triglycerides, diglycerides, monoglycerides,

cholesterols, representing the more apolar or neutral

lipids and free fatty acids, and phospholipids, sphingo-

lipids, etc. representing the polar lipids (Lovern, 1957).

Solvents to extract lipids must demonstrate a high sol-

ubility for all lipid compounds and must be su�ciently

polar to remove the lipids from their association with

cell membranes and lipoproteins. Chloroform/methanol

mixtures apply well as was recognised by Folch et al .(1957). This approach was adapted by Bligh and Dyer

(1959) resulting in a method which has become the

standard method for total lipid determination for over

30 years.

Chemists claiming they use the Bligh and Dyer

method for lipid extraction usually apply a modi®cation

of this method (de Boer, 1988; Booij and van der Berg,

1994; Gardner et al ., 1985). The evaluation of an in-

tercalibration exercise revealed that, to some extend,

di�erences in the outcome could be related to deviation

from the original method (Roose and Smedes, 1996).

When laboratories agree to apply exactly the same

procedure results became highly comparable (Randall

et al ., 1991).

In a previous paper (Smedes and Thomasen, 1996) the

original work of Bligh and Dyer was evaluated from a

theoretical viewpoint. The variable lipid contents found

by Bligh and Dyer applying eleven di�erent solvent

mixtures could not be explained by di�erences in the

ability to dissolve lipids nor adsorption to the tissue

residue. Further reasoning revealed that the measured

lipid content was mainly determined by the fraction of

organic phase that could be recovered. The methanol

contents in the organic phase varied only little and the

optimum methanol content cannot be derived from theBligh and Dyer experiments. However an indication was

found that methanol had a positive e�ect on the ex-

traction yield, either because less organic phase was

sticking to the tissue or better extraction kinetics, due to

higher solubility in the mono-phasic situation and in the

aqueous phase. The order solvents were added also

seemed important for the kinetics of the extraction. First

the association with cell constituents is destroyed where

after lipids are dissolved in a mono-phasic system. Then

transfer to an organic phase is performed in a bi-phasic

system created by the addition of more chloroform and

water. Although a positive e�ect on extraction kinetics isplausible no experiments are known comparing one-step

and two-step extractionÕs using the same solvent ratios.

Marine Pollution Bulletin Vol. 38, No. 3, pp. 193±201, 1999

Ó 1999 Elsevier Science Ltd. All rights reserved

Printed in Great Britain

0025-326X/99 $ ± see front matterPII: S0025-326X(98)00170-2

*Author to whom correspondence should be addressed. Present address: LEO Pharmaceutical Products, Industriparken 55,DK-2750, Ballerup, Denmark.

193



The present paper reports about a complete repetition

of the experiments as performed by Bligh and Dyer. In

addition one-step extractionÕs are compared with two-

step extraction and the in¯uence of solvent compositions

on the extraction yield is further investigated. The pat-

terns of extracted lipids were investigated by High Per-

formance Liquid Chromatography (HPLC) with an

evaporative mass detector (EMD).

Materials and Methods

Chemicals

Chloroform (Merck, Darmstadt, Germany) and

methanol (Baker Chemicals, Deventer, The Nether-

lands) were both of Pro Analyse quality. Water was

delivered by a Milli-Q system (Millipore). The lipid

standards (Sigma, St. Louis, MO, USA) used were:

Cholesteryl palmitoleate (CHOLE), acyl-sn-glycero-3-

phosphocholine (LPC), Trioliene (TG), Oleic acid(FFA), Cholesterol (CHOL), 1,2-diacyl-sn-glycero-3-

phosphoethanolamine (PE), 1,2-diacyl-sn-glycero-3-

phosphocholine (PC), Sphingomyelin (SM) and 1,2-

diacyl-sn-glycero-3-phosphoserine (PS) ± all of high

purity (>99%). Other solvents used were hexane and

tetrahydrofuran (Baker Chemicals, Deventer, The

Netherlands), both HPLC quality.

