- The natural products obtained from plants either as pure compounds or standardized extract provides a unique opportunity for the development of new drugs due to their complex nature.
- The increasing needs for the isolation and purification of bioactive compounds from crude plant extracts leads to the development and advancement in separation techniques and instrumentation.
- Plant extract usually occurs in a combination of different classes of compounds or phyto-chemicals with different solubility’s.
- The compounds which are isolated from different natural plant sources by using various solvent systems and chromatographic techniques is very important, but obtaining that compounds in a pure state can be a difficult and time consuming step in natural products research.
- Separation of each desired class of compound from crude plant extract is a difficult task for their identification and purification. Therefore it is essential to have efficient chemical and biological screening systems for rapid investigations of the plant extracts. Practically most of them have to be purified by the combination of several chromatographic techniques.
- A number of sophisticated separation techniques have been used in the recent past such as Thin Layer chromatography (TLC), Column chromatography, Flash chromatography and High Performance Liquid Chromatography (HPLC) to obtain the pure compound from mixture of compounds in the plant extract.
- The purification strategy always comprises a multistep procedure including extraction, pre-purification and then one or several chromatographic steps. Thus the fresh or dry sample will be cut up or ground to a powder before-
1. extraction with organic solvents (perhaps in a sequence of increasing polarity)
2. water or supercritical CO2 solvent partitions to remove less polar or more polar compound
3. purification by TLC and HPLC
- By employing purification techniques one can say that the purification process is over and the compounds achieved is absolutely pure. But it doesn’t mean that the compound obtained is free of other chemicals instead it can be quoted that the amount of any impurity present doesn’t exceed the acceptance level.
- In general the main objective during isolation of natural polyphenols from complex plant extracts is to obtain compounds in sufficient quantities and with high purities for structural elucidation, for further use in bioactivity studies.
- Multistep purification procedures may sometimes result in drastic sample or activity loss during the isolation of target compounds because of sample degradation, irreversible adsorption on solid support or dilution.
- The purification stage includes removing the interfering compounds from the crude extract with partitionable solvents and using open column chromatography or an adsorption- desorption process.
- Sephadex LH-20, Polyamide, Amverlite, Solid phase extraction cartridges and styrene-divinyl benzene (XAD-4, XAD-16, EXA-90, EXA-118, SP-70), acrylic resins (XAD-7, EXA-31) are examples of regularly applied materials to purify phenolics from crude sample extracts.
- SPE is widely used for pre-concentration and clean up of analytical samples and purification of medicinal plant extracts. This method offers a variety of solvents based mainly on silica. Liquid- liquid extraction is based on partition between solvents is the most commonly preparative purification method where a large proportion of extraneous constituents need to be removed.
TECHNIQUES USED FOR PURIFICATION
Flash chromatography is mainly used for rapid fractionation of crude extracts or coarsely purified extractions. This technique can be used with silica column or reverse phase-18 columns for final purification of crude extracts. So, a purification method should be employed to purify individual isolated compound from the crude plant extract.
Sephadex LH-20 is a liquid chromatography medium, which is beaded, cross linked dextran, and has been hydroxypropylated for the matrix to be both hydrophilic and lipophilic in nature. Sephadex LH-20 is useful for clean up and had also been used for the separation of compounds such as condensed tannins, catechin, and fractionation of proanthocyanidins and also for other phenolic compounds. Further Sephadex G-25 and G-50 which are generally used in gel filtration chromatography have also been used for the purification of phenolic compounds and fractionation as well.
HPLC is routinely used in phytochemical extractions to optimize the experimental conditions and to check the purity of the final isolated compound. The combination of columns used and solvent system has been widely employed and useful for the study of polyphenolic compounds. Preparative HPLC is a purification process which aims at isolation of a pure compound from a mixture. During the separation of the compounds by analytical HPLC, we consider important parameters such as resolution, sensitivity and time while during preparative HPLC degree of the solute purity is considered most. However in most of the cases semi-preparative and preparative HPLC with higher peak resolution power had to be applied for final purification.
- Tea, Maize and Petunia are known to be rich in polyphenolic compounds. Therefore, the detailed protocol for purifying the compounds from these plant systems has been provided in procedure.
