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Detectors for HPLC of lipids with special reference to evaporative light-scattering detection
DETECTORS FOR HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF

LIPIDS WITH SPECIAL REFERENCE TO EVAPORATIVE LIGHT-SCATTERING
DETECTION
William W. Christie
(formerly of) Hannah Research Institute, Ayr, Scotland KA6 5HL
A.
B.
C.
D.
E.
Introduction
Optical and Spectrophotometric Detectors
1. Differential refractometry
2. Ultraviolet spectrophotometry
3. Fluorescence detection
4. Infrared spectrophotometric detectors
5. Spectrophotometric detection with post-column chemical reaction
Some Miscellaneous Detection Systems
1. The mass spectrometer as an HPLC detector
2. Radioactivity detectors
3. Density, electrochemical and other detectors
Transport-flame Ionization Detectors
1. Apparatus
2. Applications of transport-flame ionization detectors
Evaporative Light-Scattering Detectors
1. Construction and the nature of the response
2. Applications of light-scattering detection in lipid class separations
3. Applications of light-scattering detection in separations of molecular species of lipids
4. Some miscellaneous separations
References
A. INTRODUCTION
Detectors functioning according to many
different principles are available as a means of
sensing solutes in the mobile phase as they elute
from the column during high-performance liquid
chromatography (HPLC) of lipids. Defined peaks may
be quantified directly or fractions containing the
solutes can be collected for analysis by other means.
The topic of detectors for HPLC analysis of lipids was
extensively reviewed by the author in 1987 [14] and
that work should be certainly be consulted for specific
applications. However, the much wider use of lightscattering detectors in the last few years has
changed the perspective greatly. In discussing
different detection systems here, the author has been
highly selective in his choice of examples, with
concentration on more recent papers. This review is
to some extent a supplement to the earlier work
avoiding unnecessary repetition, but applications of
light-scattering detectors are discussed at length.
Others have reviewed HPLC separations of lipids in
general [59,109,110] and phospholipids and

glycolipids in particular [68,69].
As most lipids lack chromophores that permit
specific spectrophotometric detection, this most
essential process has been the weak link in the
chromatographic system. The availability of one
particular detector in a laboratory may determine
which solvents can be used in the mobile phase,
whether they can contain ionic species, whether
gradient elution is possible, and sometimes even
which mode of chromatography and which stationary
phase are appropriate. For example, if a differential
refractometer is the only detector to which the analyst
has access, a mobile phase of constant composition,
i.e. isocratic elution, must be used and some
compromise in the quality of the separation may have
to be accepted. Most lipid classes are heterogeneous
and contain aliphatic moieties with a range of chainlengths and variable numbers of double bonds. Each
type of detector has its idiosyncrasies and responds
to such structural features of lipids in a different
manner, and it must be calibrated with appropriate
standards.
W.W. Christie, in Advances in Lipid Methodology One, pp. 239-271 [1992] [Ed. W.W. Christie, Oily Press, Ayr
For some time, it was thought that detectors

working on the transport-flame ionization principle
(see Section D below) would be the ideal answer to
the problem, but the commercial instruments have
been disappointing for a variety of reasons. They
certainly have a theoretical potential to exhibit a linear
response that may be largely independent of the
structure of the solute, and it is to be hoped that this
potential will one day be fulfilled. On the other hand,
evaporative light-scattering or "mass" detectors
(Section E below) are available now, and their virtues
are becoming increasingly evident. They are simple,
rugged and versatile in use, and afford high sensitivity
in comparison to other "universal" detectors. Almost
any solvent can be used in the mobile phase in
complex gradients. It must be admitted that the
response does not bear a simple linear relationship to
the amount of solute and varies in a poorly
understood manner with the nature of the lipid. Yet
the advantages of these detectors far outweigh the
disadvantages, and with care it is possible to live with
the latter provided that they are properly understood.
It is the author's opinion that they offer much more to
lipid analysts than any alternative, and there does not
appear to be any superior system on the horizon.
Further, the analyst now has a choice of instruments
from at least four manufacturers.
B. OPTICAL AND SPECTROPHOTOMETRIC

DETECTORS
1.
Differential refractometry
Refractive index (RI) detectors of three different

kinds are in common use, i.e. deflection, Fresnel and
interference refractometers. All can be considered as
universal in scope and can be used with any solute
with a different refractive index from that of the
mobile phase. In operation, they monitor the
difference in refractive index between the eluent and
the pure mobile phase continuously. Although
differential refractometers can in theory be used with
gradients by having a reference column in parallel
with the main one, it is almost impossible to achieve a
balance between the two and thence a stable baseline in practice. They are at their best with isocratic
elution, although programming of the flow-rate or
temperature of the mobile phase affords some limited
opportunities to enhance separations. In order to
minimize changes in solvent composition and baseline drift during a chromatographic run, volatile
solvents should be avoided. When a static reference
cell is used, it is often necessary to flush it out with
fresh mobile phase at some point during a day's
work. Lipid samples should be dissolved in fresh
mobile phase for injection onto the HPLC column. RI
detectors are very sensitive to fluctuations in
temperature, so many commercial instruments have
some means of controlling this, varying from watercirculation to a more sophisticated thermostatted
system. For this reason, they should be located away
from sun-lit windows and from draughts, such as are
found near doors or fume cupboards. Other
disadvantages are that sample peaks can be both
positive and negative, and that bubbles of gas in the
solvents, the flow-rate of the mobile phase, leaks in
the system, back pressure and pulsations of the
HPLC pump can influence base-line stability.
With simple lower-cost RI detectors, solute
components amounting to about 10 micrograms can
perhaps be detected. A 10 to 30 times improvement
in this sensitivity may be possible with precise control
of
temperature
and
other
chromatography
parameters in the best commercial instruments.
Comparatively little use has been made of RI
detectors for quantitative analysis of lipids, and there
has been some debate on whether response factors
are necessary for different lipid classes or molecular
species [14]. In analyses of molecular species of
triacylglycerols at least, the consensus appears to be
that acceptable results can be obtained by equating
detector response directly with the mass of
components, but careful calibration and calculation of
response factors will improve accuracy.
Such detectors were once relatively common in
lipid laboratories, and have been used especially for
preparative-scale chromatography and for gelfiltration, but are nowadays used much less. They are
at their best for the preparative-scale isolation, under
isocratic elution conditions, of particular lipid
components that are required for analysis by other
procedures. In this way, they have been applied to
the isolation of neutral lipids, phospholipids and
molecular species of both, and fatty acid derivatives
of various kinds [14]. The limits of development of RI
detection may have been reached by Frede et al.
[28,29], who developed a system involving
temperature programming for the separation of
molecular species of milk triacylglycerols by HPLC in
the reversed-phase mode. Programming of the flowrate of the mobile phase can also be of assistance
with related samples [3].
2.
Ultraviolet spectrophotometry
Spectrophotometric detectors in the ultraviolet

(UV)-visible range for HPLC are used more frequently
than any other by analysts in general, so they are
relatively inexpensive and tend to be one of the first
to which lipid analysts have access. Detectors
constructed specifically for HPLC use with a cell
volume of about 8 microlitres are recommended (as
opposed to UV spectrophotometers with a flow-cell
as an optional extra), and only those affording
continuously variable wavelengths are of much value
to lipid analysts. UV detectors can sometimes give
great selectivity and sometimes sensitivity in the

