Download (direct link):
Figure 1 Separation of a chromatographic test mixture by a traditional gradient method. Separation was performed on Waters Alliance® HPLC System (Waters Corporation, Milford, MA) and a 3.9 by 150mm 5 micron particle size Symmetry® C18 column at 30°C. The mobile phase consisted of 0.1% phosphoric acid as the A solvent, and acetonitrile as the B solvent, run as a linear gradient from 0-80% B over 40 minutes at 1.0 mL/min. Total analysis time does not include twenty minutes of post run reequilibration of the column and system. UV detection at 254nm, and a 20 |J,L injection was used. Peaks 1-12 (0.1mg/mL each in 50/50 methanol/water) are uracil, theophy-line, acetylfuran, acetanilide, acetyl-, propio-, butyro-, benzo-, valero-, hexano-, hep-tano-, and octano-phenone, respectively.
to generate the example chromatogram shown in Fig. 2A had a smaller particle size (3.5 |jm) and an increased internal diameter was used (4.6 mm) to accommodate the increased flow rate of 4 mL/min. A formic acid instead of a phosphate-buffered mobile phase was used for better MS compatibility.
Besides using a lot of mobile phase, analyses run at these high flow rates are not compatible with an MS detector without some sort of split to divert flow. For many LC analyses, flow splitters are a fact of life when using MS detection. However, Fig. 2B illustrates that separation of the chromatographic test sample can be further scaled to a 2.1-mm i.d. column, now running at an equivalent linear velocity (1.0 mL/min). Under the conditions used in
0.0 Time in Minutes 5.0
Figure 2 (A) Generic method optimized for high-throughput. Separation was per-
formed on a 4.6 by 50mm 3.5 micron particle size Symmetry® C18 column (Waters Corporation, Milford, MA) at 30°C. The mobile phase consisted of 0.1% formic acid as the A solvent, and acetonitrile as the B solvent, run as a linear gradient from 0100% B over 3 minutes at 4.0 mL/min. All other conditions (injection, detection, and sample) were identical to those listed in Figure 1. (B) Chromatography optimized by APCI/MS detection. Separation was performed on a 2.1 by 50mm 3.5 micron particle size Symmetry® C18 column (Waters Corporation, Milford, MA) at 1.0 mL/min. and at 30°C. Mobile phase and gradient conditions were identical to Figure 2A. All other conditions (injection, detection, and sample) were identical to those listed in Figure 1.
Fig. 2B, atmospheric pressure ionization MS can be used directly without splitting the flow. However, depending on the compound class or type in the library being analyzed, electrospray ionization might be preferred and in some cases might still necessitate a split flow, depending on the flow rate and mobile phase composition.
Figures 3 and 4 illustrate the diversity of the method. In Fig. 3, six penicillin-type antibiotics representing a somewhat more realistic sample are separated. Peak 1 is the synthetic precursor for the rest of the compounds in the
Figure 3 Separation of penicillin type antibiotic homologous series. Separation conditions are identical to those reported in Figure 2B. Peaks 1-6 are: 6-aminipenicillanic acid, amoxicillin, ampicillin, oxicillin, cloxicillin, and dicloxicillin, respectively.
mixture, and peaks 4-6 differ only by a chlorine atom, representing possible synthesis byproducts. The generic method readily resolves all of the components in this series of homologous compounds, in spite of the wide polarity range. Figure 4 shows the separation of another test mix of ‘‘drug-like’’ compounds reported in the literature for use as a chromatographic test mixture (8).
Although the improvements illustrated in Figs. 2-4 are significant, it is possible to improve sample throughput even more dramatically. When determining sample throughput capabilities for short run times, additional system timing issues must be taken into account. That is, to determine actual sample throughput, cycle-to-cycle inject times must be determined that take into account all other aspects of each individual sample analysis. Run time, while important, is only one factor in determining overall cycle-to-cycle inject times. The amount of time it takes to position the injector, aspirate a sample, load the sample loop, inject the sample, and rinse the injector apparatus is referred to as ‘‘injection overhead.’’ While injection overhead varies from instrument to instrument, times from 1 to 2 min per injection cycle are not uncommon. In
0.0 Time in Minutes 5.
Figure 4 Separation of chromatographic test mix showing generic method diversity. Separation conditions are identical to those reported in Figure 2B. Peaks 1-5 are uracil, 1-hydroxy-7-azabenzotriazole, methoxybenzenesulfonamide, methyl-3-amino-2-thio-phenecarboxylate, and 4-aminobenzophenone, respectively.