Complexing eluents

Another example of a metal ion separation on a weak-acid column is shown in Fig. 7.14. Here, spectrophotometric detection at 530 nm was used after post-column addition of pyridylazo resorcinol (PAR) as a complexing reagent. At highly alkaline pH values forms colored complexes with metal ions that are more stable than the eluent complexing reagent (Chapter 4). 

Mercaptoethanol (ME), when added to an eluent, complexes with mercury compounds to produce charge-neutral compounds that can be separated on reverse-phase columns. MacCrehan et al. described this procedure in a separation of Hg, MeHg, EtHg+, and PhHg- [32,33]. 

Uranium ores are leached with dilute sulfuric acid or an alkaline carbonate [3812-32-6] solution. Hexavalent uranium forms anionic complexes, such as uranyl sulfate [56959-61-6], U02(S0 3, which are more selectively adsorbed by strong base anion exchangers than are other anions in the leach Hquors. Sulfate complexes are eluted with an acidified NaCl or ammonium nitrate [6484-52-2], NH NO, solution. Carbonate complexes are eluted with a neutral brine solution. Uranium is precipitated from the eluent and shipped to other locations for enrichment. Columnar recovery systems were popular in South Africa and Canada. Continuous resin-in-pulp (RIP) systems gained popularity in the United States since they eliminated a difficult and cosdy ore particle/leach hquor separation step. 

FIGURE 7.17 Separation of a complex mixture on Fractogel EMD BioSEC (S) with a column dimension of 1000 X 50 mm (Superformance glass column). The sample contained ferritin (I), immunoglobulin G (2), transferrin (3), ovalbumin (4), myoglobin (5), aprotinin (6), and vitamin B, (7). Five milliliters of the mixture was injected onto the column at a flow rate of 3 ml/min (eluent 20 mAI sodium phosphate buffer, 0.1 M NaCI, pH 7.2). 

There is increasing interest in copolymer systems, which, due to their chemical heterogeneity, may require very complex eluent systems in order to dissolve the sample and ensure that the separation ensues hy a pure size exclusion mechanism. In these examples, the PLgel is also compatible with eluent systems containing mixed solvents of different polarity (including water as a cosolvent up to 10% hy volume) and in organic solvents modified with acids or bases (e.g., acetic or formic acid, triethanolamine) as it is stable in the pH range of 1-14. 

FIGURE 16.17 Nonbranched/long chain branched glucans of potato starch dissolved in hot water-steam and 0.1 M NaOH 1.2 ml of the 18-mg/ml solution was separated on Sephacryl S-1000 (95 X 1.6 cm) 3-ml fractions were collected for further analysis normalized (area = 1.0) eluogram profiles (ev) constructed from an off-line determined mass of carbohydrates for each of the fractions branching index ( ) determined from iodine-complexing potential of individual 3-ml fractions flow rate 0.40 ml/ min V ,i = 75 ml, Vtot = 162 ml eluent 0.005 tA NaOH. 

Another way to improve the analysis of complex matrices can be the combination of a multidimensional system with information-rich spectral detection (31). The analysis of eucalyptus and cascarilla bark essential oils has been carried out with an MDGC instrument, coupling a fast second chromatograph with a matrix isolation infrared spectrometer. Eluents from the first column were heart-cut and transferred to a cryogenically cooled trap. The trap is then heated to re-inject the components into an analytical column of different selectivity for separation and subsequent detection. The problem of the mismatch between the speed of fast separation and the... 

Another important parameter is the eluent composition. Binary mixtures (and obviously pure solvents) should be preferred to complex mixtures, since new systems perform an on-line analysis of the composition of binary eluents. These eluent systems allow the automatic eluent recycling, with a reduced number of controls. 

Macaudiere et al. first reported the enantiomeric separation of racemic phosphine oxides and amides on native cyclodextrin-based CSPs under subcritical conditions [53]. The separations obtained were indicative of inclusion complexation. When the CO,-methanol eluent used in SFC was replaced with hexane-ethanol in LC, reduced selectivity was observed. The authors proposed that the smaller size of the CO, molecule made it less likely than hexane to compete with the analyte for the cyclodextrin cavity. 

