Comparison of the Bligh and Dyer
`and Folch Methods for Total Lipid Determination
`in a Broad Range of Marine Tissue
`Sara J. Iverson*, Shelley L.C. Lang, and Margaret H. Cooper
`Department of Biology, Dalhousie University, Halifax, Nova Scotia B3H 4J1, Canada
`
`ABSTRACT:
`For many studies, it is important to measure the
`total lipid content of biological samples accurately. The Bligh
`and Dyer method of extraction was developed as a rapid but ef-
`fective method for determining total lipid content in fish mus-
`cle. However, it is also widely used in studies measuring total
`lipid content of whole fish and other tissues. Although some in-
`vestigators may have used modified Bligh and Dyer procedures,
`rarely have modifications been specified nor has their effective-
`ness been quantitatively evaluated. Thus, we compared this
`method with that of the classic Folch extraction in determining
`total lipid content of fish samples ranging from 0.5 to 26.6%
`lipid. We performed both methods as originally specified, i.e.,
`using the chloroform/methanol/water ratios of 1:2:0.8 and
`2:2:1.8 (before and after dilution, respectively) for Bligh and
`Dyer and of 8:4:3 for Folch, and with the initial solvent/sample
`ratios of (3+1):1 (Bligh and Dyer) and 20:1 (Folch). We also
`compared these with several other solvent/sample ratios. In
`samples containing <2% lipid, the results of the two methods
`did not differ. However, for samples containing >2% lipid, the
`Bligh and Dyer method produced significantly lower estimates
`of lipid content, and this underestimation increased significantly
`with increasing lipid content of the sample. In the highest lipid
`samples, lipid content was underestimated by up to 50% using
`the Bligh and Dyer method. However, we found a highly signif-
`icant linear relationship between the two methods, which will
`permit the correction of reported lipid levels in samples previ-
`ously analyzed using an unmodified Bligh and Dyer extraction.
`In the future, modifications to procedures and solvent/sample
`ratios should be described.
`Paper no. L8731 in Lipids 36, 1283–1287 (November 2001).
`
`The total lipid content of biological samples is an important
`quantity used in many biochemical, physiological, and nutri-
`tional studies. Thus, reliable methods for the quantitative ex-
`traction of lipids from tissues are of critical importance. Nat-
`ural lipids generally comprise mixtures of nonpolar compo-
`nents such as glycerides (primarily triacylglycerol) and
`cholesterol, as well as some free fatty acids and more polar
`lipids. Isolation, or extraction, of lipid from tissues is per-
`formed with the use of various organic solvents. In principle,
`the solvent or solvent mixture used must be adequately polar
`to remove lipids from their association with cell membranes
`and tissue constituents but also not so polar that the solvent
`
`*To whom correspondence should be addressed. E-mail: siverson@is.dal.ca
`
`does not readily dissolve all triacylglycerols and other non-
`polar lipids (1). Folch et al. (2) were one of the first to recog-
`nize this and develop the chloroform/methanol/water phase
`system (the so-called “Folch” method), which, under various
`modifications, continues to be considered the classic and most
`reliable means for quantitatively extracting lipids. In the in-
`terest of economy, less exhaustive methods have been devel-
`oped. By far the best known is the “Bligh and Dyer” method
`(3), which has become one of the most recommended meth-
`ods for determining total lipid in biological tissues (4,5) and
`indeed has become the standard for lipid determination in
`many studies of marine fish (e.g., 1, 5–12) as well as for other
`types of samples such as milks (e.g., 13,14).
`The primary advantage of the Bligh and Dyer method is a
`reduction in the solvent/sample ratio (1 part sample to 3 parts
`1:2 chloroform/methanol followed by 1 or 2 parts chloro-
`form) (1,3). In contrast, the Folch method employs a ratio of
`1 part sample to 20 parts 2:1 chloroform/methanol, followed
`by several washings of the crude extract (2). Despite this sol-
`vent reduction, the Bligh and Dyer method is nevertheless
`thought to yield recovery of ≥95% of total lipids (1). Al-
`though the procedure was developed using cod muscle, it
`states (1,3) that it can be applied to any tissue containing (or
`modified to contain) 80% water. Hence, it has been used ubiq-
`uitously. Although the Bligh and Dyer method has undergone
`rigorous and favorable evaluations (e.g., 5,9,16), virtually all
`of these evaluations have been performed on samples contain-
`ing less than 1.5% total lipid. Some studies report using a
`modified Bligh and Dyer method for lipid-rich samples; how-
`ever, the modifications are often unspecified (e.g., 15), mak-
`ing the evaluation and comparison of results difficult. In other
`cases, investigators report the use of the Bligh and Dyer
`method even with samples having high lipid contents, but do
`not indicate that any modifications have been made. In the
`course of recent studies in our laboratory, we discovered that
`samples of a known high lipid content were greatly underes-
`timated using the Bligh and Dyer method compared to the
`Folch method, although we did not detect any difference in
`the fatty acid composition under either method. Since much
`of the data published on the lipid contents of whole fish and
`other samples have been derived using the Bligh and Dyer
`method, we undertook a study to evaluate the relationship be-
`tween these methods in their estimation of total lipid content.
