Mobile phase incompatible sample solvents

Mobile phase as sample solvent. If possible, always use mobile phase as the sample solvent. This ensures the composition (e.g., percent organic, pH) of sample solution matches that of mobile phase and reduces the chance of any problem due to incompatibility of sample solvent and mobile phase. Alternatively, always use sample solvent weaker than that of the mobile phase to ensure that the chromatography is not deteriorated. For example, in reversed-phase HPLC, use less organic solvent in the sample solvent than in the mobile phase. 

The fraction of column effluent containing the analyte and internal standard can be either collected manually for subsequent reinjection onto the second (analytical) column (offline operation) or diverted directly onto the second column via a high-pressure switching valve (online operation). For manual collection, a drop-counter-fraction collecting system rather than a volume collection system has been recommended (117). The fraction is collected in a small tapered tube, and the solvent is carefully evaporated off under a stream of nitrogen. The residue is then dissolved in a small volume of a suitable solvent for the analytical separation. Because the sample is reconstituted in offline operation, the potential problem of mobile phase incompatibility between the two HPLC systems is avoided, and hence any semipreparative/analytical combination can be used. 

Sample solvent incompatible with mobile phase. Inject samples in mobile phase. 

Sample dissolved in a solvent that is incompatible with the mobile phase. —> Dissolve sample in the mobile phase. 

Incompatible sample solvents and mobile phase may lead to the sample coming out of solution within the HPLC system. 

The solvent in which a sample is dissolved can play a very important role in HPLC analysis. Immiscibility, precipitation, decomposition, and system peaks are all problems potentially caused by a sample solvent incompatible with the analysis. Ideally, the mobile phase should be identical to the reaction solvent. The addition of an internal standard permits a kinetic analysis to be conducted. 

There are two general types of multidimensional chromatography separation schemes those in which the effluent from one column flows directly on to a second column at some time during the experiment, and those in which some type of trap exists between the two columns to decouple them (off-line mode). The purpose of a trap is often to allow collection of a fixed eluate volume to reconcentrate the analyte zone prior to the second separation step, or to allow a changeover from one solvent system to another. The use of offline multidimensional techniques (conventional sample cleanup) with incompatible mobile phases, is common in the literature, and replacing these procedures with automated on-line multidimensional separations will require continuous development efforts. 

The use of extraction cartridges in the separation of azines, discussed in the last Section, is an example of on-column concentration using off-line column switching. A chromatogram can be cut off-line by collecting the zones of interest at the detector outlet followed by reinjection of the collected fraction onto a secondary column. The mobile phases used with the two columns should be compatible, eg they should be miscible and the mobile phase used with the first column should not have too high an eluting power in the second column. If the mobile phases are incompatible it may be possible to evaporate the primary mobile phase and redissolve the sample in a suitable solvent. 

Sample concentration is sometimes needed to increase the analyte concentration or to eliminate the extraction solvents, which might be incompatible with the HPLC mobile phase. Evaporation is carried out in open hoods, ovens, rotary evaporators, Kuderna-Danish evaporators, freeze driers or lyophilizers. 

The removal of triglycerides from the food sample by saponification provides the opportunity to utilize reversed-phase chromatography. The unsaponifiable matter is conventionally extracted into a solvent [e.g., diethyl ether/petroleum ether (50 50) or hexane] that is incompatible with a semiaqueous mobile phase. It then becomes necessary to evaporate the unsaponifiable extract to dryness and to dissolve the residue in a small volume of methanol (if methanol is the organic component of the mobile phase). For the analysis of breakfast cereals, margarine, and butter, Egberg et al. (153) avoided the time-consuming extraction of the unsaponifiable matter and the evaporation step by acidifying the unsaponifiable matter with acetic acid in acetonitrile to precipitate the soaps. An aliquot of the filtered extract could then be injected, after dilution with water, onto an ODS column eluted with a compatible mobile phase (65% acetonitrile in water). 

Sample solvent incompatible with mobile phase. 

Peak Fronting (Peak Asymmetry Factor < 0.9). This indicates that a small band is eluting before a large band, a wrong pH value of the mobile phase is used, an overloaded column, a void volume at the inlet, or that the sample solvent is incompatible with the mobile phase. 

The method of preparation of the crude extract pnor to loading on the solid phase extraction column will depend on the compatibility of the column and the solvent. For example, if an aqueous methanol extract is loaded onto a normal phase column (e.g., silica gel, diol, or alumina), the sample must first be evaporated to dryness to remove water, which is incompatible with normal phase adsorbents, and then the residue must be dissolved in a nonpolar solvent such as ethyl acetate, toluene, or hexane that has limited solubility for the product. Conversely, if a toluene extract is loaded onto a reverse phase column (e.g., C8, Cl8), the sample should be evaporated to dryness and the residue dissolved in a minimal volume of solvent compatible with the mobile phase. 

The secondary processes usually disturb the gel chromatographic separation or complicate the processing of the chromatographic analytical data. That is why it is necessary to remove, or at least to suppress, the secondary processes in common experimental practice by the appropriate choice of the operational variables. For example, the effects of adsorption, thermodynamic partition and incompatibility can be diminished by the choice of gel and eluent, while the ionic effects are suppressed by adding a suitable salt into eluent, and the concentration effects are not important when working with very low sample concentration or when applying the thermodynamically poor solvent as mobile phase. 

The sample solvent is incompatible with the mobile phase. 

The injection solvent must be compatible with the HPLC mobile phase. If the carotenoids are much more soluble in the injection solvent than in the mobile phase, the carotenoids will precipitate on injection, leading to peak tailing, or they will remain in the injection solvent while passing though the colunm, resulting in broad bands and doubled peaks [100], These problems are particularly pronounced if the carotenoid solution is nearly saturated. The carotenoids will not dissolve completely if the solvent is too weak. Samples can be injected in the mobile phase to avoid this incompatibility problem. However, because of the solubility range of carotenoids in food samples, another solvent may be preferred for solubilization and injection. 

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