Chromatographic methods reversed-phase

In liquid chromatographic methods, reversed-phase columns are usually employed due to hydrophobic interaction of the amphenicols molecules witli the Cs or Ci8 stationary phases. Ion-pairing liquid chromatography has been also described for the separation of florfenicol and florfenicol amine (62) or chloramphenicol and deacetylchloramphenicol residues (46) using heptanesulfonate or octylsulfate-pairing ions, respectively. 

In the liquid chromatographic methods, reversed-phase columns are commonly used for the separation of the -agonists molecules due to their hydrophobic interaction with the Cis sorbent. Efficient reversed-phase ion-pair separation of -agonists has also been reported using sodium dodecyl sulfate as the pairing ion (477). 

The LC/MS/MS method utilizes the principle of three-dimensional separation to achieve excellent selectivity based on chromatographic separation (reversed-phase, size-exclusive, ionic, etc.), the unique mass-to-charge ratio of the analyte s parent ion, and the fragment ion. A sample clean up... 

An on-line concentration, isolation, and Hquid chromatographic separation method for the analysis of trace organics in natural waters has been described (63). Concentration and isolation are accompHshed with two precolumns connected in series the first acts as a filter for removal of interferences the second actually concentrates target solutes. The technique is appHcable even if no selective sorbent is available for the specific analyte of interest. Detection limits of less than 0.1 ppb were achieved for polar herbicides (qv) in the chlorotriazine and phenylurea classes. A novel method for deterrnination of tetracyclines in animal tissues and fluids was developed with sample extraction and cleanup based on tendency of tetracyclines to chelate with divalent metal ions (64). The metal chelate affinity precolumn was connected on-line to reversed-phase hplc column, and detection limits for several different tetracyclines in a variety of matrices were in the 10—50 ppb range. 

For more specific analysis, chromatographic methods have been developed. Using reverse-phase columns and uv detection, hplc methods have been appHed to the analysis of nicotinic acid and nicotinamide in biological fluids such as blood and urine and in foods such as coffee and meat. Derivatization techniques have also been employed to improve sensitivity (55). For example, the reaction of nicotinic amide with DCCI (AT-dicyclohexyl-0-methoxycoumarin-4-yl)methyl isourea to yield the fluorescent coumarin ester has been reported (56). After separation on a reversed-phase column, detection limits of 10 pmol for nicotinic acid have been reported (57). 

Numerous high pressure Hquid chromatographic techniques have been reported for specific sample forms vegetable oHs (55,56), animal feeds (57,58), seta (59,60), plasma (61,62), foods (63,64), and tissues (63). Some of the methods requite a saponification step to remove fats, to release tocopherols from ceHs, and/or to free tocopherols from their esters. AH requite an extraction step to remove the tocopherols from the sample matrix. The methods include both normal and reverse-phase hplc with either uv absorbance or fluorescence detection. AppHcation of supercritical fluid (qv) chromatography has been reported for analysis of tocopherols in marine oHs (65). 

A six-port valve was first used to interface the SEC microcolumn to the CZE capillary in a valve-loop design. UV-VIS detection was employed in this experiment. The overall run time was 2 h, with the CZE runs requiring 9 min. As in the reverse phase HPLC-CZE technique, runs were overlapped in the second dimension to reduce the apparent run time. The main disadvantage of this yu-SEC-CZE method was the valve that was used for interfacing. The six-port valve contributed a substantial extracolumn volume, and required a fixed volume of 900 nL of effluent from the chromatographic column for each CZE run. The large fixed volume imposed restrictions on the operating conditions of both of the separation methods. Specifically, to fill the 900 nL volume, the SEC flow rate had to be far above the optimum level and therefore the SEC efficiency was decreased (22). 

The ionic or polar substances can be seperated without any reaction on specially treated chromatographic columns and detected refractometrically. This is necessary because alkyl sulfosuccinates show only small absorption in the UV-visible region no sensitive photometric detection can be obtained. Separation problems can arise when common steel columns filled with reverse phase material (or sometimes silica gel) are used. This problem can be solved by adding a suitable counterion (e.g., tetrabutylammonium) to the mobile phase ( ion pair chromatography ). This way it is possible to get good separation performance. For an explanation of separation mechanism see Ref. 65-67. A broad review of the whole method and its possibilities in use is given in an excellent monograph [68]. 

In recent years, the methods for carotenoid determination without saponification have increased. Independently of the mobile phase and food composition, there are similar patterns of chromatographic separation on reversed phase columns. A chromatograph can be divided roughly into four zones the first zone corresponds to free xanthophyUs, the second zone to monoesterified pigments, the third zone contains carotenes, and finally the fourth zone corresponds to diesterified carotenoids. - ... 

Tswett s initial column liquid chromatography method was developed, tested, and applied in two parallel modes, liquid-solid adsorption and liquid-liquid partition. Adsorption ehromatography, based on a purely physical principle of adsorption, eonsiderably outperformed its partition counterpart with mechanically coated stationary phases to become the most important liquid chromatographic method. This remains true today in thin-layer chromatography (TLC), for which silica gel is by far the major stationary phase. In column chromatography, however, reversed-phase liquid ehromatography using chemically bonded stationary phases is the most popular method. 

Radke et al. [28] described an automated medium-pressure liquid chromatograph, now commonly called the Kohnen-Willsch instrument. At present, the method is widely used to isolate different fractions of soluble organic matter (for instance, as described in Reference 29 to Reference 31). A combination of normal phase and reversed-phase liquid chromatography has been used by Garrigues et al. [32] to discriminate between different aromatic ring systems and degrees of methylamine in order to characterize thermal maturity of organic matter. 

The most significant differences (i.e. independence) in the analytical methods are provided in the final chromatographic separation and detection step using GC/ MS and LC-FL. GC and reversed-phase LG provide significantly different separation mechanisms for PAHs and thus provide the independence required in the separation. The use of mass spectrometry (MS) for the GC detection and fluorescence spectroscopy for the LG detection provide further independence in the methods, e.g. MS can not differentiate among PAH isomers whereas fluorescence spectroscopy often can. For the GC/MS analyses the 5% phenyl methylpolysiloxane phase has been a commonly used phase for the separation of PAHs however, several important PAH isomers are not completely resolved on this phase, i.e. chrysene and triphenylene, benzo[b]fluoranthene and benzofjjfluoranthene, and diben-z[o,h]anthracene and dibenz[a,c]anthracene. To achieve separation of these isomers, GC/MS analyses were also performed using two other phases with different selectivity, a 50% phenyl methylpolysiloxane phase and a smectic liquid crystalline phase. 

The PRISMA model was developed by Nyiredy for solvent optimization in TLC and HPLC [142,168-171]. The PRISMA model consists of three parts the selection of the chromatographic system, optimization of the selected mobile phases, and the selection of the development method. Since silica is the most widely used stationary phase in TLC, the optimization procedure always starts with this phase, although the method is equally applicable to all chemically bonded phases in the normal or reversed-phase mode. For the selection of suitable solvents the first experiments are carried out on TLC plates in unsaturated... 

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