Microcapillary columns

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

In the case of peptide separation by HPLC, separation modes are combined in series. This approach is called tandem LC. For instance, ion exchange associated with RP is used for peptide separation. Multidimensional protein identification technique (MudPIT) involving use of microcapillary columns (SCX cationic column and RP column) linked in series and eluted into MS is preferred for separation of complex peptide mixtures (Figure 5.4). 

LC ESI (8) mass spectra were recorded on a Finnigan TSQ700 instrument (San Jose, CA) in positive ion mode. A sample aliquot was injected into a fused silica microcapillary column with an inside diameter of 100 pm. The microcapillary was filled at the end with 10 cm of a C-18 reversed phase resin. PMP labeled oligosaccharides were eluted at ca. 1 pL/min directly into the electrospray ionization source with a 10 min gradient of acetic acid in H2O (0.5 %, v/v) to 80 % acetonitrile. Determination of experimental molecular weights was done with the deconvolution software provided by the manufacturer. 

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]. 

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. 

Willems et al. [20] proposed on-column desalting using 1,2-diaminocyclohexane-tetraacetic acid (CDTA), ammonium acetate, formic acid, or acetic acid prior to chromatographic separation on a 300-pm-ID microcapillary column. Desalting by the addition of EDTA was also reported [21],... 

Heeft, E.v.d. Hove, G.J.t. Herberts, C.A. Meiring, H.D. Els, C.A.C.M.v. Jong, A.P.J.M.d. A Microcapillary Column Switching HPLC-Electrospray Ionization MS System for the Direct Identification of Peptides Presented by Major Histocompatibility Complex Class I Molecules, Anal. Chem. 70, 3742-3751 (1998). 

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. 

Trace a methane, n-hexane, and n-pentane in a straight column (0.027 cm radius, 501 cm length) pressure drop 0.5 bar. Trace b same separation in the same column as under (a) but now tightly coiled 0.4 cm (coil radius) pressure drop 0.5 bar. Trace c methane, n-pentane, and n-hexane in a microcapillary column (0.0082 cm radius, 299 cm length) tightly coiled into a coil of 0.1 cm radius pressure drop 2 bar. 

Characteristics of microcapillary columns (i-d 100 mm) with graphitized capillary black (GCB) modified liquid phase [44]. 

Fig. 6-10 Chromatograms of high-boiling amines on microcapillary columns [44].

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