Tezuka s group (Tezuka and Ando, 1985 Tezuka et al., 1986) was able to isolate and characterize the benzenediazo ether of 1-naphthol (6.10). They stirred a solid mixture of the molecular complex 6.9 formed between an a-azohydroperoxide acid and benzene with an excess of 1-naphthol at room temperature in the dark for several hours. The separation of this solid by thin layer chromatography (silica gel, with a benzene-ethyl acetate mixture [9 1] as eluent) afforded the diazo ether 6.10 as a yellow oil in 17 % yield, together with 4- and 2-phenylazo-l-naphthol (6.11 and 6.12, 4% and 42%, respectively), 4-phenylbenzaldehyde (32%), benzoic acid (23%), and traces of other compounds (Scheme 6-6). Higher yields of the diazo ether (up... 

A new approach to the resolution of sulphoxides 242 was recently reported by T oda and coworkers282. It takes advantage of the fact that some sulphoxides form crystalline complexes with optically active 2,2 -dihydroxy-l, 1-binaphthyl 243. When a two-molar excess of racemic sulphoxide 242 was mixed with one enantiomeric form of binaphthyl 243 in benzene-hexane and kept at room temperature for 12 h, a 1 1 complex enriched strongly in one sulphoxide enantiomer was obtained. Its recrystallization from benzene followed by chromatography on silica gel using benzene-ethyl acetate as eluent gave optically pure sulphoxide. However, methyl phenyl sulphoxide was poorly resolved by this procedure and methyl o-tolyl, methyl p-tolyl, s-butyl methyl and i-propyl methyl sulphoxides did not form complexes with 243. 

Preparative planar chromatography is a very important step in the complicated procedures of isolation of group of compounds or pure substances from complex matrices. The method gives additional possibilities of using various adsorbents and eluent systems to achieve complete separation of stracmral analogs. The method also enables combining the various methods of sample application, plate development, and derivatization to achieve satisfactory separation of isolated plant extracts components. 

One of the most complex separation schemes utilizes flash liquid chromatography and PLC to obtain petropophyrins both from geochemical samples or those synthesized and used subsequently as standards [110]. Ocampo and Repeta [111] described the scheme of petroporphyrins isolation in which at the first step the sediment extract is fractionated into ten fractions on silica gel using dichlo-romethane (fractions 1 to 4), a mixture of dichloromethane-acetone with increasing acetone concentrations (for fractions 5 to 9), and, at last, dichlo-romethane methanol (4 1) (fraction 10). Next, the fifth fraction was separated on silica PLC plates using dichloromethane-acetone (97.5 2.5 v v v) as a developer. Two purple bands (with Rj 0.53 and 0.50) were recovered from silica and purified further on a silica gel column with dichloromethane-acetone (97.5 2.5, v v v) as an eluent. The emiched fraction was then separated by PLC with the same solvent mixture, and the purple bands containing two bacteriopheophytin allomers were recovered with acetone. 

Ion-exchange chromatography (lEC) is used mainly for the separation of ions and easily ionized substances (e.g., substances that form ions by pH manipulation or complexation) in which one of the principal contributions to retention is the electrostatic attraction between mobile phase ions, both sa le and eluent, for immobilized ion centers of opposite charge in the stationary phase. The sample ions are separated based on differences in their relative affinity for the stationary phase ion centers compared to that of the mobile phase counterions in a dynamic exchange system, in which sample ions and eluent ions interact with multiple stationary phase ion centers as they pass through the column. Ion-... 

Eluent selection in ion chromatography is made more complex by the need to consider detector operating characteristics. 

IEC continues to have numerous applications to the detection and quantification of various inorganic ions.1 1 This is particularly true in water analysis.5-14 Inorganic ions in a variety of other sample types, such as food and beverages,1518 rocks,19-23 biological fluids, (blood, urine, etc.),24-31 pharmaceutical substances,32 33 concentrated acids,34 alcohols,35 and cleanroom air36 have also been analyzed by IEC. IEC has also been employed in isotopic separation of ions,37 including the production of radioisotopes for therapeutic purposes.3839 Typical IEC sample matrices are complex, and may contain substances that interfere with measurement of the ion(s) of interest. The low detection limits required for many IEC separations demand simple extraction procedures and small volumes to avoid over-dilution. Careful choice and manipulation of the eluent(s) may be needed to achieve the desired specificity, especially when multiple ions are to be determined in a single sample. 

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