analysis of specific compounds, and they are
relatively insensitive to changes in ambient
temperature or the flow-rate of the mobile phase.
While they can be used in gradient elution
applications on occasion, base-line drift can be
troublesome. A detector cell can easily become
contaminated in use, although this may not be
immediately obvious.
Compounds containing conjugated double bond
systems and aromatic rings give much the best
response, but such functional groups are only rarely
found in natural lipids. There are some seed oils
containing fatty acids with conjugated double bond
systems, and these are often present in lipids
subjected to hydroperoxidation through chemical or
enzymatic action, as in many biologically important
eicosanoids for example [102]. UV detection is the
most common procedure used with autoxidized lipids
[14]. In addition to mere detection, second-derivative
UV spectroscopy has proved of value for the
identification of configurational isomers of conjugated
dienes formed in oxidized phospholipids [42].
Cis,trans- and trans,trans-dienes were easily
distinguished, for example. If lipids are defined in a
broad sense, carotenes and tocopherols can be
subjected to HPLC analysis with UV detection.
Among published applications, modern diode array
detectors, which covered a range of wavelengths,
were employed to detect and identify cholesterol
esters, sterols, dolichol, ubiquinone, alpha-tocopherol
and retinol in tissue extracts [32] and acyl-coenzyme
A esters [140] after separation by HPLC.
An alternative approach has been to convert
lipids to compounds that absorb strongly in the UV
range. For example, fatty acids have been esterified
with aromatic alcohols for analysis, the carbohydrate
moieties of glycolipids have been benzoylated, and
diacylglycerols derived from phospholipids have been
esterified with aromatic acids. Following conversion
to the phenacyl or naphthacyl esters, fatty acids are
easily resolved by HPLC in the reversed-phase mode
on chemically bonded octadecylsilyl (ODS) or
octylsilyl phases for UV detection at 242 to 254 nm
[14]. The nature of the separation is dependent both
on the chain-length of the fatty acid and the number
of double bonds, each double bond reducing the
retention time by the equivalent of about two
methylene groups. Whether this technique has any
real analytical value per se is dubious, since gas
chromatography (GC) offers much more in terms of
convenience and ease of identification of components
[17]. Reversed-phase HPLC used in this way certainly
does have advantages for the analysis of fatty acids
containing functional groups that are thermally labile,
e.g. cyclopropene fatty acids [141] and perhaps for
those with trans-double bonds [38,127], but otherwise
the technique is best considered as a micro-
preparative method for the isolation of fatty acids for

say structural analysis or radioactivity measurements.
Phenacyl esters of fatty acids have also been
separated by HPLC in the silver ion mode, i.e. by the
number and configuration of the double bonds, and
this technique may have some potential for the
analysis of fatty acids with trans-double bonds [21].
Benzoylation of glycosphingolipids for HPLC
analysis has proved its worth in terms of ease of
detection by UV spectrophotometry, and by reducing
polarity and simplifying resolution, both in the
separation of different glycolipid classes and for
molecular species of these [14,68]. The response is
dependent on the number of benzoyl groups that
react in each instance, but can usually be related to
the molar amount of the native lipid. Similarly, a wellestablished technique for the analysis of molecular
species of phospholipids consists in hydrolysing them
with phospholipase C, followed by conversion to the
benzoyl or dinitrophenyl urethane derivatives, for
separation by HPLC in the reversed-phase mode for
UV detection [14]. The technique has the advantages
that the same type of derivative can be made from
any phospholipid class, and that detection is
independent of the fatty acid compositions of
components so molar proportions are determined
directly. Naphthylethylcarbamate derivatives have
been utilized to facilitate chiral separations (see this
volume pp. 121-148). Among recent applications, UVabsorbing derivatives have been employed for the
analysis of acylcarnitines [78] and lysogangliosides
[57].
With lipids or derivatives of these kinds with
which specific wavelengths can be selected, good
quantification in direct molar terms is usually possible
with UV spectrophotometric detection. Both gradient
and isocratic elution methods can be used if the
composition of the mobile phase is chosen with care
and other chromatographic parameters are carefully
controlled and standardized.
Most natural lipids exhibit a weak absorbance in
the range 200 to 210 nm of the UV spectrum,
sometimes termed "end absorption", that is the result
of the presence of isolated double bonds
predominantly,
although
carbonyl,
carboxyl,
phosphate, amino and quaternary ammonium and
other functional moieties make some contribution
[49]. When UV detection at such wavelengths is used
in lipid analysis, several disadvantages become
apparent. Many solvents of proven value in the
chromatography of lipids, such as chloroform,
acetone, toluene or ethyl acetate, absorb strongly and
cannot be used in mobile phases. Others such as
ethers require to be carefully purified to eliminate
contaminants that absorb in the appropriate region.
Similarly, those solvents which are transparent at the
required wavelengths, e.g. hexane, isopropanol,
acetonitrile, methanol and water, must be of very high
purity since traces of extraneous materials with

appreciable extinction coefficients, such as
antioxidants or plasticisers, could seriously impair
base-line stability and give high background values.
In samples for analysis, impurities and natural
substances, such as peroxidized lipids or
tocopherols, can give disproportionately large peaks
in the HPLC trace, obscuring the components of
interest.
In spite of these difficulties, UV detection at
these low wavelengths has been very widely used in
lipid analysis, and especially in the separation of
simple and phospholipid classes [14]. Even with the
restricted range of solvents that can be employed in
mobile phases, some versatility remains to change
the selectivity in particular analyses. For example in
the separation of phospholipids, hexane-isopropanolwater or acetonitrile-methanol-water mixtures are
commonly used; the order of elution of the neutral
phospholipids is not greatly affected by the type of
mobile phase employed, but acidic phospholipids,
such as phosphatidylserine and phosphatidylinositol,
tend to elute much earlier when acetonitrile is
present.
a standard mixture very similar to those to be

analysed, direct quantification has been attempted by
many analysts. For example, calibration lines for
cholesterol and cholesterol esters at 210 nm are
shown in Figure 1 [138]. There was a good rectilinear
response for each fatty acid, but the slope of the line
varied greatly according to degree of unsaturation.
This of course means that it is necessary to be
certain of the identity of every peak (and these should
contain only one species), whenever direct
quantification is attempted. This is perhaps an
extreme example and smaller correction factors were
found to be required in analyses of lipid classes,
where the differences in the fatty acid compositions of
the separated components were less (c.f. reference
[39]). When samples are likely to be variable in
composition, it is advisable to collect the peaks as
they emerge from the detector for estimation by
appropriate micro-methods. Most analysts have
followed the latter approach, using phosphorus
analysis for phospholipids, for example. GC analysis
of the methyl ester derivatives of fatty acid
constituents, prepared from fractions, with an added
internal
standard
permits
identification
and
quantification simultaneously and has wider
applicability [14,17].
Direct detection (without response factors) and
quantification at 215 to 230 nm, where ester bonds
exhibit a distinct absorbance but double bonds do
not, has been used for analysis of molecular species
of triacylglycerols separated by reversed-phase
HPLC [40]. With mixtures of acetonitrile and hexaneisopropanol as the mobile phase and detection at 215
nm, as little as 1 microgram of triacylglycerol could be
analysed. These results have been confirmed and
extended by others [31,111], and there would appear
to be no reason why this form of detection should not
be extended to other types of sample.
3.
Fig. 1. Calibration lines for the quantification of cholesterol

esters by HPLC with detection at 210 nm [138]. The ratio
of the peak heights of the components to that of
cholesteryl heptadecanoate (internal standard) are plotted
as a function of the mass of each cholesterol ester
standard, i.e. cholesteryl arachidonate (20:4), linoleate
(18:2), oleate (18:1) and palmitate (16:0) and for
unesterified cholesterol. (Reproduced by kind permission
of the authors and of Atherosclerosis, and redrawn from
the original paper).
Because variation in the degree of unsaturation

of each component can make a substantial difference
to the response at low wavelengths, direct
quantification is difficult and relatively saturated lipids
might even be overlooked. By determining the
apparent extinction coefficient for each component in
Fluorescence detection
Only a few rare lipids exhibit natural

fluorescence, but it is possible to make use of the
high sensitivity (up to a hundred times greater than
absorption detectors) and selectivity of fluorescence
detection by preparing suitable derivatives of lipids for
chromatography. Detectors of this kind have a wide
dynamic range. Unfortunately, the response is
affected substantially by the nature of the mobile
phase, and careful calibration is necessary when
gradient elution conditions are employed [66]. The
detector has been used most often in the analysis of
fatty acids, separated by reversed-phase HPLC, after
conversion to suitable derivatives. Anthrylmethyl
esters have been used most often for the purpose,
but many different alternatives have been suggested,
some offering sensitivities to the femtomole level [14].
At these concentrations, such analytical systems offer
real competition to GC in some circumstances, and