Sample Preparation and Extraction

About 1 kg cod iced ®llet from fresh ®sh was pur-

chased from a local supplier and quickly homogenised in

a 1 liter glass jar placed on ice using an Utra Turrax,

T45 (Janke & Kunkel kg, Staufen, Germany). From this

homogenate portions of 5 and 10 g were weighed in 50

glass jars of 100 ml with a polypropylene lid. During

homogenisation the temperature did not exceed 10°C.

Samples were stored in a freezer atÀ20°C until extrac-

tion. For extraction an Ultra Turrax mixer T25 (IKA

Labortechnik) with a 18 mm shaft was used and phase

separation was achieved by centrifugation for 10 min at

2000 rpm at 20°C in a thermostated centrifuge (Sigma

3K12, Germany).

All extractions were performed in the same way as

Bligh and Dyer in 1959, except that sample and solventamounts were reduced by about a factor 10. Solvent

additions were done on weight basis, while also total

weights were recorded throughout the whole procedure

to detect possible evaporation or spilling. During ex-

traction the weight decreased approximately 0.6 g due to

evaporation and in two cases some mixture was lost by

splashing. The loss represented 1±2% for the higher

volumes and up to 3% for the smaller volumes. Up-

scaled, but otherwise identical, blank extraction mix-

tures were also put together and weight and volumes of

the formed phases were determined as well as the solvent

composition.The extraction procedure is brie¯y as follows: meth-

anol and chloroform were added to the sample followed

by 3 min mixing. This mixing was repeated for 30 s both

after adding the second portion of chloroform and after

addition of water. After centrifugation the organic

phase was isolated using a Pasteur pipette and the

weight was recorded. This extract was split in two parts

on weight basis. One part was transferred to an alu-

minium cup, evaporated to dryness and the weight of

the residue was determined after 30 min at 105°C. The

other part was ®ltered by means of 13 mm 0.5 lm PTFE

®lter (Millex, LCR13, Millipore), concentrated to obtain

a lipid concentration of 50 mg/ml and used to record a

lipid pattern by HPLC-EMD.

Applying 4 min of mixing the Bligh and Dyer ex-

tractions A±E were repeated without a stepwise addition

of solvents. This procedure was also applied to an extra

set of extractions, here called K±N wherein the metha-

nol amount was varied complementary with the water

keeping the total volume constant.

Gas chromatographic analysis

To analyse the solvent compositions a Varian 90P gas

chromatograph (GC) was used equipped with a thermal

conductivity detector. Helium was applied as a carrier

gas at a ¯ow of 140 ml/min and the compounds were

injected at 250°C on a Porapak-Q column (stainless

steel, length´diameter was 2000´2.1 mm) at an oven-

temperature of 140°C. Chromatographic data were

processed by computer utilising Turbochrom (Perkin

Elmer) software. Calibration was performed by injecting

standard solutions and quanti®cation was done on peak

area.

HPLC analysis

Lipids were separated with a normal phase column

connected to a Hewlett Packard 1050 high performance

liquid chromatographic system consisting of a solvent

degasser, a quaternary pump (¯ow rate 0.4 ml/min) and

an automatic injector. The detector was an evaporative

mass detector (PL-EMD 940, Polymer laboratories,

UK) used at a temperature of 55°C and an air¯ow of 10

l/min. In the detector the eluent is sprayed with air in a

heated tube where it evaporates. Non evaporating

compounds in the eluent remain in the air stream as

small droplets that are detected by light scattering.Standards and extracts were injected on a Chrom-

sphere CN column (150´3 mm, 5 lm, Chrompack , The

Netherlands) either in chloroform or toluene. The gra-

dient used started with 2% tetrahydrofuran in hexane

and after 0.5 min this was linearly programmed to be

86% at 6.5 min. In the next minute the composition is

programmed to 50/50 tetrahydrofuran/methanol and

subsequently to 100% methanol from 7.5 to 8.5 min.

