Derivatives for HPLC

The formation of new chromophores for the optimization of ultraviolet (UV) detection of analytes in HPLC implies the synthesis of derivatives with conjugated systems in the molecule. Compared with GC, there are no restrictions on the volatilities of these derivatives for HPLC analysis. They may be synthesized before analysis (precolumn derivatization) or after chromatographic separation (postcolumn derivatization). The latter technique is rarely used and then only for a few classes of compounds, but it permits us to... 

The parent compounds and the metabolites have very low aqueous, solubility. They have proven difficult to extract from some matrices the current method for ivermectin involves some 41 concentration and clean-up steps preceding HPLC (8). A Merck method for recovering abamectin residues from strawberries and formation of fluorescent derivatives for HPLC analysis has 18 separate steps (9). A recently published two-step solid-phase recovery procedure for ivermectin from serum indicates that it is possible to combine an abbreviated concentration and cleanup method with a sensitive and specific detection system in this case, liquid chromatography (10). An immunoassay for avermectins that could be interfaced with simplified residue recovery protocols is a promising solution to the intensifying demands on regulatory agencies to monitor these compounds. 

Phenacyl esters are prepared by heating the potassium salt with phenacylbromide in the presence of 18-crown-6 in acetonitrile. Such esters exhibit strong UV absorption near 240 nm and are useful derivatives for HPLC (393). Para-nitrophenyl esters are prepared from the acid and p-nitrophenol in the presence of A,A -dicyclohexylcarbo-diimide and absorb at about 270 nm (see Scheme 5). 

The recent development and comparative application of modern separation techniques with regard to determination of alkylphosphonic acids and lewisite derivatives have been demonstrated. This report highlights advantages and shortcomings of GC equipped with mass spectrometry detector and HPLC as well as CE with UV-Vis detector. The comparison was made from the sampling point of view and separation/detection ability. The derivatization procedure for GC of main degradation products of nerve agents to determine in water samples was applied. Direct determination of lewisite derivatives by HPLC-UV was shown. Also optimization of indirect determination of alkylphosphonic acids in CE-UV was developed. Finally, the new instrumental development and future trends will be discussed. 

Reports on the use of fluorescent derivatives abound (5). Some reagents have become widely used. The dansyl group is probably the most thoroughly studied. Dansyl chloride has been widely used as a fluorescent derivatizing reagent for HPLC (6,7). It reacts readily with primary and secondary amino groups (7) and with phenols (8), but forms derivatives of alcohols very slowly (9). The lower detection limit for dansyl derivatives of aliphatic amines is in the range of 300 femtomoles per injection. 

Quantitation is performed by the calibration technique. A standard solution containing 0.1 mgkg of both M.A3 and M.A4 is prepared and 1, 2.5, 5 and 7.5mL of this solution are pipetted into around-bottom flask separately and evaporated. Each sample is converted into the fluorescent anhydride derivative according to the procedures described above. Each sample is dissolved in lOmL of methanol for injection into the HPLC system. The calibration curves are obtained by plotting the peak heights against the amounts of M.A3 and M.A4. The derivatives for preparing the calibration curve should be freshly prepared on a daily basis prior to quantitation. 

A large number of devices have been used as detectors for hplc. The characteristics of five of the more important types are described, and examples are given of the range of samples for which they can be used. The use of derivative preparation as an aid to detection is considered. 

The values of ks/kp for partitioning of carbocations are most conveniently determined as the ratio of the yields of products from the competing nucleophile addition and proton transfer reactions (equation 1 derived for Scheme 2). The determination of these product yields has been simplified in recent years by the application of high-pressure liquid chromatography (HPLC). Typically, the product peaks from an HPLC analysis are detected and quantified by UV-vis spectroscopy. In cases where the absorbance of reactants and products is small, substrates may be prepared with a chromophore placed at a sufficient distance so that its effects on the intrinsic reactivity of the carbocationic center are negligible. For example, the aliphatic substrates [1]-Y have proved to be very useful in studies of the reactions of the model tertiary carbocation [1+].21,23... 

For example, peroxidase catalyzes the reaction of luminol derivatives with hydrogen peroxide and results in an increase of the CL reaction velocity and CL intensity. Therefore, intense CL can be obtained from the analyte labeled with luminol derivatives after HPLC separation, followed by reaction with peroxidase. 

The structures of luminol derivatives used for HPLC-CL detection are shown in Figure 7A. Analytes labeled with luminol derivatives can be detected using hydrogen peroxide and potassium hexacyanoferrate(III) under alkaline conditions after HPLC separation (Table 1). For example, ibuprofen in saliva [34], saturated... 

Amino acids labeled with DNS-C1 were determined using the Ru(bpy)32+ CL reaction after HPLC separation with a reversed-phase column [104, 105], DNS derivatives are expected to produce intense CL owing to their secondary and tertiary amino groups. The detection limit for DNS-Glu was 0.1 pM (2 pmol/ injection). Although underivatized amino acids could be detected by Ru(bpy)32+ CL, the DNS derivatives showed improved detection limits by three orders of magnitude [105], An approach to convert primary amines to tertiary amines was also reported [106], In this method, divinyl sulfone (DVS) was used for a cycloaddition reaction of primary amines (Fig. 19). The DVS derivatives after HPLC separation were sensitively detected (e.g., detection limits for propylamine and 3-aminopentane were 30 and 1 pmol, respectively). 

The use of macrocyclic antibiotics as chiral selectors for HPLC was first proposed by Armstrong et al. [50] in 1994. The most successful of the CSPs are based on the glycopeptide antibiotics vancomycin, teicoplanin and ristocetin A and are commercially available through Advanced Separation Technologies Inc. (Astec Inc.) as Chirobiotic V , Chirobiotic 1 and Chirobiotic R , respectively. More recently, a number of other derivatives of these antibiotics have also been developed offering different stereoselectivities. A comprehensive handbook is now available from Astec Inc. [51 ] alongside a number of recent review articles... 

Difficulties encountered in the postsynthetic chemical sulfation of peptides and the correspondingly low yields have led to the proposal of an alternative approach. This approach makes use of appropriate tyrosine 0-sulfate derivatives for the chain elongation steps in solution and on solid supports by applying protection strategies compatible with the acid sensitivity of the sulfate ester. Moreover, the analytical characterization of the peptides synthesized with tyrosine 0-sulfate derivatives is greatly facilitated since contaminations deriving from the preparation of the intermediates are easily detected by chromatographic (HPLC and CE) and spectroscopic methods (see Table 2). 

Several studies attempted to relate the partition coefficient P of a solute in a liquid chromatographic or a gas chromatographic system with the composition of the two phases, one of which has a varying composition [19-23]. Tijssen et al. [24] and Schoenmakers [25] derived a relation between the partition coefficient and a binary mobile phase in reversed-phase HPLC from the solubility parameter theory of Hildebrand et al. [26]. Similarly, a relation can be derived for liquid-liquid extraction with extraction liquids composed of three components ... 

Normal-phase HPLC (NP-HPLC) and reverse-phase HPLC (RP-HPLC) are used to purify the final products of these chemical derivatives. For NP-HPLC a gradient of hex-ane/MeOH/THF works well for separations. Usually, acetogenins are placed on RP-HPLC because they are more easily detected in the low-ultraviolet range. The weak UV absorbance at 210 nm for the a,p-unsaturated y-lactone is one reason to use an RP-HPLC system. This weak absorbance is often overshadowed by impurities that have larger absorbances. Another reason for choosing RP-HPLC is because the solvent cutoff for the RP solvents is lower than NP solvents. In their pure form, acetogenins are white, waxy substances. 

---