Retention data methods

If M and ML are totally excluded from the gel the following equilibria will be established everywhere in the column. 

Equation (10) Indicates that the peak shift of L does not depend on the concentration of M, but only on Its total amount. Thus small binding constants can be measired by Increasing the amount of N applied to the column. On the other hand, for very strong Interactions L tends to elute with M. Because of the prerequisite of pseudo first-order conditions, the amount of M cannot be decreased under certain limits for Improving the 

The zone-interference method was applied in the determination of binding constants between single-strand DMA and ribonuclease A. The binding constants were found to be in fair agreement with those obtained by boundary sedimentation techniques (ref. 39). 

Another related approach based on the measurement of the retention volumes was described (even somewhat earlier than the zone interference method) by Sebille et al. (ref. 40). In this method a column containing a support excluding M is thoroughly equilibrated with a solution of M. The retention volumes of L injected into the column were shown to approach a limiting value when the injected quantities of L approached zero, (i.e. when the first order conditions were approached). For these conditions equations were derived which enabled easy determination of the total affinity between M and L, 

In another modification of the HO method by Sebille al. (ref. 42, 43) the column was initially equilibrated with a mixture of both M and L in a suitable buffer. Then an aliquot of the buffer alone or of the buffer supplemented with 

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Figure 2.24. The concept of orthogonality as shown by retention plots of two sets of columns for a variety of different analytes. (A) Since the log k data of the two columns (C8 and C18) are well correlated for most analytes, these two columns are expected to yield similar elution profiles. (B) The selectivity differences of a C18 and a polar-embedded phase (amide) column lead to very scattered correlation of their respective retention data. Methods using a C18 and a polar-embedded column are therefore termed orthogonal and expected to yield very dissimilar profiles. Diagram courtesy of Supelco, Inc.

B. Sebille, N. Thuaud and J. P. Tillement, Retention data methods for the determination of drug-protein binding parameters by high-performance liquid chromatography,/. Chromatogr., 1981, 204, 285-291. 

Algebraic Comptttation This method starts with calculation of the quantities and compositions of all the terminal streams, using a convenient quantity of one of the streams as the basis of calculation. Material balance and stream compositions are then computed for a terminal ideal stage at either end of an extraction battery (i.e., at Point A or Point B in Fig. 18-81), using equilibrium and solution-retention data. Calculations are repeated for each successive ideal stage from one end of the system to the other until an ideal stage which corresponds to the desired conditions is obtained. Any solid-hquid extraction problem can be solved by this method. 

Gtickel, W., Kastel, R., Lawerenz, J., Synnatschke, G. (1982) A method for determining the volatility of active ingredients used in plant protection. Part III The temperature relationship between vapour pressure and evaporation rate. Pestic. Sci. 13,161-168. Hafkenscheid, T. L., Tomlinson, E. (1981) Estimation of aqueous solubilities of organic non-electrolytes using liquid chromatographic retention data. J. Chromatogr. 218, 409 -25. 

Methods similar to those used in GC are applicable to HPLC. Thus, comparison of retention data is the most useful means of qualitative identification, the retention factor, , generally being used in preference to... 

The method has been proposed for the prediction of retention data in isocratic systems from data measured in gradient elution and vice versa [84], Similar calculation methods may be very important in the analysis of natural extracts containing pigments with highly different chemical structure and retention characteristics. The calculations make possible the rational design of optimal separation conditions with a minimal number of experimental runs. 

Because of their versatility and simplicity, TLC methods have been frequently applied to the separation and semi-quantitative determination of carotenoid pigments in synthetic mixtures and various biological matrices. The retention of pure carotenoid standards has been measured in different TLC systems. Separations have been carried out on silica plates using three mobile phases (1) petroleum ether-acetone, 6 4 v/v (2) petroleum ether-tert-butanol 8 2 v/v, and (3) methanol-benzene-ethyl acetate 5 75 20 v/v. Carotenoids were dissolved in benzene and applied to the plates. Developments were performed in presaturated normal chambers. The chemical structure and the Rv values of the analytes measured in the three mobile phases are listed in Table 2.1. It was concluded from the retention data that mobile phase 3 is the most suitable for the separation of this set of carotenoids [13],... 

