**4. Conclusions**

128 Chromatography and Its Applications

alkaloidal extract analysis, with some minor modifications, which allowed a good separation of it majority compounds (Figure 9B). The main modification made was the addition of octane sulfonic acid to mobile phase. The presence of this component allowed that the separation to be mediated by more types of interaction, because it acts by an ion

Fig. 9. A: high performance liquid chromatography (HPLC) chromatogram of standards mixture isolated from *Aspidosperma ramiflorum*; B: HPLC chromatogram of alkaloidal extract. Peaks – 1: internal standard (tryptophol); 2: 10-methoxy-geissoschizol; 3: ramiflorine A; 4:

For HPLC analysis, the crude extract of *A. ramiflorum* (Ferreira et al., 2007) was dissolved in CH2Cl2:MeOH (80:20 v/v) and 10 μL were injected into a Waters μ-Bondapak RP-18 (reverse phase, 4.6 mm x 250 mm) column at 40°C. Solvent A was 100 mmol/L ammonium formate in 0.12% octane sulfonic acid (v/v), formic acid and acetonitrile (88:4:8, v/v), while solvent B

**3.4.1 High Performance Liquid Chromatography analysis** 

ramiflorine B.

pair formation mechanism, increasing the resolution and efficiency of analysis.

The development of the TLC solvent system for *Aspidosperma ramiflorum* alkaloids at the beginning of this article clearly shows that TLC and PTLC as a routine and classical method appreciated in alkaloid separation techniques, can still be optimized with introduction of new methodologies as desorption electrospray ionization (DESI) which can permit coupled TLC with mass spectrometry (Jackson et al., 2009). This is not necessarily true for alkaloid determination by GC, because decomposition can occur due to the high temperatures applied and however widespread application of this technique is limited by volatility of more polar alkaloids. HPLC is a classical method for alkaloids separations and allowed a good separation of majority compounds in *A. ramiflorum* alkaloids but can still be optimized. However, the pre-purification of crude alkaloid extract will be a crucial step for all analytical procedure described. The applicability of different chromatographic techniques for the separation and identification of crude mixtures of bioactive *A. ramiflorum* indole alkaloids has thus been demonstrated.
