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What is a column switching method (automated pretreatment)?
One of the complexities in manual sample pretreatment is limited reproducibility. The column switching method resolves this problem. A pre-column for pretreatment (a column filled with packing material designed for direct injection of biological samples) is connected before an analytical column via a high-pressure six-way switching valve, and by re-routing the mobile phase, the target substance can be automatically separated from its matrix and concentrated in the pre-column.
【Fig. 5】Column Switching System (two-column system)
【Fig. 5】 Column Switching System (two-column system)
A typical example of using the method is shown in Fig. 5. After A biological sample such as serum is directly injected by an autosampler; proteins in the serum are removed in the pre-column (CAPCELL PAK MF), and at the same time, the target drug is concentrated at the top of the column. Then, the mobile phase is re-routed using the high-pressure six-way switching valve, and the target drug is sent to the analytical column for separation and detection. The high-pressure six-way switching valve returns to the initial state and the pre-column is washed and re-equibrated during the analysis and then prepared for the next analysis. Repetition of this operation allows continuous analysis with good reproducibility.
Sensitivity enhancement using the column switching method with a semi-microcolumn system
In the drug development, it has been a trend to design drugs that act at a very low concentration to avoid their side effect. For analysis of low-concentration samples, some kind of efforts, such as concentrating or chemical derivatization, are necessary. Most of the detectors currently used in HPLC are concentration-sensitive (ultraviolet and visible absorption, fluorescence, etc.). All of the chromatographic processes can be viewed as a sort of dilution process, where an injected minute amount of sample will be developed over a large space. The smaller the inner diameter of the column is, the less dilution takes place as analytes pass through the detector. The reduced dilution will result in the sensitivity enhancement in concentration -sensitive detectors (e.g. UV/VIS) and also, the reduction of back ground signals in LC-MS.
For this reason, a semi-microcolumn with a typical inner diameter of 2 mm is widely used as the analytical column in the column switching method. It was a challenging subject to combine the two ideas, obtaining the maximum sensitivity by injecting a large amount of sample solution (not wasting any of it), and using a narrow diameter column. A column switching method using a relatively large-diameter column as a pre-column for a sample injection and a narrow diameter for the final separation solved this matter. Figure 6 shows the system scheme.
【Fig. 6】High-sensitivity analysis of trace amounts of drugs in blood (three-column system)
【Fig. 6】 High-sensitivity analysis of trace amounts of drugs in blood (three-column system)
Figs. 7 and 8 show examples of high-sensitivity analysis8) of melatonin in plasma, and high-sensitivity analysis9)of antidepressant in serum.
【Fig. 8】Analysis of diazepam in human serum
【Fig. 8】 Analysis of diazepam in human serum
【Fig. 7】High-sensitivity analysis of melatonin in human plasma Provided by Dr. Kenji Hamase and Dr. Kiyoshi Zaitsu, Drug Research Institute, Department of Pharmacy, Kyushu University
【Fig. 7】 High-sensitivity analysis of melatonin in human plasma Provided by Dr. Kenji Hamase and Dr. Kiyoshi Zaitsu, Drug Research Institute, Department of Pharmacy, Kyushu University
Fig. 9 shows a chromatogram of cortisol and cortisone, adrenocortical hormones present in saliva10). These are both applicable at a clinical concentration, and are considered to be typical examples of direct injection of biological sample and continuous analysis with superior reproducibility. This system enabled measurement of the human circadian rhythm. The cortisol concentration in saliva is the highest before and after rising, and then it gradually decreases from the morning toward the afternoon, except after lunch (the cortisol concentration rises to promote the secretion of gastric acid after lunch). Cortisol is known to be influenced by emotions. A significant change in the cortisol concentration in saliva (n = 5, p < 0.02) was observed in our experiment before and after watching a movie (see Fig. 10).
【Fig. 9】Analysis of cortisol and cortisone in saliva
【Fig. 9】 Analysis of cortisol and cortisone in saliva
【Fig. 10】 in-vivo concentration influenced by emotions
  【Fig. 10】 in-vivo concentration influenced by emotions
Application of column switching system
This system is extremely effective for analyzing dilute solutions. Bisphenol A and nonylphenol, which are environmental hormones found in river water, could be analyzed11) with high sensitivity up to a level of 0.5 ng/mL for bisphenol A and 10 ng/mL for nonylphenol within the detection limit of S/N = 3 using MS as the detector, and with the semi-microcolumn LC.
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