Cloud point technique

Cloud point extraction from biological and clinical samples. The most frequent use of CPE is for the separation and purification of biological analytes, principally proteins. In this way, the cloud point technique has been used as an effective tool to isolate and purify proteins when combined with chromatographic separations. Most of the applications deal with the separation of hydrophobic from hydrophilic proteins, with the hydrophobic proteins having more affinity for the surfactant-rich phase, and the hydrophilic proteins remaining in the dilute aqueous phase. The separation of biomaterials and clinical analytes by CPE has been described [105,106,113]. 

The phase diagrams were determined by the cloud point technique. At a constant temperature (Sodev temperature controler,... 

On the left-hand side of Fig. 4 we have the normal phase diagram of the binary BE-H2O system. The addition of DEC shifts the two phase equilibria to higher BE concentrations and to lower temperatures. If the addition of a third component is continued beyond the cloud point, eventually three distinct phases appear. Unfortunately the cloud point technique gives us the initial concentration or temperature where unmixing begins but is not suitable to distinguish between the coexistence of two phases and three phases. Also the three phase region depends quite critically on temperature. 

Based upon the use of nonionic surfactant systems and their cloud point phase separation behavior, several simple, practical, and efficient extraction methods have been proposed for the separation, concentration, and/or purification of a variety of substances including metal ions, proteins, and organic substances (429-441. 443.444). The use of nonionic micelles in this regard was first described and pioneered by Watanabe and co-workers who applied the approach to the separation and enrichment of metal ions (as metal chelates) (429-435). That is, metal ions in solution were converted to sparingly water soluble metal chelates which were then solubilized by addition of nonionic surfactant micelles subsequent to separation by the cloud point technique. Table XVII summarizes data available in the literature demonstrating the potential of the method for the separation of metal ions. As can be seen, factors of up to forty have been reported for the concentration effect of the separated metals. 

Concentration and separation of dyes in nonionic surfactant anploying a cloud point technique has been explored by Tatara et al. (2(X)4). The researchers used oxyethylated nonionic surfactants and investigated their potential to separate two direct dyes and one basic dye for recovery. It was found that the method had some potential however, separation occurred slowly by accumulation of the organic solute in the surfactant-rich phase. Both surfactant and dye as well as other reaction parameters had to be selected appropriately for reasonable results. Cloud point extraction was also explored by Purkait et al. (2(X)4) for the direct dye Congo Red. Nonionic surfactant was used and recovered from the dilute phase by solvent extraction with heptane. 

BOU Boutris, C., Chatzi, E.G., and Kiparissides, C., Characterization of the LCST behaviour of aqueous poly(V-isopropylactylamide) solutions by thermal and cloud-point techniques. Polymer, 38, 2567, 1997. 

Turbidimetry is ideally suited to detect the temperature at which a transparent polymer solution turns opaque. The temperature corresponding to the onset of the increase of the scattered light intensity is usually taken as the cloud-point temperature, TcP, although some authors define the cloud point as the temperature for which the transmittance is 80% (or 90%) of the initial value. This technique is commonly known as the cloud-point method [199]. Turbidimetry was employed, for instance, to show that the cloud-point temperature of aqueous PNIPAM solutions does not depend significantly on the molar mass of the polymer [150]. 

The theta (0) conditions for the homopolymers and the random copolymers were determined in binary mixtures of CCl and CyHw at 25°. The cloud-point titration technique of Elias (5) as moaified by Cornet and van Ballegooijen (6) was employed. The volume fraction of non-solvent at the cloud-point was plotted against the polymer concentration on a semilogarithmic basis and extrapolation to C2 = 1 made by least squares analysis of the straight line plot. Use of concentration rather than polymer volume fraction, as is required theoretically (6, 7 ), produces little error of the extrapolated value since the polymers have densities close to unity. 

This phenomenon can be exploited for separation and concentration of solutes. If one solute has certain affinity for the micellar entity in solution then, by altering the conditions of the solution to ensure separation of the micellar solution into two phases, it is possible to separate and concentrate the solute in the surfactant-rich phase. This technique is known as cloud point extraction (CPE) or micelle-mediated extraction (ME). The ratio of the concentrations of the solute in the surfactant-rich phase to that in the dilute phase can exceed 500 with phase volume ratios exceeding 20, which indicates the high efficiency of this technique. Moreover, the surfactant-rich phase is compatible with the micellar and aqueous-organic mobile phases in liquid chromatography and thus facilitates the determination of chemical species by different analytical methods [104]. 

The most important advantage of cloud point extraction is that only small amounts of nonionic or zwitterionic surfactants are required and consequently the procedure is less costly and more environmentally benign than other conventional extraction techniques such as liquid-liquid extraction and solid liquid extraction [107,108]. Moreover, CPE offers the possibility of combining extraction and preconcentration in one step. 

