**6. Thin-layer chromatography**

TLC of bile acids has been used to differentiate feces of several mammal species, mainly carnivores such as the lesser grisson (*Galictis cuja*), guiña cat (*Leopardus guigna*), red fox (*Lycalopex culpaeus*), grey fox (*Lycalopex griseus*), pampas's fox (*Lycalopex gymnocercus*), puma (*Puma concolor*)*,* jaguar(*Panthera onca*), snow leopard(*Panthera pardus ciscaucasica*), pandas and different species of bears [6, 7, 32-37]. It had also been applied to a wide variety of other species: manatees [38], sperm whales [32], storks and herons [39]. Our study was the first one which reported the use of TLC to differentiate Xenarthra species, through their fecal bile acid patterns [8].

TLC is a type of planar chromatography in which the stationary phase is a solid adsorbent of fine particles, and the mobile phase is liquid. Aluminum or glass plates are used and they are covered with a fine and uniform layer of the stationary phase which is generally, silica. In some cases, it is chemically modified so as to provide suitable resolution conditions depending on the analyte. The correct choice of the mobile or stationary phase is essential to obtain an efficient separation of compounds to be analyzed [40-42]. Mobile phase generally consists of a solvent system composed of 2, 3 or 4 components which vary in their polarity and selectivity and they can include water, organic solvents and buffers [43, 44]. Silica gel is the most common adsorbent used in TLC as stationary phase, especially for the identification and separation of steroids such as bile acids in several biological samples, due to its adsorptive properties, great active surface and high pore size [45].

TLC separation mechanism consists of the differential migration of the mixture components through the chromatographic plate, dragged by the moving solvent; the distance travelled by each compound depends on its chemical structure and its affinity for both phases. Several mechanisms are involved in the separation process and predominant forces depend on the phases and solutes properties [43, 44].

Although various separation techniques are commonly applied for the determination of bile acids in biological samples, TLC offers practical advantages, mainly its simplicity, economical equipment needed, ease of operation, short analysis time and high efficiency in analysing simultaneously a large number of samples [43, 46]. It enables reliable separation and analysis of a wide variety of compounds from different types of biological samples. Moreover, this technique is versatile because it can be modified using different types of mobile and stationary phases and visualizing methods [43, 45].

The choice of the correct stationary and mobile phases is of great importance and it depends upon the type of sample and the objectives of the study. Thus, the greatest problem in using TLC in specific cases, such as bile acid analysis, is the selection of suitable mobile phases [47].
