RESUMO
Three different HPLC column technologies (i.e., monolith, fused-core particles, and sub-2 microm particles) were evaluated, comparing van Deemter plots, speed of analysis, back pressure, and mobile phase consumption. Very high linear velocities (approximately 12 mm/s) were achieved with the monolithic column using modest pressure (110 bar) at the expense of high mobile phase consumption. The minimum plate height of the monolith was similar to that of a 3 microm-particle packed column (i.e., h = 8 microm), operated at optimal linear velocities; the monolithic column showed substantially lower mass transfer dependence, however. The 2.7 microm fused-core packing material yielded efficiencies closer to the sub-2 microm material than to the 3 microm-particle packed column and could be operated at high flow rates. The fused-core column was able to achieve linear velocities similar to those attained on the sub-2 microm column, staying below 620 bar instead of almost near 1030 bar required by the sub-2 microm material. The lack of pH stability of the monolithic column prevented its use to separate basic compounds (i.e., tricyclic antidepressants) at high pH. Best separation of these components at high pH was achieved using the column packed with 1.7 microm hybrid material.
RESUMO
The increased interest in HPLC at elevated pressures, beyond the conventional 6000 psi (400 bar), has created a demand for injection systems capable of withstanding pressures beyond the 20,000 psi (1380 bar). To achieve high-resolution separations, an appropriate length of columns packed with sub 2-microm packing materials, a 30,000-40,000 psi (2070-2760 bar) pressure range is desirable. A new air-actuated needle valve injection system rated to withstand pressures of up to 40,000 psi (2760 bar) has been evaluated. Under isocratic chromatographic conditions, injecting 200 nL and operated at approximately 20,000 psi (1380 bar), the system showed a peak area reproducibility of approximately 2.5% RSD, contrasting the 5% RSD of a pressured-balanced injection system operated under similar conditions. Programmed for partial loop injections using injection times of 300-700 ms (injection volumes in the range of 1-2.5 microL) and operated at pressures close to 30,000 psi (2070 bar), the reproducibility in peak area for the amounts injected was approximately 1.5% RSD or lower, while an injection time of 100 ms resulted in a reproducibility of 3-4% RSD. The new injection system did not show any significant carryover, and after thousands of injections, the system has not shown sign of wear, loss of pressure during injection, or loss in chromatographic performance.
