UV-absorbance detectors

Fluorescence detectors can be made much more sensitive than uv absorbance detectors for favourable solutes (such as anthracene) the noise equivalent concentration can be as low as 10 12 g cm-3. Because both the excitation wavelength and the detected wavelength can be varied, the detector can be made highly selective, which can be very useful in trace analysis. The response of the detector is linear provided that no more than about 10% of the incident radiation is absorbed by the sample. This results in a linear range of 103-104. 

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. 

Refractive index detectors are not as sensitive as uv absorbance detectors. The best noise levels obtainable are about 1CT7 riu (refractive index units), which corresponds to a noise equivalent concentration of about 10-6 g cmT3 for most solutes. The linear range of most ri detectors is about 104. If you want to operate them at their highest sensitivity you have to have very good control of the temperature of the instrument and of the composition of the mobile phase. Because of their sensitivity to mobile phase composition it is very difficult to do gradient elution work, and they are generally held to be unsuitable for this purpose. 

With uv absorbance detectors, we have to consider the uv absorption of the mobile phase, which always increases as the wavelength decreases. The uv cut-off of solvents indicates the useful wavelength range of the solvent and means the wavelength below which the solvent has an absorbance of 1 or more when measured in a 1 cm cell. Aliphatic hydrocarbons cut off at about 210 nm the best polar solvents for low wavelength work are methanol and acetonitrile, which cut off at 205 and 190 nm, respectively, provided they are pure. Acetonitrile is difficult to purify, and is consequently expensive. 

FIGURE 13.9 The HPLC diode array UV absorbance detector. When a mixture component elutes from the column, not only the chromatography peak but the entire UV absorption spectrum for that component can be recorded. 

In Chapter 12, we discussed the need to calculate response factors, specifically when a TCD detector is used (Section 12.8.2). Would response factors need to be calculated in HPLC when a UV absorbance detector is used Explain. 

Molecular weights were determined using a Waters high-pressure GPC instrument (Model 6000 A pymp, a series of five p-Styragel columns (10s, 10s, 10, 103, 500 A), Differential Refractometer 2401 and UV Absorbance Detector Model (440) and a calibration curve made by well fractionated polyisobutylene standards. 

The parameters that require qualification for a UV absorbance detector are wavelength accuracy, linearity of response, detector noise, and drift. These determine the accuracy of the results over a range of sample concentrations and the detection limits of the analysis. 

The wavelength accuracy and detector linearity and detector noise have the same effect on laser-induced fluorescence, as those of a UV absorbance detector. 

Chromatographic System. The isocratic liquid chromatograph used was a Waters Associates (Milford, MA) Model 24A alc which included a Model 6000A Solvent Delivery System, a Model 401 Differential Refractometer and a Model 440 Absorbance Detector operating at 254 nm and was fitted with a WISP automatic injector. The analog outputs of the UV absorbance detector or differential refractometer were recorded with a Model 730 Data Module (printer, plotter, integrator)(Waters). Eluent flow rate was 1.0 ml/min unless otherwise noted. 

This work was done with a Waters Model 244 liquid chromatograph having two Du Pont Blmodal IIS columns (29,000 plates/meter) and a Linear dual-pen recorder. Also used was a Waters Model 440 UV absorbance detector. Samples were run at 0.1% (w/v) using an Injection volume of 25-pL and a flow rate of 1 mL/mln. The system was calibrated with polystyrene standards from Pressure Chemical Co. according to the universal callbaratlon procedure. Data collection and computation were done with an Intel 80/30 microprocessor. 

UV absorbance detector, it is noted that the calibration constants for TGMDA and DDS are quite similar and that the 1-1 product has a different constant which is essentially identical to those of the higher MW products. For components having retention times less than 30 min, their total weight percentage may be calculated from the sum of area segments Aj between 28 and 32 min taken at 0.1 min intervals i.e.,... 

Liquid chromatographic separations were performed on a Waters Model ALC/GPC 204 liquid chromatograph equipped with two model 6000 pumps, a model 660 solvent programmer, and a model 440 dual UV absorbance detector. 

Pure fluids. Carbon dioxide is often the mobile phase of choice for SFC, since it has relatively mild critical parameters, is nontoxic and inexpensive, chemically inert, and is compatible with a wide variety of detectors including the flame ionization detector (FID) used widely in GC and the UV absorbance detector employed frequently in HPLC (7). The usefulness of carbon dioxide as a mobile phase in many instances is somewhat limited, however, because of its nonpolarity (8), and many polar compounds appear to be insoluble in it. For a sample containing polar compounds, pure carbon dioxide may not be the proper mobile phase. The elution of polar compounds is often difficult and the peak shapes for these polar compounds are sometimes poor. This latter difficulty is commonly observed with nonpolar supercritical fluids and may be due to active sites on the stationary phase rather than any inherent deficiency in the fluid itself. 

