**Author details**

54 Chromatography – The Most Versatile Method of Chemical Analysis

to correctly identify compounds.

resolved by TLC, in Xenarthra feces.

bile acids.

acids not resolved by TLC.

their fecal bile acid patterns.

diseases such as colon cancer [46, 96, 97].

conservation strategies in the long term.

Diet of animals, and particularly in the case of Xenarthra species, can have an effect on the detectability of some bile acids. In our study, in feces with high contents of vegetal material, pigments appeared as colored bands in the TLC chromatographic plates and unidentified peaks in HPLC. However, they did not interfere with bile acid detection and we were able

With the application of HPLC we corroborate TLC results, not only confirming the presence of the compounds found by TLC but also identifying new compounds which were not

Bile acid detection constituted an essential step in both techniques. The choice of the detection system is governed mainly by bile acid structures. In TLC, spraying the plates with the revealing reagent is the critical step, in which it is important the ability and experience of the operator. Spraying must be uniform to avoid uncolored areas that later interfere with the correct identification of spots. In HPLC, different classes of bile acids have different absorption capabilities to UV light; this is a critical factor especially for free bile acids, showing limitations for their detection. However, in our study we could detect free

In relation to the components of the chromatographic system, the choice of the mobile and stationary phase is very important, as they depend on the type of sample and the objectives of the study. In TLC we used silica gel as stationary phase, which is the most used phase for lipid analysis [47]. The solvent system composed of toluene:acetic acid:water (5:5:1.5 v/v) proposed by [5] was the most adequate for our aims, achieving a good resolution of the great majority of the compounds, except for some tauro-conjugated bile acids such as TDCA and TLCA. In HPLC we used a reversed-phase column with a ODS stationary phase, and the mobile phase composed of ammonium carbonate and acetonitrile; under these conditions we were able to separate almost all bile acids, including tauro-conjugated bile

Thus, our work demonstrates that both techniques, TLC and HPLC, are complementary, and they should be used together to take advantage of the positive aspects of each one.

The analysis of fecal bile acids is a useful tool not only for ecological and biological studies, but also is of great clinical interest. These determinations can be helpful in the evaluation of intestinal, biliary and hepatic functions, and in the diagnosis and treatment of some related

Chromatographic techniques have been widely used to identify bile acids in different biological materials, mainly in gallbladder bile of mammal species. However, our study is the first one to report the use of TLC and HPLC to differentiate Xenarthra species through

Finally, our work established the validity of fecal bile acids to differentiate close related species, being useful to assess habitat use and to study food habits of sympatric species. The determination of these species-specific patterns offers robust data for the elaboration of Emma B. Casanave *Cátedra de Fisiología Animal, Dpto. de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, Bahía Blanca, Buenos Aires, Argentina Consejo Nacional de Investigaciones Científicas y Técnicas, (CONICET) Argentina* 

M. Soledad Araujo *Cátedra de Fisiología Animal, Dpto. de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, Bahía Blanca, Buenos Aires, Argentina* 

Gustavo H. López *Cátedra de Bioanalítica II, Dpto. de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, Bahía Blanca, Buenos Aires, Argentina* 
