3.2 Types of Analytes, Samples and Sample Introduction
Gas chromatography is somewhat limited in that the analyte has to be volatile below 300 C and thermally stable with molecular weights less than 1000 Daltons. HPLC greatly expands the range of possible analytes and with reverse phase HPLC and can include many of the same analytes as GC (but with less resolution as compared to capillary column GC). Analysis of compounds commonly used in HPLC increases the analyte range of molecular weights to just less than one million Daltons. Many chemicals, such as petroleum hydrocarbons, solvents, illicit drugs and environmental chemicals, can be analyzed by both GC and HPLC. But with the ability of HPLC to analyze non-volatile chemicals many bio-molecules can be analyzed, including sugars, amino acids, proteins, and a large variety of other non-volatile compounds.
Samples analyzed by HPLC are always in liquid form, in either aqueous or organic solvents. Little to no sample preparation is needed, except that it is good practice to filter all samples through a 0.2 μm low-volume cartridge filter prior or during injection. Filtration of samples and solvents avoids the buildup and eventual clogging of the in-line filter or guard column. Samples containing high concentrations of analytes may need to be diluted in order to avoid overwhelming the capacity of the stationary phase and remaining in the linear range of the detector (governed by Beer’s law in UV-Vis applications). Solvent exchange of the samples may be necessary depending on the solvent gradient conditions needed for separation. Samples containing compounds with significantly different chemical structures may interfere with the detector of the analyte(s) and may require removal with a micro-column clean up (such as silica gel or alumina) or may require the use of a specialty clean up cartridge (such as those used in GC for concentration of the analytes from aqueous samples described in Section 2.2). Samples containing relatively low concentrations of analytes may need to be concentrated using one of these same micro-resin columns.
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