Chromatographic peak splitting

Figure 10.8. An example of chromatographic peak splitting possibly caused by column voiding.

Chromatographic artifacts and apparent peak splitting Solvent incompatibility... 

When reversed-phase columns are used for the analysis of enzymatic reactions, many of the components of the reaction may become bound to the packing material. As a result, the debris may alter the retention time, chromatographic profiles, or both of subsequently injected molecules. Types of column malfunction include peak splitting or the appearance of a shoulder, loss of baseline resolution, broadening of peaks, particularly at their base, or both and an increase in back pressure. To some extent, all these symptoms may be traced to material that adhered to the column and was not removed during the methanol wash. 

The liner volume affects the shape of the chromatographic peak (larger volumes cause peak tailing). Split-splitless injectors should be operated in the splitless mode. 

Vouros and co-workers recently demonstrated the effectiveness of nanospray to improve both the S/N quality concurrent with a reduction in matrix effects such as ion suppression [102]. Rather than use a nanobore LC column, the authors constructed a unique postcolumn flow that allowed for nanospray flow rates with conventional column formats. A cylindrically symmetric flow splitter was shown to preserve chromatographic peak shape at split ratios as high as 2000 1 thus, effluent from a conventional 2.1-mm Cl8 column at 200 pi. /min was reduced to 100 nL/min. The heart of the flow splitter was a fused-silica needle emitter with an orifice diameter of 5 pm. Tlie nanoflow splitter was applied to the in vitro metabolite analysis of a test... 

In the split/pool method of combinatorial synthesis, mixtures of compounds are made that are difficult to characterize. The LC/CLND of a nominally equimolar pool (based on nitrogen) should yield equal-sized chromatographic peaks of compounds. In the early stages of a lead development project, weighable quantities of authentic pure samples of a compound are not available, and yet quantitative measurements such as IC50, solubility, or plasma stability need to be made. LC/CLND can be used to calibrate solutions made from submilligram synthetic samples. LC/CLND is an important new technique to add to the arsenal of the organic analytical laboratory. 

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