Columns, reversed HPLC

Chemical stabiUty studies are monitored by siUca gel thin-layer chromatography (dc) or by high performance Hquid chromatography (hplc) using a reverse-phase C g coated column (24). Hplc peaks or dc spots are visualized by thek uv absorption at 245 nm the tic spots can also be detected by ceric sulfate or phosphomolybdic acid staining. 

HPLC allows a quantitative determination with relatively simple extractions. In many cases, extraction only involves a heating of the commodity with water, followed by filtration and injection onto an HPLC column. In the determination of caffeine, theobromine, and theophylline in cocoa, coffee, or tea, as well as in other foods, there is scarcely a month that passes without a new paper on this assay. Kreiser and Martin provide typical conditions for analysis.28 In their studies, samples were extracted in boiling water and filtered prior to injection onto the HPLC column. The HPLC conditions used a Bondapak reversed phase column and a mobile phase of water methanol acetic acid (74 25 1) with detection at 280 nm. This method is accurate, precise, and conserves time. It has also been adopted by the AOAC as an official method for the determination of theobromine and caffeine in cocoa beans and chocolate products.29... 

Fig. 2.31. Chromatogram of crude lutein from the microalga Chlorella vulgaris by HPLC analysis, A = lutein. Conditions column reversed-phase C18 column (250 X 4.6 mm i.d., 5 pm) mobile phase methanol-dichloromethane-acetonitrile-water (67.5 22.5 9.5 0.5, v/v) flow rate l.Oml/min detection at 450 nm (a). Chromatogram of crude lutein from the microalga Chlorella vulgaris by preparative HSCCC separation, A = lutein. Conditions column multilayer coil of 1.6mm i.d. PTFE tube with a total capacity of 230ml rotary speed 800rpm solvent system ra-hexane-ethanol-water (4 3 1, v/v) mobile phase lower phase (ethanol-water) flow rate lml/min detection at 254 nm sample size 200 mg retention of the stationary phase 58 per cent (b). Reprinted with permission from H.-B. Li el al. [70].

C18 reversed-phase column for HPLC separations of chlorophylls narrow-bore (2.1 -mm i.d.) column at flow rate of 50 to 300 p.l/min without splitting the flow, or analytical column (4.6-mm i.d.) at 1 ml/min with post-column solvent splitting of 5 1 (200 p,l/min entering the mass spectrometer) for APCI electrospray interfaces are available for use without solvent splitting over all flow rates from nl/min to 1 ml/min... 

Table 4 Survey of Commercially Available Reversed-Phase Prepacked Columns for HPLC of Proteins11...

Mattila et al. (205) described a two-dimensional HPLC procedure for determining vitamin D3 and 25-hydroxyvitamin D3 in meat and milk products. Samples were saponified in the presence of vitamin D2 and 25-hydroxyvitamin D2 as internal standards, and the extracted un-saponifiable matter was subjected to normal-phase semipreparative HPLC to obtain a fraction containing 25-hydroxyvitamin D2 + 25-hydroxyvitamin D3 and a fraction containing vitamin D2 + vitamin D3. The collected fractions were evaporated and purified by reversed-phase HPLC. Fractions were again collected, after which vitamin D3 was quantified by tandem-column reversed-phase HPLC and 25-hydroxyvitamin D3 by tandem-column normal-phase HPLC. Analytical chromatograms of a purified extract of chicken are shown in Fig. 12. 

Recent advances in chromatography have made it possible to employ microbore HPLC for the determination of NOC. Its main advantage is that it uses a very low mobile-phase flow (20-100 /rl/min). This might make the TEA compatible with a reversed-phase system. Massey et al. (73), in fact, have successfully used reversed-phase chromatography for the HPLC-TEA determination of V-nitroso-V, 7V -di methylpiperazinium iodide. A 500-mm X 1-mm microbore ODS column and a mobile phase consisting of 0.1 M ammonium heptane-sulfonate in methanol water (70 30) (flow rate 20 /zl/min) was used for the HPLC separation. In another study, Riihl and Reusch (74) used a microbore Spherisorb 3 SW column for HPLC-TEA determination of volatile V-nitrosamines. The mobile phase was a mixture of 2-propanol and n-hexane (2.5 97.5). Further application of such techniques for the determination of various polar NOC, especially A-nitrosamides, in foods is desirable. 

Aqueous samples or aqueous extracts of nonaqueous samples directly injected onto a C-18 reverse phase column for HPLC analysis with UV detection at 195 nm (U.S. EPA Method 8316, 1992) mobile phase, water flow rate 2 mL/min pressure 38 atm. 

Work has recently been undertaken at the Home Office Central Research Establishment at Aldermaston on a procedure for the specific measurement of THC and its metabolites. Following an initial HPLC separation stage, the RIA is used as a sensitive quantatative method for screening column eluants. HPLC separation was performed on a reverse phase column system - highly polar compounds being eluted first followed by substances of decreasing polarity. Methanol extracts of plasma or hydrolyzed urine samples were chromatographed. 

Because acidic solvents slowly destroy the reverse-phase columns, the HPLC system should be left filled with MeOH when not in use. 

Several HPLC methods have been reported for the determination of xanthine and hypoxanthine. Different RP-HPLC methods, using gradient elution and UV detection, have been described to determine these metabolites and other methylated purines.To improve xanthine determination in urine samples, analyses were carried out with two columns—reversed-phase column and anion exchange column—connected by a column switch. With this method, urinary hypoxanthine and xanthine can be measured without any sample preparation other than filtration. ... 

