Appendix 4 Column Packing

Chromatographic System (See Chromatography, Appendix IIA.) Use a suitable high-performance liquid chromatograph equipped with a detector measuring at 210 nm and a 250- x 4.6-mm (id) column packed with octadecyl silanized silica (10-p.m Partisil ODS-3, or equivalent), and operated under isocratic conditions at 40°. The flow rate of the Mobile Phase is about 2 mL/min. 

Chromatographic System (See Chromatography, Appendix IIA.) Use a liquid chromatograph equipped with a refractive index detector that can be maintained at a constant temperature of 25°, a 25-cm x 4.6-mm (id) column packed with 10- im porous silica gel bonded with aminopropylsilane (Alltech 35643, or equivalent), and a guard column that contains the same packing. Maintain the column at a constant temperature of 25° 2°, and the flow rate at about 2.0 mL/min. Inject 20 pL of System Suitability Preparation into the chromatograph, and record the peak responses as directed under Procedure. The relative standard deviation for replicate injections is not more than 2.0%, and the alpha-Cyclodextrin and beta-Cyclodextrin peaks exhibit baseline separation, the relative retention times being about 0.8 and 1.0, respectively. 

Procedure (See Chromatography, Appendix IIA.) Use a liquid chromatograph suitable for size-exclusion chromatography and equipped with a refractive index detector and a 60-cm x 7.5-mm (id) column packed with 5-p.m, 500A porosity PL-Gel, or equivalent, both operated at 40°. Operate the chromatograph at 500 to 1500 psi at a flow rate of 1.0 mL/min. 

Procedure Use a high-performance liquid chromatograph (see Chromatography, Appendix IIA) equipped with an ultraviolet detector that measures at 254 nm. Under typical conditions, the instrument contains a 300- x 4-mm (id) column packed with 10-p.m octadecylsilanized silica (jxBon-dapak C 18, or equivalent). Set the flow rate at about... 

Chromatographic System Determine as directed under Chromatography, Appendix HA, but use a liquid chromatograph equipped with a differential refractometer detector, a 7.8-mm id x 30-cm column packed with 25-pm silver bonded to sulfonated divinyl benzene-styrene copolymer (Aminex HPX-42A, Bio-Rad Laboratories, or equivalent). Maintain the column at a constant temperature of 65° + 10°, and the flow rate at 0.3 to 1.0 mL/min. Use water as the mobile phase. 

Appendix A2.2 gives a detailed description of different column packing techniques and their standard operating procedures. 

The results obtained were probably as accurate and precise as any available and, consequently, were unique at the time of publication and probably unique even today. Data were reported for different columns, different mobile phases, packings of different particle size and for different solutes. Consequently, such data can be used in many ways to evaluate existing equations and also any developed in the future. For this reason, the full data are reproduced in Tables 1 and 2 in Appendix 1. It should be noted that in the curve fitting procedure, the true linear velocity calculated using the retention time of the totally excluded solute was employed. An example of an HETP curve obtained for benzyl acetate using 4.86%v/v ethyl acetate in hexane as the mobile phase and fitted to the Van Deemter equation is shown in Figure 1. 

Only tray-type columns were considered because of the difficulty of incorporating an effective cooling circuit into a packed column. Sieve trays (as opposed to bubble or valve-type trays) were preferred because of the ease of installing cooling coils and also their low unit cost. Details of tray selection are included in Appendix G.l. 

Procedure (See Chromatography, Appendix IIA.) Use a high-performance liquid chromatograph capable of separating acesulfame potassium and 4-hydroxybenzoic acid ethyl ester with a resolution of 2. Use a chromatograph equipped with a UV or diode array (227 nm) detector and a 25-cm x 4.6-mm (id) stainless steel column, or equivalent, packed with 3 to 5 p,m of reversed phase C18 silica gel, or equivalent. The elution is isocratic. Use a 40 60 (v/v) solution of acetoni-trile 0.01 MlL tetrabutyl ammonium hydrogen sulfate (TBAHS) in water as the mobile phase, with a flow rate of about 1 mL/min. 

Procedure (See Chromatography, Appendix IIA) Use a gas chromatograph equipped with an electrolytic conductivity detector operated in the halogen mode and fitted either with a capillary injector operated in the splitless mode or with a purged, packed injector with a glass insert. Use a 30-m x 0.53-mm (id), fused-silica column, or equivalent, coated with l-(xm Supelcowax 10 or an equivalent bonded carbowax column fitted with a 50-cm retention gap of 0.53-mm, deactivated, fused silica, or equivalent. Set the column temperature to 170° for 5 min, raise the temperature at a rate of 5°/min to 250°, and hold it at that temperature for 10 min. Maintain the injector temperature at 225°. Use helium as the carrier gas at a flow rate of 8 mL/min. 

Chromatographic System (See Chromatography, Appendix BA.) Use a gas chromatograph equipped with a flame-ionization detector and containing a 1.8-m x 2-mm (id) stainless-steel column, or equivalent, packed with 10% silicone GE XE-60, or equivalent. Maintain the column isothermally at a temperature between 175° and 185°, and use helium as the carrier gas at a flow rate of 30 mL/min. Chromatograph a sufficient number of injections of the Standard Preparation,... 

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