Micro LC column

There is a need for smaller columns in order to reduce solvent consumption and analysis time. Following Vissers et al. (57), we suggest different names for packed micro LC columns according to the internal diameter ... 

Many of these problems have been resolved or minimized through developments in the last two decades. This section discusses key modem trends, including high-purity silica, hybrid particles, and novel bonding chemistries, and describes their impacts on column performance. The benefits of Fast LC and micro LC columns are also discussed with their salient applications. 

Micro-LC columns are used for better compatibility with specific sample preparation methods (e.g. on-column focusing) or interfaces for MSD. The use of microcolumns also enables temperature gradient as an additional selectivity/efficiency tuning factor. Applications on triazines use temperature gradients ranging from subambient conditions up to 70°C. 

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. 

Another advantage of the micro-LC approach is that the required sample size is minimal, so the sample can be drawn from a 1-1 laboratory scale reactor without influencing the reactor composition. The ISCO pLC-500 microflow syringe pump has proven to be reliable and reproducible in evaluations in our laboratory. Capillary liquid columns have been fabricated on planar devices such as silicon to form a miniaturized separation device.19... 

Table 7.87 shows the main features of on-line micro LC-GC (see also Table 7.86). The technique allows the high sample capacity and wide flexibility of LC to be coupled with the high separation efficiency and the many selective detection techniques available in GC. Detection by MS somewhat improves the reliability of the analysis, but FID is certainly preferable for routine analysis whenever applicable. Some restrictions concern the type of GC columns and eluent choice, especially using LC columns of conventional dimensions. Most LC-GC methods are normal-phase methods. This is partly because organic solvents used as eluents in NPLC are compatible with GC, making coupling simpler. RPLC-GC coupling is demanding water is not a suitable solvent for GC, because it hydrolyses the siloxane bonds in GC columns. On-line RPLC-GC has not yet become routine. LC-GC technology is only applicable to compounds that can be analysed by GC, i.e. volatile, thermally stable solutes. LC-GC is appropriate for complex samples which are difficult or even impossible to analyse by a single chromatographic technique. Present LC-GC methods almost exclusively apply on-column, loop-type or vaporiser interfaces (PTV).

The current status of chromatography is shown in Table 10.25. Since reducing separation time is a major issue, there is a pronounced trend toward shorter columns filled with small particles. The current trends for lower flow (micro- and nano-LC) columns, and great strides to achieve (ultra-) fast chromatographic... 

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. 

Due to the limited peak capacity of the 15 cm analytical column utilized in 2-D nano LC-MS, several elution steps are required to achieve the required separation. The 15 cm analytical column can be replaced with a 100 cm nano LC column to increase the resolution of sample in each step. As shown by Yang,20 a 100 cm column allows the one-step separation of more than 2000 polypeptides from trypsin digest of mouse brain lysate, P2 fraction using XtremeSimple ultrahigh pressure nano LC (Micro-Tech Scientific, Vista, California) and LTQ MS (Thermo Electron, San Jose, California) in 6 hr (Figure 14.16). In addition to the improvement of resolving power with a 100 cm column, it... 

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... 

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. 

Controlled by the MS-MS software, the PE Series 200-micro LC pump operates iso-cratically using 80% acetonitrileiwater at 40 pl/min. A 50 x 1 mm C18 column (Keystone) is used between the pump and autosampler to provide back-pressure. The syringe/system flush solution is the 80% acetonitrileiwater used as the mobile phase. The autosampler is connected directly to the MS-MS Turboion Spray source. The injected sample volume is 20 pi. 

At present, reciprocating pumps are in use for conventional column LC. In micro LC both syringe and reciprocating pumps are the favored solvent delivery systems. 

In the syringe-type pump the liquid is enclosed in a cylinder. A piston moves at a constant speed to push the liquid. Eluent compressibility induces time-consuming flow equilibrium. Nevertheless, the flow from a syringe pump is pulse free. For micro LC, flow rates of 50 yuL/min are utilized in spite of the drawback of column pressurization. With very low flow rates (in the nanoliter range) the use of pumps is tedious, and split-flow techniques are required. 

Different approaches utilizing multidimensional LC or SFC systems have been reported for the analysis of middle distillates in diesel fuel. A method, based on the LC separation of paraffins and naphthenes by means of a micro-particulate, organic gel column has been described (23, 24). The complete system contained up to four different LC columns, a number of column-switching valves and a dielectric constant detector. However, the LC column for the separation of paraffins and naphthenes, which is an essential part of the system, is no longer commercially available. 

Recently, Alexander et al. [44] reported the use of an automated separation system developed for micro-LC and CEC using both isocratic and gradient elutions. The complete system is shown in Fig. 2.15. An enlarged view of the coupling of the column to the injection valve presents Fig. 2.16. The mobile phase was delivered by two micro-LC pumps at a flow rate of 30 pL/min to a post injection splitter that houses the column inlet. In the CEC mode, pressure was not applied (no restriction on... 

Fig. 2.15. Schematic automated isocratic and gradient elution nemo-liquid chromatograph/ capillary electrochromatograph according Alexander et al. (reproduced from Ref. [44] with permission of the publisher). 1, high-voltage power supply (negative polarity) 2, platinum electrode 3, outlet reservoir vial 4, UV detector with on-column flow cell 5, nanocolumn 6, two-position switching valve 7, jack stand 8, fused-silica make-up adapter (split device) 9, ground cable 10, internal loop micro-injection valve 11, plexiglas compartment 12, autosampler 13, dynamic mixer 14, micro-LC pumps.

For most applications to CWC-related analysis, conventional and narrow bore columns have been used with APCI and pneumatically assisted ESI, using a split eluent if combined with ESI. The TNO Prins Maurits Laboratory routinely uses micro LC, particularly for biomedical analysis. 

Hooijschuur et al. (54) also reported the analysis of the hydrolysis products of sulfur mustard homologues (11), using micro-LC/ESI/MS (triple quadrupole) and micro-LC/FPD in sulfur mode. To improve sensitivity, large-volume injection was used with peak compression by adding suitable coeluting alcohols. LC employed a 0.28-mm ID packed C18 column, eluted isocratically with water-methanol (80 20 v/v)-0.2% formic acid, flow rate 6 xl/min. Spectra were dominated by MH+, [MH — H20]+,... 

Figure 15 CEC and micro-LC separations of nine analytes achieved on the same column entrapped with composition O (see Table 1 for detailed description), (a) The CEC separation was achieved by applying 10 kV on 26-cm-long packed bed. (b) The micro-LC separation was performed on the same instrument (HP3DCE by applying El-bar gas pressure on the inlet vial (L = 24 cm). Conditions mobile phase, 80% aceto-nitrile/20% Tris buffer, 25 mM. pH 8.0. (Reprinted from Ref. 21, with permission.)...

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