Chromatography of Alkaloids: Part B: Gas-Liquid Chromatography and High-Performance Liquid Chromatography

431 Pages·1984·9.819 MB·iii-iv, 5-457\431

by  R. Verpoorte and A. Baerheim Svendsen (Eds.)

-
JOURNAL OF CHROMATOGRAPHY L!6RARY volume 23B
chroma tograph y
of alkaloids
part B: gas-liquid chromatography and
high-performance liquid chromatography
R. Verpoorte
and
A. Baerheim Svendsen
Department of Pharmacognosy, State University Leyden, Leyden, The Netherlands
E LSEVl ER
Amsterdam - Oxford - New York - Tokyo 1984
ELSEVIER SCIENCE PUBLISHERS B.V.
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Distributors for the United States and Canada:
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Library of Coagreir Cataloging in Publication Data
(Revised for part B)
Baerheim-Svendsen, A.
Chromatography of alkalcias.
(Journal of ChrODIatOgrapkii librvy ; v. 23-23B)
Includes bibliogryhical references and indexes.
--
Contents: pt. A. Thin-layer chromatography pt. B.
Oae-liquid chromatography and high-perfomence liquid
chromatography.
1. Alkaloids--Analysis. 2. Chromatographic analysis.
I. Verpoorte, R. 11. Title. 111. Seriee.
QD421.Bl25 1983 547.7'2C46 82-20976
ISBN 0-444-42145-9 (U.S. : pt. A)
ISBN 0-444-42265-X (U.S. : pt. B)
ISBN 044442265-X (Vol. 238)
ISBN 044441616-1 (Series)
0 Elsevier Science Publishers B.V.. 1984
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6
PREFACE
Most naturally occurring alkaloids are fairly high-molecular-weight compounds. As such
they are usually only slightly volatile or non-volatile. The application of gas chromatogra-
phy to the analysis of alkaloids is, therefore, limited.
When working with compounds of high molecular weight and low volatility in gas chromato-
graphy, it is often necessary to increase their volatility or to reduce their polarity by
converting them to special derivatives. Usually it is also necessary to work with gas chroma-
tographic column with a low percentage of stationary phase, in order to be able to operate
1.
at relatively moderate column temperatures
The injection of an alkaloid or a mixture of alkaloids into the injection port of the gas
chromatograph may be of vital importance for the further course of the analysis. One has to
pay attention to the fact that the substance to be analyzed may be in many cases degraded
during the analysis due to catalytic effects of the metal parts of the gas chromatograph,
especially to the injection port, where the temperature has to be relatively high to obtain
an instant and complete evaporation of the compounds.
The high temperature of the injection port required for the evaporation of the alkaloids
may sometimes lead to decomposition. such as dehydration, hydrolysis or transesterification.
Atropine can under certain conditions be dedydrated to apoatropine. The degree of dehydration
has been found to be associated with the amount of glass wool on the top of the column mater-
ial. Diacetylmorphine is eluted as a sharp well-defined peak when chromatographed alone. In
mixtures with codeine, morphine or other phenolic or alcoholic substances transesterifica-
tions take place in the injection port, giving rise to several new esters not.present in the
original solution. 6-0-Acetylmorphine gives peaks of morphine, 6-0-acetylmorphine and di-
acetylmorphine. 3-0-Acetylmorphine is more stable and may be gas chromatographed with little
2 .
or no decomposition
To prevent an undesirable degradation of the compounds to be analyzed, glass columns have
mostly been used for gas chromatography of alkaloids because they are indifferent to the
compounds. Possible catalytic decomposition of sensitive compounds and adsorption phenomena
caused by metal columns, e.g. copper, aluminium and stainless steel, may. however, be elim-
inated in some cases by a simple coating of the tubing material with the stationary phase
used in the analysis. The decomposition and adsorption phenomena di~appear~'C~o.d eine and
4
noscapine3 and ephedrine were successfully gas chromatographed on coated, packed metal col-
umns without decomposition or adsorption.
REFERENCES
1 E.C. Horning, E.A. Moscatelli and C.C. Sweeley, Chem. rnd. (London), (1959) 751.
2 E. Brochmann-Hanssen and A. Baerheim Svendsen, J.Phann. sci., 51 (1962) 1095.
3 J.E. Arnold and H.t1. Fales. J. cas Chromatop.. 4 (1965) 131.
4 A.M.J.A. Duchateau and A. Baerheim Svendsen, Pharm. weekbl., 107 (1972) 377.
9
Chapter I
PACKED COLUMNS
...............................................
