The UV Detector

A low volume (0.2 pi) Valeo sample valve was employed with one end of the open tube connected directly to the valve and the other connected directly to the sensor cell of the detector. The UV detector was the LC 85B manufactured by Perkin Elmer, and specially designed to provide low dispersion with a sensor volume of about 1.4 pi. The total variance due to extra-column dispersion was maintained at... 

The pressure sensitivity of a detector will be one of the factors that determines the long term noise and thus can be very important. It is usually measured as the change in detector output for unit change in sensor-cell pressure. Pressure sensitivity and flow sensitivity are to some extent interdependent, subject to the manner in which the detector functions. The UV detector, the fluorescence detector and the electrical... 

The UV detector is the most popular and useful LC detector that is available to the analyst at this time. This is particularly true if multiwavelength technology is included in the genus of UV detectors. Although the UV detector has definite limitations, particularly with respect to the detection of non-polar solutes that do not possess a UV chromaphore, it has the best combination of sensitivity, versatility and reliability of all the detectors so far developed for general LC analyses. 

The popularity of the UV detector, the electrical conductivity detector and the fluorescence detector motivated Schmidt and Scott (5,6) to develop a trifunctional detector that detected solutes by all three methods simultaneously in a single low volume cell. 

Quantitative accuracy and precision (see Section 2.5 below) often depend upon the selectivity of the detector because of the presence of background and/or co-eluted materials. The most widely used detector for HPLC, the UV detector, does not have such selectivity as it normally gives rise to relatively broad signals, and if more than one component is present, these overlap and deconvolution is difficult. The related technique of fluorescence has more selectivity, since both absorption and emission wavelengths are utilized, but is only applicable to a limited number of analytes, even when derivatization procedures are used. 

This classification is concerned with whether the detector monitors a property of the solute (analyte), e.g. the UV detector, or a change in some property of the solvent (mobile phase) caused by the presence of an analyte, e.g. the refractive index detector. 

Like the UV detector, the mass spectrometer may be employed as either a general detector, when full-scan mass spectra are acquired, or as a specific detector, when selected-ion monitoring (see Section 3.5.2.1) or tandem mass spectrometry (MS-MS) (see Section 3.4.2) are being used. 

The great advantage of the mass spectrometer is its abihty to use mass, more accurately the mass-to-charge ratio, as a discriminating feature. In contrast to, for example, the UV detector, which gives rise to broad signals with little selectivity, the ions in the mass spectrum of a particular analyte are often characteristic of that analyte. Under these conditions, discrete signals, which may be measured accurately and precisely, may be obtained from each analyte when they are only partially resolved or even completely umesolved from the other compounds present. 

The most widely used LC detector, and the one which, other than the mass spectrometer, gives the most insight into the identity of an analyte, is probably the UV detector, although a UV spectrum very rarely allows an unequivocal identification to be made. It may allow the class of compound to be identified and this, together with the retention characteristics of the analyte, can provide the analyst with a better indication of the identity of the analyte. In the vast majority of cases, however, identification with complete certainty cannot be achieved. 

There are a number of features worthy of note in this figure. For example, there is a difference in retention times, determined by the two detectors, of ca. 0.32 min, and this reflects the fact that they are used in series, i.e. the column effluent passes through the UV detector on its way to the mass spectrometer. 

Solute-property detector A detector which monitors a property of the analyte, e.g. the UV detector. 

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. 

Ability to analyze unreacted monomers was dependent on detector selectivity. The UV detector was operated at 254 nm for analysis of AN/S latex solutions. Styrene is a strong UV abosrber at this wavelength while acrylonitrile has no measurable absorbance at 254 nm. Thus, the UV detector was entirely selective to monomeric styrene. The refractometer detector was sensitive to both acrylonitrile and styrene when each was present in the desired copolymer proportions (70/30). However,... 

Unreacted monomers In AN/M latex solutions were measured similarly. Methylacrylate Is a relatively strong UV absorber In the 225-250 nm range while acrylonitrile does not significantly absorb UV radiation at wavelengths above about 220 nm. At 230 nm, the wavelength used, the UV detector responded only to methylacrylate. And the refractive Indices of the two monomers are such that. In the 75/25 AN/MA proportions used, the refrac-tometer was Insensitive to methylacrylate. The reiErac tome ter therefore functioned as a selective detector for acrylonitrile In the presence of methylacrylate. 

Similarly, estimation of chemical composition of soluble polymer was also dependent on selectivity of the UV detector. Polymerized acrylonitrile has no significant UV absorbance at 230 and 254 nm. Thus, UV chromatograms were used to estimate amounts of polymerized methylacrylate and styrene In each resin system. The refractometer detector was sensitive to polymerized methylacrylate and styrene, as well as to polymerized acrylonitrile. It was therefore necessary to calculate comonomer contribution to refractometer peak areas In order to estimate concentration of polymerized acrylonitrile. This was done by obtaining a refractometer calibration for all three homopolymers. Quantity of polymerized comonomers measured by UV were then converted to equivalent refractometer peak areas. Peak areas due to polymerized acrylonitrile were then calculated by difference, and used to calculate amount of polymerized acrylonitrile. 

The homopolymers of styrene and acrylonitrile were not soluble In the acetonitrile mobile phase. Calibration factors thus had to be derived from a combination of literature data and experimental measurements. To calibrate the UV detector for polystyrene, 254 nm absorbance of both monomer and polymer was measured with a conventional spectrophotometer, using chloroform... 

The equipment consisted of two Waters (Waters Corp. Milford, MA) M-45 pumps, a Waters 481 UV detector, a six-port Valeo sampling valve (A2L6P) with 0.08" holes in the valve body and rotor, a Rheodyne Model 7413 injection valve with a 1-pl loop, a valve interface box, and a Digital Equipment LSI-11/23-based microcomputer system. The microcomputer was used to control all valves, collect raw data from the UV detector, integrate the chromatogram, and store and plot results. 

Although they are more sensitive (and cheaper) than uv absorbance detectors, ec detectors are not as easy to use, and have a more limited range of applications. They are chosen for trace analyses where the uv detector does not have a high enough sensitivity. Fig. 2.4k shows some examples of compounds for which ec detection has been used. 

The instrumental setup for capillary HPLC-NMR coupling is shown in Figure 4.6. The capillary pump is connected via 50 pm capillaries between the capillary HPLC pump, the UV detector, and the NMR flow probe. 

Online LC-ESI-TOF-MS experiments are carried out in a very similar fashion to the off-line NPS-HPLC separations described above, with a few notable exceptions. Firstly, 0.3% (v/v) formic acid is added to each mobile phase to counteract the ionization suppression induced by TFA. Because of the formic acid UV detection must be carried out at 280 nm (as opposed to 214 nm). To aid in normalization between runs 1 jag of Bovine insulin (MW = 5734 Da) is added to each chromatofocusing fraction prior to injection onto the column. Finally, the flow is split postcolumn directing 200 JlL/min into the ion source and the remaining 300 JlL/min through the UV detector and fraction collection. 

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