Page 363 - Academic Press Encyclopedia of Physical Science and Technology 3rd Analytical Chemistry

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Encyclopedia of Physical Science and Technology EN0011A-541 July 25, 2001 17:27

466 Organic Chemistry, Compound Detection

however, most of the sample co-extractives are transpar- chiral phases vary by the size of cyclodextrin macro-
ent and no longer interfere with the detection of aflatoxins. molecules and by substituents used to derivatize them.
Although much work is perfomed with detectors that op- Both factors greatly affect a column’s ability to resolve
erate in the UV region of the spectrum a good deal is also the enantiomers of different chiral compounds.
practiced in the visible region.
G. Enantiomeric Analysis of Amino
C. Fluorescence Detection Acids by HPLC

Fluorimetry is well known for its very high selectivity Most of the protein amino acids, except glycine, have at
and sensitivity to very small quantities of some samples least one asymmetric (chiral) carbon atom and can exist
occurring in biological fluids while being completely in- as two isomers, designated D and L enantiomers. When
sensitive to many other materials such as durgs, vitamins, the D and L enantiomers each react with a chiral (optically

and steroids. GC detectors such as electrical conductivity active) molecule of, for example, the L configuration, they

and electron capture are also used in LC. form the diastereoisomeric compounds DL and LL .

Amino acids of L configuration are more abundant in
nature; the D-enantiomers are usually found in bacte-
D. Radioactivity Detectors
rial cell walls, antibiotic compounds, and rare biological
There are many applications in the studies of the molecules. The DL-form (the racemate) are found in geo-
metabolism of drugs, pesticides, etc., in which radioactive logical specimens such as fossil shells and bones, or they
samples are employed to enable the compounds of interest are formed by abiotic synthesis.
to be detected at very low concentrations. The important Methods for resolving amino acids into their respec-
current trends in HPLC are the use of super critical fluids tive enantiomers are of importance in the preparation of
as eluants and the coupling of HPLC with NMR, MS, or peptides, drugs, and food additives as well as the extreme
FTIR. complex amino acid mixtures in meteorites in which the
An equally important trend is a reduction in column ready determination of optical purity is essential.
size and a concomitant increase in analytical speed. Short Two main approaches using HPLC are (1) covalent
columns can reduce analysis time, and efficiency may be bonding of chiral ligands (which can complex copper (II)
as high as 5000 theoretical plates since the particle size of ions) to solid supports (such as polystyrene and polyacry-
the packing material is 3 µm. The analysis time is about lamide) and resolution of amino acids by eluting with a
1 minute. A standard HPLC column has a diameter of mobile phase containing copper (II) ions; (2) introduc-
4.6 mm as compared to 2, 1, or even 0.5 mm for the new tion of chirality into the mobile phase. Metal ions such
columns. The small size of these columns allows the use as Cu(II), Zn(II), Co(II), and Mg(II), in conjunction
of more expensive solvents; deuterated solvents might be with chiral ligands are added to the mobile phase. Thus,
used when it is necessary to analyze the collected samples a Cu(II)–L proline complex as the chiral additive can be
by NMR. The use of ultramicrobore (50-µm diameter) operated in conventional cation-exchange resin.
columns allows feeding of the eluted peaks directly into a A good enantiomeric resolution of α-amino acids was
mass spectrometer of FTIR instrument. recently achieved by using chiral complexes of copper (II)
with N,N-di-n-propyl-L-alanine (DPA) as the additive in
the mobile phase. Actually, the mixture of amino acids is
E. Preparative Liquid Chromatography
separated into four groups by conventional ion-exchange
Most preparative HPLC columns are capable of separating chromatography and then resolved by means of the chiral
as much as 10 g of sample at a time. Industry often utilizes DPA reagent (Fig. 5).
columns 13 to 22 cm in diameter and 53 cm long having The relationship between temperature and enantiomeric
flow rates of 3 to 20 L/min. They can separate from 1 to separation may not always follow the same rules as con-
10 kg of sample per hour. ventional chromatography. Although the common liquid
stationary phases used in GC do not possess adequate
selectivity for enantiomeric separation, the addition of
F. Chiral Separations Using Capillary GC
derivatized cyclodextrin macromolecules to common sta-
Chiral capillary chromatography has increasing utility in tionary phases often creates the ability to separate volatile
flavor, fragrance, pharmaceutical, and industrial chemical enantiomers. Figure 6 shows that linalool and linalyl ac-
applications. The proper selection of a column is essential etate have symmetrical peak shapes and excellent chiral
since chiral stationary phases illustrate different capabil- separation on a Rt βDEXse at 25 mg per component on
ities and applications. The compositions of commercial column.