Elution with methanol is maintained till 15 min and then

the gradient is programmed back to 100% tetrahydro-

furan and ®nally back to the initial composition. To

obtain equal column activity and subsequently constantretention times a ®xed equilibrium time (30 min) was

required before each injection. An automatic injector is

194

Marine Pollution Bulletin



prerequisite to accomplish this. The ®rst injection

should be ignored since it will always have a deviating

equilibration history. Chromatographic data were pro-

cessed as for GC analysis and quanti®ed by peak area.

Results and DiscussionIn Table 1, part I, the solvent weights used (in ac-

cordance with the experiments of Bligh and Dyer) are

listed. Solvents were added on weight basis and calcu-

lated back to the equivalent for 10 g intake although in

some cases only 5 g sample was processed. Honeycut

et al. (1995) evidenced that scaling down to 5 g did not

in¯uence the extractable weight. The added solvent

weights for the extra experiments are given in part II of

Table 1. For all extractionÕs the recovered organic

phase, the lipid content determined from that organic

phase and the organic phase volumes measured from

blank mixtures are listed.The phase compositions determined from the blank

mixtures are given in Table 2 and were in agreement

with earlier work (Smedes and Thomasen, 1996). A

check of a few mixtures in the presence of sample

showed that compositions were not in¯uenced.

The obtained data are evaluated below in relation to

the di�erent variables.

Multiple-step extractions

In the extractions A through E solvent compositions

are extremely close while the organic phase volume is

increasing over a factor 10. At the same time the aque-

ous phase varies only a factor 2 what allows one to studythe in¯uence of the organic phase volume on the lipid

yield from the mass balance according to Smedes and

Thomasen (1996) is given by the mass balance T:

m � g Org w Org � g Aq w Aq � g Tis s TisY �1�

where m is the total amount of lipid, C Org, C Aq and C Tisare the lipid contents respectively in the organic-, water-

and tissue phase, and M Org, M Aq and I Tis are theamounts of the subsequent phases. To relate the lipid

content in the organic phase, C Org, to the amount of

organic phase, M Org, eqn (1) is rewritten as follows:

1

g Org�s Tis

mÁ w Org

s Tis�g Aq w Aq

g Org�g Tis s Tis

g OrgX �2�

In this equation the last two terms are nearly constant.

The amount of aqueous phase varies only little in the

second last term and the ratio between the concentration

in aqueous and organic phase equals the partition co-

e�cient, which is a constant provided the solvent com-

position is ®xed. For the last term a similar reasoning isvalid. In the formula I Tis/m equals the reciprocate value

of the lipid content in the tissue (C L). Regression ana-

lyses with l/C Org as y-variable and the amount of organic

phase per gram tissue, M Org/I Tis, as x-variable will return

the reciprocate value of C L as the slope.

When an intercept is detected the extraction is not

complete and aqueous phase or tissue still contains lipid.

In Fig. 1 the above mentioned variables are plotted and

regression analyses of the repeated Bligh and Dyer ex-

periments revealed a non-signi®cant (P>0.3) negative

intercept. The regression line drawn in Fig. 1 is therefore

forced through zero. The absence of an intercept proves

that, neither the solubility of lipids in the aqueous phase

nor sorption onto the tissue is of any signi®cance. Be-

TABLE 1

Weights of solvents applied for multiple step lipid extractions as performed by Bligh and Dyer (part I) and additional experiments (part II). Fur-thermore the recovered organic phase and the lipid content measured in it are given. For each extraction the weight of the organic phase measured

from a blank mixture is given.