`
`Copyright © 2001 by AOCS Press
`
`1283
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`Lipids, Vol. 36, no. 11 (2001)
`AKBM 1105
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`1284
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`METHODS
`
`MATERIALS AND METHODS
`
`Fish and invertebrates were chosen to represent a wide range
`of lipid contents based on previous species estimates. A total
`of 36 individuals were used, which included pollock, herring,
`rock sole, rock fish, sculpin, octopus, and squid. Each whole
`animal was thoroughly ground and homogenous subsamples
`were taken for extraction. To increase the range of lipid con-
`tents evaluated, we also used weighed aliquots (n = 9) of a
`homogenous mixture of ground commercial fish (originally
`containing 2% fat) and commercial fish oil. Weighed quanti-
`ties of oil were added to produce mixtures ranging from an
`estimated 21 to 26% lipid. Our primary interest was to evalu-
`ate the Bligh and Dyer method compared to the Folch
`method, but because of the high solvent volumes used in the
`Folch, we also evaluated the performance of a reduced-
`solvent Folch using a subset of these samples. Within each
`method, all samples were extracted and lipid contents were
`quantified in duplicate.
`The Bligh and Dyer extraction was performed as originally
`outlined using the following ratios (1,3): Briefly, 100 g sam-
`ple containing (or adjusted to contain) 80 g water (as deter-
`mined by oven drying separate aliquots) is homogenized with
`100 mL chloroform and 200 mL methanol (monophasic sys-
`tem). The solution is rehomogenized with 100 mL chloro-
`form, following which 100 mL of either distilled water (3) or
`weak salt solution (e.g., 0.88% NaCl or KCl) (1,9) is added.
`After filtration is performed under suction, the final biphasic
`system is allowed to separate into two layers and the lower
`(chloroform) phase is collected. For quantitative lipid extrac-
`tion (3), the tissue residue is then rehomogenized with 100
`mL chloroform, filtered, and the filtrate added to the lower
`phase collected. Lipid content is then determined gravimetri-
`cally after evaporating a measured aliquot of the combined
`chloroform phase to dryness under nitrogen (see below). As
`Bligh and Dyer stated (3,16), the above volumes can be
`scaled down, as long as the critical ratios of chloroform,
`methanol, and water (1:2:0.8 and 2:2:1.8, before and after di-
`lution, respectively) and of initial solvent to tissue [(3 + 1):1]
`are kept identical. Thus, we followed the above procedures
`but reduced the scale of all components (i.e., keeping all ra-
`tios the same) for use with a smaller sample amount (4 g sam-
`ple in a 40 mL conical glass centrifuge tube), to allow both
`centrifugation of the final biphasic system and collection of
`the entire lower phase for evaporation and subsequent lipid
`estimation. Instead of applying manual pressure (3) to the
`small filter cake, we performed a second chloroform wash to
`improve removal of residual lipid during filtration.
`The Folch extractions were performed as described, using
`the original extraction ratio of 20 parts 2:1 chloroform/
`methanol to 1 part tissue, which can be done on any scale that is
`technically feasible (2). A weak salt solution (e.g., 0.58–0.88%
`NaCl or KCl) is then added to achieve a final ratio of 8:4:3 chlo-
`roform/methanol/water after including the water contained in
`the tissue (1,2). We also compared the original ratio against a
`modified version using 30 parts 2:1 chloroform/methanol to 1
`
`Lipids, Vol. 36, no. 11 (2001)
`
`2
`
`FIG. 1. Estimates of total lipid content determined in replicate aliquots:
`(A) samples (n = 27) extracted using both a 20:1 and a 30:1 solvent/sam-
`ple ratio Folch and (B) all samples (n = 45) using the Bligh and Dyer
`method in comparison with the original Folch method. The last nine
`samples on the x-axis represent the homogenates of commercial fish
`and oil, which were produced to contain a range of 21–26% lipid. All
`samples were analyzed in duplicate in each of the extraction methods
`and are presented in approximate order of increasing lipid content.
