Micro-HPLC-column

Use of FID and SCD are compatible with SFE-HPLC, since they are flame-based and unaffected by gases in the mobile phase. Unfortunately, SCD can only be used with micro-HPLC (column i.d. <320 (tm), which requires miniaturised equipment not commonly found in most analytical laboratories. When following SFE with HPLC analysis using a spectroscopic detector, a medium-purity grade is usually sufficient. 

Mlcrodevlces. The sol-gel technique allows the reproducible preparation of capillary electrochromatography (CEC) microcolumns that could be used for high performance separation in microfabricated devices (microchips). Eluid hydrolyzed silica sols can be injected into the microchip-channels. Condensation occurs inside the device micro-HPLC column made of silica gel (Constantin, 2001). 

MacMillan and Wright [133] identified and measured saturated and unsaturated 1,3- and saturated 1,4-sultones in anionic surfactants by a series of separation maneuvers. Ion exchange treatment separates sultones from the bulk of the ionic surfactant. TLC concentrates the sultones systems for HPLC analysis. They found that pentane-ether is preferable to the usual hexane-ether system and that the addition of a little methanol sharpens the separations. Finally, HPLC using a micro-Porasil column with 90 1 isooctane/ethanol provides quali-... 

The catalytic reaction was carried out at 270°C and 101.3 kPa in a stainless steel tubular fixed-bed reactor. The premixed reaction solution, with a molar ratio catechol. methanol water of 1 1 6, was fed into the reactor using a micro-feed pump. To change the residence time in the reactor, the catechol molar inlet flow (Fio) and the catalyst mass (met) were varied in the range 10 < Fio <10 mol-h and 2-10 < met < 310 kg. The products were condensed at the reactor outlet and collected for analysis. The products distribution was determined quantitatively by HPLC (column Nucleosil 5Ci8, flow rate, 1 ml-min, operating pressure, 18 MPa, mobile phase, CH3CN H2O =1 9 molar ratio). 

P 21 ] Palladium on alumina was employed as catalyst [26]. Hydrogen and organic reactant were mixed in the micro mixer and fed to a Merck Superformance HPLC column of 100 mm length and 5 mm inner diameter, which was used as a hydrogenator. No further details are given in [67] or [26]. 

Trisciani, A. and Andreolini, F, Evaluation of a micro-HPLC system dedicated to packed capillary column liquid chromatography, /. HRC CC, 13,270,1990. 

FIGURE 16.5 Schematic of instrumental setup for 2D micro-RPLC-CZE. A split injection/ flow system is used to deliver a nanoliter per second flow rate to the micro-RP-HPLC column from the gradient LC pump. The HPLC microcolumn has 50 pm i.d. and 76 cm length, and the electrophoresis capillary has 17 pm i.d., L — 25 cm, and/= 15 cm. The valve is air-actuated and controls the flow of flush buffer (reprinted with permission from Analytical Chemistry). 

Column diameter is an important parameter to consider in life science applications in which sample amounts are very limited and the components of interest may not be abundant. Researchers have reviewed micro HPLC instrumentation and its advantages.910 Nano LC-MS offers 1000- to 34,000-time reductions in the dilution of a sample molecular zone eluted from nano LC columns of 25 to 150 [Mi IDs in comparison to a 4.6 mm ID column. This represents a large enhancement of ion counts in comparison to counts obtained for the same amount of sample injected into a conventional 4.6 mm column. Solvent consumption for an analysis run or sample amount required for injection in a nano LC application may be reduced 1000 to 34,000 times compared to amounts required by an analytical column operated at a 1 mL/min flow rate. 

Fig. 3.107. Comparison of micro-HPLC separations of aromatic sulphonic acids in different mobile phases (a) 0.005 M tetrabutylammonium hydrogensulphate (TBAS) in 15 per cent (v/v) methanol in water (1) Laurent acid, (2) amino-F-acid, (3) Cleve-1,6- and Peri acids, (4) unidentified impurity, (5) Cleve-1,7-acid and (6) unidentified impurity, (b) 0.005 M tetrabutylammonium hydrogensulphate (TBAS) in 15 per cent (v/v) methanol in water with 0.01 M /Lcyclodextrin (CD) (1) Laurent acid, (2) amino-F-acid, (3) Cleve-1,6-acid, (4) Peri acids, (5) unidentified impurity, (6) Cleve-1,7-acid and (7) unidentified impurity. Column, Biosphere Si C18, 162 X 0.32 mm i.d. flow rate 5 pl/min, column temperature ambient, detection, UV, 220-230 nm. Reprinted with permission from P. Jandera et al. [164].

FIGURE 1.30 Micro-HPLC separation of all 4 stereoisomers of the dipeptide alanyl-alanine as FMOC derivatives (a) and DNP-derivatives (b), respectively, on a 0-9-(tert-butylcarbamoyl)quinine-based CSP. Experimental conditions Column dimension, 150 X 0.5 mm ID mobile phase (a) acetonitrile-methanol (80 20 v/v) containing 400 mM acetic acid and 4 mM triethylamine, and (b) methanol-0.5 M ammonium acetate buffer (80 20 v/v) (pHa 6.0) flow rate, 10 ixLmin temperature, 25 C injection volume, 250 nL detection, UV at 250 nm. (Reproduced fromC. Czerwenka et al., J. Pharm. Biomed. Anal., 30 1789 (2003). With permission.)... 