I Bligh and Dyer extraction mixtures A B C D E F Ga H I J P

Weights of solvents in ®rst step (g) Methanol 11,8 16,4 18,6 21,9 28,9 9,8 14,2 7,1 6,5 6,2 16,4Chloroform 3,5 8,2 14,3 24,5 44,8 14,7 7,6 8,2 14,5 26,5 15,2

Waterb 8,0 8,0 8,0 8,0 8,0 8,0 16,4 8,0 8,0 8,0 8,0Weight in second step Chloroform 9,6 23,9 39,4 67,6 88,7 30,4

Weight in third step Water 17,0 24,1 27,0 31,5 41,5 18,4Recovered organic phase (g) 2,2 14,3 30,8 56,2 71,4 11,0 1,3 3,4 9,8 21,7 23,5Lipid content in organic phase (mg/g) 8,52 3,27 1,93 1,07 0,80 5,55 17,2 10,3 5,11 2,55 2,41Organic phase in blank mixture (g) 8,3 22,6 38,4 67,2 88,5 13,8 7,7 7,4 14,2 26,7 29,6Lipid content in tissue (mg/g) 7,03 7,40 7,42 7,18 7,05 7,64 13,2 7,57 7,26 6,81 7,14

II Additional single step extractions A B C D E Kc L M P N O

Weights of solvents Methanol 11,8 16,4 18,6 22,0 29,0 0,0 8,2 12,2 16,4 17,9 20,3Chloroform 9,8 24,0 39,3 67,5 88,9 30,4 30,6 30,3 30,4 30,2 30,1

Water 17,1 23,9 27,0 31,5 41,6 39,0 29,0 23,6 18,5 16,3 13,2Recovered organic phase (g) 1,8 14,4 29,5 57,7 76,2 17,3 20,7 25,3 24,6 22,3Lipid content in organic phase (mg/g) 7,57 2,65 1,74 0,95 0,75 1,42 2,00 2,35 2,56 2,51Organic phase in blank mixture (g) 8,3 22,6 38,3 67,2 88,7 30,4 30,5 30,4 29,6 31,5 33,2Lipig content in tissue (mg/g) 6,29 5,97 6,65 6,37 6,63 4,32 6,09 6,96 8,06 8,36

a Strong emulsion, organic phase di�cult to recover.b First 8 g of water origins from the sample.c No recovery of organic phase possible.

195

Volume 38/Number 3/March 1999



cause of this outcome, the fact that the aqueous phase

volume was not constant becomes irrelevant too. The

extraction of lipids seems a simple solubility process and

for all extractions, A±E, the lipids were equally su�cient

extracted to the organic phase. In Fig. 1 the slope of the

regression line implies a lipid content of 7.14 (�0.06)

mg/g wet weight [see eqn (2)]. The increasing lipid yields

from A through E found by Bligh and Dyer are not

related to di�erent solvent compositions but entirely due

to increasing recovery of organic phase. A larger organic

phase results in a more complete recovery.

Assuming that the lipid yield for the experiments A±E

found by Bligh and Dyer re¯ects the recovered organic

phase, it can be calculated that the yield of organic

phase is 8±18% lower for the experiments described in

this report. This can easily be explained by the di�erence

in procedure. Bligh and Dyer ®ltered the tissue and

squeezed it for maximal yield. Apparently squeezing is

more e�ective to obtain maximal yield than centrifuga-

tion. This paper was focusing on the determination of

C org and because solvent evaporation cannot be con-

trolled during ®ltration, centrifugation in closed jars was

more appropriate to obtain phase separation.

Single step extractions

Concluding that the lipid yield is predominantly de-

termined by the recovery of the organic phase, the im-

portance of using a multiple step approach for the

extraction becomes questionable. The lower lipid con-

tents found by Bligh and Dyer for F±J cannot unam-

biguously be attributed to the fact that extraction was

performed in a single step. Therefore these single step

extractions were repeated and, in addition, also single

step extractions were performed with the mixtures A±E.

