Page 362 - 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

Organic Chemistry, Compound Detection 465

Rotating Coil Method to provide adequate information for unequivocal identifi-
cation. HPLC is particularly suitable in the separation of
The introduction of these potentially useful techniques is
high-molecular-weight (up to 6 million) substances and
largely due to Ito in 1981. These methods involve CCD
thermally unstable biologically active products that can-
and eliminate the need for solid supports. One of the prin-
not be volatilized without decomposition; aqueous and
cipal advantages is the speed of operation—the separation
nonaqueous samples can be analyzed. HPLC has been
time (and consumption of solvent) approximating the level
applied to the analysis of natural and synthetic products
of HPLC in one instance involving the isolation of plant
such as amino acids, antibiotics, antioxidants, flavonoids,
hormones by toroidal coil planet centrifugation.
carotenoids, lipids, flavoring and aroma compounds, her-
No direct comparisons with DCCC or RLCC have been
bicides, pesticides, hormones, steroids, mycotoxins, pol-
reported, and it will be of interest to see whether planet
yaromatics, proteins, sugars, purines, dyes, vitamins, and
and toroidal coil centrifuges have the possibility of com-
water and air polutants. An example of HPLC applica-
plementing or supplementing DCC or RLCC. Separation
tions includes the detection of the highly toxic aflatoxins
of amino acids and peptides have recently been reported
sometimes found in peanuts, wheat, corn, and other grain
utilizing the rotating coil method.
crops. They are produced by fungus on the grain and are
considered to be dangerous to human health when their
concentration exceeds 20 ppb.
V. HIGH-PRESSURE LIQUID
CHROMATOGRAPHY HPLC is an ideal system in pharmaceutical analysis,
examination of drug formulation, degradation products of
drugs that might be to toxic to humans, and detection of
Although the number of compounds detected by GLC
drugs in human fluids and tissues. HPLC is useful for polar
could be increased by derivatization of polar functional
and ionic compounds of medium to high molecular weight
groups, it has been suggested that only some 15% of all
such as drugs and their metabolites.
chemicals are capable of existing in the vapor phase.
HPLC has emerged as an instrumental technique
offering rapid separations with simultaneous sensitive A. Reversed-Phase HPLC (RP/HPLC)
monitoring of the course of the analysis. HPLC in its most This technique utilizes a nonpolar stationary phase, usu-
modern form is able to achieve separations in a matter of ally a fully porous microparticulate chemically bonded
a few minutes which by previous techniques may have alkylsilica and a polar mobile phase are now recognized
taken hours or days or may not have been possible. as the technique par excellence for the separation of po-
Of particular importance in HPLC development has lar, ionogenic solutes. For the separation of free amino
been the availability of specialized chromatographic col-
acids, their derivatives, peptides, and proteins, the octyl
umn packings and sensitive on-line detection systems for (C 8 )andoctadecyl(C 18 )hydrophobicphasesboundedinto
continuous monitoring of the separations being carried 5- and 10-µm fully porous silicas with pore diameters in
out. These developments have led to systems which in the range of 60–100 A and 300–500 A are being used.
˚
˚
favorable instances can on the one hand detect parts per
9
billion (1 in 10 ) levels of organic compounds, and on the
B. Detection Systems
other hand be used for collecting gram quantities of pure
chemicals by preparative HPLC. The lack of truly univer- Photometric detectors with variable wavelength capabil-
sal detectors has resulted in the development of several ity are most widely used currently in LC. As an example,
selective detectors as described in Table I. the carcinogenic aflatoxins in cereal products absorb light
Although LC detectors are sufficiently sensitive for strongly at both 254 and 365 nm. Many other compounds
trace analysis, they are nevertheless limited in their ability also absorb light in the former wavelength. At 365 nm,

TABLE I Comparison of Various LC Detectors
Refractive MS
UV index Fluorescent Electrochemical Fullscan SIM a

Range of application Selective Universal Very selective Very selective Universal Very selective
Minimum detectable quantity, g. 10 −9 10 −6 10 −12 10 −12 10 −9 10 −11