Microcapillary packed columns

These have mainly been developed by McGuffin and Novotny [120] and coworkers and are characterised by low column diameter to particle size ratios of 2 to 5. This is much less than small bore packed columns (50-200) or conventional columns (500-2000). Below ratios of 2, it has been reported [101] that the packing structure collapses under the viscous flow and causes clogging of the column. The microcapillary columns are prepared by extruding a heavy walled glass tube, 0.5-2 mm i.d., packed with 10-50 pm particle size high temperatures resistant silica or alumina. For reverse phase work the stationary phases have then to be bonded in situ. 

Chromatographic performance in terms of speed of analysis and resolving power has been found to be poor relative to conventional small particle packed columns. Although packed capillary columns have larger capacities their permeability is reduced. 

1 Summary. Knox and Gilbert [119] and Halasz [121,122] have considered the theoretical limits on the separation performance of OTC. They conclude that in order to match the performance and speed of analysis of conventional HPLC systems ultra low flow-rates and dead volumes are required and that it is unlikely that OTCs will offer any significant advantage. However, until the very stringent instrumental requirements are satisfied the theoretical potential of OTCs cannot be satisfactorily examined. Many workers, on the other hand, have already demonstrated the advantages of microbore and narrow bore columns in terms of low solvent consumption, increased sensitivity and in the latter, dramatically increased performance. 

Despite the numerous advantages the instrumental demands of microcolumn LC are considerable, and these demands are further accentuated as the requirements vary from one column type to another. A consequence of the reduced flow rates is that the detector flow-cell volume should be reduced to 10nl for OTCs, 0.1 pi for packed microcapillaries and 1 pi for microbore columns. An additional demand of the detector is that it should have a rapid response, 0.5 s. Development of suitable detectors is paramount if the potential of micro-LC is to be realised. Study of detector systems has focused in two areas firstly, the miniaturisation of ultraviolet, fluorescence and electrochemical systems, using in the former two systems LASERS as excitation sources and ultraviolet fibre optic and on-line cells to reduce band broadening and increase sensitivity [123,124] secondly, the direct interfacing with systems which previously required transport and/or concentration of the eluant. Interfacing of HPLC with mass spectroscopy has been undertaken by Barefoot et al. [125] and Lisek et al. [126] and flame systems (FPD and TSD) have been reviewed by Kientz et al. [127]. Jinno has reviewed the interfacing of micro-LC with ICP [128]. 

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Where solvent costs are an important consideration the reduced solvent consumption of microbore columns will allow the use of more exotic solvents. For the other column technologies there is no substantial applications literature as the investigative work has been carried out on individually prepared columns. The following chromatograms illustrate the potential of small bore packed and microcapillary packed columns, respectively. Shown below. Figure 6.56 is an example of a highly efficient separation of... 

An example of the separation obtained of an aromatic fraction of coal tar using a microcapillary packed column is shown below (Figure 6.57 [131]) the chromatogram was obtained using a step-wise gradient in the reversed phase mode. 

The instrumentation of HdC, including a pump, an injector, a column (set), a detector, and a recorder or computer, is very similar to size exclusion chromatography SEC). The essence of this technique is the column. There are two types of HdC columns open microcapillary tubes and a nonporous gel-packed column. This chapter emphasizes column technology and selection and the applications of this technique on the molecular weight analysis of macromolecules. 

HdC separation also occurs in the interstices of a packed column, although the configuration of channels is not as simple as a microcapillary tube. The... 

An improvement over 2D PAGE involves two-dimensional microcapillary HPLC coupled with mass spectrometry (see Fig. 24.24). In this technique, called MudPIT (multidimensional protein identification technology, developed by John Yates and co-workers at The Scripps Research Institute), a microcapillary HPLC column is used that has been packed first with a strong cation-exchange resin and then a reversed-phase (hydrophobic) material. The two packing materials used in sequence and with different resolving... 

Menet, H. Gareil, P. Rosset, R. Dead-volume free termination for packed columns in microcapillary liquid chromatography. Anal. Chem. 1984, 56, 2990. 

Recent developments in fused-silica capillary technology has led to such columns being used for HDC. The microcapillaries are normally 1-10 pm diameter with column volumes not exceeding 20 nl. The corresponding flow rates required for such systems are thus much lower than those of packed-column HDC. This technology has recently been commercialised with the... 

Tsuda, T. and Novotny, M., Packed microcapillary columns in high performance liquid chromatography, Anal. Chem., 50, 271, 1978. 

Cf-FAB in all its forms is a low flow-rate technique, i.e., 1-15 pl/min. Therefore, one should use either a microbore or packed microcapillary column, or a conventional colunm in combination with a post-column splitting device [47-48]. 

From a practical point of view, the discussion on flow-rate can be summarized as follows. In LC-APCI-MS, the typical flow-rate is 0.5-1.0 ml/min. For routine applications of LC-ESI-MS in many fields, extreme column miniaturization comes with great difficulties in sample handling and instrument operation. In these applications, LC-MS is best performed with a 2-mm-ID column, providing an optimum flow-rate of 200 pFmin, or alternatively with conventional 3-4.6-mm-ID columns in combination with a moderate split. In sample limited cases, further reduction of the column inner diameter must be considered. Packed microcapillary and nano-LC columns with micro-ESI and nano-ESI are rontinely applied inproteomics stndies (Ch. 17.5.2). 

The column inner diameter is determined by the amount of sample available and the LC-MS interface selected, tn general, flow-rates between 200 and 400 pl/min are considered optimum for (pneumatically-assisted) ESt. This explains the frequent use of 2-mm-ID columns, tn sample-limited analysis, e.g., in the analysis of mouse plasma samples, microbore (1 mm ID) or packed-microcapillary columns (320 pm ID) are applied at relatively low flow-rates [12-13]. For APCt, 4.6-mm-tD columns are preferred, operated at typically 1 ml/min. The LC system should provide symmetric peaks with a width that enables the acquisition of tO-20 data points for each compound in order to enable an accurate determination of the peak area. 

In early RPLC-MS studies on tryptic digests, typically 1-mm-ID columns were used. Further column miniaturization led to the introduction and use of packed microcapillary columns (typically 250-320 pm ID) and nano-LC columns (typically 75-150 pm ID). Nano-LC is nowadays routinely applied in proteomics studies. 

The volume that can be injected onto a packed microcapillary or nano-LC column is very limited, e.g., less than 0.1 pi for a 100-pm-lD column. This seriously compromises the achievable concentration detection limits, unless on-column preconcentration would be performed. However, for dilute sample solution, the injection volume is restricted by external peak broadening, and not by column loadability (typically -50-200 pg/g of porous packing material). Therefore, on-line SPE can be applied for sample preconcentration. 

Of the numerous methods developed to pack stationary porous medium/column in a microcapillary, two widely used approaches involve the packed beads column and the in situ polymerized monolithic column [5]. The preparation of the packed beads column includes the fabrication of retaining frits within a microcapillary as well as subsequent packing of micro-sized silica particles into the capdlary. To avoid the problems associated with frit... 

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