Lipid contents calculated from C Org and the organic

phase volume from blank mixtures were summarised in

Fig. 2. The open squares represent the lipid contents for

a single extraction and the solid squares the multiple

step extractions. A horizontal dotted line represents the

lipid content calculated above by linear regression

(Fig. 1). The vertical bars indicate the water respectivelythe methanol contents in the organic phase. The ex-

tractions A±E clearly show a higher lipid yield for the

multiple step approach, which means that a stepwise

addition of solvents promotes the extraction kinetics. F,

G, H and I, however, demonstrate that a single step

extraction can result in a high yield too. The G extrac-

tion is an artefact here. Due to a di�cult phase sepa-

ration, hardly any organic phase could be recovered

(<1 ml) what made weighing very inaccurate. For the P

extraction a single step extraction performs nearly equal

to multiple step.

Based on observed precipitation Bligh and Dyerconcluded that co-extraction of non-lipids took place for

the F extraction. Although precipitation occurred in-

TABLE 2

Actual solvent compositions (in % w/w) of both phases in blank extractions.

Aqueous phase Organic phase

Water Methanol Chloroform Water Methanol Chloroform

A 58.2 38.6 3.2 0.4 3.8 95.5B 58.9 37.7 3.4 0.4 3.5 96.0C 59.4 37.8 2.8 0.4 3.3 97.1D 59.4 37.3 3.3 0.3 3.1 96.9E 59.4 37.3 3.3 0.3 3.1 96.9F 45.4 45.2 9.4 2.4 12.1 85.6G 52.2 43.1 4.8 1.0 7.1 93.2H 52.4 42.4 5.2 0.9 6.3 93.1I 56.0 39.7 4.3 0.5 4.5 95.5J 58.6 37.6 3.8 0.3 3.4 96.1P 53.3 41.6 5.0 0.8 6.0 93.0K 99.7 0.0 0.0 0.0 0.0 99.0L 77.3 21.4 1.3 0.1 0.7 98.6M 65.8 32.0 2.2 0.2 2.0 97.6N 48.5 43.8 7.8 1.5 9.6 88.8O 38.5 45.7 15.8 4.7 17.9 77.1

Fig. 1 Relation between the reciprocal lipid content in the organicphase ( y-axis) and weight of organic phase applied per gram

sample. No signi®cant intercept was found and therefore theline is forced through zero. The slope equals the reciprocallipid content i.e. 7.14 (�0.06) mg/g.

196




deed, not co-extraction but azeotropic distillation was

responsible for this e�ect. Because chloroform forms an

azeotrope (Weast, 1979) with methanol of 13% (v/v),evaporation of the organic phase of F (20% methanol)

ends in water/methanol what causes lipids to precipitate.

Repeating the evaporation after addition of extra chlo-

roform results in a clear solution. Since for F, H and I

the lipid contents are equal or higher than P the multi-

step approach does not seem to be obligatory for a

quantitative extraction in all cases.

Methanol content

As mentioned before the methanol contents did not

really vary in the Bligh and Dyer experiments. However

the results in Fig. 2 do indicate a positive in¯uence of themethanol on the extraction yield although phase vol-

umes varied at the same time. Therefore the extractions

K±O were performed which, together with P, cover the

entire possible range of 0±20% (w/w) methanol in

chloroform. Addition of more methanol results in a

mono-phasic system. In the extractions the amount of

chloroform used was kept constant. Without addition of

methanol (situation K) a very strong emulsion was

formed, which could not be broken even by prolonged

centrifugation. As a result no organic phase could be

recovered.

In Fig. 3 the measured lipid yields (solid squares) are

plotted against the methanol content and show a linear

relation with the log methanol content. It appears that

composition P, selected by Bligh and Dyer, does not give

the optimum yield, although the possible increase in

yield at higher methanol contents is not very large.Considering the importance of the methanol content

all the other obtained lipid yields are also shown in

Fig. 3. All single-step extractions, indicated by a square-

framed letter, are grouped around this line and seem to

follow the same relation as L±O. The multiple-step ex-

tractions, indicated with a circled letter, show a higher

yield than expected from their methanol content. Ap-

parently the much higher methanol content they were

subjected to in the ®rst step, i.e. around 50% which is

higher than O, dissolves lipids which are not (com-

pletely) re-adsorbed when in the following steps the

methanol content is decreased again by addition of more

chloroform and water. This elevated extraction yield athigher methanol contents explains the better extraction

capability of multiple-step procedures and con®rms the

view of Bligh and Dyer on this.