especially for the analysis of the biologically important
free fatty acid fraction in small samples of plasma.
The wide variety of lipid classes found in animal
tissues but including analogues containing pyrenyl
-13
fatty acids, which are detectable to levels of 10
mole by their fluorescence, have been separated by
HPLC and quantified following correction for
quenching by the mobile phase [43]. The ternary
gradient elution procedure was based on one
developed originally for use with an evaporative lightscattering detector (see Section E.2 below) [12].
Among other recent applications, fluorescence
detection has been applied for the analysis of aminophospholipids (phosphatidylethanolamine, phosphatidylserine and their lyso-forms) [50], phosphatidic acid
[142], platelet-activating factor [79] and its lyso-form
[108], diacylglycerols derived from phospholipids
[104] and monosialogangliosides [144].
4.
Infrared spectrophotometric detectors
Infrared (IR) detectors have been used to a

limited extent only for the analysis of non-polar lipids,
with the specific absorbance for the carbonyl function
between 1650 and 1860 cm-1 (or at about 5.75
microns) being the spectral region of value [35]. The
detector is not sensitive to variations in ambient
temperature or the flow-rate of the mobile phase, but
most solvents tend to absorb to some extent at least
at the key wavelengths, so causing high background
values. A consequence is that marked base-line drift
is seen whenever gradient elution is used. Although
the effect is diminished in cells with a short pathlength (1 to 2 mm), the sensitivity is then reduced.
Hexane, acetonitrile, chloroform, methylene chloride
and tetrahydrofuran appear to be the most useful
solvents for IR detection. No quantitative studies have
been performed with lipids, but the detection limit was
about 1 microgram [35].
Others used a purpose-built infrared detector
with a helium-neon laser operating at 3.39 microns,
the CH2- stretching frequency, in separations of a
limited range of model simple lipids [117]. However,
few solvents are permissible in the mobile phase,
chloroform being utilized in this application, so that
this form of detection has no obvious practical value.
5.
Spectrophotometric detection
column chemical reaction
with
post-
The content of particular analytes in an HPLC

mobile phase can be monitored by incorporating a
reaction chamber after the column, where mixing with
specific chemical or enzymatic reagents takes place
so that the products can be quantified by
spectrophotometric detection. Among the more
familiar applications to lipids that have been
described are chemical determination of phosphate in

phospholipids and enzymatic determination of
triacylglycerols and cholesterol in lipoproteins or lipid
extracts [14]. While such detection systems can offer
high specificity, they are inherently limited in scope.
More recent applications of reaction and postcolumn detection in the analysis of lipids by HPLC
include separations of molecular species of
monoacylglycerols [128] and mono-, di- and
triacylglycerols [58], fluorescent detection using the
probe 1,6-diphenyl-1,3,5-hexatriene for quantification
of molecular species of phospholipids [101] and an
iron-thiocyanate assay for hydroperoxides [87]. In
addition, hydroperoxides have been detected with
high specificity and sensitivity (picomolar levels) by
making use of the hydroperoxide-dependent
chemiluminescence produced by luminol oxidation
during the reaction of hydroperoxides with
cytochrome c-haeme [46,80-82,145-147]. Polyphosphoinositides have been detected at low
concentrations by means of a novel method of this
type involving a "metal-dye" interaction [74,75]. As
inositol phosphates emerged from the HPLC column,
they encountered an yttrium salt with which they
rapidly formed pH-dependent complexes. A "reporter"
dye, 4-(2-pyridylazo)resorcinol, was used to sense
formation of the complex by producing a negative
adsorption peak at 520 to 550 nm. The method was
reportedly usable down to picomole levels.
C. SOME MISCELLANEOUS DETECTION

SYSTEMS
1.
The mass spectrometer as an HPLC detector
Mass spectrometry (MS) is a powerful analytical

tool that can supply both structural information about
compounds and quantitative data relating to mass.
Under optimum conditions, it can provide the
molecular weight, the empirical formula and often the
complete structure of an unknown compound in
addition to giving a measure of the amount present.
For some years it was necessary to volatilize the
sample in the ion source of the instrument before
ionizing it by electron-impact or chemical-ionization
techniques, but recently methods have been
developed for producing ions from materials in the
condensed phase (e.g. by fast-atom bombardment
techniques). While combined GC-MS has been
available for many years, it has proved more difficult
to marry HPLC with MS, because of problems in
removing the solvent prior to ionization. Many of the
difficulties have now been overcome; several different
HPLC-MS interfaces are available on commercial
instruments, and the construction and properties of
these have been reviewed [51]. One of the first
methods of transferring an HPLC solute to the ion
source of the mass spectrometer was the field-
desorption technique, in which the sample is

deposited on a wire emitter in liquid solution, dried
and then inserted into the instrument in order to
obtain the spectrum. The technique can give good
results but is not a continuous one. A second
approach to interfacing an HPLC eluent with a mass
spectrometer makes use of a mechanical transport
system, similar to that used with flame-ionization
detection (see Section D below), in which the sample
is deposited on a moving belt and the solvent is
evaporated before the solute reaches the ion source.
A spray deposition technique is usually favoured to
promote even deposition and evaporation. In its
simplest form, the vaporization-ionization principle is
field desorption, but electron-impact and chemical
ionization techniques can also be employed. Of
greater value is a "thermospray" method in which a
supersonic jet of vapour carrying entrained particles
or droplets of solute is produced by controlled rapid
heating of the capillary tube connecting the HPLC
column to the mass spectrometer. The solute
droplets travel rapidly through the ion source, where
they continue to vaporize and are ionized by standard
chemical-ionization techniques. Modifications for fast
atom bombardment MS and an atmospheric pressure
ionization technique are also available. Such
equipment has been applied to some relatively
involatile lipids, such as phospholipids and
gangliosides. In general, direct-inlet interfacing
methods are better suited to microbore or capillary
column HPLC applications where the volume of
mobile phase is relatively limited.
With HPLC-MS systems, most structural
information is obtained with lipids of comparatively
low molecular weight; with compounds of lower
volatility, it is sometimes possible to obtain molecular
weights only (although this information is not
negligible). At this time, the equipment is still very
costly, but such is the pace of technological advance
that this may not always be so. The use of systems of
this kind in the analysis of lipids, especially for the
identification of molecular species within specific lipid
classes, is certainly becoming much more
widespread [14,60]. Among recent applications, a
moving-belt interface was used in conjunction with
chemical ionization in analyses of neutral
glycosphingolipids
[27],
HPLC-fast
atom
bombardment-MS interfaces were used to study
acylcarnitines [94], coenzyme A esters [93] and
glycosphingolipids
[124,125],
thermospray-MS
interfaces have been utilized in analyses of
phospholipids
[1,53,54,56,96],
diradylglycerol
derivatives prepared from phospholipids [10,63],
eicosanoids [1,55,56], fatty acid hydroperoxides [55]
and acylcarnitines [76,77], and HPLC linked to
atmospheric pressure-ionization MS has been
employed for serum cholesterol [129], fatty acid
anilides [61] and glycolipids [62].
A recent paper in which an HPLC micro-column

(100 mm x 0.3 mm i.d.) of silica gel was used for the
separation of glycosphingolipids with analysis by
mass spectrometry may indicate an important new
direction for the technique [126]. Flow rates as low as
6 microlitres/min were possible and components
amounting to only 160 ng were separated and
identified.
2.
Radioactivity detectors
Methods for detecting radioisotopes are often

