High performance LC

Classical vs. High Performance LC. Most workers are familiar with classical LC, a tool that has been predominantly used for preparative scale clean-up of samples. In order to appreciate more fully HPLC, Table I compares some of the column characteristics of classical vs. HPLC. 

The analysis of ILs may afford considerable insight into the physicochemical properties underlying the rich potential interaction chemistries of ILs [14] and suggest possibilities for future applications. Simultaneously, the unique features of ILs provide some intriguing new possibilities in the area of separations that have yet to be realized. Hence, topics to be covered in this chapter include analysis of ILs by LC, applications of ILs in liquid-phase microextraction (LPME), in high-performance LC (HPLC) as mobile-phase additives, and in capillary electrophoresis (CE) as buffer additives as well as applications of surface-confined ILs (SCIL) as novel stationary phases for LC. 

In an attempt to avoid interactions with residual silanol groups, Abidi and Mounts investigated the separation of the molecular species of PC, PE, and SPH on polymeric C18 columns by RP-HPLC (103). Of the three polymer columns evaluated, the best HPLC results were obtained with an octadecanoyl polyvinyl alcohol (ODPVA) stationary phase. High-performance LC on ODPVA with an A/M/W mobile phase provided significantly faster analysis and greater detection sensitivity than assays with C18 silica columns. 

Finally, this section would remain incomplete without a few comments on the applications of HPLC to a particular group of carbohydrates—polysaccharides—whose determination, both qualitatively and quantitatively, has received much less attention than the rest (56). This may be surprising when the importance of these compounds, in terms of both functional properties and nutrition, is considered, but it is not so surprising when the difficulty of the analyses required is studied. High-performance LC can be used in this field, either to characterize the polysaccharides per se or to study their carbohydrate composition and the nature of bonding after acid or enzymic hydrolysis. 

The nutritional evaluation of the vitamin E-rich vegetable oils and the products made from them using a nonbiological assay necessitates the determination of the individual tocopherols and to-cotrienols, for these vitamers vary widely in biological activity. High-performance LC is ideally suited for this purpose, and the overall vitamin E value of such foods can be estimated by applying appropriate factors based on relative biological activities. For the analysis of those animal products known to contain predominantly a-tocopherol, only this vitamer need be determined. In vitamin E-fortified foods it is usually sufficient to determine either the added a-tocopheryl acetate or the total a-tocopherol. 

Cleanup or fractionation procedures that have been used in the more recent fat-soluble vitamin assays include sterol precipitation, open-column chromatography, solid-phase extraction, and high-pressure gel permeation chromatography. High-performance LC has been used on a semipreparative scale in vitamin D and vitamin K assays to obtain purified fractions of sample extracts. This technique is discussed in Sec. V.B.3. 

High-performance LC is by far the most studied technique, and it is used whenever possible for aflatoxins, ochratoxin A, and zearalenone, especially in view of the improved rapidity attributable to the use of immunoaffinity in the cleanup step. 

High-performance LC is by far the most frequently used technique to monitor aspartame, intermediates formed during its synthesis, and decomposition products formed during manufacture and storage of food products. It is also used to check for the amounts declared on food labels (14,23). 

High-performance LC is routinely used for the analysis of stevia sweeteners in plant material or in food and beverages. The first successful HPLC separation of stevioside and rebaudioside A... 

High-performance LC was also used for determination of TBZ after its extraction from marmalades and curds with ethyl acetate (13). The use of a buffered mobile phase improved the response of the UV detector, and column performance remained constant throughout 2 months of daily use with a detection limit of 100 ppb. Three detectors (UV, fluorimetric, and electrochemical) were used for the determination of OPP, BP, and TBZ in plant materials (45). The compounds were extracted with dichloromethane and separated on an RP-18 column with a methanolic formic acid buffer as eluent. It was not possible to determine TBZ using an electrochemical detector, although the extraction recovery varied between 80 and 95%. 

High-performance LC is also a suitable method for separating BHA isomers. Commercially available BHA is a mixture of two positional isomers, an approximately 85 15 ratio of 3-BHA and 2-BHA. The former is approximately 2.4 times more effective as a food antioxidant than is 2-BHA, and half as effective as an inhibitor of benzo(a)pyrene-induced for stomach neoplasia in mice. For the separation, Ansari (116) used isocratic elution with 7% of 2-propanol in hexane on a Pirkle Type I-A column packed with 5- 01 y-aminopropyl packing, modified with lV-3,5-dinitrobenzoyl derivative of D-phenylglycine. Column eluent was monitored at 288 nm, with a detection limit between 1 and 2 ng. Under these conditions, isomers were separated without derivatization, where the phenolic group of 3-BHA was sterically hindered by an o-rm-butyl group and therefore could not interact with stationary phase that resulted in its rapid elution. 

High-performance LC systems used for the determination of SPA are presented in Table 9. 

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-... 

High-performance LC is the technique of choice for residue determination in foods of nonvolatile, thermally labile carbamate and urea pesticides. National programs for monitoring pesti-... 