Lipid patterns with HPLC

Besides determining the weights of the extracted lipid

material, all extracts were analysed with HPLC to de-

termine the lipid composition. For the separation of

lipids, Christie (1985) used normal silica and a gradient

from isooctane/isopropanol, via isopropanol/chloro-

form, ending in isopropanol/water. The necessity to

apply water to elute the phospholipids is a severe dis-

advantage as it needs extensive washing to activate the

column for the next analysis. A CN bounded column

Fig. 2 Lipid contents (left y-axis) in cod ¯esh using di�erent solvent mixtures as indicated on the basis.The ®lled squares (n) represent the results using multiple extraction. Values obtained by single stepextraction are shown by an open square (h). A dotted line is drawn at the lipid content found byregression (Fig. 1). The methanol and water contents in the organic phase are indicated by re-spectively open and closed bars (right y-axis).

197




allows elution of phospholipids with only methanol.

Using hexane, tetrahydrofuran and methanol the lipids

could be separated in the di�erent groups. It should be

noted that also with this type of gradient the separation

is strongly in¯uenced by the activity of the column and

the equilibration period before each injection should be

remained constant. The use of an automated injection

and gradient system is therefore inevitable.

In Fig. 4 a typical chromatogram is presented. The

upper one is a 10 times expansion of the lower one in

order to show the details. The CHOLE and LPC elute at

the same time and are only partly separated from thelargest TG peak. Two medium large peaks were attrib-

uted to FFAÕs. Although most lipid peaks are not pure

compounds (di�erent alkyl groups) this is not true for

the cholesterol, which is a pure compound. Diglycerides

show two peaks; the 1,2 acylated and the 1,3 acylated

glycerol. The same distinction occurs for monoglyce-

rides; acylation at the 1 or at the 2 position of the

glycerol. All phospholipids elute at the end of the

chromatogram and are separated in a peak representing

PE, another representing the PC and SM, which elute

together, and a small one representing the PS. From the

chromatogram one can see that the phospholipids rep-resent the major fraction in cod ¯esh followed by cho-

lesterol, the triglycerides and free fatty acids. In Table 3

the measured concentration of the di�erent lipid groups

are given for all extracts.

The contents of minor constituents are not very ac-

curate. Although the chromatogram shows nice peaks

without any baseline noise one should consider that the

signal is the result of light scattering of small lipid

droplets. Response diminishes rapidly when droplets get

small and their diameter range approaches the wave-

length. Secondly, not only the solvent evaporates in the

detector but, depending on the properties, also the

compounds of interest can evaporate. This evaporation

is independent of the amount present and is only in¯u-enced by the detector temperature and air ¯ow. The

result of both e�ects is that the amount of compound

has to exceed a certain threshold and the obtained signal

is only a measure of the amount above it. Furthermore

the signal of an EMD is not linear with the concentra-

tion. These shortcomings can be accounted for by using

a second order calibration polynome, but nevertheless a

relatively high error can occur especially for the lower

contents. Total lipids by HPLC-EMD can be calculated

by adding up. A comparison of the yield found by

evaporation to dryness with the sum of lipids deter-

mined by HPLC reveals that the latter are slightly butconsistently higher, 13% (�2.6). This is probably an

e�ect of the calibration although it is also possible that

Fig. 3 Presentation of the lipid contents in relation to the methanol content (log scale). The®lled squares are the one step extractions performed to see the in¯uence of themethanol content. All other extractions, indicated by a letter, are shown too. Squaresare the one-step and circles are the two-step extractions.

