Capillary LC

The development of new fiber coatings in the near future should further improve the specificity of SPME and overcome some of the observed matrix effects. Quantification by stable isotope dilution gas chromatography/mass spectrometry (GC/MS) may assist in improving analytical performance. Along with the possible application of micro LC and capillary LC columns to in-tube SPME, the development of novel derivatization methods and the potential for the analysis of fumigant pesticides, SPME appears to be a technique with a future in the analysis of pesticide residues in food. 

Figure 7.27 Constructed Gram-Schmidt chromatogram of a temperature-programmed packed-capillary LC separation of 4.8 ig Irgafos P-EPQ dissolved in DMF temperature programme 50°C for 8min, 4°Cmin-1 up to 140°C. Legend 1, mono-P-EPQ 2, Irgafos 168 3, 4,3 -P-EPQ 4, oxidised 4,4 -P-EPQ 5, 4,4 -P-EPQ. After Bruheim et al. [511]. From I. Bruheim et al., Journal of High Resolution Chromatography, 23, 525-530 (2000). Wiley-VCH, 2000. Reproduced by permission of Wiley-VCH.

Chen, H.S., Rejtar, T., Andreev, V., Moskovets, E., Karger, B.L. (2005). High-speed, high-resolution monolithic capillary LC-MALDI MS using an off-line continuous deposition interface for proteomic analysis. Anal. Chem. 77, 2323-2331. 

The ability to resolve and characterize complicated protein mixtures by the combination of 2DLC and online mass spectrometry permits the combination of sample fractionation/simplification, top-down protein mass information, and bottom-up peptide level studies. In our lab, the simplified fractions generated by 2D(IEX-RP)LC are digested and analyzed using common peptide-level analysis approaches, including peptide mass fingerprinting (Henzel et al., 1993 Mann et al., 1993), matrix-assisted laser desorption/ionization (MALDI) QTOF MS/MS (Millea et al., 2006), and various capillary LC/MS/MS methodologies (e.g., Ducret et al., 1998). 

Lee, S.W., Berger, S.J., Martinovic, S., Pasa-Tolic, L., Anderson, G.A., Shen, Y., Zhao, R., Smith, R.D. (2002). Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR. Proc. Natl. Acad. Sci. USA 99, 5942-5947. 

FIGURE 14.4 Chromatograms of high speed isocratic capillary LC elution of three components. Column 15 cm x 320 /im inner diameter, 5 /im C18 particles. Column head pressure 6800 psi at 48 /(L/min flow rate. System XTS two-dimensional splitless ultrahigh pressure nano UHPLC, Micro-Tech Scientific, Vista, California. 

D nano LC-MS-MS can identify approximately 100 components per salt step within 100 min. Ten-step 2-D SCX capillary LC-MS can separate approximately 1000 polypeptides in about 17 hr. 

FIGURE 14.18 Flow diagram of split flow capillary LC system. 1. Solvent reservoirs. 2. Model 5000 syringe pump (Varian, Walnut Creek, California). 3. Static mixer. 4. Injection port. 5. Column. 6. Detector. 7. Pressure transducer. 8. Pulse dampener. 9. Purge valve. 10. U-flow controlling device. 11. Waste. 

Many commercial split flow capillary LC systems incorporate a nano flow sensor mounted online to the capillary channel. The split flow system can be easily modified from a conventional system and performs satisfactorily for capillary LC applications. However, the split flow system may require thermal control and the LC solvent requires continuous degassing. In addition, the system may not work reliably at a high flow split ratios and at pressures above 6000 psi due to technical limitations of the fused silica thermal conductivity flow sensor. The split flow system based on conventional check valve design may not be compatible with splitless nano LC applications. The conventional ball-and-seat check valve is not capable of delivering nano flow rates and is not reliable for 7/24 operation at low flow. 

FIGURE 8.12 Separation of model compounds using hybrid monohthic columns, (a) and (c) hybrid allyl-monolith before hydrosilylation reaction, (b) and (d) hybrid allyl-monohth after modification with Cg-DMS. (a) and (b) show separations by capillary LC at 90 psi— mobile phase of acetonitrile/4 mM borate buffer (pH = 9.3) (40/60)—while (c) and (d) show separations by CEC at 25 kV—mobile mobile phase of acetonitrile/4 mM borate buffer (pH = 9.3) (20/80). Solutes are (in order of elution) DMSO, benzene, toluene, and ethylbenzene. 

Syringe pumps driven by screw mechanisms were popular in the 1960s because of their inherent precision and pulseless flow characteristics. Their disadvantages are a higher manufacturing cost and the problems associated with syringe refill cycles. Syringe pumps are currently used in specialized systems for microbore and capillary LC. ... 

This new hyphenated analytical system integrates capillary LC with NMR detection. The capillary LC-NMR system is comprised of an NMR spectrometer equipped with a capillary flow probe and the capillary LC. The capillary flow probe has a flow-cell design with an active sample volume of only 1 or 1.5 pL. This volume is chosen to match the typical peak volumes of capillary LC separation. 

Both the capillary LC and NMR are controlled by the interface software, which enables the operator to use the UV-detector output for peak selection. Only peaks of interest can be subjected to NMR analysis, while minor or unimportant compounds can be directed to waste. NMR acquisition can take place in either on-flow or stop-flow mode. The combination of capillary LC and NMR is suitable for sample-limited applications (e.g., proteomics) and allows for low nanograms detection. 

The present pneumatically assisted ESI interface is optimised around flow rates of 50-300 pl7min. The use of analytical columns 3. 6 mm i.d. with flow rates between 0.5 to 2 ml/min and narrow bore columns of 1-2 mm id with flow rates of 0.2-0.5 ml7min is routine in most pharmaceutical laboratories for HPLC analysis [24]. Capillary LC columns, because of their limited commercial availability and special practical considerations are used more where there is limited sample available or when sensitivity issues are present [25]. 

Timperman, A. T. and Aebersold, R., Peptide electroextraction for direct coupling of in-gel digests with capillary LC-MS/MS for protein identification and sequencing. Analytical Chemistry 72( 17), 4115-4121, 2000. 

The effect of the dwell volume on the retention times of analytes increases with decreasing retention factor at the start of gradient elution and with increasing ratio VpIV, and becomes very significant in the instrumental setup with the dwell volume comparable to or larger than the column hold-up volume, which is more likely to occur in micro- or in capillary LC than in conventional analytical LC (see Figure 5.4) [12]. 

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