Separation conditions

The goals of the ion chromatographer are first, to achieve a satisfactory separation of the sample components of interest and second, to perform the separation as quickly as possible. Several parameters can be manipulated to fulfill these objectives. 

Resin structure. Changes in the chemical composition may alter the selectivity. For example, an ethanolamine group instead of the usual methyl groups in the quaternary ammonium function increases the affinity of the OH for the ion exchanger and makes NaOH or KOH a more powerful eluent. 

Resin capacity. A lower exchange capacity will result in faster elution of analyte ions or will permit elution with an eluent of lower anion concentration. Divalent anions are affected more relative to monovalent anions. 

Column diameter. Use of a column with i.d. of 2 mm instead of 4.0 or 4.6 mm will result in a faster elution at the same volume flow rate. 

Eluent strength. Anion eluents vary considerably in their ability to elute sample anions. Several anions commonly used for IC with suppressed conductivity detection are listed in Table 6.5. An anion of higher charge, such as carbonate, has greater eluting strength than an eluent containing a monovalent anion. 

Detectors fall into either of two classifications. Conductivily detectors may be classed as general detectors because aU ions (cations as well as anions) contribute 

When direct detection is used, the eluent anion has a significantiy lower equivalent conductance than the sample ions to be detected. As an example, a benzoate salt (limiting equivalent conductance = 32 S cm equiv ) can be used for direct detection of ions such as chloride, bromide, iodide, nitrate and sulfate, which have a limiting equivalent conductance between 71 and 80 S cm equiv . A significant increase in overall conductivity is obtained as each of the sample ions passes through the detector, provided that the eluent concentration is not too high. 

A highly conductive anion such as hydroxide (limiting equivalent conductance = 198 S cm eq ) would normally be used for detection of anions by indirect conductivity. This would establish a chromatographic baseline at a high conductance level. When a sample anion that has a much lower equivalent conductance passes through the detector, a peak of much lower conductance is observed. This occurs because the total ionic concentration (sample anion plus OH plus cations) remains constant but the zone containing the sample anion has a lower conductance that that of the background eluent. 

With direct spectral detection, the detector is set at a wavelength where the sample anions absorb but the eluent cation and anion do not. A number of inorganic anions including nitrate, bromide, iodide, thiocyanate, bromate and thiosulfate, as well as aromatic organic anions, can be detected. Sodium chloride, sodium perchlorate, or a number of other anions could be used in the eluent 

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Figure 5.23 shows the phase envelopes for the different types of hydrocarbons discussed, using the same scale on the axes. The higher the fraction of the heavy components in the mixture, the further to the right the two-phase envelope. Typical separator conditions would be around 50 bara and 15°C. 

Direct property prediction is a standard technique in drug discovery. "Reverse property prediction can be exemplified with chromatography application databases that contain separations, including method details and assigned chemical structures for each chromatogram. Retrieving compounds present in the database that are similar to the query allows the retrieval of suitable separation conditions for use with the query (method selection). 

The overall distribution of stresses and strains in the local and global directions is shown in Fig. 3.23. If both the normal stress and the bending are applied together then it is necessary to add the effects of each separate condition. That is, direct superposition can be used to determine the overall stresses. 

Column dimensions mainly determine the quantity of sample to be separated. However, because the SEC process is driven by size separation and is diffusion controlled, special care has to be taken to keep optimized separation conditions, especially when going to smaller internal diameter columns. Overloading and excessive linear flow rates can be observed quite often in these typese of columns. For this reason, standard 8-mm i.d. columns are commonly used, as they are rugged and have a good tolerance toward separation conditions. 

Table 9.8 shows examples of preparative separation conditions that allow a simple transfer of one method to a different column dimension (6). 

Table 9.9 (6) gives some guidelines for proper SEC separation conditions when analyzing polymer standards with narrow molar mass distribution on a single 30-cm column. The conditions have to be adjusted when running industrial polymers (which are normally much wider in molar mass distribution). Depending on the width of the MMD, concentrations can be increased by a factor of 3 to 10 for such samples. As a general rule, it is advisable to keep the concentration of the injected solution lower than c [ j] < 0.2. 

TABLE 23.1 Separation Conditions by Columns of Different Diameters... 

A rule of thumb has been developed after a large number of analytes were tested. Once the selectivity was observed on the coupled column, a baseline separation can always be achieved on a 25 cm column under optimized conditions. Since the screening procedure already indicates the separation conditions, optimization is straightforward and requires a minimum amount of time. 

Table 9.1. Retention times and accurate masses for TBDMS derivatized amino acids (separation conditions Chapter 9.1.A)...

Figure 1. Chromatogram showing separation of alkyl bromide derivatives of Reagent III. Conditions as described in the text under "HPLC Separation Conditions."...

HPLC Separation Conditions. Separations of alkyl bromide and alcohol derivatives were performed using an Altex Ultrasphere Cig S Mio>... 

Figure 2. Chromatogram of the PSP toxin standards. Separation conditions from Table I (A) Method 1 (B) Method 2.

In the case of two analytes able to participate in the mixed lateral interactions (i.e., able to form the hydrogen bonds of the AB. .. A,AB. .. B, or AB,... ABj type) and chromatographed in mild chromatographic systems (i.e., those composed of a low-active adsorbent and a low-polar mobile phase), mixed lateral interactions can even prevent a given pair of analytes from a successful separation (whereas under the slightly more drastic separation conditions, resolntion of a given pair of analytes can be perceptibly worsened, at the least). 

Multiple development techniques using stepwise solvent gradients enable a subset of optimal separation conditions to be used to separate a mixture of wide polarity that cannot be separated using a single mobile phase (117,119,120,125). As an example of this approach the separation of 20 common protein amino acid PTH-derivatives is shorn in Figure 7.12 (126). Five... 

Mass transfer in either the stationary or mobile phase is not instantaneous and, consequently, complete equilibrltui is not established tinder normal separation conditions. The result is that the solute concentration profile in the stationary phase is always displaced slightly behind the equilibrluM position and the mobile I se profile is similarly slightly in advance of the equilibrium position. The combined peak observed at the column outlet is broadened about its band center, which is located where it would have been for instantaneous equilibrium, provided the degree of nonequllibrluM is small. The stationary phase contribution to Mass transfer is given by equation (1.25)... 

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