- A basic method for purifying Catechins from Tea plant is shown in Fig 1
- Derivatization is the process of chemically modifying a compound to a new product with same chemical structure called derivative that are suitable for the analysis using GC or HPLC.
- The main aim of this method is to modify the functional groups of the reacting compound so that it can be easily detectable and systematically analyzed.
- The process allows enhancing the chromatographic qualities by fixing the chemical and physical properties of the analyte to get better volatility and more importantly lower polarity of the derivatized adduct.
- The use of derivatization process during sample extraction can be simply and friendly to automated throughput for sample analysis. Further the derivatization step can be carried out before in combination with or after sample pre-treatment and can be used for different analytes.
- During the analysis no interference of excess reagents should be present and they should be removed easily. Further care should be taken that no additional impurities are present during analysis and the reaction must be quantitative.
- The selection of appropriate derivatization reaction depends on-
1. Nature (polar, non-polar) and stability of the compounds to be extracted.
2. Nature of the solvent use.
3. Reaction conditions maintained during extraction.
Advantages of Derivatization
2. Eliminates the presence of polar OH, NH and SH groups
3. Increases stability and detectability
4.Reduces the adsorption of polar samples on active surfaces of column walls.
- Derivatization reactions are needed in GC and HPLC. The aim of derivatization reactions for use with GC analysis is to improve peak symmetry, resolution, selectivity and sensitivity of the analytes and their thermal stabilities.
- Usually analytes are converted by derivatization either into volatile compounds able to be analyzed by GC with sensitive detectors. The derivatization technique generally consists of substitution of the active H2 atom in –NH, - COOH, -OH or –SH using alkylation, acylation or silylation reactions.
a) Silylation: Silylation is the most common derivatization technique used in the conversion of the analyte to its trimethylsilyl derivative.
Examples of silylation for functional groups are listed below:
- It is the most prevalent derivatization process which volatizes the sample readily.
- They react with water and alcohol and produces silyl derivatives which are volatile. They replace active hydrogens with TMS (trimethyl silyl groups).
- Nearly all functional groups which present a problem in gas chromatographic separation (hydroxyl, carboxylic acid, amine, thiol, phosphate) can be derivatized by silylation reagents.
- The derivatives of the silylation reactions are generally less polar, more volatile and more thermally stable.
- The introduction of a silyl group(s) can also serve to enhance mass spectrometric properties of derivatives, by producing either more favorable diagnostic fragmentation patterns of use in structure investigations, or characteristic ions of use in trace analyses in other related techniques.
b) Alkylation: This method replaces active hydrogen with alkyl groups thereby reducing molecular polarity. They make esters, ethers, alkyl amides and alkylamines. Reagents commonly used are acidified methanol, boron trichloride and boron trifluoride in methanol or butanol.
c) Acylation: It reduces the polarity of the reactive functional groups such as amino, hydroxyl and thiol groups. The acylating reagents targets highly polar, multifunctional compounds such as carbohydrates and amino acids. Commonly used reagents are – acetic anhydride, TFA etc.
d) Chiral Derivatization: The reagents used in this method target one specific functional group and produce individual diastereomers of each of the enantiomers. Enantiomers can be separated in chromatography by allowing separation on an optically active stationary phase.
- Pre-column derivatization involves the reaction of the analyte of interest with a reagent and performs sample clean up before chromatographic separation and detection are carried out.
- When derivatization of analytes is performed prior to loading the sample into GC or LC column, this mode is called pre-column derivatization. The post- extraction derivatization type is the most common used. Once the extract has been obtained, it’s derivatized before chromatographic analysis.
- Derivatization mode can be considered:-
1. Off-line derivatization using manual direct addition of the derivatization reagent to sample extract in a fume hood
2. On-line derivatization, as an attractive alternative to manual derivatization because it avoids preparative steps accelerates reaction rates and reduces evaporation losses.
- Post column derivatization is carried out after the separation of components. For post column mode, derivatization takes place only after the analytes are separated in GC or LC columns they are then converted to a form more amenable to detection.
- Derivatization is affected by some drawbacks such as complexity, loss and contamination of analytes and time consuming. Sometimes the derivatization is carried out after chromatographic separation and before the final detection in order to increase the selectivity and sensitivity of determinations.
Fig 3: Derivatization Strategies in Sampling/Extration Techniques