Buszewski, B., Szumski, M., and Sus, S. (2002). Methacrylate-based monolithic columns for micro-HPLC and CEC. LC-GC Europe 15, 792-798. 

Fig. 5.9 Design of the chip-based enzyme ESI-MS assay. MS instrument Ion-trap mass spectrometer (LCQ Deca, Thermo Electron). I Sample components/inhibitors injected by flow injection or eluting from capillary HPLC column. E Infusion pump delivering the enzyme cathepsin B. S infusion pump delivering the substrate Z-FR-AMC. Micro-chip design Vrije Universiteit Amsterdam. Micro-chip production Micronit Microfluidics BV (Enschede, The Netherlands).

High-performance liquid chromatography (HPLC) has become a standard separation technique used in both academic and commercial analytical laboratories. However, there are several drawbacks to standard HPLC, including high solvent consumption, large sample quantity, and decreased detection sensitivity. Micro-HPLC (pHPLC) is a term that encompasses a broad range of sample volumes and column sizes (as shown in Table 3.1), but Saito and coworkers provided narrower definitions in their review based on the size of the columns. ... 

Today nearly all of the major HPLC companies offer a pHPLC system or at least the possibility to modify a standard instrument to accept micro-bore columns. In our laboratory, we routinely use the pHPLC systems Ultimate from Dionex/LC Packing (Ligure 3.2), the Extreme Simple 4-D... 

In addition to the commercially available systems, several authors have described laboratory-built systems using commercially available components from companies such as Upchurch Scientific (Oak Harbor, WA). One of the first reported laboratory-built micro-bore HPLC systems was described by Simpson and Brown, which was a simple adaptation of a standard HPLC system to accept micro-bore columns built from guard columns. A complete system has been described based on dual microdialysis syringe pumps (CMA Microdialysis, Chelmsford, M A) or dual syringe pumps (Harvard Apparatus, Inc., Holliston, MA), a microinjection port, and a micro-column the latter components being obtained from Upchurch scientific (Figure 3.5). This system was coupled with a laser-induced fluorescence (LIF) detector and used to measure neuropeptides in sub-microliter samples. A further modification of this system was built to perform immunoaffinity isolations of biomedically important analytes from clinical samples. ... 

Mitulovic, G., Smolnch, M., Chervet, J. R, Steinmacher, I., Kungl, A., and Mechtler, K., An improved method for tracking and redncing the void volnme in nano HPLC-MS with micro trapping columns. Analytical and Bioanalytical Chemistry 376(7), 946-951, 2003. 

Micro-HPLC operation sets special demands on the gradient instrumentation. As the internal column diameter, d, decreases, lower flow rates should be used at comparable mean linear mobile phase velocities, u = 0.2-0.3 mm/s. At a constant operating pressure, the flow rate decreases proportionally to the second power of the column inner diameter, so that narrow-bore LC columns with 1mm i.d. require flow rates in the range of 30-100pL/min, micro-columns with i.d. 0.3-0.5mm, flow rates in between 1 and lOpL/min, and columns with 0.075-0.1 mm i.d. flow rates in the range of hundreds nL/min. Special miniaturized pump systems are required to deliver accurately mobile phase at very low flow rates in isocratic LC. 

New concepts presented in this edition include monolithic columns, bonded stationary phases, micro-HPLC, two-dimensional comprehensive liquid chromatography, gradient elution mode, and capillary electromigration techniques. The book also discusses LC-MS interfaces, nonlinear chromatography, displacement chromatography of peptides and proteins, field-flow fractionation, retention models for ions, and polymer HPLC. 

As mentioned earlier, that it is possible to simultaneously apply a high hydraulic pressure at the inlet vial to drive the solvent through the column and an electrical field. This hybrid method combining both pressure and electrodriven liquid chromatography was first introduced by Tsuda [46] and is referred to as pressure assisted CEC or field assisted micro HPLC. Verheij et al. [47] used this the technique to couple CEC with MS. These authors also proposed the name pseudo-CEC for this technique. 

Fig. 2.19. Illustration of selectivity gain in CEC compared with micro HPLC (taken from Ref. [56] with permission of the publisher). Column Waters Spherisorb ODS I, 3 pm, 250 (335) x 0.1 mm. Mobile phase, 80% acetonitrile-20% 25 mM phosphate, 0.2% hexylamine, pH 3.8. Voltage 25 kV, temperature 20°C. Sample 1, procaine 2, timolol 3, ambroxol 4, metoclopramide 5, thiourea 6, naproxene 7, antipyrine.

Liquid Chromatograph. The liquid chromatograph was comprised of a Waters 660 Solvent Programmer, two Waters 6000A pumps, a Waters U6-K Injector and a Waters 440 absorbance detector (254 nm). Whatman micro-capillary tubing (0.007" ID) was used to transfer the HPLC column effluent from the 254 nm absorption detector to the fluorescence detector. 

Apply the sample to a micro-Mono S HPLC column and elute proteins with a salt gradient. Turn the UVdetector on 215 nm. Separate peaks manually according to absorbance at 215 nm. 

---