Preparation of Fused-Silica Capillary Columns

Most users of a modern capillary column regard it as a sophisticated high-precision device and purchase columns from a vendor. Few give any thought to the steps involved in column preparation. Their highest priority is understandably the end result of accurate and reproducible chromatographic data that the column can provide for quality assurance/quality control protocols. In this section, deactivation and coating of a fused-silica column with stationary phase are discussed. 

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FIGURE 9 Column drawing apparatus for the preparation of fused silica capillaries. [From Lipsky, S. R., McMurray, W. J., Hernandez, M., Purcell, J. E., and Billeb, K. A. (1980). J. Chro-matogr. Sci. 18,1.]... 

CZE has been employed for the analysis of another set of dyes in foodstuffs. The chemical structures, numbers and names of the dyes included in the investigation are listed in Fig. 3.142. A fused-silica capillary column of 57 cm length (50 cm effective length 75 jum i.d.) was employed for the separations. The capillary was conditioned by 1.0 M NaOH for 20 min followed by 10 min wash with water and 10 min wash with the running buffer. The buffer was prepared by adding NaOH to 10 mM phosphoric acid to reach pH 11.0. The capillary was thermostated at 25°C and the separation voltage was 20 kV. A hydrodynamic injection mode was applied (0.5 psi, 4 s, 21 nl) and spectra of... 

The solution, 10 pL, prepared as outlined in Note 18 was loaded an a Chirasil-Val fused-silica capillary column of Machery-Nagel (25 m, 0.4 mm) in a Carlo-Erba-Fraktovap 4160 HR GC. After 5 min at 160°C, the column temperature was increased by 2°C per min up to 200°C. 

Determination of CholesteroL For meat extraction, the procedures for determining the cholesterol of extracted lipid samples were described Chao et al. (2i). For edible beef tallow extraction, the preparation of samples for cholesterol content was based on the AOAC (22) method Section 28.110. The prepared sample was then injected into a Supelco SPB-1 fused silica capillary column of 30 meters x 032 mm i.d. in a Varian Model 3700 gas chromatograph equipped with dual flame ionization detectors. The initial holdup time was 4 min at 270°C and then programmed to a temperature of 300°C at a ramp rate of 10°C/min. Helium flow rate and split ratio were 13 ml and 50 1, respectively, while the injector/detector temperature was 310°C. 

Capillary gas chromatography (GC) using modified cyclodextrins as chiral stationary phases is the preferred method for the separation of volatile enantiomers. Fused-silica capillary columns coated with several alkyl or aryl a-cyclo-dextrin, -cyclodextrin and y-cyclodextrin derivatives are suitable to separate most of the volatile chiral compounds. Multidimensional GC (MDGC)-mass spectrometry (MS) allows the separation of essential oil components on an achiral normal phase column and through heart-cutting techniques, the separated components are led to a chiral column for enantiomeric separation. The mass detector ensures the correct identification of the separated components [73]. Preparative chiral GC is suitable for the isolation of enantiomers [5, 73]. 

Figure D1.2.2 Sample GC chromatogram of the FAME from butter fat (Sweet Cream Butter, Wisconsin Grade AA, Roundy s, Milwaukee, Wise.) prepared using the sodium methoxide method (see Basic Protocol 2). Equipment DB-23 fused silica capillary column, 30 m x 0.32 mm i.d., 0.25 pm film thickness, FID detector. Temperature, injector 225°C detector 250°C. Column (oven) temperature program 100°C initial, hold 4 min, ramp to 198°C at 1.5°C/min, hold 10 min. Total run time was 80 min. Split injection.

A stereoselective GC method for determination of etodolac enantiomers in human plasma and urine was first reported as a preliminary method [35], and then as a validated method [36]. Sample preparation involved addition of (S)-(+)-naproxen (internal standard) and sodium hydroxide to diluted plasma or urine. The samples were washed with diethyl ether, acidified with hydrochloric acid, and extracted with toluene. ( )-(+)-naproxen was used as a derivatizing agent to form diastereomeric derivatives of etodolac. The gas chromatograph system used in this work was equipped with fused-silica capillary column (12 m x 0.2 mm i.d.) coated with high-performance cross-linked methylsilicone film (thickness 0.33 pm) and a nitrogen-phosphorous detector. The operating conditions were injector 250°C detector 300°C column 100-260°C (32 °C/min). 

Procedure Inject 1-pL aliquots of the Standard Preparation and the Sample Preparation into a gas chromatograph equipped with a split injector, a flame-ionization detector, and a 25 -m fused silica capillary column coated with a 2-pm film of 7% cyanopropyl-7% phenyl-85% methyl-1% vinylpolysiloxane (CP-Sil 19 CB, Chrompack Middelburg, or equivalent). Maintain the column at 100°, raising the temperature at 8°/min to a final temperature of 300°. Set the injector temperature to 270°and the detector temperature to 270°. Use a mixture of helium and methane, at a split ratio of 1 100, as the carrier gas, flowing at 120 mL/ min. Run the chromatogram for 27 min. 

A Hewlett Packard 5880-A gas chromatograph equipped with a flame ionization detector, a 5880-A series level four computerized data system and a DB-5 (30 m x 0.25 mm I.D.) fused silica capillary column (J W Scientific, Folsom, CA) were used. Puree was centrifuged at 10,000 x g and the supernatant was injected onto the column (direct aqueous injection). Ultrafiltered juice was injected directly with no sample preparation. Sample size in all cases was 5uL split 15 1 with helium as the carrier gas at a flow rate of 1 ml/min. The oven temperature was held at 60°C for the initial seven minutes of the injection time. It was then increased to 200°C at a rate of 5°C/min and was held there for twenty five minutes. Total volatiles were obtained by summation of integrated peak areas on the chromatograms. Percent areas of each peak were also calculated. 

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