Fused silica column micro HPLC

Micronization in the field of column hardware and packing materials led to the development of microbore and short columns the latter allow separation times sometimes to be shortened by a factor of 10 (83, 385). The advent of microbore columns created a whole new methodology in HPLC with detection limits lowered into the femto-mole range (224, 260, 291). Special micro-metering pumps give pulseless flow rates down to pl/min, and allow direct interfacing of the LC apparatus to a mass or infrared spectrometer. They also open the field of fused silica columns for HPLC use, and it might be not too optimistic to expect theoretical plate numbers of >500000 per LC column for the near future. 

Fig. 22.1 Separation of bile acids using micro-HPLC. (Reproduced with permission from D. Ishii, S. Murata and T. Takeuchi, J. Chromatogr., 282, 569 (1983).) Conditions sample, 11 nl of solution containing 20 ng each of the acids guard column, 5cm x 0.2 mm i.d. fused silica column, 20cm x 0.26 mm fused silica stationary phase, silica ODS CS-01, 5 im mobile phase, 60 mM phosphate buffer containing NAD and acetonitrile, gradient with increasing acetonitrile content, 2.1 pi min - detector, derivatization with immobilized enzyme, then fluorescence, 365/470 nm. For abbreviations, see text.

Additionally there are fused-silica columns with i.d. ca. 0.2 mm. Representing all other small-bore techniques, Fig. 22.1 illustrates the performance of micro-HPLC using this kind of column. Fifteen bile acids which are vital to any assessment of possible liver disease were identified in body fluids. The total solvent consumption of 210 pi, the ability to mix a gradient in this small volume and reproduce it and the refined reaction detector already described in Section 19.9 are special features of this particular system. The following abbreviations are used UDC = ursodeoxycholic acid C = cholic acid CDC = chenodeoxycholic acid DC = deoxycholic acid LC = lithocholic acid G (prefix) = glycine conjugate T (prefix) = faurine conjugate. 

Insert the exit column of the micro-capillary HPLC column into the 26-gauge stainless-steel electrospray needle until the fused silica capillary exits the... 

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