1.1. Deactivation of solid support ........... 10
1.1.1. Deactivation of solid support by acid and alkaline wash.in.g. ............ 10
1.1.2. Deactivation of solid support by chemical deactivation 10
1.1.3. Deactivation of solid support by chemical bonding of stationary phase. 11
1.1.4. Deactivation of solid support by precoating with small amounts of a
...............................................
polar stationary phase 13
..............................
1.2. Coating of solid support with stationary phase 13
..................................................................
1.3. References 14
A successful gas chromatography depends to a great extent upon the quality of the columns
used. For packed columns Roman et al.' showed that the quality of such columns is proportion-
al to the care bestowed upon the preparation procedure. In comparing some commercially avail-
able solid supports the authors found that poor gas chromatographic performance was often
caused by adsorptive sites on the surface of the solid support, e.g. by incomplete deactiva-
tion of the support. However, the quality could be improved by more careful, eventually re-
peated, deactivation of the support.
Most authors prefer to make their columns themselves. In the literature a number of recom-
mendations for preparation of packed columns for high boiling, slightly volatile compounds
are given. Most of them are based on empirical observations, made when performing gas chroma-
tography to solve specific analytical problems. When satisfactory results were achieved, no
need for further evaluation of the column was found to be necessary, and the procedure used
was therefore assumed to be generally applicable. When adopted by others, they became grad-
ually transformed into some kind of magic!
For gas chromatography of alkaloids low load packed columns are usually used. In such
cases "tailing", caused by active adsorptive sites, is often observed.
With gas-liquid chromatography a separation is achieved by the differences in the parti-
tion coefficients of the various compounds between the gaseous mobile phase and the station-
ary liquid phase. However, in gas-liquid chromatography with low load packed columns, the
gas-liquid partition equilibrium is influenced by the properties of the solid support, be-
cause of active adsorptive sites on the surface of the support, and inhomogeneous coating
of the support, which leaves parts of the support uncoated. In both cases adsorption of the
solutes to the support material can take place. The effect is especially troublesome with
small sample sizes. Various techniques have been developed to deactivate the active sites
on the solid support as well as to assure a homogeneous coating. The deactivation has mostly
been achieved by acid and alkaline washing, often followed by chemical deactivation, lastly
by precoating of it with a very small amount of a polar stationary phase.
R8lenne.r p. 14
10
1.1. Deactivation of solid support
1.1.1. Deactivation of solid support by acid and alkaline washing
The methods which have mostly been used to deactivate the active adsorptive sites on solid
supports of diatomaceous earth type include acid washing and alkaline washing. Acid washing
of such support material was recomnended by James and Martin2. They suspected metallic oxides
to be at least partly responsible for the adsorptive properties. Washing with concentrated
hydrochloric acid followed by rinsing with water until neutral, was intended to remove these
metallic oxides. Several other authors have clearly shown that the acid washing is an import-
ant step prior to silanization.
In some cases an alkaline washing has been applied in addition to the acid one, as a pre-
treatment for silanization. The combination was thought to be more effective for removing
3
amphoteric ions, such as aluminium, than acid washing alone. Holmes and Stack prepared
packing material with low adsorptive properties in this way, so also Brochmann-Hanssen and
Baerheim Svendsen for their analysis of barbiturates, sympathomimetic amines and a1 kal~ids~-~.
Comparative studies to investigate the influence of acid and alkaline washing on a solid
support, such as Chromosorb W, prior to silanization and coating with the stationary phase
by a) acid washing, b) acid washing followed by alkaline washing and c) acid washing followed
by alkaline washing and then again acid washing, were carried out by Meilink7. He recomnended
the following procedure to achieve optimum deactivation:
Suspend Chromosorb W in hydrochloric acid (25 X), filter off after two days by suction and
rinse the Chromosorb with distilled water until all acid is removed. Remove most of the water
by washing with methanol, filter and dry the product in a rotary evaporator under reduced
pressure at 90°C. Suspend the acid washed material in 1 mole per liter KDH in methanol. Fil-
ter off after 15 hours and rinse with methanol until free of KOH. Dry as described above.
Treat the acid and alkali washed material once more with hydrochloric acid as described above,
rinse it with water and dry it as described above.
