Ultraviolet absorbance detectors

High performance liquid chromatography is used to determine the purity of calcitriol, and to separate it from related compounds. Using a 10 micron silica column of 25 cm length, and a mobile phase of spectroquality heptane ethyl acetate. methanol (50 50 1) at a flow rate of 1.7 ml/ minute, separation and quantitation are achieved. p-Dimethyl-aminobenzaldehyde may be used as an internal standard to compensate for variations in injection technique and instrumental conditions. With a 254 nm ultraviolet absorbance detector, 0.01 ug of calcitriol may be detected (3). 

Panderi and Parissi-Poulou developed a microbore liquid chromatographic method for the simultaneous determination of benazepril hydrochloride and hydrochlorothiazide in pharmaceutical dosage forms [30]. The use of a BDS C-18 microbore analytical column was found to result in substantial reduction in solvent consumption and in increased sensitivity. The mobile phase consisted of a mixture of 25 mM sodium dihydrogen phosphate buffer (pH 4.8) and acetonitrile (11 9 v/v), pumped at a flow rate of 0.4 mL/min. Detection was effected at 250 nm using an ultraviolet absorbance detector. The intra- and inter-day relative standard deviation values were less than 1.25% (n = 5), while the relative percentage error was less than 0.9% (n = 5). The detection limits obtained according to the IUPAC definition were 0.88 and 0.58 pg/mL for benazepril hydrochloride and hydrochlorothiazide, respectively. The method was applied to the quality control of commercial tablets and content uniformity test, and proved to be suitable for rapid and reliable analysis. 

Experimental. Absorbances of selected polymer and copolymer samples were measured with a Waters 440 ultraviolet absorbence detector equipped with a 254 nm filter, and a Beckman 25 ultraviolet/visible spectrophotometer. The Water s instrument was used on-line with the size exclusion chromatograph (16). The Beckman 25 was used to calibrate and standardize the Water s 440 UV detector. THF was used as standard solvent. The extinction coefficients were estimated from absorbance measurements at several polymer concentrations. The standard deviation for the absorbance measurements was typically . 001 Au for replicates on the same sample. All measurements were done at room temperature. 

Whilst spectroscopy techniques can be used on their own to obtain spectral information about a sample they are also commonly incorporated as a detector as part of another technique, for example the use of an ultraviolet absorbance detector as part of a liquid chromatography system. In recent years there has been much enthusiasm for the research and development of hyphenated techniques, that is the interfacing/linking together of two or more techniques, because of the enhanced additional data that can be generated. 

Wavelength at which solvents become opaque in ultraviolet, for use with ultraviolet absorbance detectors. 

Fig 2. Overall scheme of S/LALLS experiment. P = polymer molecules CS = chase solution F = membrane filter UV = ultraviolet absorbance detector LS = low-angle light scattering detector C = computer. (Reprinted with permission from Macromolecules 422-426. Copyright 1986 American Chemical Society)... 

A UV/Vis absorbance detector can also be used if the solute ions absorb ultraviolet or visible radiation. Alternatively, solutions that do not absorb in the UV/Vis range can be detected indirectly if the mobile phase contains a UV/Vis-absorbing species. In this case, when a solute band passes through the detector, a decrease in absorbance is measured at the detector. 

Collect fractions starting from the bottom of the gradient, while recording an absorbance profile at 254 nm (which is dominated by the very abundant ribosomal RNA). We use a setup consisting of a peristaltic pump, ultraviolet (UV) detector, and fraction collector commonly used for chromatography experiments (GE Healthcare Life Sciences). 

CZE = capillary zone electrophoresis EC = electrochemical detector GC = gas chromatography HCD = Hall conductivity detector HPLC = high performance liquid chromatography IDMS = isotope dilution mass spectrometry MS = mass spectrometry RSD = relative standard deviation SEE = supercritical fluid extraction SPE = solid phase extraction UV = ultraviolet absorbance detection... 

In the beginning, the refractive index detector was the most used detection system, although it has two important drawbacks (1) solvent gradients cannot be used, and (2) it has low sensitivity and different responses to saturated and highly unsaturated TGs (112). Moreover, use of the ultraviolet (UV) detector is difficult, because the most adequate solvents also absorb in the same range and therefore cause an important baseline drift with gradient elution systems (106). 

Ultraviolet absorbance detection is the most prevalent type of detection in CE, and UV detectors operate in both the direct and indirect modes. Laser-induced fluorescence detection is often used for high-sensitivity work. Conductivity detection, suppressed conductivity detection, and mass spec-... 

An ultraviolet-laser based thermo-optical absorbance detector for micrometer capillaries was used by Qi et al. [76] to monitor the separation of a mixture of 13 phenylthiohydantoin-amino acids. A modulated pump laser beam periodically illuminated the capillary at a point near its end. Complex deflection and diffraction effects occur at the capillary-solution interface. Perturbation of the refractive index at this interface changes the intensity of the probe beam that is measured using a small photodiode. 

As far as the conversion of the analytical response is concerned, the most used detectors in GrFFF have been, until now, conventional ultraviolet (UV) detectors commonly used for HPLC. With this type of detector, the amount of particles with diameter di is proportional to the detector response at the zth point. With particulate samples, in fact, because of UV detector optics, the response is a turbidity signal read within an angle between the incident light and the photosensor (i.e., usually smaller than —10°) rather than the absorbance. This turbidity signal can be assumed to be directly proportional to the sum of all cross-sectional areas of the particulate sample components at any time. The validity of the above assumption, in the case of particles which are about 10-fold larger than the incident wavelength, is discussed elsewhere [6]. The mass frequency function can thus be expressed as [7]... 

The UV detector is the most widely used detector for LC. It is a solute property detector that is suitable for those solute compounds that absorb radiation in the UV range ( 190-400 nm). Ultraviolet-photometric detectors are relatively insensitive to temperature and flow rate fluctuations. The sensitivity to solute detection is high (noise equivalent concentration 10 °g/ml).f Ultraviolet-photometric detectors are also well suited to applications that use gradient elution, given that many common LC solvents have low UV absorptivities. 

BAA = 2-buto vacetic acid BE = 2-butoxyethanol BEA = 2-butoxyethanol acetate ECD = electron capture detector FID = flame ionization detection 6GC = gas chromatography HCl = hydrochloric acid HPLC = high performance liquid chromatography MS = mass spectrometry NaCI = Sodium chloride RSD = relative standard deviation UV = ultraviolet absorbance detection v/v = volume/volume w/v = weight/volume... 

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