Absorbance detection, indirect

Similar to indirect absorbance detection, indirect fluorescence has been employed to detect MPA, EMPA, IMPA, and PMPA, using tetrakis(4-sul fopheny 1 iporphine (TSPP) as the indirect probe (19). A violet diode laser operating at 415nm was used for excitation. Using an electrolyte composed of 50 tM TSPP and 5mM [Bios(2-hydroxyethyl)-amino]tris-(hydroxymethyl)methane (Bistris) at pH 7.2 under normal polarity, baseline separation was achieved in less than 2 min. A limit of detection of 0.1 xM (9ppb) for MPA was achieved. 

A UV/Vis absorbance detector can also be used if the solute ions absorb ultraviolet or visible radiation. Alternatively, solutions that do not absorb in the UV/Vis range can be detected indirectly if the mobile phase contains a UV/Vis-absorbing species. In this case, when a solute band passes through the detector, a decrease in absorbance is measured at the detector. 

Indirect UV absorbance detection in capillary zone electrophoresis has been used to analyze sodium alcohol sulfates. Excellent reproducibility was obtained when veronal buffer was used as UV-absorbing background electrolyte [302],... 

Foret, F., Fanali, S., Nardi, A., and Bocek, P., Capillary zone electrophoresis of rare earth metals with indirect UV absorbance detection, Electrophoresis, 11, 780, 1990. 

In suppressed-ion chromatography, a separator column separates ions of interest, and a suppressor membrane converts eluent into a nonionic form so that analytes can be detected by their conductivity. Alternatively, nonsuppressed ion chromatography uses an ion-exchange column and low-concentration eluent. If the eluent absorbs light, indirect spectrophotometric detection is convenient and sensitive. Ion-pair chromatography utilizes an ionic surfactant in the eluent to make a reversed-phase column function as an ion-exchange column. 

Figure 3.20. Analysis of carboxylic acids and alcohols by reversed phase HPLC, with indirect UV detection, (a) Carboxylic acids. Chromatography conditions mobile phase, 3 X 10 4 M l-phenethyl-2-picolinium in acetate buffer (pH 4.6) column, ju-Bondapak phenyl detection, indirect UV absorbance at 254 nm. Peaks 1, acetic acid 2, propionic acid 3, butyric acid 4, valeric acid 5, caproic acid S, system peak, (b) Aliphatic alcohols. Chromatography conditions mobile phase, 4 x 10 4 M nicotinamide in water column. Ultrasphere ODS detection, indirect UV absorbance at 268 nm. Peaks 1, methanol 2, propylene glycol 3, ethanol 4, 2-propanol 5, 1-propanol 6, system peak 7, 2-butanol 8, 2-methyl-l-propanol 9, 1-butanol. (Redrawn from Refs. 23 and 24 with permission.)...

The first report demonstrating the feasibility of indirect detection in CE was published in 1987 by Hjerten et al.45 who employed indirect UV absorbance detection for the analysis of both inorganic ions and organic acids. The UV-background-providing electrolyte was 25 mM sodium veronal, pH 8.6, and detection was monitored on-column at 225 nm. In 1990, the first separation of alkali, alkaline earth, and lanthanide metals was reported by Foret et al46 Indirect UV detection at 220 nm was employed to detect 14 metals in 5 min, with baseline resolution achieved between all but two of the components. The baseline showed a reproducible upward drift between 1 and 3 min. The UV-absorbing component of the electrolyte was creatinine, with a-hydroxyisobutyric acid introduced to complex with the lanthanides and improve resolution. 

Figure 6.9 Indirect UV detection of inorganic cations by CE. Conditions electrolyte, UVCat2 with tropolone as the complexing agent (pH 4.4) detection, indirect UV absorbance at 190 nm capillary, 60 cm X 75 /urn I.D. fused silica. Peaks 1, Potassium 2, Barium 3, Strontium 4, Calcium 5, Sodium 6, Magnesium 7. Lithium. (Reprinted from Ref. 10 with permission.)...

Ultraviolet absorbance detection is the most prevalent type of detection in CE, and UV detectors operate in both the direct and indirect modes. Laser-induced fluorescence detection is often used for high-sensitivity work. Conductivity detection, suppressed conductivity detection, and mass spec-... 

When compounds are not optically active and are not easily derivatized, indirect detection is sometimes the best alternative. In indirect detection, an absorbing or fluorescing probe is added to the buffer. Displacement of the probe by the analyte produces a decrease in signal (10,11). Because of its universality and simplicity, indirect UV absorbance detection has been the predominant detection scheme for phos-phonic acid analyses by CE (12 17). 

In a recent series of papers, Nassar et al. demonstrated the utility of indirect UV-absorbance detection for environmental samples. In their first study, samples were collected from an area known to have been exposed to nerve agents and subsequently cleaned up (16). Using the same buffer system as described above (Section 3.1.2), MPA, IMPA, and PMPA were detected in soil and surface wipe leachates. Interference from common anions was minimal, likely owing to the acidic buffer conditions employed (pH 4), which can keep weak acids such as carboxylic acids and carbonic acid primarily in their protonated form. Further, interference from fluoride may have been reduced as it is known to adsorb to the capillary surface at low pH. 

