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Shiseido's Semi-microcolumn LC
Osamu Shirota, Ph.D.
Vice Chief Researcher
Laboratory of Chromatographic Technologies
Shiseido Research Center
The prefix "micro-" frequently used in analytical chemistry generally has no direct relation to the size of an instrument. "Micro" means that the observed object is very small or that information of higher resolution about the object can be obtained. Microcolumn high performance liquid chromatography (hereafter referred to as "microcolumn LC", using a column with an inner diameter of less than 1 mm) discussed in papers by Scott1) et al. and Tsuda2)) et al. in the 1970s, is commonly called micro LC, reflecting the column size. The real value of this technology is that it can measure micro-sized objects. The benefits of reducing the inner diameter of the column from 4.6 mm frequently used for general high performance liquid chromatography (hereafter "conventional LC"), to a micrometer range are: 1) Improvement in absolute sensitivity of a concentration-sensitive detector 2) Reduction in required amount of mobile phase solvent 3) Easy connection to instruments such as a mass spectrometer from which solvents must be removed 4) Advantageous for microanalysis of a substance causing irreversible adsorption due to its small amount of packing materials
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. 1】 Advantages of semi-microcolumn LC (conceptual figure)
【Fig. 2】  Sensitivity comparison with UV detector
 【Fig. 2】 Sensitivity comparison with UV detector
【Fig. 3】  Comparison of semi-micro HPLC NANOSPACE with conventional HPLC of Company B
 【Fig. 3】 Comparison of semi-micro HPLC NANOSPACE with conventional HPLC of Company B
【Fig. 4】  Reproducibility of gradient elution by semi-microcolumn LC
 【Fig. 4】 Reproducibility of gradient elution by semi-microcolumn LC
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