Table Of ContentANALYSIS OF
TRIGLYCERIDES
CARTER LITCHFIELD
Department of Biochemistry
Rutgers University
New Brunswick, New Jersey
1972
A C A D E M IC P R E SS N ew York and London
Copyright © 1972, by Academic Press, Inc.
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Leslie Froomes
Tex Isbell
Woolsey Motl
Raymond Reiser
teachers in lipid research
PREFACE
Perhaps in no field of study have the tools of the lipid chemist been more
inadequate for the task presented (than) in the study of glyceride structure.*
H. J. Button
Dutton wrote these words in 1955 as he announced the first successful
resolution of a natural triglyceride mixture (linseed oil) by countercurrent
distribution. How true they were then! From Chevreul's initial character-
ization of natural fats as glyceryl esters of fatty acids in 1815 up until
1955 only two relatively unsophisticated techniques were available for
determining the triglyceride composition of natural fats: fractional crystal-
lization and permanganate oxidation. Because of the limitations of these
methods, natural fats were usually thought of as mixtures of trisaturated,
disaturated-monounsaturated, monounsaturated-diunsaturated, and triun-
saturated types of triglycerides rather than as made up of individual
molecular species as is truly the case.
Between 1955 and the present, the powerful new analytical techniques
of silver ion adsorption chromatography, liquid-liquid partition chromatog-
raphy, gas-liquid chromatography, pancreatic lipase hydrolysis, and stereo-
specific analysis were introduced and have completely revolutionized the
methodology of the field. Using these new tools, it is now possible to dis-
tinguish individual molecular species of triglycerides such as src-l-palmito-
2-oleo-3-stearin and sn-l,2-dipalmito-3-olein in cocoa butter.
* H. J. Dutton, /. Amer. Oil Chem. Soc. 32, 652 (1955).
xvi PREFACE
This rapid growth of a new and complex methodology brings with it
the need to decide which techniques can best accomplish a given type of
analysis for a specific research problem. This monograph was written to
provide a comprehensive reference source for those seeking more infor-
mation on the subject. All reported methods are discussed, and the relative
merits and limitations of each are evaluated. Numerous illustrations of
practical examples are provided, and applications of both individual
techniques and appropriate combinations are described.
Carter Litchfield
ACKNOWLEDGMENTS
No one writes a first book without realizing that there is more work
to bookwriting than he originally thought. It has been my good fortune to
have numerous friends and co-workers who have made the task much
easier.
I am especially grateful to Frank Gunstone and to Carol Litchfield who
read the entire first draft of the manuscript and made many helpful
suggestions for its improvement. I also extend my personal thanks to Bob
Ackman, Hans Brockerhoff, Bill Christie, Mike Coleman, Earl Hammond,
Bob Harlow, Bob Jensen, Fred Padley, Madhu Sahasrabudhe, A. G.
Vereshchagin, and Herbert Wessels who reviewed individual chapters in
their own areas of specialization. Their advice and comments proved
invaluable.
Linda Fisher aided immensely with the editorial work in assembling
the final copy; and Diane Cranfield, Anne Greenberg, Susan West, Karen
Whitworth, and Dolores Young helped in typing and proofreading the
manuscript at various stages in its progress.
To all of these helpful friends and to the many others who answered
my innumerable questions along the way, I extend my hearty thanks.
xvii
1
INTRODUCTION
The vital role of triglycerides in human life and activities is familiar
to almost all who read these lines. Triglycerides are a major form of energy
storage for both plants and animals. Man draws upon these sources to
provide fatty foods for himself and to obtain fats and oils as industrial
raw materials. To better understand these biosynthetic, metabolic, and
technological processes involving triglycerides, chemists have developed
numerous analytical techniques for characterizing complex triglyceride
mixtures.
Two factors make the analytical chemistry of natural fat triglycerides
exceptionally difficult: (i) the extremely large number of possible molecu-
lar species (Section I,B)> and (ii) the very similar chemical and physical
properties of most of these molecules. Using the classical techniques of
fractional crystallization and permanganate oxidation, only simple separa-
tions of groups of triglycerides were possible, and most analyses were semi-
quantitative in nature. Between 1956 and 1965, however, a series of new
chromatographic and enzymatic techniques revolutionized the field, and
many of the earlier difficulties have now been overcome. With this pro-
liferation of analytical methods, the former question, "Can I analyze for
XYZ triglyceride content?" has now changed to, "Which method should
I use to analyze for XYZ triglyceride content?"
The purpose of this monograph is to provide a comprehensive and criti-
cal review of the entire field of triglyceride analysis so that the reader can
select the best technique or techniques for solving his own specific problem.
By devoting an entire book to the subject at a time when the field has
reached considerable maturity, triglyceride analysis can now be viewed
1
2 1. INTRODUCTION
with a broader perspective than was possible in earlier review papers
(186,240,365,494,550,585,700,898,909). It is assumed that the reader is
already familiar with the fundamental chemistry of fatty acids and the basic
techniques of organic analysis. Therefore, discussions in this book will cen-
ter on the types of analyses possible and the specific operating conditions
necessary when dealing with certain types of triglyceride molecules and
their derived diglycerides. Particular emphasis is placed on the experi-
mental details of such work.
Analytical techniques for triglyceride analysis are conveniently subdi-
vided into those for sample preparation (Chapters 2 and 3), molecular
fractionation (Chapters 4-8), and positional analysis (Chapters 9-11).
Since the analysis of derived diglycerides is an integral part of positional
analysis, diglyceride characterization procedures are also covered in Chap-
ters 4-11. Chapter 12 describes the various fatty acid distribution theories
for estimating the composition of natural triglyceride mixtures. Finally,
Chapter 13 outlines useful combinations of analytical techniques for ob-
taining maximum compositional information.
