Detection by ultraviolet absorbance

In these detection procedures, which are certainly not the most popular, but most accurate, the absorbancy of the supporting medium plays an important role. Thus, monomeric acrylamide has a UV absorption peak at 280 nm which decreases upon polymerization. Polyacrylamide gel has a rather low extinction coefficient below 270 nm, but absorption rises rapidly, and at 270 nm is characterized by the extinction coefficient, 0.569. Therefore unpolymerized acrylamide must be absent in the gel if spectrophotometric evaluation of the gel is to be applied. To ensure this Watkin and Miller [193] recommended to use only 0.2% Bis in 7.5% gel. 

Scanning of polyacrylamide gel is done by using specifically constructed densitometers [196-200]. In many instances the marketed scanners are multi-purpose scanning devices suitable both for the evaluation of polyacrylamide gel electro-phoretograms as well as flat bed chromatograms. 

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The systems reported above all rely on detection by ultraviolet absorbance, and require some separate means of identifying the desired product among the various components (and thus collected fractions) of the reaction mixture. Efficiency and throughput can be considerably enhanced by real-time detection and identification of products. Two groups have now reported preparative HPLC systems where fractionation decisions are based upon output from a mass spectrometer detector. The first preliminary report of such a system came from a collaboration between CombiChem and Sciex [21]. This system bases fractionation decisions on the output of a single ion mass chromatogram for the predicted molecular ion of the desired product. Reverse phase preparative HPLC is used in conjunction with electrospray ionization mass detection. A full report of this work has appeared in early 1998 [22]. The second report of such a system came from a collaboration between Pfizer and Micromass [23]. This system uses a flexible combination of UV and/or ion chromatograms to control fractionation. Unlike other systems, fractionation parameters are set by the mass spectrometer control software. Variants of both of the above systems will probably become commercially available in late 1997 [24]. 

Melting and helix formation of nucleic acids are often detected by the absorbance of ultraviolet light. This process can be understood in the following way The stacked bases shield each other from light. As a result, the absorbance of UV light whose wavelength is 260 nanometers (the Amo) of a double-helical DNA is less than that of the same DNA, whose strands are separated (the random coil). This effect is called the hypochromicity (less-color) of the double-helical DNA. 

Quantitation of metabolites is challenging in the absence of radiolabel or authentic standards. The most commonly used methods for metabolite monitoring are detection by ultraviolet (UV) absorbance and by mass spectrometry. When UV absorbance is being used for detection, the peak response will be approximately proportional to the amount of metabolite if the chromophore is not altered, such as hydroxylation of an aliphatic side group. However, if the molecule is cleaved, altering conjugated systems, relative UV peak responses may not accurately represent the relative amounts of parent compound and each metabolite. Mass spectrometry is even less reliable than UV absorbance in the absence of authentic standards, as ionization efficiency for each detected molecule will differ. In practice, a combination of UV and mass spectrometry is most appropriate. However, unlike the substrate depletion approach, the possibility would still exist of missing metabolites if the detection method does not pick up the metabolite of interest. 

In contrast to HPLC, where polar effects dominate molecule separation, gel permeation chromatography (GPC) separates molecules according to their molecular size. Types of stationary phase and eluents used for GPC are HIBAR columns (Merck) [33] and Microgel (Chrompack) columns [215] of different pore size with tetrahydrofurane as eluent [33,214,215] Sephadex LH 20 gel and a sodium acetate solution [216] orN,N-dimethylformamide [217] as mobile phase Styragel columns and dimethylformamide eluent containing 2% acetic acid [218] and micro-packed, fiised-silica columns with tetiahydrofiirane as eluent [219]. Detection is by ultraviolet absorbance at the same wavelength discussed in HPLC analysis. 

Analytical Techniques. Sorbic acid and potassium sorbate are assayed titrimetricaHy (51). The quantitative analysis of sorbic acid in food or beverages, which may require solvent extraction or steam distillation (52,53), employs various techniques. The two classical methods are both spectrophotometric (54—56). In the ultraviolet method, the prepared sample is acidified and the sorbic acid is measured at 250 260 nm. In the colorimetric method, the sorbic acid in the prepared sample is oxidized and then reacts with thiobarbituric acid the complex is measured at - 530 nm. Chromatographic techniques are also used for the analysis of sorbic acid. High pressure Hquid chromatography with ultraviolet detection is used to separate and quantify sorbic acid from other ultraviolet-absorbing species (57—59). Sorbic acid in food extracts is deterrnined by gas chromatography with flame ionization detection (60—62). 

He, Y., Yeung, E.S., Chan, K.C., Issaq, HJ. (2002). Two-dimensional mapping of cancer cell extracts by liquid chromatography-capillary electrophoresis with ultraviolet absorbance detection. J. Chromatogr. A 979, 81-89. 