much more sensitive than many spectrophotometric
and other physical procedures, and the use of lipids
14
3
32
labelled with the beta-emitters C, H and P has
revolutionized the study of lipid biochemistry. Indeed,
the ease and accuracy of quantifying radioactive
lipids are such that these are frequently used in the
development of new methods to test recoveries. It
can even be advantageous to convert lipids to
isotopically labelled derivatives for quantification
purposes. It is of course possible to use
discontinuous methods of liquid-scintillation counting
for estimating radioactivity in fractions collected from
HPLC columns, but continuous methods are more
relevant to this review. Methods for direct monitoring
of radioactivity in HPLC mobile phases can be
classified as either homogeneous or heterogeneous
counting systems. In the latter, the eluent is passed
through a flow cell, packed with a solid scintillator
such as yttrium silicate or cerium-activated lithium
glass, and positioned in a scintillation counter. An
advantage is that solutes are easily recovered for
analysis by other methods. In homogeneous
counting, the eluent from the HPLC column is mixed
with a liquid scintillant before passing into the
counter. One difficulty is that there are limitations on
the range of solvents that can be incorporated into
mobile phases with this technique; chloroform
especially will cause strong quenching. The major
disadvantage, however, is that the residence time of
a sample in the flow cell must be short to maximize
resolution, and this greatly reduces the number of
counts that can register. Depending on the mode of
HPLC and the type of counting procedure, the
minimum limits of detection can be as high as 800
14
3
dpm and 27,000 dpm for C and H respectively.
Better results can be obtained with the higher energy
32
emitter P, as in separations of labelled
phospholipids.
Conventional
liquid-scintillation
counters equipped with flow cells can be used as
HPLC detectors, but purpose-built counters/detectors
are preferable in that they are designed to eliminate
or at least minimize chemiluminescence effects due
to flow phenomena.
Because of the sensitivity and specificity of
radioactivity detection, preparation of isotopically
labelled derivatives is sometimes undertaken as an
aid to quantification of specific lipids [14]. In a recent

application, glycosphingolipids were oxidized with
3
galactose oxidase and reduced with H-labelled
sodium borohydride for HPLC separation and
detection with a flow-through scintillation counter [88].
Others have used the technique for analyses of acylcoenzyme A esters [140], molecular species of
32
phospholipids after conversion to
P-labelled
dimethylphosphoric acid esters [52] and molecular
species of cholesterol esters [130]. Reviews of
radiotracer techniques and HPLC in the analysis of
fatty acids and eicosenoids [5] and phospholipids [83]
have appeared.
3.
Density, electrochemical and other detectors
A detector in which the changes in the density of

a mobile phase on passage of a solute is sensed by
variations in the frequency of a quartz oscillator has
been applied in the analysis of simple lipid classes
[131]. It appears to lack sensitivity and shares many
of the disadvantages of differential refractometry.
An electrochemical (tensammetry) detector for
phospholipids has been described, that has novelty
value but apparently little else to commend it [6].
Much more valuable is the use of electrochemical
detection with a mercury drop or glassy carbon
electrode for the detection of lipid hydroperoxides
[30,72,143]. The detector is sensitive and highly
specific, since it is dependent on the controlled
reduction of hydroperoxides to the analogous hydroxy
compounds and can be used to quantify the former
even when the two types of compound have coeluted as is often the case. With the glassy electrode,
the detection limit was ten times better than UV
detection of the conjugated double bond systems
[72].
An electron-spin resonance spectrometer has
been used as a detector for an HPLC system to study
the formation of free radicals from polyunsaturated
fatty acids [123].
D. TRANSPORT-FLAME IONIZATION
DETECTORS
1.
Apparatus
A transport-flame ionization detector was first

introduced by Pye Unicam Ltd and, as with the
evaporative light-scattering detector (Section E
below), it is of universal applicability. In this
commercial model, the eluent was fed continuously
onto a clean moving wire that was passed into an
evaporator oven to remove the solvent and then into
a flame ionization chamber, similar to that in a gas
chromatograph, where the solutes were combusted
and detected. Subsequently, a more sensitive model
was constructed in which the solute was pyrolysed to
carbon dioxide and reduced to methane before

entering the detector. These instruments were soon
discontinued, apparently because they were
unreliable and were not sufficiently sensitive,
although second-hand equipment was in demand in
some quarters for many years.
Many other detectors of this type have been built
in laboratories but never manufactured commercially.
For example, Privett and colleagues constructed and
described a detector of improved design of this type,
in which the eluent was entrained on a helical wire, so
that more of the sample was combusted and the
sensitivity of detection was greatly increased [122].
They later replaced the helical wire with a stainlesssteel belt of perforated structure that allowed most or
all of the eluent to be trapped and carried into the
detector [103]. In addition, after evaporation of the
mobile phase, the solute was converted to
hydrocarbons in a stainless-steel reactor before being
combusted and detected in a hydrogen flame. It has
been shown to have a good linear response with
respect to sample size although it was not as
sensitive as some alternatives. The author was
favourably impressed on seeing it in operation in
1985. Further development has been undertaken by
a Danish manufacturer, who appears to have
encountered some technical difficulties but has
promised to release a commercial version soon.
The only commercial detector based on this
principle, for which there is a body of users, is
manufactured by Tracor instruments (Austin, Texas,
U.S.A; UK distributor - Kemtronix Ltd, Compton,
Berks], and details of its construction and some
applications have been published [26]. A key
component is a fibrous quartz belt round the
circumference of a rotating disc, and enclosed in a
heated ventilated housing, containing the solvent
applicator and a dual-flame ionization detector. In
operation, the mobile phase from the HPLC column is
applied to the rotating belt and the volatile solvents
are vaporized and eliminated by a vacuum pump,
before the involatile solutes are transported into the
twin flames of the detector, where they are
combusted and ionized. As in a gas chromatograph,
the total ion current is amplified and transmitted to a
recorder and integrator. Any residual solute is burnt
off in a pyrolyser unit before the belt picks up fresh
eluent. While this detector is costlier than many
others, this would not be a serious disadvantage if
reliable results were possible. Regretfully, there
appear to be difficulties (see below).
An interesting detector of the transport-flame
ionization type has been described in which the
transport mechanism was in effect a spoked wheel;
the eluent from the HPLC column was deposited in
discrete packets on the spokes, which were made
from a material of low conductivity and thermal mass
(quartz rods) and allowed the solvent to be
evaporated in an air-flow of controlled temperature

[71]. Eventually each rod was passed through the
centre of the flame of the detector and ions were
generated.
Most investigators, though working with a range
of models, have reported that the response of the
transport-flame ionization detector is linearly related
to the amount of solute, although some correction
factors might have to be introduced to compensate
for non-combustible moieties in lipids. The only
limitations on the choice of solvent for mobile phases
are that they must be reasonably volatile and should
not contain inorganic ions. Although the solute is
destroyed by the detector, a stream-splitter can be
introduced to divert a substantial proportion of the
eluent to a collection system.
The author once gave his opinion that "if a
reliable and relatively inexpensive detector of this
type were to be made available commercially, it
would fulfil a long-felt need. Indeed, it would not be an
overstatement to suggest that it would revolutionize
the practice of lipid analysis" [14]. This is probably
true still, but there may yet be a long wait for such a
detector. In my opinion, evaporative light-scattering
detectors have reached a higher state of
development and are a better investment.
2.
Applications of transport-flame ionization

detectors
The early literature on the subject of applications

of transport-flame ionization detectors has been
reviewed elsewhere [14] and is of limited relevance,
since most workers used instruments that are no
longer available commercially. Therefore, only
TM
applications of the Tracor
detector need be
considered here (although this too is no longer
available commercially, it is still in use in some labs).
Hammond [36] has briefly described his experience
of this instrument in different forms of lipid analysis.
In particular, he stresses the importance of setting up

the applicator jet and the flame in the pyrolyser oven
to the optimum values in order to realise the full
potential of the instrument. Difficulties were
experienced with lipids of relatively low molecular
weight, which tended to evaporate before detection.
Ions of sodium or potassium in solvents or samples
were particularly troublesome, because of ionization
of the flame and irreversible contamination of the belt.
Perhaps surprisingly, silver ions eluting onto the belt
did not appear to cause difficulties. A need for careful
distillation of even "HPLC-grade" solvents in order to
eliminate involatile impurities, as a prerequisite for
acceptable base-line stability, has been reported by
others [95]. In more recent instruments, the
manufacturers appear to have improved background
noise levels by improved electronic filtering.
TM
The first published application of the Tracor
detector to lipids was in the separation of molecular
species of phosphatidylglycerol and galactosyldiacylglycerols from plant chloroplasts by means of
HPLC in the reversed-phase mode [91,95,112]. For
the former the mobile phase, methanol-acetonitrileethylpropylamine-acetic acid (34.7:64.5:0.3:0.5 by
volume), contained ionic species but gave a stable
base-line. An application to molecular species of
digalactosyldiacylglycerols is illustrated in Figure 2.
Minor constituents, i.e. those present at a level of as
low as 1.2 nmol, were quantifiable. Although the
linearity of the detector response was not determined
rigorously, direct quantification by integration of the
signal gave results which were comparable to those
obtained by alternative methods, and there did not
appear to be any requirement for calibration factors to
compensate for differences in chain-length or degree
of unsaturation. Similar methods were utilized to
separate molecular species of the relatively
uncommon
diacylglyceryltrimethylhomoserine
of
algae [92].
Fig. 2. Separation of molecular species of