The surfactant selected for CPE technique should not have too high a cloud point temperature. In practice, it is possible to obtain almost any desired temperature by choosing an appropriate mixture of surfactants, as cloud point temperatures of mixtures of surfactants are intermediate between those of the two pure surfactants, or by the choice of an appropriate additive (i.e., salts, alcohols, organic compounds) [105]. 

Cloud point extraction has been applied to the separation and preconcentration of analytes including metal ions, pesticides, fungicides, and proteins from different matrices prior to the determination of the analyte by techniques such as atomic absorption, gas chromatography, high performance liquid chromatography, capillary zone electrophoresis, etc. 

Cloud point extraction of metal ions. The use of cloud point extraction as a separation technique was first introduced by Watanabe for the extraction of metal ions forming sparingly water soluble complexes [109], Since then, the technique has been applied successfully to the extraction of metal chelates for spectrophotometric, atomic absorption, or flow injection analysis of trace metals in a variety of samples [105-107,110]. Other metal complexes such as AUCI4 or thiocyanato-metal complexes can be extracted directly using nonionic surfactants such as polyoxyethylene... 

O.OOl C) the BE-H2O systems were titrated (2.5 ml Gilmont syringe) with DEC until a slight cloudiness appeared, corresponding to the formation of two or more phases. For temperature studies, the temperature of a known mixture of BE-DEC-H2O was varied until a cloud point was observed. The temperature was read to 0.01 C with a pre-calibrated thermistor. In general this technique is fast and quite reproducible but difficulties are often encountered. 

Most proteins must be folded into a specific three-dimensional conformation to express their specificity and activities, which comphcates the DSP [212]. Researchers in the area of RME of proteins/enzymes have reafized this and directed more efforts in developing novel and imaginative techniques in RME as well as coupling the existing techniques such as chromatography, electrophoresis, and membrane extractions with RME. Such promising techniques developed in the recent past have been discussed in this section. Apart from these techniques, use of novel surfactants in the RME and surfactant based separation processes (e.g., cloud-point extraction) are also considered. 

A plot of the temperatures required for clouding versus surfactant concentration typically exhibits a minimum in the case of nonionic surfactants (or a maximum in the case of zwitterionics) in its coexistence curve, with the temperature and surfactant concentration at which the minimum (or maximum) occurs being referred to as the critical temperature and concentration, respectively. This type of behavior is also exhibited by other nonionic surfactants, that is, nonionic polymers, // - a I k y I s u I Any lalcoh o I s, hydroxymethyl or ethyl celluloses, dimethylalkylphosphine oxides, or, most commonly, alkyl (or aryl) polyoxyethylene ethers. Likewise, certain zwitterionic surfactant solutions can also exhibit critical behavior in which an upper rather than a lower consolute boundary is present. Previously, metal ions (in the form of metal chelate complexes) were extracted and enriched from aqueous media using such a cloud point extraction approach with nonionic surfactants. Extraction efficiencies in excess of 98% for such metal ion extraction techniques were achieved with enrichment factors in the range of 45-200. In addition to metal ion enrichments, this type of micellar cloud point extraction approach has been reported to be useful for the separation of hydrophobic from hydrophilic proteins, both originally present in an aqueous solution, and also for the preconcentration of the former type of proteins. 

Another new approach combines MAE with the use of an aqueous surfactant solution as the extracting phase. This new technique is called microwave-assisted micellar extraction (MAME). This procedure is based on the well-known solubilization capacity of aqueous micellar solutions toward water-insoluble or sparingly soluble organic compounds. As a general rule, nonionic surfactants are usually the most effective, showing greater solubilization capacities that rapidly increase with the solubilization kinetics as the cloud-point temperature of the solution is raised. 

As can be noted, the correlation between the calculated and actual results, using a very simple curve fitting technique, yields good fits concerning molar volume, which is examined in terms of density, and in cloud point data, examined in terms of tai jerature. For those not familiar with the cloud point phenomena, nonionic surfactants exhibit inverse solubility characteristics per taiperature increases, i.e., as the... 

Bezerra, M.A., Arruda, M.A.Z., Ferreira, S.L.C. Cloud point extraction as a procedure of separation and pre-concentration for metal determination using spectroanalytical techniques a review. Appl. Spectrosc. Rev. 40, 269-299 (2005)... 

For this reason we determined in this study, for the third time and with the highest accuracy of our studies to date, the cloud point curves for PS(100.000)/PVME and PS(1,800,000)/PVME blends. Using the turbidimetric technique described in the Experimental Section, we determined cloud point temperatures for each of the PS molecular weights over the whole concentration range. The results are shown in Figure 4, where the solid points represent the experimentally determined cloud point temperatures. As proof of the closer approach to equilibrium for these measurements as compared to the previous determinations, both sets of data were approximately parallel to each other and did not cross at high PS concentration. 

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