198




solvents evaporate di�erent from mixed lipids in sample

than from the pure lipid compounds in standards. In-

complete evaporation will cause larger droplets and

consequently more scattering. Al these uncertainties

were, to a large extend, surpassed by adjusting the in-

jected amount to about the same level for all the sam-

ples, so the underlying di�erence would not hamper the

comparison of the di�erent extractions.

Lipid composition

Evaluating the lipid compositions in Table 3 learns

that no spectacular di�erences are present between the

extractions as applied by Bligh and Dyer. A deviating

composition is only observed for the one step extrac-

tionÕs with lower methanol contents (L and M). Espe-

cially extraction of the phospholipids is incomplete

while the neutral lipids and cholesterol show much less

dependency on the methanol content. This is visualised

by a bar graph in Fig. 5 where the sum of glycerides and

cholesterol for the di�erent methanol contents are

compared with the sum of the phospholipids (note thedi�erent scales). The presence of methanol especially

favours the extraction yield of the latter group while the

glyceride group is nearly equal for each methanol con-

Fig. 4 Typical HPLC chromatogram. The upper one is a 10 times expansion of the lowerone. For abbreviations see text.

TABLE 3

Individual lipid contents in the di�erent extracts in mg/g extract. For abbreviations see text. The last two columns on the right allow comparison of the total content measured by HPLC and gravimetrically.

CHOLE/LPC TG's FFA's CHOL DG's MG's PE PC/SM PS Total Gravi-metrical

AS 0.06 0.34 0.32 0.38 0.07 0.04 1.0 4.7 6.9 6.3BS 0.05 0.32 0.34 0.36 0.06 0.04 1.0 4.6 0.07 6.9 6.0

CS 0.06 0.37 0.41 0.40 0.07 0.04 1.1 5.1 0.10 7.6 6.7DS 0.05 0.30 0.36 0.35 0.07 0.04 1.0 5.0 0.12 7.3 6.4ES 0.39 0.22 0.43 0.08 0.04 1.1 4.9 0.15 7.2 6.6FS 0.06 0.40 0.29 0.44 0.08 0.04 1.2 5.9 0.17 8.5 7.6GS 0.11 0.74 0.64 0.77 0.14 0.07 1.8 10.4 0.19 14.8 13.2HS 0.07 0.45 0.35 0.48 0.08 0.05 1.2 6.0 0.14 8.8 7.6IS 0.06 0.41 0.37 0.43 0.07 0.04 1.2 5.7 0.13 8.3 7.3JS 0.05 0.37 0.30 0.44 0.07 0.04 1.1 5.3 0.12 7.8 6.8PS 0.06 0.37 0.34 0.40 0.06 0.04 1.1 5.4 0.14 7.8 7.0LS 0.05 0.32 0.33 0.33 0.06 0.04 0.7 3.0 4.8 4.3MS 0.06 0.33 0.35 0.42 0.07 0.04 1.0 4.6 0.07 7.0 6.1NS 0.45 0.27 0.48 0.06 0.05 1.2 6.4 8.9 8.1OS 0.06 0.48 0.32 0.47 0.07 0.05 1.2 6.7 0.16 9.5 8.4AM 0.07 0.39 0.44 0.41 0.06 0.04 1.1 5.7 8.2 7.0BM 0.07 0.40 0.43 0.41 0.07 0.04 1.1 6.1 8.6 7.4CM 0.06 0.41 0.44 0.39 0.06 0.04 1.1 6.1 0.09 8.7 7.4

DM 0.07 0.39 0.42 0.37 0.06 0.04 1.1 5.6 0.08 8.2 7.2EM 0.07 0.43 0.43 0.38 0.06 0.04 1.1 5.7 0.12 8.3 7.0PM 0.06 0.37 0.60 0.39 0.06 0.05 1.0 4.9 0.19 7.6 7.1

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tent. This ®nding is very important for the validation of existing lipid extraction methods and likewise for the

development of new methods. Validation of an extrac-

tion method using tissue in which triglycerides are the

major compounds is only of limited value and does not

necessarily apply to all sample types.

