DEVELOPING THE SEPARATION

The final stage in the simple step-by-step approach to method development is to develop the separation. When the separation technique has been chosen and 

Finding an appropriate mobile phase Developing a mobile phase to improve selectivity Using nonaqueous mobile phases Developing a separation Summary 

Tactics for separating ionic compounds Reverse phase for ionic compounds Ion suppression chromatography Paired-ion chromatography 

General approach to the separation of ionic compounds Models for ion-pair retention 

Finding an appropriate mobile phase References Acknowledgment 

When developing a separation, the most influential force is the chemical interaction of the mobile phase, sample, and stationary phase. This is a three-way interaction (1) mobile phase-sample (2) mobile phase-station-ary phase and (3) sample-stationary phase. The ability to alter the selectivity of a column by changing the composition of the mobile phase is a major reason why LC is such a versatile and, hence, powerful separation tool. When doing retention chromatography, the mobile phase is interactive since it is involved in the three-way chemical interactions. Only in the GPC (size separation) mode is the mobile phase noninteractive. With experience, scientists can easily develop an intuitive knowledge of those chemical interactions that are mainly dependent upon mobile phase and sample properties. At the point when an individual understands the basis for the mechanisms of separation, he/she becomes a chromatographer. 

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The column used for blood serum analysis was 100 cm long, 1 mm in diameter and packed with RP 18 reversed phase having a particle size of 10 pm. A concave gradient program was used to develop the separation over a period of 45 min. at a flow rate of 50 pl/min. The initial solvent was 75% methanol 25% water and the final solvent was pure methanol. 

Finally, it is worth emphasizing once again that the C8 reverse phase, with a 3 p particle size, packed in a column 3 cm long and 4.6 mm in diameter is an excellent scouting column. A column of this size can be made to provide very rapid separations and subsequently can be quickly reconditioned to another mobile phase. By using such a column, and employing a gradient from pure water to pure acetonitrile to develop the separation, the complexity of the sample will often be revealed, and from the results an improved phase system can be educed. 

The spot capacity for t io-dinensional TLC is less than the product of two unidimensional developments but is still considerably greater than the value for column chromatography. Two reasons contribute to the loss in separation potential in two- r dimensional development. At the start of the second development the separated spots have increased in size due to the first development and are thus always larger than the initial starting -v size, this decreases the spot capacity in the second development 1 compared to the first. Also, during the second development the spots spread laterally, and consequently, they must be separated with a resolution greater than unity at the beginning of the second development if they are to have a resolution of unity at the end. 

Selection of solvents for adsorption chromatography. The choice of solvent for transferring the mixture to be chromatographed to the column will naturally depend upon the solubility characteristics of the mixture. If it is already in solution, for example as an extract, this is usually evaporated to dryness under reduced pressure and the residue dissolved in the minimum volume of the most non-polar solvent suitable. As concentrated a solution as possible is desirable to achieve a compact band at the top of the column of adsorbent, so that during subsequent development the separation will hopefully proceed with formation of discrete bands. 

A valuable application of LC is to rapidly establish optimum chemical reaction conditions. After developing the separation, the first step is to collect and identify the individual reaction products. Most detectors have a linear response of peak height to amount present therefore, once the characteristic retention time and detector response versus concentration are known, it is easy to establish the optimum reaction conditions. In chromatography the retention time establishes the absence or presence for each compound of interest. The calibration curves indicate the amount of each component. Comparison of the chromatograms of reaction mixtures is a simple and rapid method of determining the preferred operating reaction conditions and concentrations. Since HPLC is much faster than TLC and is carried out in a closed system, a minor benefit is that it is easier to prevent oxidative degradation in HPLC than in TLC. 

When a salt is added as a modifier (1) it will drastically change k because adding a salt to water increases the polarity of the water and (2) the salt will also change the chemistry so that there will be a different a. Thus, by adding a salt to the aqueous portion of the mobile phase, we have an entirely new mobile phase and it is necessary to redevelop the separation using the same rationale as was used in Figures 5-10 through 5-12. Two new, important considerations must now be kept in mind. First, as the new eluent is used to develop the separation, the concentration of the phosphate buffer must be... 

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