Detection UV absorption

Enami, T. and Nagae, N., UV absorption detection with a packed flow cell in microcolumn liquid chromatography, Am. Lab., 30(24), 1998. 

This chapter describes use of solid-surface room temperature phosphorimetry (SSRTP) as a detection technique in the liquid chromatographic (LC) analysis of caffeine, theophylline, and theobromine. Measurements were made in a continuous mode, using a 2-nebulizer automatic system for SSRTP analysis (previously optimized for LC detection). Use of SSRTP and UV absorption detection was compared under identical experimental conditions.38... 

If appropriate cleanup is accomplished and if the analyte or analytes have sufficient absorption, UV-absorption detection is a highly reliable method of quantification. However, the UV-absorption detector... 

Two analytical methods for priority pollutants specified by the USEPA (38) use HPLC separation and fluorescence or electrochemical detection. Method 605, 40 CFR Part 136, determines benzidine and 3,3-dichlorobenzidine by amperometric detection at +0.80 V, versus a silver/silver chloride reference electrode, at a glassy carbon electrode. Separation is achieved with a 1 1 (v/v) mixture of acetonitrile and a pH 4.7 acetate buffer (1 M) under isocratic conditions on an ethyl-bonded reversed-phase column. Lower limits of detection are reported to be 0.05 /xg/L for benzidine and 0.1 /xg/L for 3,3-dichlorobenzidine. Method 610, 40 CFR Part 136, determines 16 PAHs by either GC or HPLC. The HPLC method is required when all 16 PAHs need to be individually determined. The GC method, which uses a packed column, cannot adequately individually resolve all 16 PAHs. The method specifies gradient elution of the PAHs from a reversed-phase analytical column and fluorescence detection with an excitation wavelength of 280 nm and an emission wavelength of 389 nm for all but three PAHs naphthalene, acenaphthylene, and acenaphthene. As a result of weak fluorescence, these three PAHs are detected with greater sensitivity by UV-absorption detection at 254 nm. Thus, the method requires that fluores-... 

Figure 9. Illustration of the use of extracted ion-current profiles obtained with LC-MS, moving-belt interface, for the detection of carbamate and other pesticides. T op, extracted ion-current profile for 17 major ions second from top, extracted ion-current profile for m/z = 151 to m/z = 181 third from top, extracted ion-current profile for m/z = 86 to m/z = 305 bottom, UV absorption detection at 220 nm. (Reproduced with permission from reference 53. Copyright 1982 Preston Publications.)...

Nishimoto, T., Fujiyama, Y., Abe, H., Kanai, M., Nakanishi, H., Arai, A., Micro-fabricated CE chips with optical slit for UV absorption detection. Micro Total Analysis Systems, Proceedings of the 4th TTAS Symposium, Enschede, Netherlands, May 14-18, 2000, 395-398. 

Figure 9.67 Isocratic HPLC separation and UV absorption detection of UMP, UDP-Gal, and UDP as sequentially eluted. A mixture containing 500 pmol of each compound was injected. (From Hymes and Mullinax, 1984.)...

Yes. But there is a problem in that kind of experiment. What happens is that there is a pressure broadening of the HO2 line on addition of H2O vapor. The sensitivity of the experiment is derived from the narrowness of the rotational line. In the presence of tiny amounts of water vapor, an enormous broadening of the HO2 line develops, making it impossible to measure the HO2 accurately. This is an indication perhaps of a strong interaction between the HO2 molecule and the water vapor. So in our Laser Magnetic Resonant experiment we cannot study the association reaction. The previous work on these complexes has all been done at high pressure with uv absorption detection. 

LFP/UV, Excimer laser photolysis, UV absorption detection of hydrocarbon. LFP/PIMS, Excimer laser photolysis, photoionization mass spectrometric monitoring of hydrocarbon. RR, Relative rate determination. DF/MS Discharge flow production of radicals, mass spectrometric monitoring of hydrocarbon radicals. PR/UV, Pulsed radiolysis in 760 Torr H2, UV absorption detection of ethyl and ethyl peroxy. 

The formation of PTH-amino adds by the Edman degradation [4] of peptides and proteins or by successive modifications of the method constitutes the most commonly used technique for the study of the structure of biologically active polypeptides today. Identification of PTH-amino acids in mixtures may be successfully achieved by TLC. Quantitative determination is based on UV absorption (detection limit 0.1 jLg at 270 nm). An alternative is offered by the chlorine/toUdine test, which is very useful because the minimal amount required for detection is about 0.5 (xg. 

The stunning sensitivity of huorescence detection arises from two areas (a) detection is performed against a very dark background and (b) the use of the laser as an excitation source provides a high photon hux. The combination of the two can yield single-molecule detection in exceptional circumstances, although picomolar (10 M) is typically obtained. Under conditions that are easy to replicate, LIF detection is often 10 times more sensitive compared to ultraviolet (UV) absorption detection. 

