Analytical and Preparative Separation

The general goals of separation can be divided into two broad categories preparative and analytical [5]. 

Small scale preparative work is important for recovering valuable products like recombinant proteins from complex mixtures. Because of its high resolving power, preparative scale liquid chromatography (LC) has moved rapidly into the biotechnology industry where it is used for purifying such 

Preparative methods, both large- and small-scale, are used widely for the simple purpose of removing low-level contaminants from products of value (e.g., sulfur from coal). There is major emphasis now on developing better methods to remove pollutants from air and water. 

Analytical goals, by contrast to those of preparative separation, are based upon the information generated in the process of separation, or realized by 

The sequence, position, and distribution of separated components contain a good deal of information on the mixture. If properly measured and interpreted, this can serve many analytical goals without further tests. The quality of this information naturally improves as the system is better understood, characterized, and controlled. Informational content is greatest when, through theory and/or calibration, one can identify zones or peaks located at defined positions in the sequence with specific molecular species At that point, using a suitable sensor (detector), both qualitative and quantitative analyses follow. One can, at the same time, often measure certain physicochemical constants for the components, such as partition coefficients and diffusion constants. 

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Multi-column switching can be an effective approach for the determination of high and low concentrations of sample components in complex mixtures. This is a very powerful technique for the analytical and preparative separation of components... 

Tennikova TB, Freitag R (1999) High-Performance Membrane Chrommatography of Proteins. In Aboul-Einen HY (ed) Analytical and Preparative Separation Methods of Macromolecules. Marcel-Dekker Inc, New York-Basel, p 255... 

Turkova J (1999) In Aboul-Enein HY (ed) Analytical and preparative separation methods of biomolecules. Marcel Dekker, New York Basel, pp 99-166... 

The precise measurement of competitive adsorption isotherms not only of theoretical importance but may help the optimization of chromatographic processes in both analytical and preparative separation modes. The methods applied for the experimental determination of such isotherms have been recently reviewed [90], Frontal analysis using various flow rates can be successfully applied for the determination of competitive adsorption isotherms [91]. 

Various liquid chromatographic techniques have been frequently employed for the purification of commercial dyes for theoretical studies or for the exact determination of their toxicity and environmental pollution capacity. Thus, several sulphonated azo dyes were purified by using reversed-phase preparative HPLC. The chemical strctures, colour index names and numbers, and molecular masses of the sulphonated azo dyes included in the experiments are listed in Fig. 3.114. In order to determine the non-sulphonated azo dyes impurities, commercial dye samples were extracted with hexane, chloroform and ethyl acetate. Colourization of the organic phase indicated impurities. TLC carried out on silica and ODS stationary phases was also applied to control impurities. Mobile phases were composed of methanol, chloroform, acetone, ACN, 2-propanol, water and 0.1 M sodium sulphate depending on the type of stationary phase. Two ODS columns were employed for the analytical separation of dyes. The parameters of the columns were 150 X 3.9 mm i.d. particle size 4 /jm and 250 X 4.6 mm i.d. particle size 5 //m. Mobile phases consisted of methanol and 0.05 M aqueous ammonium acetate in various volume ratios. The flow rate was 0.9 ml/min and dyes were detected at 254 nm. Preparative separations were carried out in an ODS column (250 X 21.2 mm i.d.) using a flow rate of 13.5 ml/min. The composition of the mobile phases employed for the analytical and preparative separation of dyes is compiled in Table 3.33. 

The approach to method development is similar to the one described for HPLC and can be characterized as a rapid stationary phase screen using column and solvent switching with gradient elution followed by development of an isocratic preparative method. SFC has been successfully applied to the analytical and preparative separation of achiral and chiral compounds. 

Wessler E (1968) Analytical and preparative separation of acidic glycosaminoglycans by electrophoresis in barium acetate. Anal Biochem 26 439-444... 

Giddings, J. C., Unified Separation Science, Wiley, New York, 1991. (Undergraduate level. A textbook on chromatographic and other analytical and preparative separation techniques. Some of the techniques discussed are relevant to the concepts we introduce in Chapters 2 and 3 of this book.)... 

The molecular-sieve dextran gels are widely used in chemistry, biochemistry, and pharmacy for the analytical and preparative separation of metabolites and other biological products. 

