**Author details**

266 Chromatography – The Most Versatile Method of Chemical Analysis

where HPLC and other costly analytical systems are not available.

content of contaminated puffer extracts without any pretreatment.

TLC is a very commonly used technique in synthetic chemistry for identifying compounds, determining their purity. In TLC analysis, TTX is spotted onto a silica gel 60 precoated plate (Merck). The plate is developed in two different solvent systems of pyridine-ethyl acetate-AcOH -water (15:5:3:4) and 3-BuOH-AcOH-water (2:1:1) in a sealed chamber. The solvent rises by capillary action and an ascending chromatographic separation is obtained. The plate is then sprayed with 10% KOH followed by heating at 100℃ for 10 minutes. The toxin is visualized as a yellow fluorescent spot under UV light (365nm). In TLC analysis, the Rf values of TTX are around 0.71(0.65) and 0.50, respectively [10, 51]. It is also possible to detect TTX on the TLC plate using the Weber reagent that gives pink spot of the toxin. The detection limit is about 2 μg of TTX (10 MU). TLC is a useful technique in those laboratories

Electrophoresis is a relatively simple and rapid method with high resolution detection of polar compounds like TTX [10, 11, 51]. When 1 μl of TTX (10 MU, corresponding to 2 μg) is applied onto a 5 x 18 cm cellulose acetate membrane (Chemetron, Milano), the ion molecules of TTX move toward the cathode with a mobility (Rm) clearly smaller than that of authentic of STX. The analysis is performed for 30 minutes in an electrolytic buffer solution of 0.08 M Tris-HCl (pH8.7), under the influence of an applied electric field with a constant current of

Capillary isotachophoresis proved to be a very effective technique for the analyses of organic acids, carbohydrates, drugs and amino acids. It is a rapid and accurate detection technique for TTX [52]. It is performed using a cationic system, as TTX exists as cation under acidic and neutral conditions. Conditions for capillary isotachophoresis composed of a leading electrolyte of 5 mM potassium acetate (pH6.0), containing 0.2% Triton X-100 and 0.5 volume of dioxane, and a terminating electrolyte of 10mM β-alanine adjusted to pH 4.5 with acetic acid. When TTX is applied to isotachophoretic analyzer (Shimadzu IR-2A) equipped with a potential gradient 0.32, it is eventually monitored by the detector. The quantitative detection limit by this method is about 0.25μg of TTX. It was possible to quantify TTX

In an attempt to protect consumers from TTX-intoxication, the mouse bioassay has historically been the most universally applied tool to determine the toxicity level in monitoring programs. This bioassay, however, shows low precision and requires a continuous supply of mice of a specific size. These potential drawbacks and world-wide pressure to refrain from the unnecessary killing of live animals subsequently led scientists to

0.8 mA/cm width. The toxin is visualized in the same manner as described for TLC.

**8. Thin-layer chromatography** 

**9. Electrophoresis** 

**11. Conclusion** 

**10. Capillary isotachophoresis** 

Manabu Asakawa\* and Keisuke Miyazawa *Department of Bioresource Science and Technology, Department of Bioresource Science and Technology, Graduate School of Biosphere Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima, Hiroshima, Japan* 

Yasuo Shida *Clean Energy Research Center, University of Yamanashi, Takeda, Kofu, Yamanashi, Japan* 

Tamao Noguchi *Tokyo Healthcare University, Setagaya, Tokyo, Japan* 
