Liquid chromatographic columns of 5-to 10-µ TLC grade silica gel have been packed by a high pressure, balanced density slurry technique. The columns have yielded HETP values of less than 0.1 mm at a linear velocity (v) of 1.18 cm/sec. For nitrobenzene (k' = 4.3), 15 effective plates per second were generated at 1.18 cm/sec. The HETP vs. v curve was lower than that obtained on Corasil II, a porous layer bead (PLB) adsorbent. The columns showed little loss of efficiency at high v's. The sample loading was greater than for an equal weight of PLB, but not so great as predicted by the ratio of surface areas.Porous adsorbents, such as silica and alumina, in large particle sizes (150+ µ ) have been used for many years in liquid chromatography. As the adsorbent particle diameter (dp) decreases, column efficiency should increase because of more rapid mass transfer. Snyder (1) verified experimentally for silica gel that column efficiency expressed as the HETP, (or H, Height Equivalent to a Theoretical Plate) was dependent on (dp)o s, for 20 5$ dp ^200 µ in a regularly packed column. Particles of silica smaller than 40 µ have proved difficult to pack reproducibly by dry packing techniques (/).Using small diameter kieselguhr, Huber (2) successfully packed 5-to 15-µ irregular particles by a dry-packing procedure with a controlled amount of tamping. Scott (3) preferred packing 5-to 7-µ spherical, highly cross-linked ion exchange resins in a concentrated aqueous slurry.Slurry packing has been applied to 20-µ silica gel (1), but little additional work has been reported on the successful application of the technique. This paper reports the preparation and use of columns of finely divided silica gel of a 5-to -µ particle size range which are packed by a high pressure, balanced density slurry procedure. A balanced density solvent is one that has a density equal to that of the silica so there is a minimum of particle size segregation during packing. Polystyrene-based steric exclusion chromatography packings are sometimes packed by a balanced density procedure (4).
EXPERIMENTALApparatus. Columns were packed using a high pressure slurry packing apparatus shown schematically in Figure 1. A reservoir supplied liquid to a Whitey Laboratory Feed Pump Model LP-10 (Whitey Research and Tool Co., Emeryville, Calif.). This diaphragm-type pulsating pump forced the slurry from the slurry reservoir into the chromatographic column at very high flow rates and pressures. Equipped with an 8-mm plunger and 30:1 gear ratio, this pump can deliver a maximum flow rate of 1400 ml/hr and a maximum output pressure of 5000 psi.
The potential of high-speed analyses by rapid resolution liquid chromatography (RRLC) and RRLC/MS on 1.8-microm porous particles packed into short columns operated at high flow-rate was investigated and compared with the performance of 5-microm porous particles packed into conventional columns. Using similar chemistries, the ease of conversion from conventional HPLC to an RRLC method was demonstrated. In order to display the practicality of RRLC separations, the analysis of pesticides in crops and catechins in Japanese green tea was selected. Using the Japanese Food Hygiene Law method, which employs a conventional 5-microm RP column (250 mm x 4.6 mm) for quantification of pesticides in crops, the analysis time was 25 min under isocratic conditions. Using the RRLC method on the short (50 mm x 4.6 mm) column packed with 1.8-microm porous particles, the same separation could be performed in 0.8 min with the RRLC/MS method without a loss in resolution. At the highest flow rate, compared to the conventional method, the time could be reduced by a factor of 31. In gradient elution, the fastest separation of catechins in Japanese green tea was achieved by RRLC on 50-mm x 4.6-mm id or 50-mm x 2.1-mm id RRLC columns packed with 1.8-microm particles. The analysis time at 5 mL/min was less than 1 min. Compared to the conventional HPLC method on a 150-mm column packed with 5-microm particles, time was reduced by a factor of 15. The effect of other experimental parameters such as the column temperature, acquisition rate of the detector and the influence of cell volume on chromatographic performance was also investigated. After the optimization, the analysis precision under the fastest RRLC conditions was examined. RSDs of retention time and peak area were 0.2% and 0.47%, respectively.
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