I. TRIGLYCERIDE MOLECULES
A. Nomenclature
A proper nomenclature for triglycerides must accurately describe the
myriad ways in which three fatty acids, either alike or different, can be
esterified to glycerol. A number of different systems have been proposed
to meet this need, and several are in current use.
Any discussion of triglyceride nomenclature must begin with an under-
standing of the distinctive stereochemical nature of glycerol. By itself,
glycerol is a completely symmetrical molecule.* However, if only one of
H
I
H—C—OH
HO—C—H plane of symmetry
I
H—C—OH
I
H
the primary hydroxyl groups is esterified or if the two primary hydroxy Is
* The two ends of the glycerol molecule are not stereochemically identical in
many enzymatic reactions, however. The sn-1- and s/i-3-hydroxyls are easily
distinguished when the molecule forms a three-point attachment to any surface
(754). This stereochemical nonidentity of the two —CH OH groups is demon-
2
strated by glycerol kinase, which only esterifies phosphate at the sn-3-position.
I. TRIGLYCERIDE MOLECULES 3
are esterified to different acids, then the plane of symmetry is destroyed,
and the central carbon atom acquires chirality. Therefore, an unambiguous
H H H
I I I
H—C—OOCR H—C-OH H—C—OOCR
I * I * I *
HO C H HO-C—H HO-C—H
I I I
H—C—OH H—C—OOCR' H—C—OOCR'
I I I
H H H
C*= an asymmetric carbon atom
convention is needed for numbering the three hydroxyl groups so that the
attachment of specific acids at specific hydroxyls can be clearly designated.
Since both ends of the molecule are —CH OH groups, any numbering con-
2
vention is essentially arbitrary.
The convention of Hirschmann (381) has now been universally adopted
for numbering the three hydroxyl groups of glycerol. If the central carbon
atom of the glycerol molecule is viewed with the C—H bond pointing
away from the viewer, then each of the three remaining bonds leads to
an hydroxyl group (Fig. 1-1). Hirschmann has proposed that the three hy-
droxyl groups viewed in this manner be numbered in clockwise order, with
the 2-position already defined as the hydroxyl attached directly to the cen-
tral carbon atom. This is equivalent to a standard Fischer projection in
which the middle hydroxyl group is located on the left side of the glycerol
carbon chain (Fig. 1-2). A more simple view of the same concept is to
state that all triglycerides are named as derivatives of L-glycerol and that
Fig. 1-1. Schematic diagram illustrating the Hirschmann stereospecific numbering
convention (381) for the three hydroxyl groups of glycerol. The central carbon
atom of the glycerol molecule is viewed with the C—H bond pointing away from
the viewer. The three remaining bonds then lead to the hydroxyl groups, which
are numbered in clockwise order with the 572-2-position already defined as the
hydroxyl attached directly to the central carbon atom.
CH2OH sn-1-position
HO C —"H sn- 2-position
CH2OH s«-3-position
Fig. 1-2. Stereospecific numbering convention applied to the usual Fischer planar
projection of glycerol. When the middle hydroxyl group is located on the left
side of the glycerol carbon chain, then the carbon atoms are numbered 1 to 3
in the conventional top-to-bottom sequence.
4
1. INTRODUCTION
the carbon atoms are numbered in the conventional top-to-bottom se-
quence. The prefix "sn-" (for stereospecifically numbered) is included in
the names of all glycerol compounds in which the Hirschmann numbering
convention is used (406). This sn- nomenclature is preferred over the con-
ventional D and L or R and S notations, since it can describe the stereo-
chemistry of glycerolipid reactions in the most simple and unambiguous
manner (406).
A number of other prefixes are also commonly used to designate the
positioning of substituents in glycerides: "a-" refers to the two primary
hydroxyl groups, the sn-1- and sn-3-positions; "β-" designates the sec-
ondary hydroxyl group, the sn-2-position; "rac-" (for racemic) precedes
the names of glycerides which are equal mixtures of two enantiomers.
When no prefix or "X" is used, then the positioning of substituents is either
unknown or unspecified.
The various systems of triglyceride nomenclature in current use are
listed and illustrated in Table 1-1. The alcohol-acid, simplified, and ab-
breviated systems have received the widest usage and have been adopted
throughout this book.
The abbreviated system of triglyceride nomenclature merits a detailed
explanation, since it is extensively employed in this book to avoid the
use of lengthy systematic names. The abbreviated system is based first of all
on the standard letter and number fatty acid abbreviations listed in Table
1-2. Triglyceride abbreviations are then formed by combining the ap-
propriate fatty acid abbreviations in groups of three (Table 1-3). The
positioning of the fatty acids within the triglycerides is indicated by the
presence or lack of a prefix. An "sn-9 prefix specifies that the sn-1-,
sn-2-, and sn-3 -positions are listed in order, thus identifying a single
molecular species. A ''rac-' prefix indicates that the middle fatty acid in
the abbreviation is attached to the ^π-2-position, but the remaining two
acids are equally divided between the sn-l- and sn-3-positions, producing
a racemic mixture of two enantiomers. Α "β-" prefix designates that the
middle fatty acid in the abbreviation is esterified at the β- or ^-2-position
and that the positioning of the other two acids on the sn-1- and sn-3 -posi-
tions is unknown. Thus "β-" specifies a mixture of the two enantiomers
in any proportion. The lack of a prefix indicates that all positional isomers
that may exist are being referred to.
The complexity of natural triglyceride mixtures has prompted classifica-
tion of the many molecular species into simple groups according to the
kinds of fatty acids they contain. Widely used terms of this nature include:
Monoacid. Triglyceride molecules containing only one fatty acid
(triolein, trioctanoin, etc.).