digalactosyldiacylglycerols from Dunaliella salina by
HPLC in the reversed-phase mode with flameionization detection [112]. A column of Rainin
TM
Microsorb was employed with methanol-water
(24:1, v/v) at a flow-rate of 0.8 mL/min as the
mobile phase. (Reproduced by kind permission of
the authors and of the Journal of Chromatography,
and redrawn from the original paper).
Molecular species of triacylglycerols from milk

fat have also been resolved by HPLC in the reversedphase mode with flame-ionization detection with the
Tracor instrument [95]. Once a number of practical
difficulties associated with the detector were
resolved, the response was found to be linear over a
wide range of solute levels above 5 micrograms.
Below this value, there was some drop in response
thought to be associated with evaporation of the
sample prior to combustion. In addition, the response
was dependent on the nature of the fatty acid
constituents in the molecular species. Similar results
were described for separations of molecular species
of triacylglycerols separated by silver ion
chromatography [36]. While substantial differences in
response according to degree of unsaturation were
noted in this study, no such problems were described
in a more extensive description of the technique [47].
The response of the Tracor detector was linear over
a wide range and it was possible to use the
instrument for routine analyses of confectionery fats
in an industrial context.
A further important area for HPLC is the
separation of lipid classes. Maxwell et al. [73] appear
to have been the first to describe an application of the
TM
Tracor
detector to this problem. They used a
column of silica gel and a complex gradient system of
dichloromethane-hexane
to
dichloromethanechloroform followed by the introduction of ammonia in
order to separate lipids ranging in polarity from sterol
esters to phosphatidylcholine. The repeatability of
injections was found to be good, and the detector
response was linear for each component over a wide
range of practical concentrations. Some variation in
response that depended on the nature of the lipid
classes was observed. Others [33] used a related
system but with a gradient of chloroform to
chloroform-methanol-ammonia
to
separate
triacylglycerols and the individual phospholipids of
soybean lecithin. Moreau and colleagues [84,85]
adapted a method described earlier by Christie [12]
for evaporative light-scattering detectors (discussed
in greater detail in Section E.2 below) to the
separation of plant lipids with flame-ionization
detection. With a column of silica gel and a complex
ternary gradient system, individual simple lipid
classes were eluted first followed by glycolipids and
then by each of the phospholipids. Although some
base-line drift was evident, the progress of the
separation was clearly monitored (see also Figure 7
below). The response of the detector was close to
linear over a range of 1 to 200 micrograms for most
lipid classes, but this did not appear to be true for
triacylglycerols or free fatty acids. Some loss through
evaporation of the last appears inevitable. Hammond
has reported some success with similar methodology
without giving full details [36].
TM
The Tracor
detector was used to quantify
monomer, dimer and polymeric acids in commercial
"dimer" mixtures following HPLC separation on a
column of silica gel [137]. With careful calibration, it
was possible to obtain results of good reproducibility,
but the response factors for each component were
very different. That for the dimer was 2.35 times
greater than the factor for the monomer, for example.
On the other hand, linear responses and small
differences only with structural features were
observed for cholesterol and its oxidation products,
separated by HPLC on silica gel and with flameionization detection [70].
It is easy to exaggerate the problems of using
TM
the Tracor and related detectors for quantification
of lipids, especially those associated with the
variation in response due to small structural
differences in the nature of the fatty acyl constituents.
For example, spectrophotometric detectors have very
little to offer in comparison, other than ready
availability in laboratories [85]. After calibration with
suitable standards and careful purification of mobile
phases, transport flame-ionization detectors are
being used for routine quantification in analyses of
various kinds. It is also possible to insert a stream
splitter between the end of the column and the
detector to collect fractions for analysis and
quantification by other means, and this is a
satisfactory approach in many research applications
especially.
E. EVAPORATIVE LIGHT-SCATTERING
DETECTORS
1.
Construction and the nature of the response
An optical detection system for HPLC that is now

assisting lipid analysts to make important advances
has been variously termed a "mass detector",
"evaporative analyser" or "light-scattering detector".
The term "evaporative light-scattering detector"
seems most accurate if rather lengthy; the pithy
expression "mass detector" is easily confused with
mass spectrometry by the unwary. With the
instrument first described by Charlesworth [11] in
1978, the solvent emerging from the end of the
column is evaporated in a stream of air or nitrogen in
a heating chamber (Figure 3); the solute does not
evaporate, but is nebulized and passed in the form of
minute droplets through a light beam, which is
reflected and refracted. The scattered light is
measured by a photomultiplier tube, set at an
appropriate angle, and bears a relationship to the
amount of material in the eluent. Such detectors are
truly universal in their applicability, in that they will
respond to any solute that does not evaporate before
passing through the light beam.
Fig. 3. A schematic diagram of an evaporative lightscattering detector.
The first commercial detector based on this

principle is very similar in construction to the
illustration and is available at a cost comparable to
that of other optical detectors from Applied
Chromatography Systems (ACS) Ltd (Macclesfield,
Cheshire, U.K.). There are at least three competitors
now including instruments from Varex Corp.
(Burtonsville, MD, U.S.A.), Cunow S.A. (Cergy St.
Cristophe, France) and Sedere (Vitry-sur-Seine,
France), some of which have distinctive design
features with the reported objectives of improving
sensitivity and linearity. For example in the Cunow
detectors, the larger droplets in the spray from the
nebulizer are condensed out before they reach the
heater chamber; the consequence is a more uniform
particle size and improved linearity. The Varex
detector has a laser light source and a photodiode
detector instead of a photomultiplier tube. Real
differences therefore exist among commercial
instruments and no objective comparison has yet
been made. There are no special wavelength
requirements for the light source, and in the first
commercial instruments, it was a simply a lamp for a
slide projector. On the other hand, a prototype
experimental detector has been described that uses a
laser light source [118,120], and at least one
commercial instrument has this feature.
Evaporative light-scattering detectors can be
considered to be universal in their applicability, in that
they will respond to any solute that does not
evaporate before passing through the light beam.
Almost any solvent, including ketones, esters and
chlorinated and aromatic compounds, can be used in
complex gradients; up to 20% of water and small
amounts of ionic species are also permissible. In

designing an elution system to effect a particular
separation, the analyst can therefore make use of the
complete range of solvent selectivity groups as
defined by Snyder [113,114] and recently reevaluated [107]. Further advantages are that such
detectors are simple, versatile and rugged in use,
requiring little time for the base-line to stabilize when
started up at the beginning of the day or when
columns and mobile phases are changed; little or no
adjustment may be required during lengthy periods of
operation even with complex gradients. The
sensitivity in the newer instruments is at least equal to
that of the very best refractive index detectors, and is
better than in transport-flame ionization detectors or
in UV spectrophotometric detectors operated at low
wavelengths. A further advantage is that evaporative
light-scattering detectors are not affected by changes
in ambient temperature or small variations in the flowrate of the mobile phase.
As with all detectors, there are some
disadvantages. Although the detector is destructive in
that the sample is lost in the stream of air, it is
possible to insert a stream splitter between the end of
the column and the detector to divert much of the
eluent to a collection device. A source of dry, filtered
compressed air, that is capable of delivering 5
litres/min, is required and in practice, this means that
an air compressor must be used; a standard cylinder
of air or nitrogen is emptied in about 4 hours at this
rate. For safety reasons (and to avoid the risk of
litigation), most manufacturers recommend the use of
nitrogen or carbon dioxide as evaporator gas when
inflammable solvents are employed, but this is
impracticable for prolonged usage. In practice, the
concentration of organic solvent in the gas stream at
any given moment should be very low and the risk of
explosion with air as the carrier gas is minimal. The
stream of air containing the evaporated solvent must
be conducted directly to the outside of the laboratory
or into a fume cupboard. There are few limitations on
the range of solvents that are suitable, and in
particular they must be sufficiently volatile to
evaporate in the heating chamber; the author [12] has
used isopropanol containing 15% water without
difficulty, but formic or acetic acids at a level of about
1% caused "spikes" on the base-line. Small but
significant amounts of salts or ionic materials can be
incorporated as ion suppressants into the mobile
phases, without adversely affecting base-line stability,
when the detector is operated at optimum sensitivity
levels [13]. Indeed, there has even been a report of
the use of 0.1M aqueous ammonium acetate as a
mobile phase, though at the cost of a ten-fold
reduction in detector sensitivity [86]. Limited
experience in the author's laboratory with the Cunow
detector indicates that the aqueous component and
any ionic species condense out preferentially in
advance of the evaporator chamber, with the result

