Page 361 - Academic Press Encyclopedia of Physical Science and Technology 3rd Analytical Chemistry

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464 Organic Chemistry, Compound Detection

the use of 10- to 100-fold higher flow rates and lower new methods such as droplet countercurrent chromatogra-
cycle times without significant losses in either resolution phy (DCCC), rotation locular countercurrent chromatog-
or capacity compared with the conventional soft gels and raphy (RLCC), and coil planet centrifugation. These
HPLC columns. methods have the advantage of being more rapid and less
solvent consuming than traditional CCC. Furthermore, the
advent of commercially available, compact apparatus has
III. FLASH CHROMATOGRAPHY
led to a widespread acceptance of these new liquid–liquid
techniques as standard laboratory procedures for the sep-
The traditional method for preparative separations of sim-
aration of natural products. The detection of compounds
ple mixtures of organic compounds by means of col-
that are eluted from a DCCC can be performed by three
umn chromatography is giving way to flash chromatogra-
methods: (1) UV detection for suitable UV-active sub-
phy (FC) or low-pressure liquid chromatography (LPLC)
stances, (2) monitoring of the fractions that are collected
which was initially carried out by Still in 1978. FC is a
by TLC, and (3) weighing of fractions after evaporation of
simple, cheaper, and faster technique for routine purifi-
solvent. The majority of DCCC separations involve polar
cation of mixtures, allowing preparative separations from
compounds, especially glycosides, which are often dif-
10 mg to more than 25 g. The resolution associated with
ficult to purify. Chloroform–methanol–water systems of
FC on a standard 5 in. high column of 40 µm silica gel
varying compositions remain the most widely used, in
is as great as 200 theoretical plates. This amount of sepa-
view of the good formation of droplets and the convenient
rating power effectively means that compounds having R f
viscosity of this combination. The most notable develop-
values on analytical TLC as close as 0.1 may be reliably
ments in the application of DCCC have occurred in the
separated with sample recovery of at least 90%. FC is ba-
field of polyphenols, in particular in the separation of tan-
sically an air-pressure-driven hybrid of medium-pressure
nins. DCCC has also been applied in the separation of
and short-column chromatography which has been opti-
natural products such as alkaloids, triterpene glycosides,
mized for rapid separations. Elution of the components
steroid glycosides, basic steroid saponins, and glycosides
is extremely rapid, usually taking about 5 to 10 min. As
of flavonoids. Rotation locular countercurrent chromatog-
with other forms of chromatography, sample size is one
raphy (RLCC) relies on the percolation of one layer of a
of the most important variables. The amount of sample
two-phase solvent system through compartments (loculi)
that can be separated on a given column is proportional to
that contain the second layer. During passage of the mo-
its cross-sectional area and the degree of separation of the
bile phase, the loculi (connected into tubes) are constantly
components as indicated by TLC. Still et al. successfully
rotated, to increase contact between the two phases. Ba-
separated a 1-g mixture of epimeric alcohols I and II with
sically, RLCC has the same advantages as DCCC. As in
only a 65 mg mixed fraction in 7 min on a 40-mm diameter
DCCC, the apparatus can be run in either ascending or de-
column using 500 mL of 5% ethyl acetate/petroleum ether.
scendingsolventmodesbuttheformationofdropletsisnot
a necessary condition of RLCC. Consequently, a broader
range of solvent system is possible, and a system con-
taining ethyl acetate (often incompatible with DCCC) has
been used, for example, in the separation of flavonoids—
Reversed-phase systems with nonpolar bonded C 8 and an important application of this method has been the
C 18 silica are used in the separation of sugars. A mixture separation of enantiomers of (±)-norephedrine on an in-
of 200 mg of fructose and sucrose were completely strument consisting of 16 columns and each column con-
separated in the 3/1 acetonitrile/water mobile phase in- taining 37 loculi. The stationary phase was sodium hex-
corporating a silylamine bonded phase with silica in less afluorophosphate solution at pH 4, and the mobile phase
than 1 h. was (R, R)-di-nor-5-yl tartrate in 1,2-dichloroethane. Pre-
This method has been used for the separation of various sumably, the enantiomers of (t)-norephedrine form dif-
products of organic synthesis and small biomolecules. ferent diastereotopic complexes with the tartrate ester,
and these complexes are then partitioned differently be-
tween the two solvent phases. Separations by RLCC of
IV. DROPLET COUNTERCURRENT a range of natural products, including flavones, xanthone
CHROMATOGRAPHY glycosides, and antitumor antibiotics have been reported.
RLCC provides a useful complementary method to DCCC
The technique of countercurrent chromatography (CCC) in instances in which suitable solvent systems are not
has seen a rapid expansion following the introduction of available.