Capillary Columns

Capillary columns offer a significant improvement in separation, in comparison to conventional packed columns, and have been used for the separation of complex mixtures and components closely related chemically and physiologically. As shown in Table 2, various glass and fused silica capillary columns have been used for free amine analysis. Fused silica capillary columns provide strength, flexibility and a more inert surface for improved performance and less adsorption. Cross-linked or bonded-phase columns can be washed with solvents, prolonging their lifetime. Furthermore, the advance of commercially available cross-linked and bonded-phase capillary columns and precise temperature-controlled GC ovens has meant that the retention times are extremely reproducible. This is critical when using automated date-handling equipment for identification and quantification. Typically, 10-30 m long columns, coated with either nonpolar or polar stationary phases, have been used for amine analysis. Many of the phases used today are specifically manufactured by the column supplier, and give excellent performance, low bleed and high efficiency. However, there is the drawback that a col-

Figure 2 Aniline compounds on packed column. GC conditions: column, 3% SP-2250 on Supelcoport (1.83 m x2 mm i.d. glass); column temperature, 4°C min~1 from 80 to 230°C; injector and detector temperatures, 250 and 300°C, respectively; He carrier gas flow rate, 30mLmin~1; detector, NPD. Peaks: 1, aniline; 2, 2-chloroaniline; 3, 3-chloroaniline; 4,4-chloroaniline; 5, 4-bromoaniline; 6, 3,4-dichloroaniline; 7, 2,4,6-trichloroaniline; 8, 2-nitroaniline; 9, 2,4,5-trichloroaniline; 10, 3-nitroaniline; 11,4-chloro-2-nitroaniline; 12, 4-nitroaniline; 13, 2,6-dichloro-2-nitroaniline; 14, 2-chloro-4-nitroaniline; 15, 2-bromo-6-chloro-4-nitroaniline; 16, 2,6-dibromo-4-nitroaniline; 17, 2-chloro-4,6-di-nitroaniline; 18, 2,4-dinitroaniline; 19, 2-bromo-4,6-dinitroaniline. (Reproduced with permission from Riggin etal. (1983) Analytical Chemistry 55: 1862.)

Figure 2 Aniline compounds on packed column. GC conditions: column, 3% SP-2250 on Supelcoport (1.83 m x2 mm i.d. glass); column temperature, 4°C min~1 from 80 to 230°C; injector and detector temperatures, 250 and 300°C, respectively; He carrier gas flow rate, 30mLmin~1; detector, NPD. Peaks: 1, aniline; 2, 2-chloroaniline; 3, 3-chloroaniline; 4,4-chloroaniline; 5, 4-bromoaniline; 6, 3,4-dichloroaniline; 7, 2,4,6-trichloroaniline; 8, 2-nitroaniline; 9, 2,4,5-trichloroaniline; 10, 3-nitroaniline; 11,4-chloro-2-nitroaniline; 12, 4-nitroaniline; 13, 2,6-dichloro-2-nitroaniline; 14, 2-chloro-4-nitroaniline; 15, 2-bromo-6-chloro-4-nitroaniline; 16, 2,6-dibromo-4-nitroaniline; 17, 2-chloro-4,6-di-nitroaniline; 18, 2,4-dinitroaniline; 19, 2-bromo-4,6-dinitroaniline. (Reproduced with permission from Riggin etal. (1983) Analytical Chemistry 55: 1862.)

umn from one supplier may not give the same separation as the nominally equivalent column from another supplier.

Glass capillary columns In early work, glass capillaries were employed for the separation of aromatic amines using alkaline PEG as the stationary phase. Although a disadvantage of this phase is its tendency to deteriorate at temperatures slightly above 200°C, it has been used for the separation of methylanilines and methylpyridines in coal-tar light oil. Carbowax 20M columns have been used for the determination of airborne aromatic amines with an NPD. The necessary inertness of glass capillary columns may be achieved by deactivation with octamethylcyclo-tetrasiloxane (OMCTS). The glass or fused silica columns were silanized using OMCTS and trifluoro-propyl(methyl)cyclosiloxane, and coated with various phases (SE-30, SE-52, SE-54). Test mixtures containing about 1 ng of such difficult substances as primary mono- and diaminoalkanes gave symmetrical peaks on some of these phases. As shown in

Figure 3 Drug standard mixtures on (A) AR glass and (B) fused silica capillary columns coated with SE-54 and with flame ionization detector. Temperature programmes are shown within the figure. Peaks: 1, amphetamine; 2, phentermine; 3, propylhexedrine; 4, methamphetamine; 5, ethylamphetamine; 6, propylamphetamine; 7, ephedrine; 8, phenmetrazine; 9, phendimetrazine; 10, amfep-ramone; 11, benzocaine; 12, phenacetin; 13, methyl phenidate; 14, procaine; 15, methaqualone; 16, cocaine; 17, codeine; 18, ethylmorphine; 19, morphine. (Reproduced with permission from Blomberg etal. Journal ofChromatography 239 (1982) 51).

