Wavelength absorbance detection

The analysis of cigarette smoke for 16 different polyaromatic hydrocarbons is described in this experiment. Separations are carried out using a polymeric bonded silica column with a mobile phase of 50% v/v water, 40% v/v acetonitrile, and 10% v/v tetrahydrofuran. A notable feature of this experiment is the evaluation of two means of detection. The ability to improve sensitivity by selecting the optimum excitation and emission wavelengths when using a fluorescence detector is demonstrated. A comparison of fluorescence detection with absorbance detection shows that better detection limits are obtained when using fluorescence. 

A variety of formats and options for different types of applications are possible in CE, such as micellar electrokinetic chromatography (MEKC), isotachophoresis (ITP), and capillary gel electrophoresis (CGE). The main applications for CE concern biochemical applications, but CE can also be useful in pesticide methods. The main problem with CE for residue analysis of small molecules has been the low sensitivity of detection in the narrow capillary used in the separation. With the development of extended detection pathlengths and special optics, absorbance detection can give reasonably low detection limits in clean samples. However, complex samples can be very difficult to analyze using capillary electrophoresis/ultraviolet detection (CE/UV). CE with laser-induced fluorescence detection can provide an extraordinarily low LOQ, but the analytes must be fluorescent with excitation peaks at common laser wavelengths for this approach to work. Derivatization of the analytes with appropriate fluorescent labels may be possible, as is done in biochemical applications, but pesticide analysis has not been such an important application to utilize such an approach. 

In uv absorbance detection, it is often useful to be able to detect different peaks in the chromatogram at different wavelengths. This may be because certain solutes have only a small absorptivity at the wavelength selected. At other times, a wavelength change can be used to edit out unwanted peaks. It is also useful to be able to record the spectrum of each component this can tell us the optimum detection... 

The recognition of the importance of MP in maintaining the health of the retina has led to the development of a number of methods for determining its concentration in situ. These methods, necessarily noninvasive, are routinely employed in dietary supplementation studies with lutein or zeaxanthin to monitor the uptake of the carotenoids into the retina. Every method exploits the optical properties of lutein and zeaxanthin, specifically their absorbance at visible wavelengths. The detection of a light signal, modified by the carotenoids, is accomplished either by the retinal photoreceptors themselves (psychophysical methods) or by a physical detector such as a photomultiplier,... 

Compared to absorbance detection, direct detection of proteins rich in aromatic amino acids by the intrinsic fluorescence of tryptophan and tyrosine residues provides enhanced sensitivity without the complexity of pre- or postcolumn derivatization. The optimal excitation wavelengths for these amino acids are in the 270- to 280-nm range. 

Fig. 7 Comparative HPLC separations of a standard solution of six vitamins using (A) 250 X 4.0-mm-ID standard-bore and (B) 250 X 2.0-mm-ID narrow-bore columns. Stationary phase (both columns), 5-/tm Nu-cleosil-120-5 C8 (octyl) mobile phase, methanol/water (92 8). Flow rate (A) 0.7 ml/min, (B) 0.2 ml/min. Injection volume, 1 fi 1. Wavelength-programmed absorbance detection. Peaks (1) retinol (2) retinyl acetate (3) vitamin D3 (4) a-tocopherol (5) a-tocopheryl acetate (6) retinyl palmitate. (From Ref. 108.)...

Fluorescence detection is more selective than absorbance detection because two wavelengths are required in the measurement, and the structural features necessary for a molecule to fluoresce are more limited. Most lipids, including glycerides and sterols, do not fluoresce. Maximum sensitivity is obtained by selecting the wavelengths corresponding to the intensity maxima... 

Thompson and Hatina (135) showed that the sensitivity of a fluorescence detector toward unesterified vitamin E compounds under normal-phase conditions was at least 10 times greater than that of a variable-wavelength absorbance detector. The relative fluorescence responses of the tocopherols at 290 nm (excitation) and 330 nm (emission), as measured by HPLC peak area, were a-T, 100 /3-T, 129 y-T, 110 and 5-T, 122. The fluorescence responses of the corresponding to-cotrienols were very similar to those of the tocopherols, and therefore tocotrienol standards were not needed for calibration purposes. The fluorescence detector also allows the simultaneous monitoring of ubiquinone derivatives for example ubiquinone-10 has been detected in tomato (136). 

For the determination of supplemental vitamin E in infant formulas, Woollard and Blott (222) employed a radially compressed Radial-PAK cartridge. This enabled lipid material to be rapidly cleared by stepping up the mobile-phase flow rate from 2 ml/min to 10 ml/min after elution of the a-tocopheryl acetate. Fluorescence detection, using a filter-type fluorometer, allowed the indigenous a-tocopherol to be conveniently estimated, while UV absorbance detection was used to quantify the a-tocopheryl acetate. Supplemental retinyl acetate could be assayed simultaneously with either added or indigenous vitamin E using the appropriate detection mode. With the aid of a dual-monochromator spectrofluorometer, a-tocopheryl acetate and a-tocopherol could be determined simultaneously with wavelengths of 280 nm (excitation) and 335 nm (emission), but the increased selectivity eliminated detection of the vitamin A esters (233). 

TNT vapor in air was determined by a colorimetric method involving a Na sulfite-hydroxide color complex (Ref 15). A method is presented for the quant spectrophotometric analysis of polynitroaromatics as their Meisenheimer complexes in ethylene diamine dimethylsulfoxide solns (Ref 32). Detection by formation of colored reaction complexes determined by wavelength absorbances and absoiptivities of the reagent/expl samples are described (Refs 92 132). A semi-quant colorimetric determination... 

Advantages of the technique are its relatively low cost combined with quickness and ease of operation. One can work with dilute solutions (indeed they are usually a prerequisite) and water is a good solvent as it does not absorb in the UVMsible range. Quartz cells are required for observation of absorptions in the UV range, otherwise glass or plastic (for aqueous solutions) can be used for visible frequencies. Flow cells are available for coupling to separation equipment and absorption can either be monitored at a fixed wavelength for detection of a specific compound or class of compounds, or, if a diode array is available, a complete spectrum can be recorded for each fraction on-line. 

This paper is a progress report on the use of simultaneous dual wavelength (215nm, 280nm)absorbance detection for HPLC analysis of cannabinoids in biological fluids. Work in progress on fluorescence detection of cannabinoids will be the subject of a later report. 

Separations in Protocol 9 are comparable to those of SDS-PAGE but each sample run is less than 20 min and more accurate quantitation can be achieved. Limits of protein impurity detection in CGE systems with absorbance detection at a low UV wavelengths are approximately 0.1% (1000 ppm). This is about 10-fold lower than the detection limit of SDS-PAGE. 

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