Reduction of the extra-column band dispersion by a slow transport and splitting of a sample band in isocratic reversed-phase liquid chromatography.

Sample introduction method was studied to reduce the extra-column effect in reversed-phase HPLC. Slow transport of a sample band (SToSB) in the pre-column space followed by the introduction of the band into the column at a near-optimum flow rate resulted in larger plate counts for a 1.0 mmID, 5 cm long column as much as 1.4-1.6 times for solutes with a retention factor (k) of 0.5-1.8 compared to a conventional elution method. Further reduction of the extra-column effect was possible by orthogonally splitting the sample band (SplSB) by flow switching during its slow transport followed by the introduction of the leading part of the band into the column. In this case, increased plate counts of up to 2-3 times for solutes with k of 0.5-1.8 were observed for a 1.0 mmID, 5 cm column. The sample introduction method, SToSB in the injector and the pre-column tube of a few µL, was found to reduce the extra-column band variance by 0.4-0.5 µL2 for an UHPLC system with the extra-column volume (Vextra) of ca. 4.6 µL and the system variance (σextra2) of 1.1 µL2 at flow rate of 100 µL/min, while SToSB and subsequent SplSB were found to be more effective, reducing σextra2 by about 0.8 µL2. With an UHPLC instrument with Vextra of about 10 µL and σextra2 of ca. 3.6 µL2 at flow rate of 300 µL/min, 1.4-2.1 times as many plate counts were observed with SToSB and SplSB compared to the normal elution method for early-eluting solutes with k=0.25-1.7 for a column, 2.1 mmID, 5 cm long. With this UHPLC instrument, SToSB and/or SplSB resulted in the reduction of σextra2 by 1.2-2.2 µL2.

Reduction of the extra-column band dispersion by a slow transport of a sample band from the injector to the column in isocratic reversed-phase liquid chromatography.

Extra-column band dispersion during the transport of a sample band from the injector to the column can be reduced by a flow rate program starting with a low flow rate until the sample band has approached to, or just entered into the column, followed by an increased flow rate suitable for the solute separation in the column. Such a sample introduction method increased the plate counts of a 50 mm long column, 1.0 or 2.1 mmID, especially for early-eluting solutes by up to several times compared to a conventional elution method, when a 0.254 mmID, 15.2 cm connection tubing was used. Increase in plate counts of up to 50-70% was possible for solutes with retention factors smaller than 1.0 for the columns connected with a 0.13 mmID, 15 cm tube. The method also seems to reduce the contribution of the void space at the column inlet to the band dispersion. The elution method including a slow transport of the sample band in the pre-column space of 10 µL or less may require a little longer separation time than normal elution, but it was shown to be effective for increasing the observed efficiency of a small column for solutes with small retention factors.

Efficiency of short, small-diameter columns for reversed-phase liquid chromatography under practical operating conditions.

Prototype small-size (1.0mm I.D., 5cm long) columns for reversed-phase HPLC were evaluated in relation to instrument requirements. The performance of three types of columns, monolithic silica and particulate silica (2µm, totally porous and 2.6µm, core-shell particles) was studied in the presence of considerable or minimal extra-column effects, while the detector contribution to band broadening was minimized by employing a small size UV-detector cell (6- or 90nL). A micro-LC instrument having small system volume (<1µL) provided extra-column band variance of only 0.01-0.02µL(2). The three columns generated about 8500 theoretical plates for solutes with retention factor, k>1-3 (depending on the column), in acetonitrile/water mobile phase (65/35=vol/vol) at 0.05mL/min, with the instrument specified above. The column efficiency was lower by up to 30% than that observed with a 2.1mm I.D. commercial column. The small-size columns also provided 8000-8500 theoretical plates for well retained solutes with a commercial ultrahigh-pressure liquid chromatography (UHPLC) instrument when extra-column contributions were minimized. While a significant extra-column effect was observed for early eluting solutes (k<2-4, depending on column) with methanol/water (20/80=vol/vol) as weak-wash solvent, the use of methanol/water=50/50 as wash solvent affected the column efficiency for most analytes. The results suggest that the band compression effect by the weak-wash solvent associated with partial-loop injection may provide a practical means to reducing the extra-column effect for small-size columns, while the use of an instrument with minimum extra-column effect is highly desirable.

Separation of benzene and deuterated benzenes by reversed-phase and recycle liquid chromatography using monolithic capillary columns.

An alternate pumping-recycle system utilizing a commercially available low dead-volume switching valve was developed for microcolumn LC. The recycle system had two separation columns, and the dead volume of the recycling lines was kept to a minimum by avoiding passage of the sample through the pump chamber, sample injector, and the normal path length of a conventional UV detector. The drawback of the high total back pressure caused by the second column that is placed after the detector was overcome by on-column detection, and this eliminated the need for a high pressure flow cell. The system was used for the separation of an authentic mixture of benzene, benzene-1,3,5-d3, and benzene-d6. Baseline separation was accomplished after six cycles and the calculated theoretical plate number for benzene was 230,000. It was observed that the theoretical plate number (N) increased linearly with increasing number of cycles, and the N per unit time increased with increasing inlet pressure. The separation conditions were optimized and the separation of benzene and benzene-d6 was accomplished within 75 min at 2.5 MPa inlet pressure.

Sample enrichment by using monolithic precolumns in microcolumn liquid chromatography.

An on-line sample enrichment system was designed using monolithic precolumns in microcolumn LC. The monolithic ODS capillary columns were prepared via in situ sol-gel processes. The enrichment efficiency of the monolithic columns was tested by using phthalates as the analytes. The relative standard deviations (n = 6) for the retention time, peak area and peak height were between 0.4 and 1.2%, 0.9 and 5.5% and 0.4 and 3.9%, respectively. The system was linear (R2 > 0.99) within the working sample concentration and sample volume ranges. Comparing to 0.2 microl injection with a typical sample injector, the theoretical plate number of a same separation column was increased by 3-6-fold when the precolumn unit was used for sample injection. The recoveries of the analytes were between 88 and 120%, and the sample volume that could be injected into the system was increased up to 5000-fold. The limits of detection were improved by more than 2000-fold and were between 0.21 and 0.87 ng ml(-1) even with a UV absorbance detector. This system was applied to the determination of phthalates contained in laboratory distilled water and tap water samples.

Split flow and bypass flow systems for monolithic capillary columns in liquid chromatography.

Split flow and bypass flow systems were assembled using Nano Y Connectors with low dead volume commercially available for capillary liquid chromatography (LC). The split ratio could be controlled by changing the dimension of restriction tubing and applied back pressure to the restriction tubing. The split flow system allowed us to use valve injectors and pumps commercially available for capillary LC. The reproducibility of the present split flow system was acceptable. The relative standard deviation for six successive measurements was 0.4% for the retention time, whereas that for the peak height and peak area was 1-3% depending on the analytes. The bypass flow system uses two Nano Y Connectors, where the eluent split at the first Nano Y Connector, which is located in the inlet of the separation column, is merged again into the effluent from the column at the second Nano Y Connector. The bypass flow system could avoid on-column detection and allowed us to use flow cells, leading to an approximate three times improvement in signal-to-noise. The present flow systems were evaluated by using aromatic hydrocarbons and alkylbenzenes as test analytes.