that the base-line remains stable even at high
sensitivity settings.
A key factor with all detectors is their linearity
with respect to sample size, and investigators
working with different evaporative light-scattering
detectors have described the response variously as
linear, sigmoidal or exponential. In the most
comprehensive
practical
and
theoretical
investigations, use was made of the ACS detector but
the results appear to be applicable to other
commercial systems [90,97], and they compared well
with data obtained from a custom-built detector with a
laser as the light source [118,120]. It was observed
that the detector response increased sigmoidally with
increasing sample concentration, in a manner that
could be predicted by changes in the size distribution
of particles in the aerosol. Thus at low solute
concentrations, the solute particles scattered light to
a proportionately lesser extent. As the diameter of the
droplets began to approach the wavelength of light,
they no longer affected its passage and the response
fell off rapidly. The detector response was close to
linear over a concentration range of about two
decades before tending to plateau. To maximize the
response and the linear range, it was necessary to
adjust the flow-rate of the nebuliser gas and the
temperature of the evaporator chamber to the
optimum to give aerosol particles that were relatively
uniform in size. However, small variations from the
optimum temperature were found to have
comparatively little effect on the response, which was
relatively constant at fixed gas pressures. The same
response was obtained for a given solute when the
mobile phase was toluene, 2-butanone or
tetrahydrofuran. In general, it was concluded that
solvent viscosity was of relatively little importance,
although anomalous results were obtained with
dichloromethane, apparently because of its much
greater density. Finally, the response was dependent
on the refractive index of the sample and thus might
be expected to vary with changes in structural
features of lipids. Different lipid classes therefore
require different response factors, although it is the
author's experience (and that of others [119]) that the
chain-length and degree of unsaturation of the acyl
constituents does not have a significant effect on
response.
In summary, good quantitative results could be
obtained if the instrumental parameters were
adjusted carefully, and if the calibration conditions
were rigidly set to be the same as in the analysis of
real samples. An important note for manufacturers
was that the design of the nebuliser was crucial, and
this should be constructed to produce particles of
uniform size in the optimum range. For lipid analysts,
the eventual test is how these conclusions are borne
out in practice with real samples, and happily they
have been confirmed by such empirical studies (see

below). In the custom-built detector incorporating a
laser light source, the response was found to be
related to the solute mass raised to the power of 1.35
[99,118,120]. This relationship has proved useful in a
number of analytical circumstances and at least one
manufacturer is offering a "linearizer" in which this
formula is used electronically to "improve" the results.
2.
Applications of light-scattering detection in

lipid class separations
One of the more important problems in lipid

analysis is the separation of the classes of tissue
lipids, which at the extremes of polarity consist of
cholesterol
esters
(hydrocarbon-like)
and
lysophospholipids (water-soluble) with a broad
spectrum of simple and complex lipids with differing
properties in between. Considerable progress was
made with the aid of the evaporative light-scattering
detector in the author's laboratory [12]. The initial
objective was to separate and quantify the more
abundant lipid classes in animal tissues, on the 0.2 to
0.4 mg scale and in as short a time as could
conveniently be managed. The ACS mass detector
was utilized, together with a ternary solvent delivery
system and a short (5 x 100 mm) column packed with
TM
Spherisorb
silica gel (3 micron particles). When
solvents were selected for the mobile phase, the
choice was constrained by the need for sufficient
volatility for evaporation in the detector under
conditions that did not cause evaporation of the
solute, and at first by a perceived necessity to avoid
ionic species, which would not evaporate. Similar
restrictions would have applied with detectors
operating on the transport-flame ionization principle.
To achieve the desired separation, it was necessary
to use a complicated ternary-gradient elution scheme
with eight programmed steps, starting with isooctane
to separate the lipids of low polarity and ending with a
solvent containing water to elute the phospholipids; a
solvent of medium polarity was then needed to
mediate the transfer from one extreme to the other,
and mixtures based on isopropanol gave satisfactory
results. The three solvent mixtures selected by trial
and error were isooctane-tetrahydrofuran (99:1,
v/v)(A), isopropanol-chloroform (4:1, v/v)(B) and
isopropanol-water (1:1, v/v)(C). In the first successful
elution scheme, a gradient of B into A was created to
separate each of the simple lipids, then a gradient of
C into A plus B was produced to separate each of the
complex lipids; finally, a gradient was generated in
the reverse direction to remove most of the bound
water and to re-equilibrate the column prior to the
next analysis. A high flow-rate (2 mL/min) appeared
to
assist
the separation greatly, perhaps
compensating for the absence of strong acid or
inorganic ions, which others have found necessary
Fig. 4. Separation of lipids (0.35 mg - injected in 5 microlitres of solvent) from rat liver on a column (100 x 5 mm) of silica gel
TM
(Spherisorb - 3 micron) with evaporative light-scattering detection (ACS model) [12]. (Reproduced by kind permission of the
Journal of Lipid Research). The elution times of lipids not present in this particular sample are indicated. Abbreviations: CE,
cholesterol esters; TG, triacylglycerols; C, cholesterol; DG, diacylglycerols; FA, free acids; CER, cerebrosides; PG,
phosphatidylglycerol; DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PC,
phosphatidylcholine; SPH, sphingomyelin; MG, monoacylglycerols; LPE, lysophosphatidylethanolamine; PS,
phosphatidylserine; LPC, lysophosphatidylcholine; PMME, phosphatidylmonomethylethanolamine; PDME,
phosphatidyldimethylethanolamine.
for the separation of phospholipids. However, it was

eventually found to be advantageous to add small
amounts of organic ionic species (see below) [13].
The nature of the separation achieved with the
total lipids extracted from rat liver is shown in Figure
4. In spite of the abrupt changes in the composition of
the mobile phase at various times, no disturbance of
the base-line was apparent, and each of the main
simple lipid and phospholipid classes was cleanly
resolved in only 20 minutes. Only the highly acidic
phospholipids, phosphatidic acid and to a lesser
extent phosphatidylserine, did not give satisfactory
peaks. No "solvent peak" was apparent at the start of
the analysis, as is often seen with other detectors,
and di-tert-butyl-p-cresol (BHT) added to the extract
as an antioxidant evaporated with the mobile phase
and did not interfere. After a further 10 minutes of
elution to remove bound water from the silica gel to
restore its activity, the next sample could be
analysed.
It was subsequently shown that much better
resolution of the minor acidic components could be
obtained by adding small amounts of ionic species to
the aqueous component of the mobile phase [13]. In
addition, the lifetime of the column was greatly
extended by this simple step. The optimum results

were obtained in practice with 0.5 to 1 mM serine
buffered to pH 7.5 with triethylamine. Perhaps
surprisingly, ionic species at such concentrations had
virtually no effect on the base line of the detector,
although the response changed somewhat and recalibration was necessary. A further change was
made to replace isooctane with hexane in the mobile
phase, in order to reduce the maximum operating
pressure required.
When evaporative light scattering detector are
used directly in quantitative analysis, it is necessary
to work out the optimum conditions for the desired
separations first and then carry out a calibration with
lipid standards which are as close as possible in
composition to the material to be analysed. The
operating parameters for the instrument, such as gas
pressure, evaporator temperature, and attenuation,
must also be rigorously standardized. If the elution
conditions or detector settings have later to be
changed for any reason, a tedious re-calibration
might be necessary. Figure 5 shows calibration
curves for some of the main lipid classes in the
analysis described. A different line was obtained for
each lipid. With most, the response of the ACS mass
detector was approximately rectilinear in the range 50

