Solvent-gradient elution, micro

High-performetnce micro packed fused-silica and open-tubular glass capillary columns were prepared and applied to separations of complex mixtures. Solvent-gradient elution was quite useful for the separation of solutes with wide polarity. Instruments and some applications are described. 

As the vast majority of LC separations are carried out by means of gradient-elution RPLC, solvent-elimination RPLC-FUR interfaces suitable for the elimination of aqueous eluent contents are of considerable use. RPLC-FTTR systems based on TSP, PB and ultrasonic nebulisa-tion can handle relatively high flows of aqueous eluents (0.3-1 ml.min 1) and allow the use of conventional-size LC. However, due to diffuse spray characteristics and poor efficiency of analyte transfer to the substrate, their applicability is limited, with moderate (100 ng) to unfavourable (l-10pg) identification limits (mass injected). Better results (0.5-5 ng injected) are obtained with pneumatic and electrospray nebulisers, especially in combination with ZnSe substrates. Pneumatic LC-FI1R interfaces combine rapid solvent elimination with a relatively narrow spray. This allows deposition of analytes in narrow spots, so that FUR transmission microscopy achieves mass sensitivities in the low- or even sub-ng range. The flow-rates that can be handled directly by these systems are 2-50 pLmin-1, which means that micro- or narrow-bore LC (i.d. 0.2-1 mm) has to be applied. 

This nib system connected to a capillary was then placed at the outlet of a nanoLC column for on-line analysis after a separation step.26 The column was monitored from a nanoLC setup (LC Packings-Dionex, The Netherlands). An amount of 1 pL of sample was injected (Famos injection system, LC Packings-Dionex) onto a commercial nanocolumn (PepMap, 75 pm i.d.) and eluted in gradient conditions at a flow-rate of lOOnLmin 1 (Utlimate micro pump and accurate stream splitter, LC Packings-Dionex) using solvent mixtures based on AcCN and deionized water (solvent A AcCN H20 5 95 solvent B AcCN-H20 95 5) acidified with 0.1% TFA. Ionization HV was 1.5kV, as typically used for a standard emitter tip. The sample used for this test is a commercial cyctochrome c digest at a concentration of 800 nM. The spray was seen to be stable for several hours (several nanoLC runs) as expected with a standard emitter tip. The analysis of the solution eluted from the column was possible and the same as for routine conditions. 

In such cases, the corresponding critical mobile phase may be even precipitant for the polymer or may cause its full adsorption, if the polymer would be injected using critical mobile phase as a sample solvent [154]. It has been considered that in such cases the polymer can not exclude from the solvent peak, since eluent acts as a barrier, which is unsurpassed for the polymer. The term limiting conditions was coined for this phenomenon since the polymer elutes in a micro-gradient of... 

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