UV detection limits

Anion Retention time (min) UV active UV detection limite (mg L 1) UV detector position Wavelength (nm)... 

Vitamin K (phylloquinone), 2, 3 -dihydrophylloquinone, and menaquinones (where n = 4, 5, 6) were extracted fiom milk and infant formula and analyzed on a C,8 column (A = 243 nm, ex 430 nm, em). All compounds were well resolved and eluted in 15 min using a 90/10/0.5 methanol/dichloromethane/methanol (with lOitiM zinc chloride, 5mM sodium acetate, and 5mM acetic acid) mobile phase [344]. Note that this level of salts in the mobile phase could precipitate in various parts of the LC system. Flush the system regularly. The linear working curve extended from 2 to 50 ng/mL with a detection limit of 1.5 ng/mL. The authors noted that this compared favorably to a UV detection limit (at A = 269 nm) of 50 ng/mL. 

In a high percentage of HPLC and CE separations, monitoring is performed with ultraviolet detectors. However, many immunoassays are aimed at the determination of analytes that are present at concentrations below the UV detection limit. Furthermore, as indicated before, desorption buffers can decrease the sensitivity of immunochromatographic assays. Approaches used to enhance sensitivity in conventional immunoassay formats do the same in immuno-HPLC and immuno-CE detection and for this reason are considered together under this subheading. 

Benzalkonium chloride may be preconcentrated by liquid-liquid extraction to decrease the UV detection limit. A clever system allows for extraction into acetonitrile, which can be directly analyzed by HPLC. The separate acetonitrile phase is obtained by adding salt to an acetonitrile/water solution. The effect of pH and impurities has not yet been studied sufficiently to recommend this approach for general use (194). 

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. 

Aminophenols have been detected in waste water by investigating uv absorptions at 220, 254, and 275 nm (87). These compounds can also be detected spectrophotometricaHy after derivatization at concentrations of 1 part per 100 million by reaction in acid solution with /V-(1-napbtby1)etby1enediamine [551-09-7] (88) or 4-(dimethylainino)ben2aldehyde [100-10-7] (89), and the Schiff base formed can be stabilized in chloroform by chelation to increase detection limits (90). 

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). 

After the dipped or sprayed chromatogram has been dried in a stream of cold air long-wave UV light (2 = 365 nm) reveals fluorescent yellow zones (flavonoids). Sterigmatocystine, which can be detected without derivatization on account of its red intrinsic fluorescence (detection limit 0.5 pg), also fluoresces pale yellow after being heated to 80°C [9] or 100°C [13] for 10 min on the other hand, citrinine, zearalenone and vomitoxin fluoresce blue. 

In situ quantitation The in situ fluorimetric analysis was made under long-wavelength UV light (2eic = 365 nm, An > 560 nm) and is illustrated in Figure 1. The detection limits for maltose, glucose and fructose were ca. 10 ng substance per chromatogram zone. 

Sinigrin (JiRt 35—40) appeared as a yellow fluorescent chromatogram zone on a dark background under long-wavelength UV light (2 = 365 nm). The detection limit was 25 — 50 ng substance per chromatogram zone. 

In situ quantitation Fluorimetric analysis was made with long-wavelength UV light (2exc = 365 nm, X(, > 430 nm). The detection limit on HPTLC plates that were analyzed in a moist state was 25 ng cholesterol per chromatogram zone (Fig. 1). 

Folic acid is detected by irradiating the chromatogram with broad-spectrum UV light for 30 min before reaction with Bratton-Marshall reagent (detection limit 200ng)[12]. 

Detection and result The chromatogram was dried in a stream of warm air for 10 min, immersed in the reagent solution for 3 s and then subjected to intense UV radiation (high pressure lamp, A = 365 nm) for up to 10 min. Terephthalic (hRf 0 - 5), pimelic (hRf 55), suberic (hRf 60), sebacic (hRf 65 — 70) and benzoic acids (hRf 70 — 75) together with sorbic, malic, adipic, citric, tartaric, lactic and fumaric acids only exhibited a reaction on silica gel layers at higher concentrations. 4-Hydroxybenzoic, salicylic and acetylsalicylic acids fluoresced light blue after irradiation. The detection limit per chromatogram zone was 0.5 pg for salicylic acid and more than 5 pg for benzoic acid. 

In situ quantitation The fluorimetric analysis was carried out in long-wavelength UV light (A c = 365 nm An > 560 nm). The detection limit for fatty acids was ca. 100 ng per chromatogram zone. 

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