**3. Conclusion**

gel column (2.5 × 65 cm). The column was eluted with 0.1 M NaCl at a flow rate of 0.3 mL/min. The major fraction was collected and then freeze dried. All of these fractions were assayed for sugar content by the phenol–sulfuric acid method using glucose as

and inactivates enzymes, and refluxed by hot distilled water for 4 h at 90 °C. The aqueous extract was concentrated to 30% of the original volume under reduced pressure in a rotary evaporator, and proteins were removed with Sevag method. The obtained solution was precipitated with 40% ethanol. The supernatant was added by ethanol up to 60%, and kept at 4 °C overnight. The polysaccharide pellets were obtained by centrifugation at 4000 rpm for 15 min, and completely dissolved in appropriate volume of distilled water followed by intensive dialysis for 2 days against distilled water (cut-off *M* w 3500 Da). The retentate portion was then concentrated, and centrifuged to remove insoluble material. Finally the supernatant was lyophilized to give crude extract. The crude extract was dissolved in 0.2 mol/L tris (hydroxymethyl) aminomethane hydrochloride buffer solution, and filtered through a filter paper. The solution was passed through an anion-exchange chromatography column. After ion exchange chromatography other chromatographic methods was used for further

**Extraction:** The whole dried plant was soaked with 95% ethanol to remove the pigments, defats

**Stationary Phase:** DEAE-Sepharose fast flow anion-exchange chromatography column (10 × 300 mm) **Eluent:** The polysaccharides were eluted with Tris–HCl buffer solution, followed with gradient elution of 0.1–0.8 mol/L NaCl at a flow rate of 0.8 ml/min.

**Extraction:** After fermentation process, ammonium sulphate was added to the supernatant to give

in acetate buffer (20 mM - pH 6.0) and passed through a column

**Eluent:** Acetate buffer (20 mM - pH 6.0)equilibrated with the same buffer. The solution was

a final concentration of 80% saturation. The ammonium sulphate was added with constant stirring at 4ºC and the mixture stood overnight at 4ºC. The precipitated proteins were separated by centrifugation at 10000 rpm at 5ºC for 30 min. The separated proteins were then re-suspended in a minimum amount of distilled water and the solution dialyzed (using cellulose dialysis tubing) for 24 hrs against distilled water and concentrated by freeze-drying. The partially purified enzyme was dissolved

passed through the column at a flow rate of 1 mL.mim-1 with acetate buffer (20 mM pH 6.0), followed by a linear gradient from 0-1M NaCl in the acetate buffer. The eluted fractions were collected in an automated fraction collector (Pharmacia Biotech) and the absorbance of the fractions was measured at 280 nm. The major peak fractions were then assayed for tannase activity, and only the fractions possessing tannase

standard

separations.

**Analyte(s):** Water soluble polysaccharides [30].

**Source:** *Paecilomyces variotii*

**Detection:** UV Detector, 280 nm

**Detection:** UV Detector, 486 nm (phenol–sulfuric acid method)

**Stationary Phase:** Diethylaminoethyl (DEAE) Sepharose column (0.7 x 2.5 cm)

activity were pooled.

**Detection:** UV Detector, 490 nm **Analyte(s):** Polysaccharides [29].

**Source:** *Ornithogalum caudatum* Ait.

**Sample 12:**

54 Column Chromatography

**Sample 13**

Since the isolation of pharmacologically active substances which are responsible for the activity became possible at the beginning of the 19th century drug discovery researches have increased dramatically [33]. Therefore within the last decade there has also been increasing interest in the liquid chromatographic processes because of the growing pharmaceutical industry and needs from the pharmaceutical and specialty chemical industries for highly specific and efficient separationmethods.Severaldifferenttypesofliquidchromatographytechniquesareutilizedfor isolation of bioactive molecules from different sources [25]. Ion exchange chromatography is probablythemostpowerfulandclassictypeofliquidchromatography.Itisstillwidelyusedtoday for the analysis and separation of molecules which are differently charged or ionizable such as proteins, enzymes, peptides, amino acids, nucleic acids, carbohydrates, polysaccharides, lectins byitselforincombinationwithotherchromatographictechniques[34].Additionallyionexchange chromatographycanbeappliedforseparationandpurificationoforganicmoleculesfromnatural sourceswhichareprotonatedbasessuchasalkaloids,ordeprotonatedacidssuchasfattyacidsor amino acid derivatives [35]. Ion exchange chromatography has many advantages. This method is widely applicable to the analysis of a large number of molecules with high capacity. The technique is easily transferred to the manufacturing scales with low cost. High levels of purifica‐ tion of the desired molecule can be achieved by ion exchange step. Follow-up of the nonsolvent extractable natural products can be realized by this technique [17,35]. Consequently ion ex‐ change chromatography, which has been used in the separation of ionic molecules for more than half a century is still used as an useful and popular method for isolation of natural products in modern drug discovery and it continue to expand with development of new technologies.
