Sample application solvent selection

The separation and identification of natural dyes from wool fibers using reverse-phase high-performance liquid chromotog-raphy (HPLC) were performed on a C-18 column. Two isocratic four-solvent systems were developed on the basis of the Snyder solvent-selectivity triangle concept (1) 10% acetonitrile, 4% alcohol, and 2% tetrahydrofuran in 0.01 M acetic acid and (2)7% acetonitrile, 8% alcohol, and 5% tetrahydrofuran in 0.01 M acetic acid. Samples were also eluted in 30% acetonitrile. Spot tests and thin-layer chromatography were performed on all samples to confirm HPLC results. The systems also were found to be potentially useful in the identification of early synthetic dyes. A system of sample preparation that minimizes the reaction of samples was discussed. The application of this HPLC separation technique to samples from 20th century Caucasian rugs and American samples unearthed from the foundation of Mission San Jose was examined. 

In order to utilize separation power of the layer in TLC or HPTLC, it is very important to restrict the dimension of the sample initial size, in the direction of development, to a minimum. The choice of solvent for the sample also affects the size of the sample zone. That is, we will obtain good resolution only if the development chromatographic conditions are optimally selected. For TLC plates where the desirable initial spot size is about 5.0 mm, this corresponds to a sample volume of 0.5 to 10 pL. For HPTLC plates where starting spot size is about 1.0 mm, the corresponding sample volume is 100 to 200 nL. The sample solvent must be a good solvent for the sample compounds to allow quantitative transfer from the sample application device to the thin layer. It must be of low viscosity to be easily evaporated from the thin layer. Moreover, the sample solvent must be able to wet the sorbent layer adequately to produce good penetration to... 

A chromatograph can employ a column selection valve (49) and a solvent selection valve with automation to empirically find the best column, and to a lesser extent, mobile phase, for a particular application. Samples are usually run overnight and the chromatograms evaluated in the morning. After the column giving the best separation is found, it usually only takes a few runs to further develop the method to the level needed for the separation goals. 

There are also other trial and error approaches, the simplest of which is the so-called spot test. The sample is applied as several spots on a TLC plate. Then specified volumes of different solvents are applied to the centers of the spotted samples. The resulting circular chromatograms can give preliminary information about solvent strength and selectivity required for separation of the sample. With modern instruments for sample application this test can be automated. However, actual optimization of the mobile phase must still be performed in a suitable chromatographic chamber. 

Once the sorbent type has been selected, extraction of the sample should be confirmed. The cartridge should be conditioned with 1-2 bed volumes of a solvent such as methanol, then prepared for sample introduction by passage of 10-20 bed volumes of the sample solvent. If an ion-exchange phase is used, it should first be converted to the proper counterion, then equilibrated at the appropriate pH. Because of the slow diffusion rates of macromolecules, samples should be applied at relatively low flow rates, preferably less than lOmL/min a cartridge syringe device can generate flow rates of 200 mL/min [3]. After sample application, the bed should be washed twice with 10-20 bed volumes of sample solvent followed by analysis of both washes by HPLC. Absence of sample components in the chromatogram indicates adequate extraction by the sorbent, whereas appearance of samples in the wash implies that a more retentive sorbent is needed. 

The. selection of a solvent for application of the sample can be a critical factor in achieving reproducible chromatography with distortion-free zones. In general, the application solvent should be a good solvent for the sample and should be as volatile and weak as possible. For silica gel TLC, a weak solvent is nonpolar for reversed-phase TLC, it is polar. High volatility promotes solvent... 

The optimized analytical TLC mobile phase obtained in an unsaturated chromatographic chamber may generally be transferred from analytical to preparative OPLC without modirication (57,58). To eliminate the adsorbed air and/or gas in and on the stationary phase, a prerun has to be performed after sample application and closure of the chromatographic chamber (59). The prerun must be performed with a solvent in which the substance zones to be separated do not migrate. Thus, hexane may be used for apolar compounds. An appropriate solvent miscible with the mobile phase must be used for polar compounds such a solvent must be selected during mobile phase optimization. After a prerun to drive all bubbles from the layer, the separation may be started with the optimized mobile phase. 

The first critical step in TLC is the sample application. It is well-known that circular chromatography phenomena may occur when manual spots are applied (6), especially if the volume is too large or if an unsuitable solvent for the layer adsorption has been used due to its choice for the dissolution of samples. Such phenomena induce spot diffusion. Location errors can then be observed when detection is performed by a classical scanner. For example, a systemic position error of 0.1 mm for each spot produces a final location error of 1.0 mm at the 10th spot. Such a variation is not acceptable for quantitative postchromatographic evaluation. To prevent this, the most suitable laboratory equipment should combine sample application and densitometry within the same mechanical system. The automatic spraying system for sample applications is equipped with a microprocessor (7), which ensures very reproducible repetitive applications, (Figure 1). It also allows the selection of the application speed and the form of application as a spot or a narrow band. 

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