High-performance LC is widely used, offline or online, in the determination of pesticides, either as a final measurement step or as a separation technique. The increase in the use of HPLC is mainly the result, on the one hand, of its suitability for determining thermally labile and polar pesticides that require derivatization prior to GC, and, on the other, of its compatibility with online precolumn extraction and cleanup and with MS systems (19). 

High-performance LC determination is also compatible with the extraction of environmental water with an organic solvent such as methylene chloride or ethyl acetate (31,32) and solid-phase extraction (SPE) (33,34). Solid-phase extraction and liquid-liquid extraction (LLE) have been compared with respect to their ability to preconcentrate pesticides prior to HPLC analysis. The reproducibility of the method is better when C,8 cartridges are used than with conventional LLE, but LLE sometimes gives better recoveries, for example, for dimethoate, chlor-pyriphos ethyl, and carbofenothion (35). 

High-performance LC coupled to capillary GC is a technique in which fractions from one or more LC columns are transferred into the GC column for further separation. This coupled technique is used more to separate a particular compound and/or class of compounds from an unknown matrix. Another field in HPLC serves for the preseparation of closely related classes or subclasses of compounds (64). 

High-performance LC methods for pesticide residue analysis were first developed for nonvolatile or thermally labile compounds, such as carbamate insecticides. Because HPLC offers a simpler and/or faster approach to analysis for a wide range of other compounds, it is becoming more and more widely accepted, and its applications are steadily increasing in number. Although HPLC has been used in the analysis of OCPs and OPPs, the literature on its application in food is scarce. The methods reported have been summarized in Table 4. 

High-performance LC in the reversed-phase mode (RP-8 or RP-18 column) with UV detection (254 nm) and isocratic conditions was evaluated for the analysis of 15 organophosphorus pesticides. Typically the method could be used to analyze azinphosmethyl in water at 0.5 /rg/L. This compares favorably with GC, since this compound is very difficult to analyze by that method. For other pesticides, such as fenitrothion, which is readily analyzed by GC, the HPLC method can be used for confirmation purposes (31). 

High-performance LC was used to determine residue levels of p,p-DDE in marine mammal blubber. This compound can absorb UV well enough at shorter wavelengths. With normal-phase LC after derivatization to convert the p,p-DDE to / ,/ -DCPB (dichlorobenzophenone), a semiquantitative determination of this compound was done in the presence of PCBs (62). 

High-performance LC analysis of phenolics in apples and pear juices are often performed by gradient procedures (49-54), due to the presence of several phenolic classes. Most of the HPLC methods applied to apple phenolics are very similar to that of Oleszek et al. (55). 

High-performance LC with H and 13C NMR and FAB-MS techniques was utilized to isolate and identify the four esters of hydroxycinnamic acids from fresh and raw broccoli florets... 

High performance LC is commonly employed for separation and quantifying phenolic compounds in cereal grains (126), especially due to the existence of considerable evidence linking the presence of phenolic acids in grain to disease resistance and resistance to mold damage. 

High-performance LC techniques are often applied to various fruit juices and drinks (158-161) to evaluate the antioxidative activity, which is attributed largely to the phenolics, such as flavonoids and phenylpropanoids. Analysis of food phenolics is gaining popularity with the growing evidence of possible health-promoting benefits of phenolics in foods, such as antioxidative, antimicrobial, tumor-inhibiting, free-radical scavenging, and other clinically relevant activities. 

Several countries have fixed the maximum allowed concentration for some of the BAs in wine e.g., Switzerland recommends 4 mg/L for His, Netherlands 5 mg/L, Germany 2 mg/L, and France 8 mg/L. High-performance LC techniques are largely used to determine BAs content in wines, with ion exchange (96,107) or RP columns withpre- (100,108-110) or post- (10) column derivative formation or without derivatization (98,4,111,112) with different detection means, mainly UV (50,110,113,114) or fluorescence (97,100,50,108,109). Nevertheless, if BAs are to be determined at low levels with no interference from other compounds, e.g., AA, previous cleanup and preconcentration steps are required. 

Methods for the analysis of aromatic amines in synthetic azo dyes and in food and beverages colored with these dyes have been developed. High-performance LC or GC methods are generally employed, often with the help of derivatization reaction and fluorimetric detection, with HPLC generally regarded as the best technique for the determination of aromatic amines. 

The novel approaches in LC-MS-based lipidomics, such as ultra-high performance LC (UHPLC), combined with high-resolution MS methodologies allow fast and reliable analysis of various classes of lipids (33). The modem instruments, with fast duty cycles, allow acquisition of high and low collision energy data simultaneously to provide both data on precursor ions and fragmentation data for all detectable molecular ions (34). 

Beside column dimension the size of stationary phase particles is a matter of recent progress. More traditional columns are packed with 3.0-5 pm particles enabling satisfying resolution and reasonable column back pressure of solvent suitable to be processed by conventional HPLC pumps. In contrast, sub 2-pm particles (e.g. 1.7 and 1.8 pm) as applied in rapid or fast LC or ultra high-performance LC (UHPLC) allow better resolved separations in shorter run times. Column back pressure (>12,000 psi) is remarkably high demanding more robust solvent pumps. 

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