Figure 3 Drug standard mixtures on (A) AR glass and (B) fused silica capillary columns coated with SE-54 and with flame ionization detector. Temperature programmes are shown within the figure. Peaks: 1, amphetamine; 2, phentermine; 3, propylhexedrine; 4, methamphetamine; 5, ethylamphetamine; 6, propylamphetamine; 7, ephedrine; 8, phenmetrazine; 9, phendimetrazine; 10, amfep-ramone; 11, benzocaine; 12, phenacetin; 13, methyl phenidate; 14, procaine; 15, methaqualone; 16, cocaine; 17, codeine; 18, ethylmorphine; 19, morphine. (Reproduced with permission from Blomberg etal. Journal ofChromatography 239 (1982) 51).

Figure 3, the separation of some underivatized drugs is equally good on alkali-resistant (AR) glass and fused silica capillaries, although alkali-resistant (AR) glass has a basic character that can be reduced by careful leaching.

On the other hand, interesting results dealing with the separation of free amines and other nitrogen compounds were reported in glass capillary columns with stationary phases polymerized in situ.

Fused silica capillary columns For the analysis of amines, capillary columns with a nonimmobilized PEG-type stationary phase have been specially prepared and are commercially available. For the analysis of volatile amines, aromatic and heterocyclic amines and other amino substances, CAM, CP-Wax, HP-20M, Carbowax 20M and Carbowax Amine capillary columns have been recommended. These columns are generally deactivated with KOH to elute basic compounds with good peak shapes and responses. Three types of fused silica capillary columns, Supelcowax 10 (PEG), CP-Sil-19CB (methylphenyl-cyanopropylsilicone) and HP-1 (methylsilicone) have also been used for the separation of aliphatic and aromatic amines. Ammonia as a carrier gas can dras tically affect the retention factors and improve the peak symmetry for aliphatic amines. A porous polymer fused silica capillary column, PoraPLOT Amine, has been used to separate very volatile amines. By using this column and ELCD, C1-C6 amines in aqueous and methanolic solution were analysed. The separation of aniline and its halogen and nitrogen derivatives in waste water were evaluated using several glass and fused silica capillary columns of polysiloxane type (SE-30, SE-52, SE-54, SP-2100) and NPD. Each of the capillary columns gave excellent peak shape for all the anilines, but failed to resolve at least one compound pair (e.g. the SE-30 completely resolved 3- and 4-chloroaniline that co-eluted on SE-54, but failed to resolve the 2,6-dibromo-4-nitroaniline and 2,4-dinitroaniline which were completely resolved on SE-54). Figure 4 shows a chromatogram of an aniline mixture on an SE-54 fused silica column. The NPD sensitivities for many anilines are substantially better with the SE-54 capillary column (Figure 4) than with the 3% SP-2250 packed column (Figure 2), primarily because less peak tailing is observed at low concentration. Interestingly, the fused silica and glass capillary SE-54 columns gave different elution patterns for the various anilines. Using both SE-54 and SE-30

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Figure 4 Aniline compounds on fused silica capillary column. GC column, SE-54 (30 m x 0.25 mm i.d.); He carrier gas flow rate, 30 cms-1; split ratio, 10:1. Other conditions and peak numbers are the same as Figure 2. (Reproduced with permission from Yang et al. (1993) Analytical Chemistry 65: 572.)

Figure 5 Aromatic amines on fused silica capillary column. GC conditions: column, DB-35ms (25 m x 0.20 mm i.d. glass); column temperature, initially hold at 50°C for 2 min, increase to 340°C at 20°C min-1 and then hold at 340°C for 10 min; injector and detector temperatures, 280 and 320°C, respectively; He carrier gas flow rate, 35 cm"1; splitless injection; detector, NPD. Peaks: 1, o-toluidine; 2, 4-chloroaniline; 3, 2-methoxy-5-methylaniline; 4, 2,4,5-trimethylaniline; 5, 4-chloro-2-methylaniline; 6, 2,4-diaminotoluene; 7, 2,4-diaminoanisole;8, 2-aminonaphthalene;9, 2-methyl-5-nitroaniline; 10, 4,4'-oxydianiline; 11, 4,4'-methylenedianiline; 12, benzidine; 13, 2-aminoazotoluene; 14, o-tolidine; 15, 4,4'-thiodianiline; 16, 3,3'-dimethoxybenzidine; 17, 3,3'-dichlorobenzidine. (Reproduced with permission from Catalog and Technical Reference, C407, J & W Scientific, California.)

