Guard columns conditioning

HPLC condition — A Waters reversed-phase HPLC column (Symmetry Shield RP Cl8, 5 pm, 2.1 x 50 mm) was used in conjunction with a Regis SPS guard column (ODS, 5 pm, 100 A,... 

The United States Pharmacopeia (USP) test (467) describes three different approaches to measuring organic volatile impurities in pharmaceuticals. Method I uses a wide-bore coated open tubular column (G-27, 5% phenyl-95 % methylpolysiloxane) with a silica guard column deactivated with phe-nylmethyl siloxane and a flame-ionization detector. The samples are dissolved in water and about 1 p is injected. Limits are set for benzene, chloroform, 1,4-dioxane, methylene chloride, and trichloroethylene. Methods V and VI are nearly identical to method I except for varying the chromatographic conditions. For the measurement of methylene chloride in coated tablets, the headspace techniques described above are recommended. 

Once suitable ionization conditions have been established, LC separation can be optimized. As with any LC system, attention needs to be paid to column choice, correct tubing diameters, zero dead volume connections, use of guard columns, and mobile phase filtration and de-gassing. Modem LC pumps can deliver reliable gradients at low flow rates but for capillary LC, precolumn flow splitting or specialized pumps may be necessary. Mobile phase composition and pH should be chosen to... 

The separation of the substrate testosterone from the numerous reaction products was accomplished by reversed-phase HPLC on a Ci8 column preceded by a guard column. The compounds were separated by a combined isocratic and gradient elution procedure. The eluent was monitored at 240 nm. The separations obtained with these HPLC conditions are shown in Figure 9.127. 

When calculating the effects of changing column dimensions and gradient conditions, it is important to remember to include the retentive and volumetric effects of any pre-column or guard column, as these devices, whilst useful insofar as they protect the more valuable main column from the worst effects of sample matrices, will also serve to delay the effect of the gradient on the analytical column. 

Figure 11.5 Separation of normal coal-tar pitch on a nitro phase [reproduced with permission from G.P. Blumer, R. Thoms and M. Zander, Erdol Kohle, Erdgas, Petrochem., 31, 197 (1978)]. Conditions columns, two 20cm x 4mm i.d. plus a guard column stationary phase, Nucleosil NO2, Spm mobile phase, hexane-chloroform, gradient elution UV detector 300 nm sensitivity, 1 a.u.f.s. and, after 30 min, 0.4 a.u.f.s. 

Figure21.2 Preparative separation ofthe reaction mixture. Conditions sample, 1 ml of solution containing 250 mg guard column, 5 cm x 4.5 mm i.d., dry-packed with silica (40gm) column, 25cm x 21.5mm i.d. stationary phase, silica, 7 xm ...

The HPLC system consisted of five LC-lOA pumps, an SIL-lOA auto sample injector, a DGU-14 degasser, a CLD-lOA chemiluminescence detector, an SCL-lOA system controller, a C-R4A integrator, an HIC-6A and a CTO-IOAC column oven (all Shimadzu, Kyoto, Japan). The clean-up colunm (4.6 i.d. X 150 mm), concentrator column (4.6 i.d. X 30 mm), separator colunm (4.6 i.d. X 250 + 150 mm), guard columns 1 (4.6 i.d. X 30 mm) and 2 (4.6 i.d. X 50 mm) were packed with Cosmosil 5C18-MS (Nacalai Tesque, Kyoto, Japan), and the reducer column (4.0 i.d. X 10 mm) was a Nitroarene Reactor Column (Shimadzu). All other conditions were the same as those given in our previous report. ... 

Chromatographic separation was performed using a reversed phase Intersil ODS-3 analytical column (100 x 4.0mm i.d, 3 pm particle diameter) preceded by guard column Phenomenex Ci8 (ODS Octadecyl) (4 x 3.0 mm i d). The LC mobile phase was consisting of (A) water and (B) acetonitrile/methanol (70 30). The LC gradient elution conditions are shown in Table 1. 

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