`
`part tissue (1). After verifying that the 20:1 and 30:1
`solvent/sample ratios produced similar results in our samples
`(n = 27, all <25% lipid; Fig. 1A), we analyzed the rest of the
`samples using only the 20:1 ratio as follows: 1.5 g tissue was
`homogenized with 30 mL 2:1 chloroform/methanol. Although
`Christie (1) reports improvement by first homogenizing with 10
`mL methanol followed by 20 mL chloroform, we have tested
`both procedures without detecting differences (Iverson, S.J,
`Lang, S.L.C., and Cooper, M.H., unpublished results). The mix-
`ture was filtered and then washed several times with 2:1 chloro-
`form/methanol, and 0.88% NaCl in water was added to the com-
`bined filtrate at a final ratio of 8:4:3 chloroform/methanol/water.
`Finally, we used a “reduced-solvent” Folch, where the ratios of
`solvent to sample were 7.5:1.0 (i.e., closer to that of the Bligh
`and Dyer method), but the chloroform/methanol/water ratio was
`kept the same (i.e., 8:4:3).
`In all the above extractions (both Bligh and Dyer and
`Folch), the final biphasic system was centrifuged, and the en-
`tire lower phase (along with washings) was collected into a
`
`

`
`METHODS
`
`1285
`
`preweighed glass tube and evaporated to dryness in an ana-
`lytical high-speed nitrogen evaporator (24-position N-EVAP
`112, Organomation Associates, Inc., Berlin, MA) fitted with
`stainless steel 14-in. × 19-gauge needles and equipped with a
`thermostatically controlled water bath maintained at
`25–30°C. The nitrogen stream was continually moved so that
`it actively disturbed the evaporating surface of the sample
`until all detectable traces of solvent were gone. To remove all
`final traces of solvent and water, the sample tube was then
`wiped dry and placed in a sealed glass vacuum tube and
`flushed with nitrogen, and vacuum suction was applied for 5
`min (BOC Edwards model RV3 vacuum pump; Crawley,
`West Sussex, United Kingdom). Lipid content was then de-
`termined gravimetrically. Since results of the Folch method
`using 20:1 or 30:1 solvent/sample ratio did not differ, we used
`the results from the 20:1 Folch method as the basis for com-
`parison with and evaluation of the other extraction methods.
`
`RESULTS
`
`In general, duplicate analyses within each extraction method
`were very consistent, although more so for Folch extractions
`(n = 45, Fig. 1B). In samples containing <2% lipid (n = 11),
`results for the Bligh and Dyer method did not differ from
`those obtained by the Folch method (P = 0.150, paired t-test).
`However, for samples containing >2% lipid (n = 34), the
`Bligh and Dyer estimates of lipid content were significantly
`lower than those of Folch (P < 0.0001). In our nine samples
`of fish oil–supplemented homogenates, lipid content esti-
`mates (20.6–26.6%) using Folch extraction concurred with
`our estimated lipid contents (21–26%, as discussed in the Ma-
`terials and Methods section); however, lipid content estimates
`using the Bligh and Dyer extraction were 50% lower (Fig.
`1B). The next-highest lipid contents were found in herring
`samples (n = 12, 10.7–18.6% lipid by Folch), which were es-
`timated to be about 45% lower (6.1–11.6% lipid) using the
`Bligh and Dyer method.
`The underestimation of lipid content by the Bligh and
`Dyer method increased significantly with increasing lipid
`content (Fig. 2A). From 0% to approximately 2% lipid, re-
`sults of the two methods agreed well. However, with increas-
`ing lipid content, the deviation from the one-to-one reference
`line increased. We were interested in describing the predic-
`tive relationship between the two methods to allow correction
`of previous lipid content analyses that we had performed
`using the Bligh and Dyer method. Using a log–log plot, we
`found a highly significant linear relationship between lipid
`content determined by the Folch method and that determined
`by the Bligh and Dyer method (Fig. 2B).