Figure 5 Aromatic amines on fused silica capillary column. GC conditions: column, DB-35ms (25 m x 0.20 mm i.d. glass); column temperature, initially hold at 50°C for 2 min, increase to 340°C at 20°C min-1 and then hold at 340°C for 10 min; injector and detector temperatures, 280 and 320°C, respectively; He carrier gas flow rate, 35 cm"1; splitless injection; detector, NPD. Peaks: 1, o-toluidine; 2, 4-chloroaniline; 3, 2-methoxy-5-methylaniline; 4, 2,4,5-trimethylaniline; 5, 4-chloro-2-methylaniline; 6, 2,4-diaminotoluene; 7, 2,4-diaminoanisole;8, 2-aminonaphthalene;9, 2-methyl-5-nitroaniline; 10, 4,4'-oxydianiline; 11, 4,4'-methylenedianiline; 12, benzidine; 13, 2-aminoazotoluene; 14, o-tolidine; 15, 4,4'-thiodianiline; 16, 3,3'-dimethoxybenzidine; 17, 3,3'-dichlorobenzidine. (Reproduced with permission from Catalog and Technical Reference, C407, J & W Scientific, California.)

Figure 6 Chromatograms obtained from hair samples. (A) Normal hair; (B) normal hair with standard amphetamines added; (C) abuser's hair. GC conditions: column, CBJ-17 (30 m x0.53 mm i.d. fused-silica, Shimadzu); column temperature, initially hold at 100°C for 5 min, increase to 220°C at 10°C min- and then hold at 220°C for 3 min; injector and detector temperatures, 220°C; He carrier gas flow rate, 4 mL min-1; split ratio, 2:1; detector, NPD. Peaks: 1, a-phenethylamine (internal standard); 2, amphetamine; 3, methamphetamine; 4, /V-propyl-^-phenethylamine (internal standard).

Figure 6 Chromatograms obtained from hair samples. (A) Normal hair; (B) normal hair with standard amphetamines added; (C) abuser's hair. GC conditions: column, CBJ-17 (30 m x0.53 mm i.d. fused-silica, Shimadzu); column temperature, initially hold at 100°C for 5 min, increase to 220°C at 10°C min- and then hold at 220°C for 3 min; injector and detector temperatures, 220°C; He carrier gas flow rate, 4 mL min-1; split ratio, 2:1; detector, NPD. Peaks: 1, a-phenethylamine (internal standard); 2, amphetamine; 3, methamphetamine; 4, /V-propyl-^-phenethylamine (internal standard).

fused silica capillary columns, all 19 anilines can be resolved.

A polysiloxane capillary column specially designed for the analysis of basic compounds using new deacti-vation technologies has been developed. This proprietary deactivation provides both the inertness (basicity) and surface energies required to coat a 5% diphenyl/95% dimethylpolysiloxane stationary phase successfully. Using this column, C3-C10 primary amines can be separated as symmetrical peaks. This column allows lower limits of detection for basic compounds such as substituted anilines and ben-zidines. Since the column is virtually identical in polarity to the widely used ordinary columns with the same stationary phase, it can be directly substituted and run under the same temperature conditions. DB-5ms and DB-35ms columns certified for use with MS have been developed for the analysis of aliphatic and aromatic amines. These columns have very low bleed characteristics and excellent inertness. As shown in Figure 5, 17 aromatic amines were completely separated using a DB-35ms column.

Lower aliphatic tertiary amines in environmental samples were analysed by headspace GC with a mass selective detector (MSD) using a polymethylsiloxane column. The SPME method has gained popularity as a solvent-free, reliable and flexible tool for sampling a variety of volatile and semi-volatile compounds. By combining SPME with GC, these compounds can be simply and rapidly extracted, concentrated and introduced into the GC system. Using headspace SPME and GC-MSD on polysiloxane-type fused silica capillary columns such as DB-1, OV-1, SPB-1, HP-1 and HP-5, amphetamine, methamphetamine and related stimulants in urine can be analysed at the ngmL-1 level. Recently, headspace SPME and GC-NPD using a slightly polar capillary column CBJ-17 (Figure 6) has developed as a method for determining amphetamines in human hair.

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