On-column focusing

If the sample is dissolved in a solvent that is weaker than the mobile phase, then the sample can be enriched on the head of the column without penetrating into the column bed. This compression effect is particularly important for capillary LC applications, since it permits significantly larger injection volumes. A substantial increase in sensitivity results, and conventional autosamplers with 20-jnl loops can be used.16 However, sample solubility and recovery, miscibility of the sample with the mobile phase, and the maximum tolerable loss in column efficiency and resolution must all be assessed experimentally for optimum on-column focusing.16... 

P Molander,T. E. Gundersen, C. Haas,T. Greibrokk, R. Blomhoff, and E. Lundanes, Determination of retenoids by packed-capillary chromatography with large-volume on-column focusing and temperature optimization, J. Chromatogr. A 847 (1999), 59-68. 

Figure 5.2. Schematic diagram of a standard liquid chromatograph modified for use with a packed capillary column. Typical pump settings are 300-400 p,l / min with flow splitting of 1 2000 to give a column flow of 150-200 nl / min. The microinjection valve has a 40 nl internal loop and an additional T.IO split creates an injection volume of 2-3 nl (larger volumes can be injected by on-column focusing with gradient elution separations). The detector uses a U- or Z-shaped flow cell with a 3 nl volume and 8 mm path length. Fused-silica capillary tubing with an internal diameter < 20 pm and zero-dead-volume connectors are used for column connections. (From ref [8j. American Chemical Society).

The sample is loaded at atmospheric pressure into an external or internal loop, or groove in the valve core and introduced into the mobile phase stream by a short rotation of the valve. The volume of sample injected is normally varied by changing the volume of the sample loop or by partially filling a sample loop with a fraction of its nominal volume. External sample loops have volumes from about 5 p.1 up to about 5 ml, although typical injection volumes for conventional diameter columns are 10-50 xl. Injections from 1 p,l to about 40 nl require micro-injection valves equipped with replaceable internal loops [7,32-34]. Injection volumes less than about 40 nl are performed by positioning a split vent between the injector and the column. Typical injection volumes that preserve column efficiency for packed columns of different internal diameters are summarized in Table 5.1. For packed capillary columns with internal diameters < 0.2 mm direct injection will usually require the use of a split vent to minimize volume overload unless on-column focusing is possible. Injection volumes about 5 times larger than those indicated in Table 5.1 are sometimes used to increase sample detectability but with some decrease in the column separation power. 

The principal valve injection techniques are complete fill or partial fill with respect to the loop volume, timed injection and on-column focusing. The volume injected for the complete till technique is determined by the size of the loop, which has been overfilled to completely displace the mobile phase. As a sample is introduced into the loop, it displaces the mobile phase ahead of it out of the loop. As this process proceeds, the... 

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. 

Stass, H. DaUioff, A. Determination of BAY 12-8039, a new 8-methoxyquinolone, in human body fluids by high-performance Ucjuid cdiromatography with fluorescence detection using on-column focusing, J.Chromatogr.B, 1997, 702, 163-174. 

Capillary LC has become more widely accepted as commercial equipment to accommodate the low flow rates of 1-5 /ul/min, sample injection sizes of 60 nl, and the capillary detector flow cells has become available. Columns generally 100-350 fxm ID x 25 cm in length packed with 3 or 5 fxm particles are also commercially available. The main advantages of capillary LC are the small sample size and improved sensitivity as compared to analytical or microbore HPLC. Using the equation described in Section I.D, a 320-/u.m capillary could theoretically provide 200 times improvement in sensitivity as compared to a standard 4.6-mm ID column assuming the same sample size could be injected. However, for large volume injections with capillary LC (see Fig. 12), an on-column focusing... 

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