1.1.2. Deactiva.tion of solid support by chemical deactivation
An acid and base washing of the solid support is usually followed by a chemical deactiva-
tion . mostly in the form of silanization. Horning et a1.’ used gaseous dimethyl dichloro-
silane (DMDCS) for this purpose on the acid washed support. Horning et a1.’ emphasized that
acid washing was essential. Bohemen et a1.l’ pointed out the risk that DMDCS silanization
might leave active chlorine groups behind, which in their turn could be converted to hydroxyl
groups when brought into contact with water. This problem will not be encountered if hexa-
methyldisilazane (HMDS) is used as silanizing reagent.
According to Bohemen et a1.l’ acid prewashing is not necessary for HMDS silanization.
Brochmann-Hanssen and Baerheim Svend~en~o’~bt’a~in ed well deactivated support on treatment
with HMDS; neither acid nor base washing were, however, omitted. Sawyer and Barr” found
HMDS silanization very effective for deactivatlon purposes, although a slight residual adsorp-
tive activity was left.
Compared with DMDCS silanization, a disadvantage of HMDS should be mentioned. McMartin and
Street“ found that a support which had been well deactivated by HMDS was reactivated at
11
temperatures above 26OoC.
Alkaline treatment of the solid support directly prior to silanization with DMDCS seems
not to be advantageous. The presence of free hydroxyl groups on the support, obtained by
acid washings, seems to be essential for the silanization reaction with DMDCS. According to
McMartin and Street" the acid washing can be omitted when DMDCS silanization is performed
on "wet" or "damp" solid support. They assumed that hydrochloric acid, released in the reac-
tion of DMDCS with water, replaces the acid washing. They also supposed that polynerization
of DMDCS takes place in the presence of water, resulting in a chemically bonded polysiloxane
layer on the support. If higher concentrations of water in the support than about 5 X are
used, excessive formation of gaseous hydrochloric acid will be the result.
Meilink7 preferred the procedure involving acid, base and acid washing to obtain a best
possible deactivation of the support.
Recommended procedure for silanization with DMDCS:
Suspend the acid, base and again acid washed solid support in DMDCS in toluene (5 % v/v),
treat the suspension in an ultrasonic bath for 5 minutes and filter off the solvent after 24
hours. Wash with dried toluene and dried methanol under exclusion of water and dry the support
material in a rotary evaporator under reduced pressure at 90°C.
Bohemen et a1.l' recommended the following procedure for silanization with HMDS:
Dry the support under vacuum at 15OoC. Cover 25 g of this sample, while still warm. with
a mixture of 80 ml of light petroleum (b.p. 60-80°C) and 15 ml hexamethyldisilazane. Heat
the mixture on a steam-bath and reflux for 1 hr. Use a drying tube of calcium sulphate at the
condenser exit. After refluxing, add 2 ml of n-propanol; this helps materially by wetting the
support, and a1 though it reacts with unchanged hexamethyldisilazane to form SiMejDPr, this
in turn reacts with hydroxyl groups in the same way as the parent silazane. After 30 hr. heat
the mixture again and reflux for several hours. Wash the support with light petroleum (2 x
50 ml), then n-propanol (1 x 50 ml), and then again with light petroleum (2 x 50 ml). Finally,
filter off the support and dry it for 2 hr. on a steam-bath in an atmosphere of nitrogen.
1.1.3. Deactivation of solid support by chemical bonding of stationary phase
Active groups on the support can also react with other than silanizing agents. In that way
small amounts of stationary phases can be chemically bonded to the solid support as esters of
the (41-04)t ype. According to Grushka and Kikta13 such esters are liable to hydrolysis.
Chemical bonds of the (-Si-0-Si-R) type. arising from silanization with organosilanes, are
more stable. The main advantage of gas chromatographic packings with chemically bonded sta-
tionary phases, compared to physically coated ones, is their greater thermal stability. The
14.
upper temperature limit lies about 80-90' above that of the corresponding nonbonded ones
Two types of reaction procedures have been described. In the first one the coupling reac-
tion is brought about at an elevated temperature. Aue et al? stated that polyethylene
glycol 20M (PEG 20M) could not be fully washed off a support with methanol and methylene
chloride after heating to 280°C. The remaining PEG 20M layer was too thin to be measured with
analytical combustion techniques. Hastings and Aue16 demonstrated that chemical bonding of
PEG to support material leads to well deactivated products. These "polymer deactivated" prod-
ucts proved to be good supports when they were physically coated wlth conventional station-
Reference8 p. 14
12
ary phases. The stationary phase that was used, then determined the characteristics of the
packing, and not the ult ra-thin, non-extractable PEG-film. Moseman17 and Winterlin and Mose-
man18 used PEG deactivated supports coated with qV-210 for gas chromatography of pesticides.
