Incompatible sample solvents

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. 

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. 

Sample solvent incompatible with mobile phase. 

Sample solvent incompatible with mobile phase, solvent... 

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 sample solvent is incompatible with the mobile phase. 

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. 

In the separation of biomolecules, sample preparation almost always involves the use of one or more pretreatment techniques. With high-performance liquid chromatography (HPLC), no one sample preparation technique can be appHed to all biological samples. Several techiques may be used to prepare the sample for injection. For example, complex samples require some form of preffactionation before analysis, samples that are too dilute for detection require concentration before analysis, samples in an inappropriate or incompatible solvent require buffer exchange before analysis, and samples that contain particulates require filtration before injection into the analytical instrument. 

Interfacing of solution-based separation techniques with mass spectrometry has historically been a challenge because of the incompatibility of the used solvent with the vacuum system. Standard electron impact (El) ionization with techniques such as particle beam require samples to be vaporized under high vacuum for ion formation to occur. 

The low flow rate in the microbore column ensures sample volumes compatible with the secondary conventional column and permits the injection of a small volume onto the secondary column, making the transfer of incompatible solvents possible without peak shape deterioration or resolution losses [63], The possible disadvantage could be the lower sample capacity of microbore LC columns. However, in LCxLC, a sensitivity enhancement can be obtained if the formation of compressed solute bands at the head of the secondary column is achieved during the transfer from the first to the second dimension. Moreover, a larger volume can be injected into the first-dimension microcolumn, used as a highly efficient pre-separation step, and a limited decrease in efficiency due to a large injection volume can be tolerated. 

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