Conclusions

The optimisation of the total lipid determination

method done by Bligh and Dyer is limited to a variation

of the ratio between the sample and organic phase vol-

ume. Small volumes of organic phase do extract the

lipids just as e�cient as the larger volumes but the

fraction organic phase recovered is smaller what results

in an apparent lower extraction yield. The proposed

mixture P, however, is very close to the optimum yield

but using a higher methanol content the yield can be

increased by about 15%. Multiple step extraction posi-

tively in¯uences the yield but is overruled by higher

methanol contents.

As the extraction e�ciency of the individual lipids

responds di�erently to changes in the methanol content

the determination of lipid composition is essential for

the validation and development of lipid extractionmethods.

Torsten K. Askland was able to work at the National Institute forCoastal and Marine Management/RIKZ through mediation of theInternational Association for Exchange of Students for TechnicalExperience.

Bligh, E. G. and Dyer, W. J. (1959) A rapid method of total lipidextraction and puri®cation. Canadian Journal of Biochemistry and Physiology 37, 911±917.

Booij, K. and van der Berg, C. (1994) Comparison of techniques forthe extraction of lipids and PCBs from benthic invertebrates.Bulletin of Environmental Contamination and Toxicology 53, 71±76.

Christie, W. W. (1985) Rapid separation and quanti®cation of lipidclasses by high performance liquid chromatography and mass(light-scattering) detection. Journal of Lipid Research 26, 507±512.

de Boer, J. (1988) Chlorobiphenyls in bound and non-bound lipids of

®shes; comparison of di�erent extraction methods. Chemosphere 17,1803±1810.

Delbeke, K., Teklemariam, T., Cruz, de la, E. and Sorgeloos, P.(1995) Reducing the variability in pollution data : the use of lipidclasses for normalization of pollution data in marine data.International Journal of Environmental Analytical Chemistry 55,147±162.

Folch, J., Lees, M. and Stanley, G. H. S. (1957) A simple method forthe isolation and puri®cation of total lipids from animal tissues.Journal of Biological Chemistry 226, 497±509.

Gardner, W. S., Frez, W. A., Cichocki, E. A. and Parrish, C. C. (1985)Micromethod for lipids in aquatic invertebrates. Limnology and Oceanography 30, 1099±1105.

Lovern, J. A. (1957) The Chemistry of Lipids of Biochemical Signif-icance, 2nd edn, revised. Methuen, London.

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(1991) Evaluation of selected lipid methods for normalizingpollutant bioaccumulation. Environmental Toxicology and Chemis-try 10, 1431±1436.

Fig. 5 Lipid compositions in relation to the methanol content. The narrow barsrepresent the glyceride related lipids together with cholesterol and refer tothe left scale. Phospholipids are represented by the wide bars and refer tothe right scale. Clearly the methanol content is of greater in¯uence on theyields of phospholipids than on that of glycerides and cholesterol.

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Roose, P. and Smedes, F. (1996) Evaluation of a lipid intercomparisonby investigation of methodological di�erences. Marine PollutionBulletin 32 (8/9), 674±680.

Schneider, R. (1982) Polychlorinated biphenyls (PCBs) in cod tissuesfrom the western baltic: signi®cance of equilibrium partitioning andlipid composition in the bioaccumulation of lipophilic pollutants ingill-breathing animals. Helgolander Meeresforschung 29, 69±79.

Smedes, F. and Thomasen, T. K. (1996) Evaluation of the Bligh &Dyer lipid determination method. Marine Pollution Bulletin 32 (8/9), 681±688.

Weast, R. C., ed. (1979) Handbook of Chemistry and Physics, 59th edn.CRC Press, Boca Raton.

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revisiting the development of the bligh

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