to 200 micrograms, but it tended to fall off rapidly
below 10 micrograms. Rather better results have
been obtained with the newer instruments on the
market, both in terms of linearity and sensitivity (see
below). However, with careful calibration, the data
obtained in lipid analyses of this kind with the ACS
detector were found to be at least as reliable in terms
of accuracy and reproducibility as those from any
other analytical method in use with lipids [12].
resolution of phospholipid components [67]. In this

work, the Varex ELSD II light scattering detector,
which is capable of much higher sensitivity, was
employed but calibration curves were obtained that
were similar in shape to those in Figure 5, if differing
appreciably otherwise. N-oleoylethanolamine was
utilized as an internal standard. The original
procedure has been adapted somewhat for the
analysis of phospholipids alone, in this instance for
detection with a Cunow LSD Model 10 light-scattering
detector [48]. Cholesterol was employed as internal
standard, and the response of the detector for each
phospholipid relative to cholesterol was linear over a
wide range. Similar findings had been reported earlier
for a Cunow detector with a different gradient elution
system in analyses of phospholipids [7].
Fig. 5. Calibration curves of detector response against

amount of sample injected for some lipid classes
separated by HPLC with evaporative light-scattering
detection (ACS model) [12]. (Reproduced by kind
permission of the Journal of Lipid Research).
It is also possible to use an internal standard to

improve direct quantification with evaporative lightscattering detectors. For example, a synthetic
phospholipid,
phosphatidyldimethylethanolamine
(dipalmitoyl), was used in this way in order that the
absolute amount of phospholipid in an extract could
be determined in addition to the relative compositions
of each phospholipid class [23]. This lipid is only
present naturally at very low levels in tissues so
endogenous material does not interfere with the
standard. The method was applied to determine the
phospholipids of milk, which comprise only about
0.5% of the total lipids, and in order to analyse them
effectively it was necessary to first obtain a
concentrated fraction by a solid-phase extraction
method before subjecting this to HPLC as shown in
Figure 6. The chromatographic system employed the
ACS mass detector and the ternary-gradient elution
scheme described above.
Recently others described a modified gradient
elution procedure based on the above to improve the
Fig. 6. HPLC separation of a phospholipid fraction from

cow's milk on silica gel with a ternary elution scheme and
evaporative light-scattering detection (ACS model);
phosphatidyldimethylethanolamine was the internal
standard [23]. (Reproduced by kind permission of the
authors and of the Journal of the Society of Dairy
Technology). See the legend to Figure 4 for a list of
abbreviations (X is an unknown).
The author [22] modified his own method in

order to better resolve the distinctive phospholipids
and glycosyldiacylglycerols found in plant and
especially cereal tissues. To elute sequentially monoand digalactosyldiacylglycerols and N-acyl-phosph-
atidylethanolamine and their lyso forms, hexanebutan-2-one-acetic acid (35:65:0.4, v/v/v) was used
as part of a gradient before the more usual
phospholipids emerged with the elution scheme
described above. Following an earlier adaptation of
this methodology for the Tracor transport-flame
ionization detector (see Section D.2), Moreau [84]
demonstrated that much better results could be
obtained with the Varex light-scattering detector and
a comparison of the two is illustrated in Figure 7. The
improved sensitivity and base-line stability of the latter
is very evident. In this instance, no chloroform was
required in the mobile phase (as there was no need
to resolve sphingomyelin) and additional gradient
steps were introduced to separate each of the
glycolipids ahead of the phospholipids. Brief details
only of a completely different elution scheme for the
separation of both simple and complex lipids in wheat
flour have been published; a quaternary gradient
system was required, starting with toluene-containing
0.125% formic acid, and proceeding via ethyl acetate,
methanol and water-based mixtures [37]. In this
instance, the ACS mass detector was utilized.
Many lipid analysts have used evaporative lightscattering detection and columns of silica gel to
separate phospholipid classes in the absence of
simple lipids. For example, Stolyhwo et al. [121] used
a gradient of ammonia in methanol in essence to
obtain excellent resolution of phospholipid classes
from plasma, and from rapeseed and soybean

lecithins. Others used a similar elution system
containing an appreciable concentration of chloroform
[4]. In studies of both preparative [45,134,135] and
analytical
[25,136]
scale
separations
of
phospholipids, various gradients of hexaneisopropanol-water and increasing in polarity were
generated for specific purposes.
Similarly, it has proved possible to use lightscattering detection and gradients of isopropanol in
isooctane to separate all the simple lipid classes (and
not the phospholipids) in some samples of
commercial interest [8]. In other work, a column
containing an octadecylsilyl-bonded phase was
employed to separate simple lipid classes, i.e. tri-, diand monoacylglycerols and free acids, with some
simultaneous fractionation into molecular species; the
mobile phase here was a gradient of acetonitrile,
acetone and dichloromethane [25].
Much remains to be done to improve the
conditions for the separation of lipid classes and
especially for the simultaneous separation of simple
and complex lipids by means of HPLC. Many more
solvent combinations must be tried, and more column
packing materials must be tested for the purpose.
However, as evaporative light-scattering detectors
appear in more laboratories, there is now a solid
foundation upon which to build.
Fig. 7. Comparison of the analysis of lipid classes from corn coleoptiles by HPLC with transport-flame ionization detection (FID) (Tracor model)
and evaporative light-scattering detection (ELSD) (Varex model) (B), each with 125 micrograms of lipid in total [84]. Reproduced by kind
permission of the author and of Portland Press. Abbreviations a, sterol esters; b, triacylglycerols; c, sterols; d, free fatty acids; e, acylated sterol
glycosides; f, monogalactosyldiacylglycerols; g, sterol glycosides; h, digalactosyldiacylglycerols; i, cardiolipin; j, phosphatidylethanolamine; k,
phosphatidylglycerol; l, phosphatidylinositol; m, phosphatidylcholine.
3.
Applications of light-scattering detection in

separations of molecular species of lipids
Because of their commercial importance,

methods for the separation of molecular species of
triacylglycerols have been studied with particular
intensity, most analysts preferring to work with
reversed-phase systems. ODS columns have been
used universally with those with a high carbon content
generally being preferred. While acetonitrile is an
essential component of the mobile phase, it must
contain a substantial proportion of a non-polar
modifier to solubilize the lipid and displace it from the
stationary phase. There is no consensus as to which
is the best modifier solvent and the choice has often
been determined by the availability of a particular
detector. There is no such practical limitation with the
evaporative light-scattering detector, which has been
shown to be at least as sensitive as any other to have
been used for the purpose, and exhibited no baseline drift, even with lengthy gradients [34,41,98100,105,106,119,132,133]. When solvent restrictions
are removed, it is also easier to ensure that there are
no problems over the solubility of the solute in the
mobile phase as has sometimes been observed [14].
In nearly all the published work to date, the
Applied Chromatography Systems (ACS) detector
has been used. As with the lipid class separations
discussed above, Robinson and Macrae found that it
was essential for all the instrumental and
chromatography parameters, such as nebuliser inlet
pressure, evaporator temperature, mobile phase and
flow-rate, to be standardized prior to analysis, since
changes in any of these could affect the response
[105]. The detection limits were of the order of 1
microgram for most triacylglycerol standards, but
much better results can now be obtained with the
newer commercial instruments. As expected, the
response with respect to mass was found to be
sigmoidal for all triacylglycerol standards, but was
approximately linear in the range 10 to 25
micrograms for saturated compounds (limited by
solubility problems with the mobile phase selected for
the work), and in the range 10 to 60 micrograms for
unsaturated compounds. The logarithm of the
detector response was rectilinear with respect to the
logarithm of sample mass. However, the slopes of
the lines for different standards were not found to be
parallel, and there was no obvious relationship to fatty
acyl structure. This has not been the author's
experience or that of others [41,119], who have found
very little effect of the nature of the fatty acyl group on
the response. For example, with the same type of
detector, Herslof and Kindmark [41] obtained an
identical detection limit, but the reproducibility of the
analysis of molecular species of triacylglycerols from