The retention data prove that the CZE method separates the pigments in a considerably shorter analysis time than HPLC does. It was supposed that the good separation capacity and low reagent consumption make CZE a valuable alternative to RP-HPLC [272],... 

Quinones and Hydroqulnones. In the analysis of quin-ones and hydroqulnones, the use of two different dual detector systems was required. The retention data for hydroqulnones shows the normal behavior of hydroxyl groups associating with the solvent, THF. Thus octyl quinone and hydroquinone elute almost together. Similarly dioctylquinone and octyl hydroquinone elute together (Figure 7). The UV/RI response ratio for benzoquinone is 3.75. Hydroquinone and dioctylquinone show similar disparities in the UV/RI responses. This information provides a very good method for detecting impurities in dioctyl hydroquinone. 

Possible difficulties in obtaining accurate retention data for physicochemical measurements should be recognized, however. The evaluation of to, the elution time of an "unretained peak (274) is often connected with systematic error and the measurement of the retention time of asymmetrical peaks may not be accurate. Moreover, no satisfactory methods are available for the precise evaluation of the phase ratio in the column. Consequently, the measurement of the equflibrium constant proper is beset with difficulties as discussed in Section VII.B. 

In a chromatographic separation procedure the parameters of the chromatographic system (stationary phase, flow, temperature, etc.) have to be selected respectively optimized with respect to some criterion (resolution, time, etc.). In gas chromatography retention data series are published and used for the sttidy of solvent/solute interaction, prediction of the retention behaviour, activity coefficients, and other relevant information usable for optimization and classification. Several clKmometrk techniques of data anal s have been employed, e.g. PCA, numerical taxonomic methods, information theory, and j ttern recognition. 

Pattern recognition can be applied for the determination of structural features of unknown (monofunctional) compounds (Huber and Reich ). The information about the chemical structure is contained in a multidimensional gas-liquid retention data/stationary liquid phases set. The linear learning machine method is applied in a two step classification procedure. After the determination of a correction term, the skeleton number, a classification step for the determination of the functional group is executed. It is remarkable that 10 stationary phases are sufficient for the classification. 

The suitability of a stationary phase for a specific separation depends upon the selectivity of the phase. This is a measure of the degree to which polar compounds are retarded relative to their elution on a nonpolar phase. A systematic method for expressing the retention data is based on retention indices. For this sytem, the retention indices of the n-paraffins are by definition equal to 100 times the number of carbon atoms in the molecule. For example, the retention index for n-hexane is 600 and for n-octane 800. These values are defined and apply regardless of the column used and regardless of the temperature. 

Qualitative analysis by gas chromatography (GC) in the classical sense involves the comparison of retention data of an unknown sample with that of a known sample. The alternate approach involves combination and comparison of gas chromatographic data with data from other instrumental and chemical methods. 

Many factors can influence the Kovats index which can make it unreliable at times for the characterization of gas chromatographic behavior, although it generally varies less than the relative retention with temperature, flow, and column variation. However, for many it is the preferred method of reporting retention data. 

The usefulness of retention data from gas chromatography can be enhanced by reporting standardized times or retention indices (RI), which involves expressing retention in terms of a ratio of the retention time (RT) of an analyte to the RT of a standard. Retention scaling based on the Kovats (1965) method requires the chromatographic separation of a homologous series of normal paraffins, esters, and others, producing an index that is the ratio of the RT of an analyte minus the RT of a less retentive standard to the RT difference between... 

A disadvantage of simple interpretive methods is that the model to which the retention data (or other data) are fit must be fairly accurate. In other words, an interpretive approach may fail if one or more sample components exhibits anomalous retention. Although rare in SFC, such retention behavior is observed occasionally and is difficult to predict intuitively. Note, however, that by anomalous retention we do not mean behavior that is merely unusual, e.g., retention that decreases smoothly with increasing density (at constant temperature). Retention that varies in a regular (continuous) manner, even if unusual, can usually be modeled with a high degree of accuracy (vide infra). 

Table 13.7 Comparison of retention data for an environmental sample using different ultrafiltration methods...

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