They stated that the PEG deactivated support was superior to non-deactivated materials; no
comparison was, however, made with silanized supports. The method of chemical bonding was
modified by Daniewski and Aue". They refluxed the support in a solution of PEG instead of
dry heating the PEG coated support.
The coupling reaction can also be realized by using a chlorosilane as an intermediate to
"activate" the support surface. Chlorosilanes react easily with the active sites on the sup-
port. When trichloromethylsilane is used, always at least one active chlorine gmup is left,
14
which in its turn can react with alcohols. such as polyethylene glycols. In this way, Mori
prepared Chromosorb W (AW) with chemically bonded PEG, resulting in a loading of 4.2 % with
PEG 20M and of 2.0 % with PEG 3000.
Both thermally and chlorosi lane-mediated PEG-bonded supports can be used as gas chromato-
graphic packings without further coating14s17.
Because the chemically bonded PEG molecules are thought to be arranged on the support
surface like "bristles of a brush", Mori14 concluded that the rate of mass transfer should be
increased, making chemically bonded stationary phases very suitable for gas chromatographic
separations.
Recently Street et a1." acylated diatomaceous earth with benzoyl chloride in pyridine as
a pretreatment for coating. They reported that a marked reduction in adsorption could be ob-
tained, enabling polar compounds, such as morphine, to be gas chmmatographed in nanogram
amounts without derivatization. A 1000-fold improvement in chromatographic capability could
be obtained, compared to the best conventional cmercial packing.
Recomnended procedure for thermal bonding of PEG 20M:
Coat acid-base-acid washed Chranosorb W with 5 % (w/w) PEG 20M using the filtration tech-
nique of Horning et a1.8 and fill a glass gas chromatographic column with the material. Flush
the column with nitrogen (60 ml/min) for 6 hours and heat at 28OoC for 15 hours with a nitro-
gen flow of 3.5 ml/min. Empty the column and rinse the packing material thoroughly with
methanol and methylene chloride respectively, followed by extraction with methylene chloride
for 6 hours in a Soxhlet apparatus. Dry the support in a rotary evaporator at 6OoC and coat
it with the stationary phase that was chosen.
Recomnded procedure for chlorosilane-mediated bonding of PEG 4000:
Suspend 18 g acid-base-acid washed Chromosorb W in a mixture of 50 ml dried toluene and
25 ml tetrachlorosilane (TCS), treat it for 5 min. in an ultrasonic bath to remove air, fil-
ter off the solution after 4 hours at room temperature, rinse with dried toluene under exclu-
sion of water to remove the TCS completely. While it is still excluded from atmospheric mois-
ture, suspend the TCS treated support in a solution of 8 g PEG 4000 in 75 ml dried toluene
for 48 hours at 5OoC. Filter the PEG solution off and rinse the mass with toluene and methyl-
ene chloride successively, followed by extraction with methylene chloride in a Soxhlet appar-
atus. Dry the mass in a rotary evaporator at 6OoC.
13
1.1.4. Deactivation of solid support by precvating with small amounts of a polar stationary
phase
To diminish any eventual residual adsorptive activity of the support that may still be
present after deactivation by acid and alkaline washing, and by chemical procedures. Bohemen
et al.lOintroduced a precoating with 0.1 X PEG 400. The PEG molecules are thought to be ad-
sorbed tightly to the residual active sites of the support. This precoating procedure was al-
so used by Brochmann-Hanssen and Baerheiq Svendsen' in their gas chromatographic studies on
alkaloids. They preferred to apply PEG 9000 0.1 X (w/w). The higher molecular weight was used
as higher temperatures were employed. Kabot and Ettre'l recomnended to apply the main sta-
tionary phase by means of a solvent "in which the precoated polar phase (PEG) is insoluble".
Lipsky and Landowne'O dissolved both the polar phase (PEG) and the non polar phase in the
same solvent and carried out both precoating and coating simultaneously. They found that
this procedure reduced or eliminated tailing due to non-linear sorption isotherms. and attri-
buted the effect to deactivation of the support.