Harmful substances may be present in water in either undissolved or dissolved form. Some organic compounds in water can absorb the UV light in a wavelength range of 254-280 nm. Just as turbidity detection is used to measure the total solids content, UV absorption detection at a wavelength of 254 nm can be employed to measure the total content of dissolved organic matter. The typical absorbance values at 254 nm for some important types of waters are listed in Table 1. 

Capillary zone electrophoresis (CZE), with direct or indirect photometry and conductivity has become popular in wine analysis. Very little, or sometimes no sample preparation is needed and short analysis times are also apparent advantages of CE and CZE in the analysis of wine. Capillary isotachophoresis (ITP), with conductivity, thermometric, and UV absorption detection, is suitable for the separation of various anionic constituents (organic acids and inorganic anions), currently occurring in wines (Masar et al., 2001). 

Refractive index detection allows an extremely wide latitude in the selection of the eluent type, eluent pH and the ionic strength. In principle, refractive index detection can be substituted for conductance or UV absorption detection in many separations. However, in early work, refractive detection was found to be only moderately sensitive and was considered to be somewhat interference-prone [71]. Minimum detectable quantities for common anion such as chloride nitrate, or sulfate were reported to be in the 20 ng to 50 ng range (compared with 1 to 5 ng for direct conductance detection). 

The equivalent conductances of the aromatic bases listed in Table 7.2 are low enough for non-suppressed conductivity detection. However, the detection limits are generally lower with indirect UV detection and the resolution is often better also. In Fig. 7.8 chromatograms of the alkali metal cations are compared with direct conductivity and indirect UV absorption detection. With the aromatic base eluents Li elutes... 

Figure 7A Chromatogram obtained with 0.2 mM 2,6-dimethylpyridine at pH 6.35 as eluent by use of (a) direct conductivity and (b) indirect UV absorption detection. Sample 15 pL of a solution containing 2 X 10 M of each of the indicated ions. From Ref. [8] with permission.

In isotachophoresis, electrical detection is most commonly used, although thermal and UV-absorption detection are also used(3). For capillary zone electrophoresis, UV-absorption and fluorescence detection have proven most useful so far. The principles behind the optical detection modes are fairly obvious. However, the electrical and thermal detectors deserve further explanation. As described earlier, in isotachophoresis, each zone is an individual "pure" band of sample ions. The zones travel in order of decreasing mobility. To compensate for each successive... 

These electropherograms of proteins also serve to illustrate another significant problem with proteins. The signal to noise ratio in these separations is not extremely high. All three separations were monitored by on-column UV absorption detection. 

The detection unit developed for dual-wavelength UV-absorption detection has made It possible to apply fluorescence detection (see Fig. 3). 

An even more specific detection method uses radioactivity, as introduced by Kaniansky [18]. As an example of dual-wavelength UV-absorption detection, an analysis will be given performed with the computerized dual-wavelength photometric detector. 

FIGURE 30-14 Capillary gel separation of SDS donatured proteins in a polyethylene glycol column. UV absorption detection at 214 nm. Proteins (1) (i-lactalbumin. (2) soybean trypsin inhibitor, (3) carbonic anhydrase. (4) ovalbumin, (5) bovine serum albumin, (6) phosphorylase B. (Reprinted from K. Ganzier et al Anal. Cham., 1992,64.2665. Copyright 1992 American Chemical Society.)... 

UV-absorption detection at a chosen wavelength is most commonly used. Peptides are usually measured at k = 210 nm, proteins and DNA at A. = 260 nm or A = 280 nm (see section 1.3.3). The absorbance is measured directly through a detection window in the capillary approximately 1 mm long. For this, the poly-imide coating of the capillary has to be removed. The small capillary diameter, less than 100 p,m, results in a short detection path length and thus in low sensitivity. This problem may be partly overcome by use of a Z-cell (Fig. 3.13), which effectively increases optical path length 10 to 15 times. However, due to band broadening within the cell, the separation efficiency is decreased. 

Luo S, Feng J, Pang HM. High-throughput protein analysis by multiplexed sodium dodecyl sulfate capillary gel electrophoresis with UV absorption detection. J Chromatogr A 2004 1051 131-4. 

Because of the UV absorption of the CAs used in cIEF, all UV absorption detections in cIEF are performed at 280 nm. At 280 nm, the absorption of the proteins is determined by the presence of two amino acids in the proteins tyrosine and tryptophan. Thus, the sensitivity of proteins at 280 nm varies a great deal from protein to protein. It is not easy to determine the initial sample concentration. However, we found that the sensitivity of monoclonal antibodies is relatively even and predictable. For these samples and many other protein samples, 0.2 mg/mL is a good starting point for sample concentration if the samples have only one major peak (the major peak is defined as a peak having > 50% of the total protein in a sample). If the samples can be separated into several major peaks (each major peak should be >20% of the total protein in a sample), the starting point can be calculated as follows ... 

Band broadening from hydrodynamic flow in packed capillaries is independent of capillary diameter, allowing the use of capillaries with a pathlength large enough for UV absorption detection. 

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