Effect of tubing on peak efficiency for core-shell HPLC columns

Selecting the appropriate tubing inner diameter (ID) when connecting a HPLC column can be crucial to maintain high peak efficiency while not achieving too high pressure. This is especially important when the efficiency is high and the total volume is small, as is the case with core-shell columns having narrow column inner diameter and shorter lengths. The data below highlights a study of how the average peak efficiency (i.e., N, number of theoretical plates) was influenced when the tubing ID was varied between the HPLC column and the UV detector flow cell during a study at the laboratories of ChromaNik in Japan.

Based on these results, Biotech recommends tubing with 0.12 mm (0.005”) ID or less when operating core-shell columns such as the ChromaNik SunShell series. With 0.12 mm ID tubing, the peak width did not change significantly, thus preserving most of the high efficiency created by the core-shell separation column. Wider tubing diameter (0.25 mm, 0.01”) reduced the theoretical plates by almost 70% for the first eluting peak and by more than 20% for the later eluting peaks, thus making it unsuitable for the present column. The gain in theoretical plates by reducing the tubing ID further (to 0.06 mm, 0.0025”) was only a few percent for all peaks, while the backpressure increased considerably (data not shown). This confirms that tubing with 0.12 mm ID was sufficient to preserve the high separation efficiency from the ChromaNik SunShell core-shell column with the tested system and column dimension.

Biotech distributes a wide range of tubing for different purposes including HPLC instrumentation. Contact Biotech for recommendations about suitable tubing material and diameter for your particular application.

Figure 1. Recorded chromatograms with core-shell column connected with tubing of different diameters

Table 1. Average theoretical plate at different tubing diameter

Inner diameter of tubing0.06 mm0.12 mm0.25 mm
Peak (1)792785246
Peak (2)779076523535
Peak (3)10704103457998
Peak (4)1011397727689

This study was performed on peaks created from a reversed-phase HPLC separation on a 2.1 mm ID column of 50 mm length (SunShell C18 by ChromaNik), using an eluent consisting of 60% acetonitrile in water, delivered at 0.3 mL/min at ambient temperature in a X-LC HPLC instrument (Jasco), set at a detector response time of 0.01 seconds. The tubing between the column and detector cell was in all cases 30 cm long and manufactured from PEEK (poly-ether-ether-ketone), and the presented data constitutes average of three determinations.

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