soybean oil was found to be quite acceptable, even
with gradient elution; relative standard deviations of
2% were obtained for the main components. One
reason for the better results obtained in the latter
work was that the data were expressed in terms of
the relative proportions (percentages) of the different
components, and not in terms of the absolute
amounts of each. Similar data were reported by
others, but with greater variation for minor
components [34]. Again better results would be
anticipated with the newer commercial instruments.
Silver ion chromatography of triacylglycerols with
light-scattering detection has been employed to good
effect in the author's laboratory. The stationary phase
consisted of an ion-exchange medium, which was a
silica gel matrix with bonded phenyl sulphonic acid
TM
moieties (Nucleosil 5SA), and it was converted to
the silver ion form by injection of silver nitrate into an
TM
aqueous mobile phase via the Rheodyne
valve
[15]. With this column, it was possible to separate
molecular species according to the single property of
degree of unsaturation. As an example, a separation
of the triacylglycerols of rat adipose tissue on a silver
ion column is illustrated in Figure 8 [16]. The
trisaturated fraction is eluted first, followed by
disaturated-monoenes,
saturated-dimonoenes,
disaturated-dienes, trimonoenes and so forth. With
this system, one dienoic acyl group is retained by the
equivalent of about 2.5 monoenes and one trienoic
acyl group by the equivalent of about two dienes.
Species containing gamma-linolenic acid, as in
evening primrose oil, were eluted before those with
alpha-linolenic acid [18]. The mobile phase was a
gradient of dichloromethane-dichloroethane (1:1, v/v)
to acetone to elute the more saturated fractions,
before acetonitrile was introduced to bring off
polyunsaturated species. Of course, the lightscattering detector is ideal for such a combination of
solvents. By increasing the acetonitrile content, it was
possible to obtain a comprehensive separation of
triacylglycerols as unsaturated as that from linseed
oil, where the predominant molecular species had
nine double bonds, while species with up to 15
double bonds were separated from fish oils [16,65].
More subtle separations have also been reported.
Thus with meadowfoam oil, which contains a number
of different monoenoic fatty acids, species were
resolved in which the only difference was the position
of one double bond in one of the three fatty acids in
the molecule [89].
The technique has been applied similarly to the
separation of molecular species of cholesterol esters
with light-scattering detection [44].
TM
Fig. 8. Separation of molecular species of triacylglycerols from rat parametrial adipose tissue on a column of Nucleosil 5SA
(250 x 4.6 mm i.d.) in the silver ion form with light-scattering detection (ACS Model) [16]. The mobile phase consisted of a
gradient of acetone into dichloroethane-dichloromethane (1:1, v/v) to elute the more saturated fractions before acetonitrile was
introduced to bring off the polyunsaturated components. Abbreviations: S, saturated; M, monoenoic; D, dienoic; T, trienoic acyl
groups. Reproduced by kind permission of the Journal of Chromatography.
The major virtue of silver ion chromatography in

comparison
to
reversed-phase
partition
chromatography is that separation with the former is
based on the single property of degree of
unsaturation and fractions are intuitively easy to
identify. With reversed-phase chromatography in
contrast, separation is according to both the
combined chain-lengths and the total number of
double bonds in the fatty acyl groups, each double
bond reducing the retention time by the equivalent of
about two carbon atoms. Excellent resolution is
possible, but it is not easy to identify fractions when
unknown samples are analysed. When the two
techniques are used to complement each other,
analysts have a particularly powerful separatory tool.
Silver ion chromatography provides separation by
degree of unsaturation, and fractions collected can
then be resolved by reversed-phase HPLC when
resolution is then simply according to the combined
chain-lengths of the fatty acyl residues. The virtues of
this approach, for HPLC with light-scattering
detection, have been demonstrated for meadowfoam
oil [89] and for some fish oils [64].
As yet the evaporative-light scattering detector
has been little used for molecular species of
phospholipids, although the potential has been
indicated in one paper [115], and further applications
will no doubt follow.
4.
Some miscellaneous separations
HPLC with evaporative light-scattering detection

has not been used for the analysis of fatty acids per
se, as they are too volatile, but it has proved its worth
as a micro-preparative tool for the isolation of
fractions for analysis by other means. In such
circumstances, a stream-splitter must be placed
between the end of the column and the detector. For
example, picolinyl ester derivatives of fatty acids were
separated on the 1-2 mg scale by reversed-phase
chromatography by gradient elution with mobile
phases containing pyridine and acetic acid among
other components [24]; fractions were subsequently
analysed by GC-MS. It is not easy to see how any
other type of HPLC detector could have coped with
this solvent combination. Silver ion HPLC has been
applied in a similar manner to the separation of
methyl esters of fatty acids with zero to six double
bonds, again as an aid to identification by GC-MS
[2,19,20,116].
The light-scattering detector was utilized in the
detection and quantification of tocopherols and
phytosterols from seed oils separated by reversedphase HPLC [139]. The response to each component
differed somewhat but was essentially linear over the
range 10 to 100 micrograms with an error of 2% over
five analyses.
Gel permeation chromatography with lightscattering detection has been employed to separate
and quantify polymers in oxidized fish oils [9]. In this
instance, glycerol was used as an internal standard
and careful calibration was necessary; although the
response to the standard was almost linear over a
wide range, this was not true of the polymer fraction.
Abbreviations: GC, gas chromatography; HPLC,
high-performance liquid chromatography; IR, infrared;
MS, mass spectrometry; ODS, octadecylsilyl; RI,
refractive index; UV, ultraviolet.
16.
17.
18.
19.
20.
21.
22.
Acknowledgement: This work has been funded in

part by the Scottish Executive Environmental and
Rural Affairs Department.
23.
24.
25.
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Detectors for HPLC of lipids with special reference to evaporative light-scattering detection

DETECTORS FOR HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF

general [59,109,110] and phospholipids and

For some time, it was thought that detectors

B. OPTICAL AND SPECTROPHOTOMETRIC

Refractive index (RI) detectors of three different

Spectrophotometric detectors in the ultraviolet

great selectivity and sometimes sensitivity in the

preparative method for the isolation of fatty acids for

purity since traces of extraneous materials with

a standard mixture very similar to those to be

Fig. 1. Calibration lines for the quantification of cholesterol

Because variation in the degree of unsaturation

Only a few rare lipids exhibit natural

real competition to GC in some circumstances, and

Infrared spectrophotometric detectors

Infrared (IR) detectors have been used to a

The content of particular analytes in an HPLC

described are chemical determination of phosphate in

C. SOME MISCELLANEOUS DETECTION

The mass spectrometer as an HPLC detector

Mass spectrometry (MS) is a powerful analytical

desorption technique, in which the sample is

A recent paper in which an HPLC micro-column

Methods for detecting radioisotopes are often

aid to quantification of specific lipids [14]. In a recent

Density, electrochemical and other detectors

A detector in which the changes in the density of

A transport-flame ionization detector was first

carbon dioxide and reduced to methane before

evaporated in an air-flow of controlled temperature

Applications of transport-flame ionization

The early literature on the subject of applications

In particular, he stresses the importance of setting up

Fig. 2. Separation of molecular species of

Molecular species of triacylglycerols from milk

Construction and the nature of the response

An optical detection system for HPLC that is now

Fig. 3. A schematic diagram of an evaporative lightscattering detector.

The first commercial detector based on this

amounts of ionic species are also permissible. In

advance of the evaporator chamber, with the result

have been confirmed by such empirical studies (see

Applications of light-scattering detection in

One of the more important problems in lipid

for the separation of phospholipids. However, it was

extended by this simple step. The optimum results

detector was approximately rectilinear in the range 50

resolution of phospholipid components [67]. In this

Fig. 5. Calibration curves of detector response against

It is also possible to use an internal standard to

Fig. 6. HPLC separation of a phospholipid fraction from

The author [22] modified his own method in

from plasma, and from rapeseed and soybean

Applications of light-scattering detection in

Because of their commercial importance,

analysis of molecular species of triacylglycerols from

The major virtue of silver ion chromatography in

Some miscellaneous separations

HPLC with evaporative light-scattering detection

Acknowledgement: This work has been funded in

Abian,J. and Gelpi,E., J. Chromatogr., 394,