A potential alternative for determining UV response factors is to use two HPLC detectors a standard UV absorbance detector and a second detector that has a response uniformly proportional to weight or concentration. For example, if a detector could provide accurate information on the relative amounts of the impurities and parent compound, then this information, combined with the UV peak areas, would supply the desired RRF information without the need for a purified impurity sample. One could reasonably question the need for a UV detector and RRF values at all if such an alternative detector was available, as it would directly provide information on relative amounts of impurities/parent. However, UV detectors are inexpensive, rugged, and readily available therefore, RRF values, once determined, are widely applicable to situations in which no other detector is available. 

In 1986, Foret et al.41 described an on-line UV absorbance detector that employed a commercial photometer and optical fibers in direct contact with the outer walls of the separation capillary. The optical fibers (200 (im I.D. fused silica core) conducted the light beam perpendicularly across the migrating zones one fiber was connected to a mercury lamp to serve as the illumination source, and the other directed light to a photomultiplier tube for detection. The detector was found to be linear in the range of 10"5 to 10 3 M (r = 0.994 for 10 measurements), with detection limits of 1 X 10 5 M for picric acid (S/N = 2). 

The large gain in sensitivity afforded by on-line radioisotope detection in comparison with the more commonly used UV-absorbance detector is illustrated in Figure 7. In this example, a UV-absorbance detector, monitoring at 254 nm, was positioned 8.5 cm downstream from a CdTe radioisotope detector, and 2P-labeled ATP was injected at a concentration of approximately 5 x 10 M. Under these conditions, ATP is detected with an excellent signal-to-noise ratio by the radioisotope detector but is completely undetectable by UV absorbance. 

Figure 7. Electropherograms showing (A) CdTe radioisotope detector response and (B) UV absorbance detector response. The injected sample was 5 x 10" M 2P-labeled ATP. The UV absorbance detector was located 8 cm downstream from the radioisotope detector, and absorbance was monitored at 254 run.

Another example of the chromatographic resolution of a starting mixture with relatively low chemical purity is shown in Figure 4, [21. The chromatographic profile that is obtained with CD detection reveals only two peaks, allowing a safe identification of the antipodes of (R) and (S)-7-chloro-1,3-dihydro-3-i-propyl-5-phenyl-2H- l,4-benzodiazepin-2-one, (R,S)-(IV). But the profile obtained with the UV absorbance detector provides evidence for at least four different peaks, two of which are partially overlapped. Thus the enantiomeric composition is more correctly determined by CD detection. 

On-column UV absorbance detection is by far the most common method of detection in CE today. Many compounds of interest absorb light to some extent in the UV region without any chemical modification. Detector components are fairly robust and inexpensive, and little operator skill is required. For these reasons, most commercial CE instruments are equipped with a standard UV absorbance detector. However, as absorbance signals are directly proportional to the optical pathlength (Beer s Law), the 10-100 xm internal diameter of capillaries used in CE yield rather disappointing detection limits in the range of 10-5-10-7M (7). 

Figure 5.37 An example of a spectro-chromatogram recorded with a multichannel UV absorbance detector in LC. The sample contains a series of dipeptides, (a) (top) pseudo-isomeric three-dimensional plot dimensions are time, wavelength and absorption, (b) (bottom) contour plot with constant absorption lines. Figure taken from ref. [588]. Reprinted with permission.

By high-pressure liquid chromatography, using a UV absorbance detector, cyclohexaselenium has been detected in various selenium solutions in equilibrium with Se7 and Se8 (see also Section II,D) (14). Cyclohexaselenium is also formed by thermal decomposition of Se7 in inert organic solvents like CS2 according to the equation... 

Cycloheptaselenium been detected in various solutions by HPLC, using a column with octadecylsilane as a stationary phase in connection with a UV absorbance detector. The retention time of the Se7 molecule is between those of Se6 and Ses, in complete analogy to the... 

Analytical Methods. Samples were analyzed by HPLC using one of two types of column a Bio-Rad HPX87H+ (acid) column or a Bio-Rad Micro-Guard cation-H cartridge, with 0.015N phosphoric acid eluant and an UV absorbance detector. The cation-H cartridges were used to get elution of simple urea compounds in less than 15 minutes, compared to over 2 hours on the acids column. Samples were filtered (0.45 micron pores) before injection. 

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