The bonding of PEG to the active adsorptive sites on the support can take place in two
different ways. First, some chemical bonding may occur when the packing is used at higher
temperatures. This effect is comparable with the thermal bonding of PEG as described by Aue
et a1.l5. On the other hand, strong hydrogen bonding may be involved, as stated by Evans et
d3.
They used amine antioxidants as deactivators. These substances may be expected to
combat oxidative degradation of the stationary phase in addition to their deactivating prop-
erties. They suspended the support, which had not been deactivated by any previous silaniz-
ation, in a solution of the compounds. The solution was suctioned off, but the amine anti-
oxidant molecules remained bonded to the active sites of the support. The support was then
coated with a stationary phase dissolved in a solvent, which did not displace the amine anti-
oxidant from the support. In that way the amine antioxidant served as a deactivator between
the stationary phase and the support.
1.2. Coating of solid support with stationary phase
Since Horning et a1.* gas chromatographed several kinds of compounds on solid support
thinly coated with a stationary phase, the use of such packing material has beccine increas-
ingly comnon for gas chromatography of high boiling, slightly volatile compounds. The reason
is obvious: The retention times are distinctly shortened, faciliating gas chromatography of
such compounds at relatively low column temperatures. However, with a decrease in the per-
centage of stationary phase, the risk of getting uncoated areas on the surface of the support
increases. According to Bohemen et a1.l' a support with less than 5 5, of stationary phase
greatly enhances the influence of the solid support on the gas chromatographic performance.
A frequently used coating procedure for packing materials with a low percentage of station-
ary phase was described by Horning et a1.8. It is generally called the "filtration technique".
After the solid support has been suspended in a solution of the stationary phase, enough of
the solution is filtered off to ensure that the support on drying will contain the desired
amount of stationary phase. Because only small amounts of the solution are left after filtra-
tion, great differences in the concentration of the stationary phase in the moist mass during
the evaporation will not occur.
McMartin and Street" used another technique to obtain the same result: They rinsed the
References p. 14
14
support with a solution of the stationary phase and dried it on a hot plate, while stirring
gently with a glass rod. However, they did not indicate how a definite percentage of station-
ary phase on the support could be obtained. Parcher and UroneZ4 prepared packinq material by
"solution coating" and fluidized drying. The percentage of stationary phase on the support
25
depended on the concentration of the solution used. Averill coated solid supports, when
packed in gas chranatographic columns, by passing a solution of the stationary phase through
the column. Although reproducible results were obtained when the coating was carried out in
exactly the same manner, no details of the percentage of stationary phase on the support
were given.
Recomnended coating procedure:
Suspend acid washed, silanized solid support (Chromosorb W) in a solution of the station-
ary phase in a suitable solvent to obtain the desired percentage of stationary phase after
evaporation of the solvent. Treat the suspension for 5 minutes in an ultrasonic bath to re-
move air from the support and evaporate the solvent in a rotary evaporator at the boiling
point of the solvent until particles of the support begin to stick together. Stir the mass
gently with a glass rod under continous heating in a current of air. Care must be taken not
to damage the particles of the support. Continue evaporation in this way until the mass has
got flowing properties. Continue the evaporation in the rotary evaporator, and raise the
temperature gradually to about 100°C and heat at that temperature for 30 minutes.
1.3. REFERENCES
1 R. Roman, C.H. Yates and F.F. Millar, J. Chromatogr. sci., 15 (1977) 555.
2 A.T. James and A. J.P. Martin, Biochem. J., 50 (1952) 679.
3 W.L. Holmes and E. Stack, Biochim. Biophys. dcta, 56 (1962) 163.
4 E. Brochmann-Hanssen and A. Baerheim Svendsen, J. Pharm. sci., 51 (1962) 318.
5 E. Brochmann-Hanssen and A. Baerheim Svendsen, J. Pharm. Sci., 51 (1962) 938.
6 E. Brochmann-Hanssen and A. Baerheim Svendsen. J. Pharm. Sci. 51 (1962) 1095.
7 J.W. Meilink, Gas Chromatography and Cardenolides, Thesis, State University of Leiden,
The Netherland, 1980.
