The improvement in sensitivity in a microcolumn and semi-microcolumn LC (described later) is schematically shown in Fig. 1. Optical detectors often used in HPLC are concentration-sensitive, where a signal intensity is proportional to a concentration of the substance passing through a flow cell . In chromatography, a relatively small amount of a substance in the form of a sample solution is injected and then developed throughout a column during the separation (development) process. The smaller the column diameter where the dilution takes place, the lower the degree of dilution. As a result, the substance can reach the detector, keeping a relatively high concentration, thus improving the sensitivity (Fig. 2).
The microcolumn LC with such outstanding features is now often used in the field of proteomics, and its usage is expected to expand to many other fields in line with progress in:
- a system for sending solution at a stable flow rate of microliter per minute
- a packed column with excellent reproducibility, and
- an injection device with minimized band broadening
In the mid-1990s, Shiseido released a column for the semi-microcolumn LC (with inner diameter of column of 1.0 to 3.0 mm) and a dedicated instrument, NANOSPACE, ahead of other companies. The column and the instrument offer broadly the same advantages as the microcolumn LC, and are a practical technology featuring both usability and reliability equivalent to the conventional LC. Shiseido then succeeded in developing an instrument (with reduced dead volume) which can sufficiently reflect the separation efficiency of a column with an inner diameter of 1.0-1.5 mm, the efficiency equivalent to that of a column with a diameter of 4.6 mm, in final chromatograms.
The same sample was analyzed using a conventional LC and the NANOSPACE3) (Fig. 34)). A conventional LC generates some extent of band broadening in volume, which was not pronouncing with 4.6-mm column applications, but is now a serious problem with semi-microcolumn separations.
Besides the peak shape, another problem is injection accuracy within a range of injection amount (1 to 2 無) for a semi-microcolumn LC. When the injection accuracy with an injection amount of 0.1 無 was examined using NANOSPACE and a standard solution, the relative standard deviation of the area value was calculated as 0.5%5). Although there are slight fluctuations in injection accuracy in various actual cases, the data suggest that an injection accuracy (relative standard deviation of less than 1%) in the range of injection amount of a semi-microcolumn LC can be securely achieved with the current technical level.
Another problem is the reproducibility of retention time. To conduct stable analysis using the semi-microcolumn LC, a stable flow rate of approximately 60 無/min is required for a column having an inner diameter of 1 mm. Furthermore, for analysis under gradient conditions at a total flow rate of 60 無/min, each pump is required to have 1-無/min accuracy. The specifications of NANOSPACE are a maximum flow rate of the pump of 3 mL/min as opposed to a conventional flow rate of approximately 10 to 30 mL/min. Fig. 2 shows an example of the reproducibility of gradient analysis when using a column with an inner diameter of 1 mm with NANOSPACE at a total flow rate of 60 無/min 7).
【Fig. 1】 Advantages of semi-microcolumn LC (conceptual figure)
【Fig. 2】 Sensitivity comparison with UV detector
【Fig. 3】 Comparison of semi-micro HPLC NANOSPACE with conventional HPLC of Company B
【Fig. 4】 Reproducibility of gradient elution by semi-microcolumn LC