C18 reversed-phase chromatography

Figure 1. Initial methanol extraction and C18 reversed-phase chromatography of Concord grapes.

Chromatography. A number of HPLC and TLC methods have been developed for separation and isolation of the brevetoxins. HPLC methods use both C18 reversed-phase and normal-phase silica gel columns (8, 14, 15). Gradient or iso-cratic elutions are employed and detection usually relies upon ultraviolet (UV) absorption in the 208-215-nm range. Both brevetoxin backbone structures possess a UV absorption maximum at 208 nm, corresponding to the enal moeity (16,17). In addition, the PbTx-1 backbone has an absorption shoulder at 215 nm corresponding to the 7-lactone structure. While UV detection is generally sufficient for isolation and purification, it is not sensitive (>1 ppm) enough to detect trace levels of toxins or metabolites. Excellent separations are achieved by silica gel TLC (14, 15, 18-20). Sensitivity (>1 ppm) remains a problem, but flexibility and ease of use continue to make TLC a popular technique. 

C18—reverse phase adsorption chromatography—normal phase. 

Fig. 9. Reversed-phase separations of cytochrome c digests obtained with trypsin-modified beads (left) and trypsin-modified monolithic reactor (right) in a tandem with a chromatographic column (Reprinted with permission from [90]. Copyright 1996 Wiley-VCH). Conditions digestion (left curve) trypsin-modified beads reactor, 50 mm x 8 mm i.d., 0.2 mg of cytochrome c, digestion buffer, flow rate 0.2 ml/min, 25 °C, residence time, 15 min (right curve) trypsin immobilized onto molded monolith other conditions the same as with trypsin-modified beads. Reversed-phase chromatography column, Nova-Pak C18,150 mm x 3.9 mm i.d., mobile phase gradient 0-70% acetonitrile in 0.1% aqueous trifluoroacetic acid in 15 min, flow rate, 1 ml/min, injection volume 20 pi, UV detection at 254 nm...

Wise, S.A. and May, W.E., Effect of C18 surface coverage on selectivity in reversed phase chromatography of polycyclic aromatic hydrocarbons. Anal. Chem., 55, 1479, 1983. 

Aboul-Enein and Al-Duraibi (1998) employed dansyl chloride in a fluorescence assay for PUT, SP, SPD, and their acetylated derivatives by ion-pair reverse-phase chromatography. This assay could be applied to the separation of free and acetylated polyamines in biological samples. Dansyl chloride has also been used as the fluorescence reagent in the determination of polyamines in urine by Molins-Legua and colleagues (1999). Derivatization was carried out within the C18 cartridges that were used during the SPE extraction procedure. Recoveries were 80-95% for all four polyamines analyzed and the hmit of detection was 10 ng/ml. 

FIGURE 5.4 Effect of the gradient dwell volume, V7>. the elution volume, Vj, in reversed-phase chromatography. Solute neburon, retention equation (Equation 5.7) with parameters a=A, m = 4. Linear gradients 2.125% methanol/min (a) from 57.5% to 100% methanol in water in 20min ( i = 50) (b) from 75% to 100% methanol in water in 11.75 min (k = 10). Vg uncorrected calculated from Equation 5.8, Vg + Vg, Vg, added to Vg uncorrected, Vg corrected calculated from Equation 5.21. (A) A conventional analytical C18 column, hold-up volume y ,= ImL flowrate l.OmL/min. (B) A microbore analytical C18 column, hold-up volume y = 0.1mL flow rate 0.1 mL/min. 

Figure 25-20 Photodiode array ultraviolet detector for HPLC. (a) Dual-beam optical system uses grating polychromator, one diode array for the sample spectrum, and another diode array for the reference spectrum. Photodiode arrays are described in Section 20-3. (fc>) Reversed-phase chromatography (using C18-silica) of sample containing 0.2 ng of anthracene, with detection at 250 nm. Full-scale absorbance is 0.001. (c) Spectrum of anthracene recorded as it emerged from the column. [Courtesy Perkln-Elmer Corp.. Norwalk. Cl]...

High-performance LC is the favored technique for the determination of carbamates, since many of them lack the thermal stability necessary for gas chromatographic analysis. Most HPLC methods for methyl and phenyl carbamate pesticides have employed reversed-phase chromatography with C18 or C8 columns and aqueous mobile phases (47,50,105,106). Two different solvent sys-... 

Foda et al. have developed a reversed-phase chromatography method using C18 reversed-phase analytical column. 

Antidepressant separation was usually performed by reversed-phase chromatography with typical C8 or C18 alkyl chain columns, although phenyl [30, 59] or cyano [48,64, 84] stationary phases were also employed. As an exception, hydrophilic interaction liquid chromatography (HILIC), a variation of normal phase chromatography, was employed in two analytical methods for duloxetine [38] and... 

C18 Reversed phases are not necessarily limited to polycyclic and halogenated (or nitrated) hydrocarbons, but can be used for a number of oxygen-, nitrogen- and sulphur- containing heterocyclic compounds in a similar way to that used in reversed-phase chromatography. In this context, ref. 185 describes the separation of compounds of this category. A plethora of similar separations can be found in the Section devoted... 

At first glance the specifications of many stationary phases in a particular mode of separation may appear to be the same. For example, in reversed phase chromatography there are many C18 based media that will have the same carbon load, the same pore size distribution, and so on. However, stationary phases from different manufacturers will behave differently, so it is important... 

Apparatus (See Chromatography, Appendix IIA.) Use a high-performance liquid chromatograph operated at room temperature with a 10-p.m particle size, 30-cm x 4-mm (id), C18 reverse-phase column (jxBondapak C18 column, Waters Corp., 34-T Maple Street, Milford, MA 01757, or equivalent). Maintain the Mobile Phase at a pressure and flow rate (typically 2.0 mL/min) capable of giving the required elution time (see System Suitability in High-Performance Liquid Chromatography). Use an ultraviolet detector that monitors absorption at 254 nm (0.2 to 0.1 AUFS range). 

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