8 E.C. Horning, E.A. Moscatelli and C.C. Sweeley, Chem. rnd. (London), (1959) 751.
9 E.C. Horning, K.C. Maddock, K.V. Anthony and W.J.A. Vandenheuvel Anal. Chem., 35 (1963)
526.
10 J. Bohemen, S.H. Langer, R.H. Perret and J.H. Purnell, J. Chem. SOC., (1060) 2444.
11 D.T. Sawyer and J.K. Barr, Anal. Chem., 34 (1962) 1518.
12 C. McMartin and H.V. Street. J. Chromatogr., 22 (1966) 274.
13 E. Grushka and E.J. Kikta, Anal. Chem., 49 (1977) 1004 A.
14 s. Mori. J. Chrmatogr., 135 (1977) 261.
15 W.A. Aue, R.C. Hastings and S. Kapital, J. Chromatogr., 77 (1973) 299.
16 R.C. Hastings and W.A. Aue, J. Chromatogr.. 89 (1974) 369.
17 R.F. Moseman, J. Chromatogr., 166 (1978) 397.
18 W.L. Winterlin and R.F. Moseman, J. Chromatogr., 153 (1978) 409.
19 M.M. Daniewski and W.A. Aue, J. Chromatogr., 147 (1978) 119.
20 H.V. Street, W. Vycudilik and G. Machata, J. Chromatogr., 168 (1979) 117.
21 F.J. Kabot and L.S. Ettre, J. as Chromatogr., 2 (1964) 21.
22 S.R. Lipsky and R.A. Landowne, Anal. Chem., 33 (1961) 818.
23 M.B. Evans, R. Newton and J.D. Carmi, J. chromatogr., 166 (1978) 101.
24 J.F. Parcher and P. Urone, J. Gas Chromatogr., 2 (1964) 184.
25 w. Averill, J. Gas Chrmatogr., 1 (1963) 34.
16
Chapter 2
CAPILLARY COLUMNS
...........................................................
2.1. Capillary columns 15
..................................................................
2.2. References 19
2.1. CAPILLARY COLUMNS
Since Desty et a1.l introduced glass capillary columns in the pas chromatonraphic analy-
sis of petroleum products many scientist have been involved in developing procedures to pre-
pare high quality glass capillary columns. This was achieved by leaching conventional nlass,
followed by high temperature silylation.
Since most chromatographic separations of alkaloids on capillary columns employ tempera-
ture programing and often relatively high temperatures, the stability of a coated column at
higher temperatures is of the utmost importance. Depolymerization of the stationary phase
can take place because of impurities in the stationary phase itself, or in the qlass wall
(metal salts, silanol groups, straight siloxane bridqes) or in the actual silica surface
.
structure
Chemical bonding of stationary phases has been shown to increase the stability of the
stationary phase compared with conventionally coated films. A chemically bonded phase may
be regarded as one that is not extractable by solvents that do not attack the Dhase.
Conventional columns can be basic, they can be made in any internal diameter, they can be
whiskered, and they can, therefore, be coated effectively with any phase.
Fused silica columns have obvious advantages, but do not have these possibilities. The
fused silica capillary columns do not solve all the problems. The activity of silicium based
capillary columns (glass or fused silica) is due to the trace of metal ions, silanol qroups
and siloxane bridges. Whereas a leached soda-lime column is always basic, fused silica
columns are always acidic.
It may, therefore, be concluded that fused silica and leached glass columns coated with
polysiloxanes (OV-1, OV-101, OV-73 and SE-54) and polyethylene qlycols (Superox 2011) provide
a good choice for capillary gas chromatography of alkaloids. The quality of fused silica and
2 .
leached glass columns is practically equal In a number of papers the preparation of glass
capillaries for the analysis of alkaloids has been described, as well as the inactivation of
such glass capillaries, and the chemical bonding of stationary phases on glass capillaries3'
4,596
The injection of the sample of slightly volatile compounds, such as alkaloids, in capil-
lary gas chromatography can be carried out in different ways dependinq upon the kind of
column used. Verzele et a1.2 stated that the "cold on column injection" and the "falling
needle injection" were the best alternatives for quantitative gas chromatoqraphy since they
were applicable to high temperature capillary gas chromatoqraphy. The sampling system in
capillary gas chromatography has been dealt with in a series of
Although the high temperature stability of many capillary columns with non extractable
stationary phases has eliminated the need for derivatization of many compounds, derivatiza-
References p. 19