Synthetic polyelectrolytes can be separated by capillary electrophoresis applying the same rules derived for the electrophoresis of biopolymers. In the reptation regime, determination of the molecular mass and polydispersity of the polyelectrolytes is possible. Introduction of chromophores facilitates the detection of non-UV-absorbing polymers. Indirect detection techniques can probably be applied when analytes and chromophores of similar mobilities are available. 

Simunicova, E., Kaniansky, D., and Loksikova, K. 1994. Separation of alkali and alkaline earth metal and ammonium cations by capillary zone electrophoresis with indirect UV absorbance detection. Journal of Chromatography A, 665 203-9. 

Absorbance Detection in Capillary Electrophoresis Indirect Absorbance Detection... 

Absorbance detection has been applied to ion analysis through two different approaches direct detection of the sample ion and indirect detection. In some cases, a post-column, color-forming reagent can be added to the column eluate to detect sample ions. 

Nitric oxide (NO), a more recently discovered neurotransmitter and regulator of other physiological processes, has been indirectly quantified by CE of its primary metabolites. Because of the ephemeral nature of NO, nitrate and nitrite, two products of NO oxidation, have been assumed to be good indicators of NO synthase activity. These anions in single Pleurobranchaea californica and A. californica neurons have been separated by CE and quantified with UV absorbance detection." ... 

FCCE in packed capillaries can be used for many more types of analytes. Direct or indirect UVA is absorbance detection can be used to detect everything from monoatomic ions like Br to basic and acidic pharmaceuticals. 

F. Han, J.L. Fasching and P.R. Brown, Speciation of organotin compounds by capillary electrophoresis using indirect ultraviolet absorbance detection, J. Chromatogr. A, 669,103-112,1995. 

Munro et al. showed separation and detection of amino acids on microchips using an indirect fluorescence detection method. Figure 36.10 shows application of this method to urine samples with no pretreatment other than dilution in the appropriate separation buffer. Abnormal amounts of amino acids can easily be detected in the two patient samples compared to the healthy control sample. An absorbance detection based approach was utilized for the clinical analysis of calcium ion in serum, which is important in the regulation of a number of physiological processes. Beads with an immobilized calcium reactive dye were placed into the detection region, and the samples mobilized past the beads using electrophoretic flow. While a true separation was not intended, the interference... 

He J, Chen S, Yu Z (2002) Determination of poly-beta-hydroxybutyric acid in Bacillus thuringiensis by capUlary zone electrophoresis with indirect ultraviolet absorbance detection. J Chromatogr A 973 197-202... 

Several techniques have been developed for CE detection, such as indirect and direct ultravi-olet/visible (UVA is) optical methods. Laser-induced fluorescence OLIF) is widely used. Direct photometric detection based on UVA is light absorbance accounts for over half of all CE application. Absorbance detection operates at wavelength ranging from 190 to 800 nm. It covers both ultraviolet and visible regions. The usually used wavelengths for detection are 190 and 200 nm for... 

Quinine, quinidine and cinchonidine, which are amino alcohols with high chiral capability, have been used as selectors for the separation of enantiomers of acids containing a hydrogen bonding function [24]. These chiral selectors have high UV absorbance, providing indirect detection possibilities for solutes without inherent UV absorbance (Figure 6). 

CE is a separation technique that may be an alternative to LC to determine alkyl chain distribution of anionic and cationic surfactants. Most of the studies refer to alkylbenzyldimethylammonium with UV absorbance detection (Figure 2). Other uses of CE include the separation of homologs of anionic surfactants (as AS) and cationic (as alkyltrimethylammonium) by isotachophoresis with conductometric detection and homologs of non-UV absorbing surfactants (AS, alkylsulfonate, alkylsarcosinates and dialkyldimethylammonium) by capillary zone electrophoresis using indirect detection. 

Small inorganic ions of interest to the polymer chemist that can be separated and quantified by traditional CE systems include halides, alkali metals, alkaline earths, and oxyanions. Detection limits in solution are typically 50-100 ppb using indirect UV absorbance detection, but have been reported as low as 1-10 ppb for common inorganic anions, using supressed conductivity detection [12]. The detection limit in a polymer is determined by the amount of polymer used to prepare the solution as discussed in the sample preparation section. The observed precision can be less than 0.1% relative standard deviation [13]. 

Capillary electrophoresis (CE) is a separation technique for ionic or ionizable compounds. CE is particularly attractive because the instrumentation is inexpensive and separations are quick and efficient. As with GC and LC, CE can be coupled to and flame photometric detection (FED) to detect alkylphosphonic acids [30-32]. Indirect UV absorbance detection with CE has also been used for the analysis of nerve agents and their degradation products [33]. In an attempt to meet the demands of portable and efficient field instruments, miniaturized analytical systems with CE microchips have also been made for the separation and detection of alkylphosphonic nerve agents [34]. The aforementioned CE procedures all provide rapid identification without extensive sample preparation. CE is most likely to be used as a guide in order to select the appropriate methods for further analysis by more definitive techniques such as GC-MS, as most of the products detected and analysed are degradation products [35]. A review depicting various CE separation techniques, lab-on-a-chip technology and detection limits has been compiled by Pumera and is shown in Table 3.1. 

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