High-performance liquid chromatography particle size

In 1972, Kirkland at E. I. du Pont de Nemours patented porous silica microspheres (PSM) specifically for high-performance liquid chromatography (HPLC) applications (3). Prior to this development, silica particles used for chromatographic applications were simply adapted from some other use. In the 1970s, Kirkland showed that porous silica particles could be used for size-... 

Reversed-phase Cig chromatography column. Keystone Scientific Betasil, 100 x 2.0-mm i.d., 5-pm particle size, 100 A, Part No. 105-701-2-CPF TSQ 7000 LC/MS/MS system with electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) interface and gradient high-performance liquid chromatography (HPLC) unit, or equivalent Vacuum manifold for use with SPE cartridges (Varian Vac Elut 10 or equivalent)... 

It was known from gas chromatographic theory that efficiency could be improved if the particle size of the stationary phase materials used in lc could be reduced. High performance liquid chromatography developed steadily during the late 1960s as these high efficiency materials were produced, and as improvements in instrumentation allowed the full potential of these materials to be realised. As hplc has developed, the particle size of the stationary phase used has... 

Nimura, N., Itoh, H., Kinoshita, T., Nagae, N., Nomura, M. (1991). Fast protein separation by reversed-phase high-performance liquid-chromatography on octadecylsilyl-bonded non-porous silica-gel — effect of particle-size of column packing on column efficiency. J. Chromatogr. 585(2), 207-211. 

Modern high-performance liquid chromatography has been developed to a very high level of performance by the introduction of selective stationary phases of small particle sizes, resulting in efficient columns with large plate numbers per litre. 

High-Performance Liquid Chromatography. A Varian 5060 delivery system was used for this work with detection by UV absorption. Either a Varian UV-50 variable wavelength detector or a Hewlett Packard 1040A scanning diode array detector was used. All HPLC columns were packed in our laboratory (10) with 5-/um particle size Spherisorb-ODS, Spherisorb-CN (Phase Separations), or 8-pm particle size Zorbax-CN (Dupont Ltd). HPLC columns (20 or 25 cm X 4.6 mm i.d.) were coupled via short lengths of stainless steel capillary tubing (5 cm X 0.25 mm i.d.). Separation conditions were as follows ... 

Particulates from the sample matrix that are carried over during the sample preparation should be removed prior to analysis by either high-performance liquid chromatography (HPLC) or gas chromatography (GC). This is especially true for particles less than 2 /un in size. These particulates will pass through the frits on a liquid chromatographic column and settle on top of the sorbent which will eventually cause an increase in the back pressure of the chromatographic system and susequently decrease the column performance. 

High-performance liquid chromatography does similar things with more sophisticated instrumentation. It can separate closely related chemical compounds on a research scale or on a preparative scale liquid solvents, or mixtures of several solvents under positive pressure, replace the "carrier gas" of Fig. 11.3. The solid support must have small particle sizes (3- to 10-pm diameter), so that relatively high pressures can be sustained throughout the column, and it is at the interface between the liquid eluant and the solid particles that the chromatographic separation is accomplished. 

Apparatus (See Chromatography, Appendix IIA.) Use a high-performance liquid chromatograph operated at room temperature with a 10-p.m particle size, 30-cm x 4-mm (id), C18 reverse-phase column (jxBondapak C18 column, Waters Corp., 34-T Maple Street, Milford, MA 01757, or equivalent). Maintain the Mobile Phase at a pressure and flow rate (typically 2.0 mL/min) capable of giving the required elution time (see System Suitability in High-Performance Liquid Chromatography). Use an ultraviolet detector that monitors absorption at 254 nm (0.2 to 0.1 AUFS range). 

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