Separation of optically active isomers is one of the most important areas of HPLC apphcation in the pharmaceutical industry. Since most of biological systems are predominantly homochiral, different enantiomers of the same drug could have different effect and potency, and the development of enan-tioselective analytical (and preparative) separation methods is very important. Detailed description of chiral HPLC separation is given in Chapter 22 of this book here we only briefly review the specifics of distinctive types of chiral stationary phases (CSP). 

Acetylene and Deuteroacetylene. Van Hook and Phillips (36) have reported successful analytical and preparative separations of small amounts of C2H2, C2HD, and C2D2 using packed columns. They investi-... 

Analytical and preparative separation of enantiomers is of basic importance. In general, one of the two antipodes is pharmaceutically active the other one may be either inactive or toxic. There are three basic methods for the separation of enantiomeric compounds ... 

Although theory predicts that chromatographic peaks will be symmetrical, peak asymmetry is common, even in the most carefully controlled analytical and preparative separations. The most rigorous definition of peak asymmetry is given by the peak skew (y), which is related to the second (M2) and third moments (M3) of the Gaussian distribution ... 

The preparative capability of the present system is demonstrated in the separation of DNB-leucine enantiomers by varying the CS concentration in the stationary phase. The sample loading capacity is found to be determined mainly by the CS concentration or total amount of CS in the stationary phase that is, the higher the CS concentration in the stationary phase, the greater the peak resolution and sample loading capacity. Consequently, the standard HSCCC column can be used for both analytical and preparative separations simply by adjusting the amount of CS in the stationary phase. 

Both analytical and preparative separations can be performed with a standard CCC column by... 

P. J. Wyatt, in Analytical and Preparative Separation Methods of Biomacromolecules (H. Y. Aboul-Enein, ed.), Marcel Dekker, Inc., New York, 1999. 

The preponderance of analytical (and preparative) separations that precede mass spectrometric analyses are carried out using HPLC. Despite its potential for extremely high efficiencies, capillary electrophoresis (CE) remains something of a specialized application, particularly with mass spectrometric detection. The technique is hampered by its reliance on small sample volumes (typically nL injections) in order to avoid loss of separation efficiency. The consequence of these small volumes is a concentration detection limit that is substantially higher than that of techniques such as HPLC. Despite the exquisite sensitivity of mass spectrometers, characterization of related substances present at low femtomole levels (assuming a hypothetical related substance compound with molecular weight 500 daltons present at 0.1% w/w in an equally hypothetical drug substance sample... 

The application of liquid chromatography on nonracemic ( optically active ) sorbents has become a well-developed and successful method for the analytical and preparative separation of enantiomers.25,26 It is particularly useful for racemates that are difficult to resolve via diastereomeric derivatives, i.e., for molecules lacking functional groups for reaction or interaction. This is the case for many pyrans and oxazines, the first separations of which were published in... 

Thus silica with different surface chemistry is very useful for analytical and preparative separation of fullerenes. Silica with properly modified surface by deposition of carbon layer is useful adsorbent for preparative separation of fullerenes. 

Francotte, E. Novel Stationary Phases for Analytical and Preparative Separation of Drug Enantiomers, Presentation at CHIRAL EUROPE 2004, 34-36 June, Mainz, Germany, 2004. 

Analytical and preparative separation of proteins soluble in an organic phase (n-propanol) has been carried out on a LiChrosorb-Diol column. This type of chromatography has been termed normal phase (R8). It has been applied to the analysis of proteins in dialyzed fetal calf serum (R8) and in the separation of protected hydrophobic oligopeptides (Nl), but as yet, it has not been used in a clinical chemical application. 

Moreover, TLC can as a rule be very elegantly combined on-line with both spectroscopic methods and with other analytical and preparative separation methods. In this way, its abUity to separate complex mixtures and its reliability in identifying individual substances are greatly improved. Separation processes can, for example, be based on the following well-known combinations ... 

I. Kubo, S. Komatsu, T. Iwagawa and D. L. Wood, Analytical and preparative separation of bark beetle pheromones by high-performance liquid chromatography, J. Chromatogr., 363 (1986) 309-314. 

The diameter of HPLC columns may vary from several tens of micrometers to several tens of centimeters. Depending on the size of the separation column - nano-, capillary-, narrow-bore - analytical and preparative separations may be distinguished. A current trend in HPLC instrument development provides the hope that in time it may become feasible to use a column of any size with a single instrument. 

Both analytical and preparative separations have been carried out using normal phase conditions on a pelUcular silica support (Snyder and Kirkland, 1979). Most analyses are currently performed using... 

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