Halcinonide was partitioned between hexanes and methanol, and between hexanes and aqueous acetonitrile at apparent pH values of 2,4,6(unadjusted), and 10. After one hour of mixing, the steroid content was determined by ultraviolet spectrometry of both phases. In all cases, absorbance at the peak maximum of 239 nm was detected only in the acetonitrile-water or methanol layer. The aqueous acetonitrile (pH 6) result was verified- using 14C-halcinonide labeled at the 2- carbon of the acetonide moiety. Thus, the halcinonide is completely retained in either the acetonitrile-water or methanol layers, indicating the utility of these solvent systems for extracting the steroid from formulations. 

DNA is often present in amounts too small to be detected by direct spectroscopy. In this case, the fluorescent dye EtBr can be used to amplify the absorption. EtBr binds to the DNA molecule by intercalating between adjacent base pairs. It absorbs ultraviolet light at 300 nm and emits light at 590 nm in the red/orange region of the visible spectrum. The method can be used to determine the amount of DNA in a test-tube by comparing the EtBr-mediated fluorescence of the sample with that of standards of known amounts of DNA. 

J. Liu, J. Chao, G. Jiang, Y. Cai and J. Liu, Trace analysis of sulfonylurea herbicides in water by on-line continuous flow liquid membrane extraction-C18 precolumn liquid chromatography with ultraviolet absorbance detection. J. Chromatogr.A 995 (2003) 21-28. 

C.W. Huck, M.R. Buchmeiser and G.K. Bonn, Fast analysis of flavonoids in plant extracts by liquid chromatography-ultraviolet absorbance detection on poly(carboxylic acid)coated silica and electrospray ionization tandem mass spectrometric detection. J. Chromatogr.A 943 (2002) 33-38. 

Pihlainen, K. and Kostiainen, R., Effect of the eluent on enantiomer separation of controlled drugs by liquid chromatography-ultraviolet absorbance detection-electrospray ionisation tandem mass spectrometry using vancomycin and native fi-cyclodextrin chiral stationary phases, J. Chromatogr. A, 1033, 91, 2004. 

Dusci LJ, Hackett P, Fellows LM, Ilett KF. 2002. Determination of olanzapine in plasma by high-performance liquid chromatography using ultraviolet absorbance detection. 

The detection of M. aeruginosa toxins is readily achieved by HPLC and ultraviolet absorbance. Provisionally, components with identical retention times are considered identical. Confirmation is made by preparative isolation, amino acid analysis, and toxicity testing. The frequent, but not invariable, presence of toxin-LR as a principal toxin provides a useful HPLC marker. The wavelength used for detection of the toxins, 238 nm, is the principal absorbance maximum of toxin-LR. The 238 nm absorbance is probably... 

The separation and estimation of diloxanide furoate and metronidazole in solid dosage forms was reported by Bhoir et al., using packed column supercritical fluid chromatography [38], A JASCO Cig colunm (10 pm particle size, 25 cm x 4 mm) was used at 40°C, with an injection volume of 20 pL. The mobile phase consisted of 26% methanol in CO2 (flow rate of 2 mL/min), and operated at a pressure of 17.6 MPa. When detected on the basis of its ultraviolet absorbance at 230 nm, the retention time for the drug was 1.6 minutes. The linear region of the calibration graph was reported to be 20-70 pg/mL. 

Queiroz, E.F. et al., On-line identification of the antifungal constituents of Erythrina vogelii by liquid chromatography with tandem mass spectrometry, ultraviolet absorbance detection and nuclear magnetic resonance spectrometry combined with liquid chromatographic micro-fractionation, J. Chromatogr. A, 972, 123, 2002. 

Fig. 5.4.12a-d Electropherograms of pure standards containing nine BA conjugates at a concentration of 50 nmol/ml (a), a blank serum sample from a healthy subject (b), the same serum sample spiked with 50 nmol/ml of nine different BAs (c) and serum sample from a patient with chronic hepatitis infection (d) analyzed by the capillary electrophoresis technique. Ultraviolet absorbance detection at 195 nm (reprinted from [30])... 

Figure 0-4 Principle of liquid chromatography, (a) Chromatography apparatus with an ultraviolet absorbance monitor to detect analytes at the column outlet. (fc>) Separation of caffeine and theobromine by chromatography. Caffeine is more soluble than theobromine in the hydrocarbon layer on the particles in the column. Therefore, caffeine is retained more strongly and moves through the column more slowly than theobromine.

Derivatization is a procedure in which analyte is chemically modified to make it easier to detect or separate. For example, formaldehyde and other aldehydes and ketones in air, breath, or cigarette smoke25 can be trapped and derivatized by passing air through a tiny cartridge containing 0.35 g of silica coated with 0.3 wt% 2,4-dinitrophenylhydrazine. Carbonyls are converted into the 2,4-dinitrophenylhydrazone derivative, which is eluted with 5 mL of acetonitrile and analyzed by HPLC. The products are readily detected by their strong ultraviolet absorbance near 360 nm. 

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