Detection limits, HPLC

Electrochemical Detectors Another common group of HPLC detectors are those based on electrochemical measurements such as amperometry, voltammetry, coulometry, and conductivity. Figure 12.29b, for example, shows an amperometric flow cell. Effluent from the column passes over the working electrode, which is held at a potential favorable for oxidizing or reducing the analytes. The potential is held constant relative to a downstream reference electrode, and the current flowing between the working and auxiliary electrodes is measured. Detection limits for amperometric electrochemical detection are 10 pg-1 ng of injected analyte. 

Description of Method. Fluoxetine, whose structure is shown in Figure 12.31a, is another name for the antidepressant drug Prozac. The determination of fluoxetine and its metabolite norfluoxetine. Figure 12.31 b, in serum is an important part of monitoring its therapeutic use. The analysis is complicated by the complex matrix of serum samples. A solid-phase extraction followed by an HPLC analysis using a fluorescence detector provides the necessary selectivity and detection limits. 

UV/Vis detectors are among the most popular. Because absorbance is directly proportional to path length, the capillary tubing s small diameter leads to signals that are smaller than those obtained in HPLC. Several approaches have been used to increase the path length, including a Z-shaped sample cell or multiple reflections (Figure 12.44). Detection limits are about 10 M. 

A iridine traces in aqueous solution can be determined by reaction with 4-(p-nitroben25l)pyridine [1083-48-3] and potassium carbonate [584-08-7]. Quantitative determination is carried out by photometric measurement of the absorption of the blue dye formed (367,368). Alkylating reagents interfere in the determination. A iridine traces in the air can be detected discontinuously by absorption in Folin s reagent (l,2-naphthoquinone-4-sulfonate) [2066-93-5] (369,370) with subsequent chloroform extraction and hplc analysis of the red dye formed (371,372). The detection limit is ca 0.1 ppm. Nitrogen-specific thermal ionisation detectors can be used for continuous monitoring of the ambient air. 

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. 

The limits of lifetime detection and resolution in on-the-flight fluorescence lifetime detection in hplc were evaluated for simple, binary systems of polycycHc hydrocarbons (70). Peak homogeneity owing to coelution was clearly indicated for two compounds having fluorescence lifetime ratios as small as 1.2 and the individual peaks could be recovered using predeterrnined lifetimes of the compounds. Limits of lifetime detection were deterrnined to be 6 and 0.3 pmol for benzo[b]fluoranthene and benzo[k]fluoranthene, respectively. 

The analysis of mefloquine in blood, using packed-column sfc, a mobile phase consisting of / -pentane modified with 1% methanol and 0.15% -butylamine, and electron capture detection has been reported (92). The method compares favorably to a previously pubflshed hplc-based procedure having a detection limit of 7.5 ng/mLin 0.1 mL blood sample. 

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). 

There have been also found the quantitative characteristics of the methods. They are as follows for HPLC method the linearity is 0.1 ng to 2 ng the detecting limit is 0.1 ng the limit of the quantitative estimation makes up 0.0004 mg/kg a coefficient of variation is 2.74% for the chromatodensitometry method the linearity is 2 ng to 10 ng the detecting limit is 0.6 ng the coefficient of variation is 2.37%. The data obtained have been treated using a regressive analysis. 

Figure 12.1 Analysis of Tinuvin 1577 in 30% virgin olive oil (in hexane), showing (a) the gas cliromatogram comparing the pure oil with a sample at the Tinuvin 1577 detection limit concentration, and (b) the coixesponding liquid chromatogram. Reprinted from Journal of High Resolution Chromatography, 20, A. L. Baner and A. Guggenberger, Analysis of Tinuvin 1577 polymer additive in edible oils using on-line coupled HPLC-GC , pp. 669-673, 1997, with pennission from Wiley-VCH.

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. 