`The results of the reduced-solvent Folch (7.5:1.0
`solvent/sample ratio) were highly correlated with both the
`20:1 and 30:1 Folch (r = 0.999, n = 34, and r = 0.987, n = 27,
`respectively); however, the reduced-solvent method tended to
`underestimate lipid content as lipid content increased. In sam-
`ples containing ≤3% lipid (n = 19), there was no significant
`difference between the Folch extractions using the 20:1 vs.
`
`A
`
`B
`
`FIG. 2. (A) Correlation of the estimates of lipid content (duplicates aver-
`aged) in 45 samples using the Folch (20:1) vs. Bligh and Dyer methods
`(r = 0.9834, P < 0.0001); the dashed line represents the one-to-one ref-
`erence line. (B) The log–log predictive relationship between estimates
`of lipid content using the Folch (F) vs. the Bligh and Dyer (B&D)
`method.
`
`the 7.5:1 solvent/sample ratios (1.9 ± 0.16% vs. 1.9 ± 0.18%
`lipid, respectively; P = 0.9559, paired t-test), but in samples
`containing >3% lipid (n = 15), the reduced-solvent Folch sig-
`nificantly underestimated lipid content (10.7 ± 1.18% vs. 12.0
`± 1.30%, P < 0.0001). The lipid content estimates of these
`same 15 samples, using the Bligh and Dyer method, were
`even lower at 7.2 ± 0.65% lipid. In the highest-lipid natural
`fish sample tested (herring), lipid content was estimated as
`18.6, 16.4, and 11.6% using the 20:1 Folch, the 7.5:1.0 Folch,
`and the Bligh and Dyer methods, respectively.
`
`DISCUSSION
`
`In the time since the Folch (2) and the Bligh and Dyer (3)
`methods for total lipid determination were published, there
`have undoubtedly been numerous modifications to both meth-
`ods to improve the efficiency of lipid recovery from various
`tissues. However, in many publications where these methods
`have been used, modifications have been neither described
`nor validated. In other cases, investigators stated that lipids
`were quantified “according to” one or the other method, but
`they do not indicate whether any modifications were made,
`
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`1286
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`METHODS
`
`implying that the methods were applied basically according
`to the original procedures, even though that may not have
`been the case. Given that many conclusions about tissue and
`whole-body lipid and energy values are based on published
`lipid contents, our purpose was to evaluate these two meth-
`ods, as originally described, with the aim that investigators
`could evaluate previously published data and that appropriate
`modifications would be made and described in the future.
`In numerous tests with samples containing <2% lipid, the
`Bligh and Dyer method has been shown to be very effective
`and reliable (4,5,9,16). Like other investigators (5), we found
`that lipid extraction using the Bligh and Dyer method pro-
`duced estimates of total lipid content identical to those of
`Folch in samples containing <2% lipid. We also did not de-
`tect any differences in the subsequent fatty acid composition
`of duplicate samples extracted under either method, although
`this may require further investigation in very low fat samples
`that contain a higher phospholipid/neutral lipid ratio (e.g., al-
`kali hydrolysis followed by methylation and fatty acid quan-
`titation could also be used to examine any biases in total fatty
`acid recovery). However, in contrast to low-lipid samples, in
`all samples containing >2% lipid, the Bligh and Dyer method
`produced significantly lower estimates of lipid content, and
`this underestimation increased with increasing lipid content
`of the sample.
`We have several reasons to believe that the total lipid con-
`tents of all samples were accurately determined using the
`Folch extraction method. First, as stated above, in low-lipid
`samples both the Folch and Bligh and Dyer results were iden-
`tical. Second, the estimates of percent lipid in the high-lipid
`fish oil–supplemented homogenates, using the basic Folch ex-
`traction, agreed with our calculated lipid contents; further-
`more, an increased (30:1) solvent/sample ratio Folch pro-
`duced the same values. Finally, these homogenates were also
`analyzed for protein content (by macro-Kjeldahl), as well as
`dry matter (Cooper, M.H., unpublished data). The amount of
`dry matter not accounted for by protein and lipid in these
`samples was reasonably consistent with expectation at 2–4%
`using the lipid values obtained by Folch extractions, but was
`quite high (14–20%) using the lipid values obtained by the
`Bligh and Dyer extractions.
