Particulates and Solvent Filtering

Solvent-solute interactions are important in fluorescence analyses. These interactions have large effects on the reproducibility, detection limits, and sensitivity of a method. First, for n transition compounds (e.g., ketones) in nonpolar and aptotic solvents (e.g., alkanes and ethers), little or no fluorescence occurs. However, in protic solvents (e.g., methanol) these cmnpounds fluoresce. Conversely, n- - n transition compounds (e.g., polycyclic aromatic hydrocarbons) fluoresce readily in nonpolar solvehts and less in protic solvents. 

The presence of heavy atoms (e.g., halogens) in solvents generally tends to decrease fluorescence intensity. The same is true with increasing temperature, where a rC increase in temperature decreases fluorescence intensity by 2% [40], 

Special care should be taken when trying to extrapolate results from one solvent system to another, since even minor changes in the polarity or pH of the solvent oflen have a dramatic effect on the fluorescent behavior of the analyte. These changes occur because of shifts and/or changes in the and spectra due to stabilization or destabilization of the electronic structure of the analyte (e.g., protonated vs. deprotonated) or changes in the polarity of the surrounding solvent (e.g., hexane vs. methanol). 

Impurities in the solvent may also be curable of fluorescing. For example, acrylonitrile, acrolein, and acrylic acid impurities and their adducts with mobile phase components will fluoresce under the prefer conditions. This also happens when trifluoroacetic acid is a mobile phase modifier and amine impurities are in the mobile phase. The result is the appearance of spurious chromatographic peaks. As in UV absorbance techniques, these peaks are of variable size when a gradient is used and the size of the peak is directly proportional to the amount of solvent pumped onto the column between gradients. If such solvent combinations are used, then it is recommended that the fluorescence output of the solvent mixture be monitored prior to analytical use. 

Particulate materials in HPLC solvents are the potential source of many problems. These materials block the solvent reservoir inlet filters, score pistons, wear piston seals, and plug column inlet frits. Therefore, particulates must be removed from the solvent prior to use. 

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