Combining different separation methods, governed by different separation mechanisms, to multidimensional methods is suitable for multiplying the potential of the individual techniques. Reversed-phase chromatography high-performance columns (RP-HPLC) can be coupled with normal phase TLC [9,10]. 

Jinno and Kawasaki also obtained the best correlations between log k and topological index x. van der Waals volume (Vw)> and hydrophobic parameter (log P) for alkylbenzenes separated on reversed-phase C2, Cg, and Cl 8 columns by HPLC (r>0.94). In addition, Smith presented relationships between log k and connectivity index x for alkylbenzenes and n-alkylbenzenes separated by RP-HPLC on SAS-Hypersil and ODS-Hypersil columns (r>0.97). 

If an adequate separation is not obtained by the use of either the reversed-phase analytical method or the silica method, then other stationary phases are scanned for their separation capability (CN, C8, C4, phenyl, chiral, etc.). Often a scouting gradient can be employed to screen columns by HPLC.7 For reversed-phase systems (aliphatic and CN columns), a gradient of acetonitrile and water can be set up, beginning with 100% water and ending with 100% acetonitrile over approximately 30 column volumes. Of the columns that show some peak separation, the gradient can usually be... 

In the following, the synthesis of the most often employed stationary phase is discussed spherical silica with an n-octadecyl modification. The synthesis route has been chosen because all synthesis steps are well characterized and documented in standard operation procedure (SOP) protocols. The objective of this work was to develop a manufacturing process for a reversed phase C18-bonded silica column for HPLC according to standardized and validated procedures and to perform certification of the column, the tests and the mobile phases (du Fresne von Hohenesche et al., 2004). Figure 3.13 shows a scheme of the whole manufacturing process, and Table 3.7 summarizes the main steps. 

Reverse-phase (RP) methods were initially established on the basis of similarity between octanol water partitioning and retention on an RP column for HPLC... 

Meanwhile, the use of bonded reversed-phase columns in HPLC was common, and there were applications for the concentration of organic solutes directly onto the head of the HPLC columns with the term trace enrichment being used for this process (Little and Fallick, 1975). However, interferences also concentrated at the head of the column, such as proteins in biological samples and humic substances in water samples thus, column life was greatly shortened. The answer was a small precolumn positioned ahead of the analytical column to retain contaminants or to retain analytes in trace enrichment. The concept of the precolumn quickly led to the use of separate or off-line low-pressure SPE cartridges. 

Supercritical fluid chromatography, SFC a super critical fluid is produced when a gas, e.g. carbon dioxide is maintained above its critical pressure (73 atm) and temperature (3rC). The super critical fluid is used as the mobile phase with GC like capillary columns or HPLC reverse phase columns and a flame ionisation or flame photometric detector. 

Microsomal Metabolism of Trp-P-2 Trp-P-2 and AAF were metabolized by the 3-MC rat-liver microsomes in vitro. The extracts with benzene, diethyl ether, ethyl acetate and ethylene chloride were dried before injection onto a reverse-phase yBondapak C-18 column. The HPLC profiles of the metabolites of Trp-P-2 and AAF are very similar(figure 3). These metabolites of Trp-P-2 have the same fluorescence spectra with emission maximum at 332 nm. The UV spectra are identical to that of Trp-P-2, that is, with a maximum at 260 nm. 

Reversed phase HPLC is only rarely used for the chromatographic resolution of saccharides however, in instances where only occasional separations are required the wide availability of reversed phase columns in many laboratories saves the purchase of a specialised carbohydrate column. Reversed phase HPLC has been used primarily for the separation of oligosaccharides and separation is dependent on the degree of polymerisation (Cheetham et al., 1981). Organic phase modifiers such as -alkylamines can be used to provide increased capacity and selectivity for saccharides (Lochmuller and Hill, 1983). 

HPLC Conditions Columns Reverse phase, and particularly CIS columns such as Waters Symmetry Shield RP18, are most frequently used (Li et ah, 2003 Soars et ah, 2002 Zhang et ah, 2002). 

Bondapak Cjg Column Reversed Phase HPLC. A method of quantitative determination of opium alkaloids on Bondapak Cig column was standardized by Akhila and Uniyal (1983) at our Institute. It operates under the conditions noted below ... 

CN Column Reversed Phase HPLC. Another improved method of straw analysis was recently developed by Verma et al. (1990) at our Institute. This method gives better recovery of alkaloids when the sample is extracted with 2.5% aqueous acetic acid in combination with lime, instead of aqueous acetic acid alone and when the mobile phase of 1% sodium acetate in water (pH adjusted to 7.4 with glacial acetic acid)-acetonitrile-tetrahydrofuran (80 16 4) is used rather than the mobile phase of 1% sodium acetate in water (pH adjusted to 6.78 with glacial acetic acid)-acetonitrile-l,4-dioxane (75 20 5), used by Srivastava and Maheshwari (1985). 

Vitamin K HPLC has provided the first assay of the phylloquinones and menaquinones that constitute vitamin K in plasma. Phylloquinone circulates bound to lipoproteins from which it can be extracted with hexane after ethanol protein precipitation. Removal of co-eluted lipids can be achieved with normal-phase cartridge columns. Reversed-phase HPLC is almost universally used for vitamin K measurement. Either UV (270 nm) or electrochemical detection is suitable. Electrochemical detection uses the reductive mode ( —1.3 V) to convert the quinone moiety to hydroquinone the main disadvantage being the need to remove oxygen from the mobile phase. 

---