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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
W.W. Christie, in Advances in Lipid Methodology One, pp. 239-271 [1992] [Ed. W.W. Christie, Oily Press, Ayr
Detectors for HPLC of lipids with special reference to evaporative light-scattering detection
Differential refractometry
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
W.W. Christie, in Advances in Lipid Methodology One, pp. 239-271 [1992] [Ed. W.W. Christie, Oily Press, Ayr
Detectors for HPLC of lipids with special reference to evaporative light-scattering detection
W.W. Christie, in Advances in Lipid Methodology One, pp. 239-271 [1992] [Ed. W.W. Christie, Oily Press, Ayr
Detectors for HPLC of lipids with special reference to evaporative light-scattering detection
Fluorescence detection
W.W. Christie, in Advances in Lipid Methodology One, pp. 239-271 [1992] [Ed. W.W. Christie, Oily Press, Ayr
Detectors for HPLC of lipids with special reference to evaporative light-scattering detection
Spectrophotometric detection
column chemical reaction
with
post-
W.W. Christie, in Advances in Lipid Methodology One, pp. 239-271 [1992] [Ed. W.W. Christie, Oily Press, Ayr
Detectors for HPLC of lipids with special reference to evaporative light-scattering detection
Radioactivity detectors
W.W. Christie, in Advances in Lipid Methodology One, pp. 239-271 [1992] [Ed. W.W. Christie, Oily Press, Ayr
Detectors for HPLC of lipids with special reference to evaporative light-scattering detection
D. TRANSPORT-FLAME IONIZATION
DETECTORS
1.
Apparatus
W.W. Christie, in Advances in Lipid Methodology One, pp. 239-271 [1992] [Ed. W.W. Christie, Oily Press, Ayr
Detectors for HPLC of lipids with special reference to evaporative light-scattering detection
W.W. Christie, in Advances in Lipid Methodology One, pp. 239-271 [1992] [Ed. W.W. Christie, Oily Press, Ayr
Detectors for HPLC of lipids with special reference to evaporative light-scattering detection
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.
W.W. Christie, in Advances in Lipid Methodology One, pp. 239-271 [1992] [Ed. W.W. Christie, Oily Press, Ayr
Detectors for HPLC of lipids with special reference to evaporative light-scattering detection
W.W. Christie, in Advances in Lipid Methodology One, pp. 239-271 [1992] [Ed. W.W. Christie, Oily Press, Ayr
Detectors for HPLC of lipids with special reference to evaporative light-scattering detection
W.W. Christie, in Advances in Lipid Methodology One, pp. 239-271 [1992] [Ed. W.W. Christie, Oily Press, Ayr
Detectors for HPLC of lipids with special reference to evaporative light-scattering detection
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.
W.W. Christie, in Advances in Lipid Methodology One, pp. 239-271 [1992] [Ed. W.W. Christie, Oily Press, Ayr
Detectors for HPLC of lipids with special reference to evaporative light-scattering detection
W.W. Christie, in Advances in Lipid Methodology One, pp. 239-271 [1992] [Ed. W.W. Christie, Oily Press, Ayr
Detectors for HPLC of lipids with special reference to evaporative light-scattering detection
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
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.
W.W. Christie, in Advances in Lipid Methodology One, pp. 239-271 [1992] [Ed. W.W. Christie, Oily Press, Ayr
Detectors for HPLC of lipids with special reference to evaporative light-scattering detection
3.
W.W. Christie, in Advances in Lipid Methodology One, pp. 239-271 [1992] [Ed. W.W. Christie, Oily Press, Ayr
Detectors for HPLC of lipids with special reference to evaporative light-scattering detection
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.
4.
W.W. Christie, in Advances in Lipid Methodology One, pp. 239-271 [1992] [Ed. W.W. Christie, Oily Press, Ayr
Detectors for HPLC of lipids with special reference to evaporative light-scattering detection
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.
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20.
21.
22.
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W.W. Christie, in Advances in Lipid Methodology One, pp. 239-271 [1992] [Ed. W.W. Christie, Oily Press, Ayr
Detectors for HPLC of lipids with special reference to evaporative light-scattering detection
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W.W. Christie, in Advances in Lipid Methodology One, pp. 239-271 [1992] [Ed. W.W. Christie, Oily Press, Ayr
Detectors for HPLC of lipids with special reference to evaporative light-scattering detection
W.W. Christie, in Advances in Lipid Methodology One, pp. 239-271 [1992] [Ed. W.W. Christie, Oily Press, Ayr