`Bligh and Dyer (3) developed their method using fish fil-
`lets (i.e., muscle) that generally contained low levels of lipid
`and a high proportion of phospholipid. In whole animals and
`in tissue, an increase in total lipid content is due predomi-
`nantly to increases in triacylglycerol. Indeed, subsets of our
`isolated lipid subjected to thin-layer chromatography (17)
`showed that the primary component in the extract was tri-
`acylglycerol (especially as lipid content increases), followed
`by minor amounts of more polar lipid classes. Although Bligh
`and Dyer (3) stated that their method could readily be applied
`to other biological tissues, they, as well as others, acknowl-
`edged that lipid-rich samples may require modifications. For
`instance, Christie (1) suggested that very lipid-rich tissues
`such as adipose tissue and oil seeds should be extracted first
`with a nonpolar solvent such as diethyl-ether or chloroform,
`
`after which the remaining lipid could be recovered effectively
`using Bligh and Dyer methods. However, this appears to have
`often gone unrecognized. The total yield of lipids may be
`more reduced than most investigators have suspected, espe-
`cially given the widespread use of apparently unmodified
`Bligh and Dyer extractions for whole fish and other tissues.
`Even in samples containing 2–10% lipid (which is common
`for many marine fish and invertebrates), underestimation will
`still be a significant problem (e.g., Fig. 1), and this has likely
`been neglected.
`The reduced efficiency of the Bligh and Dyer method with
`increasing tissue lipid contents might be explained from sev-
`eral standpoints. One cause of reduced lipid yield at high lipid
`concentrations could be the limited solubility of the predomi-
`nantly nonpolar lipids, such as triacylglycerols, in the seem-
`ingly relatively polar solvent solution (1:2 vol/vol chloro-
`form/methanol) employed in the Bligh and Dyer method,
`which was designed chiefly to extract phospholipid effi-
`ciently. However, although the initial solvent ratios are differ-
`ent in the Bligh and Dyer vs. the Folch methods, they do not
`result in measurably different contents of methanol in the
`final organic (chloroform) phase (e.g., 16). Hence, this is not
`likely to be a significant factor. Smedes and Thomasen (16)
`found that the absorption of the organic phase by the tissue
`was one of the main causes of incomplete lipid yield. Rela-
`tively constant amounts of the organic phase are absorbed by
`the tissue such that using greater volumes of organic-phase
`solvents reduces the fraction of the organic phase that is lost
`in this manner (16). When tissues with increasing lipid con-
`tents are extracted (using the same volumes of solvents), the
`lipid concentration in the organic phase should also increase,
`assuming that limits of solubility are not reached. This would
`result in increased loss of lipid in the fraction of organic phase
`absorbed by the tissue, causing a reduction in final lipid yield.
`Thus, in addition to maintaining critical solvent and water ra-
`tios, perhaps the most important consideration is simply the
`ratio of solvent to dry-weight sample (and expected fat con-
`tent), as even with the Folch method, a reduced ratio pro-
`duced significant underestimates of lipid content.
`Our results do indicate that all methods used to estimate
`lipid contents were highly correlated. Fortunately, there is a
`highly predictable relationship between the Bligh and Dyer
`and Folch methods (Fig. 2B), potentially allowing correction
`of reported values from previous analyses that used an un-
`modified Bligh and Dyer extraction. It may also be the case
`that investigators have used a modified Bligh and Dyer ex-
`traction employing an increased solvent/sample ratio that pro-
`duced reliable results and have simply not stated this. It will
`be important in the future that investigators specify modifica-
`tions to any of these procedures, especially the precise sol-
`vent/sample ratio used. For instance, although an increase in
`the solvent/sample ratio (i.e., to 30:1) from the original Folch
`did not appear to alter the estimated lipid content significantly
`(Fig. 1A), we would not recommend making this assumption
`for tissues containing greater than 25% lipid (i.e. adipose tis-
`sue, milks of many species) unless verified. In such samples,
`
`Lipids, Vol. 36, no. 11 (2001)
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`METHODS
`
`1287
`
`a further increase in the solvent/sample ratio and/or further
`multiple extractions may be necessary for quantitative lipid
`evaluation (e.g., 1), as we have found for marine mammal
`milks (Iverson, S.J., personal communication).
`
`ACKNOWLEDGMENTS
`This study was supported by grants to Sara J. Iverson from the Exxon
`Valdez Oil Spill Trustee Council, Alaska, and by the Natural Sciences
`and Engineering Research Council (NSERC, Canada). We thank
`Lindsay Smith for assistance with some sample analyses and Suzanne
`Budge for helpful comments. We are also grateful for the suggested
`improvements to the manuscript by the editor and three anonymous
`reviewers.
`
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`[Received January 19, 2001, and in revised form October 10, 2001;
`revision accepted October 12, 2001]
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