Separation applying HPLC

The application of pressure to the liquid phase in liquid chromatography generally increases the separation (see HPLC). Also in PIC improved efficiency of the column is observed if pressure is applied to the mobile phase (Wittmer, Nuessle and Haney Anal Chem 47 1422 1975). 

Alcohol sulfates and alcohol ether sulfates separated by HPLC on a styrene-divinylbenzene copolymer column with 4 1 (v/v) methanol and 0.05 M ammonium acetate aqueous solution as the mobile phase were analyzed by simultaneous inductively coupled argon plasma vacuum emission spectroscopy (IPC), monitoring the 180.7-nm sulfur line as a sulfur-specific detector [294]. This method was applied to the analysis of these surfactants in untreated wastewaters. 

Another variation of the preceding method is to apply HPLC to fractionate the cleaned-up aliphatic-aromatic fraction from flash colurim separation of soluble organic matter as it is performed in the Chevron laboratory, for example, as described in Reference 2. A Waters HPLC system equipped with a preparative Whatman Partisil 10 silica column (9.4 X 500 mm), a HPLC pump, and two detectors for separation monitoring (a UV and refractive index detector) are used, giving three fractions of aliphatic hydrocarbons, mono-, di-, and triaromatics and polar compounds. The hrst two fractions are eluted with hexane, whereas polar compounds are eluted with... 

An improved HPLC-photohydrolysis-colorimetry method was validated for twenty-eight reference nitrosamines. These were separated by HPLC and photolytically cleaved by UV radiation. The resulting nitric oxide was oxidized and hydrolyzed to nitrite ions, which were derivatized into an azo dye with Griess reagent and measured spectrophoto-metrically. The method was applied to separate and detect hitherto unknown nonvolatile nitrosamines in biological fluids and food extracts591. 

The SEC fractions were further separated applying a conpled method of polymer HPLC, LC CC in the latter case. So far, the most important combinations are LC CC plus SEC and EG EC pins SEC. It is believed that also other combinations snch as LC LC pins SEC will find applications in 2D polymer HPLC [250],... 

Fernandez et al. [27] applied HPLC with diode array detection (DAD) to the determination of heroin, methadone, cocaine and metabolites in plasma after mixed-mode SPE. Analytes were separated using a RP8 column (250 mm x 4.6 mm i.d., 5 jam particle size) and acetonitrile-phosphate buffer pH 6.53 as mobile phase with elution in the gradient mode. The method, which provides a LLOQ of 0.1 j,g/mL for all compounds, was successfully applied to 21 plasma samples from fatal overdoses. 

Multitarget forensic applications of HPLC for other drug classes are also available in the literature. Josefsson et al. [77] applied HPLC-MS-MS to the determination of 19 neuroleptics and their major metabolites in human tissues and body fluids. Optimal separation was achieved using a cyano column within a 9 min gradient run. Detection was curried out in SRM reaching LQDs down to the lower ng/mL level, although more than a 10-fold difference in signal response was observed between analytes. The method was subjected to partial validation only. 

An example of the complex set of spectroscopic information required in natural products research is afforded by the mixture of hydroperoxides 207, 208, the endoperoxide 209 and other minor products, obtained by photosensitized addition of O2 to phytol (206), according to Scheme 10. The scheme is complicated by the fact that 208 is a mixture of the E and Z isomers. After separation by HPLC, each fraction can undergo characterization by FTIR, H and NMR, also applying the HSQC, HMBC, H- H COSY and NOESY techniques, and EI-MS . ... 

The use of HPLC for the separation of residue organics from aqueous samples for mutagenicity testing has been extended to studies of many other types of water samples from different parts of the world. Baird and co-workers (15, 16) have used such an approach in their studies of mutagenicity of residue organics from drinking water, river water, storm runoff, reclaimed waste waters, and other waste waters. Jolley and co-workers (17, 18) have applied HPLC to the separation... 

Rotaxanes were also formed when the sulfonamide macromonocycle 65 or a mixture of 32 and 65 were applied to the synthesis, but with these compounds baseline separation by HPLC could not be achieved (Figure 27) [44],... 

Since the work of Manley and Shubiak (182), who were the first to apply HPLC to anthocyanin analysis, numerous HPLC techniques have been developed for the separation and quantification of anthocyanins and anthocyanidins. Nowadays HPLC has become the method of choice, because it offers the advantage that it is a rapid, sensitive, and quantitative method. For the peak identification and quantitative evaluation of chromatograms, the use of pure anthocyanin standards is recommended however, only a limited, but constantly increasing, number of substances is avail-... 

A novel HPLC-AAS interface based upon thermochemical hydride generation (THG) was developed for the determination of AB, AC and tetramethylarsonium cations, which had been separated by HPLC (Blais et al., 1990 Momplaisir et al., 1991). This has been described in Section 15.7. The detection limits of the three species were 13.3 ng (AB), 14.5 ng (AC) and 7.6ng (tetramethylarsonium cations). Advantages of this interface included low purchase and operating costs, low detection limits, and good reproducibility of results. The method was applied to determinations of arsenic species in seafoods and human urine. 

Fig. 3.11 shows chromatograms of 2,3-dihydroxybenzoate and 2,5-dihyroxybenzoate separated by HPLC reversed-phase chromatography fitted with electrochemical detection (Halliwell et al., 1988). HPLC is carried out on a Spherisorb 50DS column (25 cm x 4.6 mm) applying 97.2% (v/v) 30 mM sodium citrate/2.7 mM acetate... 

Thiamin compounds can also be measured by HPLC, A clever techniejue has been used to facilitate the detection of thiamin compounds immediately after they have completed their chromatographic separation by HPLC, The mixture of un-separated compounds is exposed to conditions expected to provoke the conversion of thiamin to its thiochrome derivative, and then applied to the HPLC column (Tallakesen ef rt/., 1997 Rindi and Laforenza, 1997), This approach,called "pre-column derivatization/ is used widely by chemists and biochemists during the separation and detection of many compounds. 

---