HPLC has been recommended as a cleanup and fiactionation procedure for food samples prior to analysis by GC/ECD (Gillespie and Walters 1986). The advantages over the AOAC-recommended Florisil colunrn are that it is faster, requires less solvent, and gives better resolution. HPLC coupled with various detectors MS, MS/MS, UV/electrochemical detector, or UV/polarographic detection has been tested as a rapid, simplified separation and detection system to replace GC (Betowski and Jones 1988 Clark et al. 1985 Koen and Huber 1970). Recoveries, detection limits, and precisions were generally good, but further work is needed before the techniques are adopted for general use. 

The application of the fluorescence derivatization technique in an HPLC method involves utilization of a post column reaction system (PCRS) as shown in Figure 3 to carry out the wet chemistry involved. The reaction is a 2-step process with oxidation of the toxins by periodate at pH 7.8 followed by acidification with nitric acid. Among the factors that influence toxin detection in the PCRS are periodate concentration, oxidation pH, oxidation temperature, reaction time, and final pH. By far, the most important of these factors is oxidation pH and, unfortunately, there is not one set of reaction conditions that is optimum for all of the PSP toxins. The reaction conditions outlined in Table I, while not optimized for any particular toxin, were developed to allow for adequate detection of all of the toxins involved. Care must be exercised in setting up an HPLC for the PSP toxins to duplicate the conditions as closely as possible to those specified in order to achieve consistent adequate detection limits. 

The second most widely used detector in HPLC is the differential refractometer (RI). Being a bulk property detector, the RI responds to all substances. As noted in Table 3 the detection limits are several orders of magnitude higher than obtained with the UV detector. Thus, one turns to the RI detector in those cases in which substances are non-UV active, e.g. lipids, prostaglandins. In addition, the RI detector finds use in preparative scale operation. Finally, relative to the UV detector, the RI is significantly more temperature and flow sensitive and cannot be used in gradient elution. 

Five synthetic and five natural colorants were identified and quantified in lyo-philized dairy products and fatty foods using an automatic method based on solid phase extraction using a stationary phase followed by RP-HPLC C,g columns for the sequential retention of colorants and diode array detection. Lyophilization of the samples coupled with the separation procedure provided clean extracts despite the complexity of the food matrices and preserved the sample for at least 2 months without changes in colorant concentrations. The detection limits achieved for the colorants were found in a wide range from 0.03 to 75 pg/g of the lyophilized sample, according to the limits established by the European Union. ... 

The HPLC-based measurement of 8-OH-dGua is a highly sensitive method, largely because of the use of electrochemical detection, introduced by Floyd et al. (1986). Floyd et al. (1986) quote a detection limit of 20 fmol, or one 8-OH-dGua per 10 nucleosides. Shigenaga et al. (1990) quote 5-50 fmol on 40-100 / g samples of DNA. 

There are many proteins in the human body. A few hundreds of these compounds can be identified in urine. The qualitative determination of one or a series of proteins is performed by one of the electrophoresis techniques. Capillary electrophoresis can be automated and thus more quantified (Oda et al. 1997). Newer techniques also enable quantitative determination of proteins by gel electrophoresis (Wiedeman and Umbreit 1999). For quantitative determinations, the former method of decomposition into the constituent amino acids was followed by an automated spectropho-tometric measurement of the ninhydrin-amino add complex. Currently, a number of methods are available, induding spectrophotometry (Doumas and Peters 1997) and, most frequently, ELISAs. Small proteins can be detected by techniques such as electrophoresis, isoelectric focusing, and chromatography (Waller et al. 1989). These methods have the advantage of low detection limits. Sometimes, these methods have a lack of specifidty (cross-over reactions) and HPLC techniques are increasingly used to assess different proteins. The state-of-the-art of protein determination was mentioned by Walker (1996). 

In general, CE is simple, rapid, and low cost because it needs neither laborious treatment of the samples nor long times of analysis. However, its high detection limit is a major limitation of CE. CE is often poorly reproducible. Enzymatic assay is more suitable for quantifying one organic acid in honey samples because it is specific, precise, and accurate. GC is more suitable for analyzing volatile or semivolatile chemicals. HPLC is versatile and reproducible. However, common HPLC detectors such as UV-VIS are not very sensitive for organic aliphatic acids. 

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