f^ l^ltt^^p^^* V^ COLUMN CHROMATOGRAPHY, HPLC AND MASS SPECTRAL ANALYSIS OF SOME FRACTIONS OF Lasianthus lucldus Biume 8.1 Column Chromatography 8.2 HPLC 8.3 Preparatory TLC 8.4 Mass Spectral Analysis 8.5 Results and Discussion 8.6 Conclusion References 166
8.1 COLUMN CHROMATOGRAPHY The procedure of column chromatography was developed during the early stages of chromatography discovery. From that time the principle of this type of chromatography was formulated and followed till date in the most advanced modes of chromatography. Several other types of chromatographic techniques have been developed with column chromatography as a module and with slight changes. Column chromatographic technique is basically a type of adsorption chromatography. Here the extent of adsorption to stationary phase determines the extension of separation of its various components. In a vertical column made of glass or metal the stationary phase is packed. The stationary phase should be a solid material (generally silica is preferred) (Laurence and Christopher, 1989). Based on the amount of various extracts obtained as well as their separation on TLC plates, it was decided to try, to separate the individual constituents of only three of the extracts, these are petrol ether (PE), acetone (AC) and ethyl acetate (EA) extracts of leaves of Lasianthus lucidus Blume. 8.1.1 Materials and methods Isolation of the different components from leaf of L. lucidus was carried out by column chromatography, where different combination of solvents like petroleum ether, ethyl acetate, acetone, methanol, etc were used as mobile phase and silica gel ( 60-120 mesh) as stationary phase to yield different fractions. 8.1.1.1 Procedure for Gravity Column Chromatography A piece of glass wool was placed in the bottom of the column and gently tamped the glass wool down with a glass rod. The column was attached to a ring stand and it was made sure that the column was securely fastened in a vertical position. A pinch clamp was attached to the bottom of the column and closed the clamp. The column was filled about half-way with a non-polar solvent, such as petroleum ether. Before preparing the slurry, the adsorbent i.e. silica gel was activated in an oven at 300 C for an hour. loogm of silica gel 167
was weighted into a beaker. 500 ml of petrol ether was placed in a 1 litre Erlenmeyer flask and slowly added the silica gel, a little at a time, while swirling. A glass rod was used to mix the slurry and then quickly poured the slurry onto the column. Then a 500 ml Erlenmeyer flask was placed under the column, the pinch clamp was opened and the liquid was allowed to drain into it. This method was repeated until all the silica gel was added. The solvent collected in the Erlenmeyer flask can be reused to add more silica gel to the column. When finished packing, the excess solvent was drained until it just reaches the top level of the silica. The pinch clamp was closed. 8.1.1.2 Loading and eluting gravity chromatography columns The sample to be analysed was dissolved in a very small amount of solvent ether) and added to the top of the column. The pinch clamp was opened and the solvent was allowed to drain just to the top of the column. A small amount of the eluting solvent was added and allowed to drain in until the mixture was a little way into the adsorbent and then the column was filled to the top with eluting solvent. The column was then ready to run. Solvent was continuously added on the top and the fractions were collected at the bottom in 100 ml conical flasks until the compounds elute at the bottom. Gradually the eluting solvent was changed to a more polar solvent (ethyl acetate, acetone or methanol) during the eluting process. The solvent level should not drop below the top of the adsorbent. The process was discontinued when the compound(s) desired was (were) off the column. The fractions collected were concentrated by placing in a water bath and spotted on thin layer chromatographic plates to see the extent of separation of various mixtures of compounds (http://www.orgchcm.colorado.edu/labs/3361.html). 8.1.2 Results Fractions collected after performing column chromatography were further subjected to TLC analysis to see the number of visible spots in a fraction and the similarities in the fractions. In case of PE extract, fraction 1 to fraction 5 did not show any trace of compounds, so these were rejected. 168
Fraction 6 to fraction 10 showed the same Rf value, these were combined and named as AlKDC. Similarly, A2KDC is the combination of fraction 11 to 13, A3KDC from fraction 14 and 15 and lastly A4KDC from fraction 16 to 19. Other fractions were not considered due to maximum number of impurities. Similarly, in case of EA extract, fraction 1 to 5 had no trace of compounds as the solvent completely evaporated, EAIKDC was formed from fraction 6 to 9, EA2KDC from fraction 10 to 12 and EA3KDC from fraction 13 to 15 and others were rejected. In case of AC extract only the first fraction ACKl was selected and all other fractions were rejected as the acetone fractions had traces of chlorophyll and the samples appeared green in colour. The Rf values obtained after TLC analysis and other properties of the fractions are presented in table 8.1. Table 8.1:- The Retention factor and other chemical properties of various fractions of Lasianthus lucidus extracts obtained from column chromatography Leaf Fractions Mobile Appearance Colour Rf values Extract obtained solvent (9:1; Petroleum ether : ethyl PE AlKDC 98:2 0.42, 0.94 A2KDC 95:5 0.42, 0.76 0.95 A3KDC 93:7 0.35, 0.63, 0.95 169
A4KDC 9:1 ish 0.24, 0.43, green 0.57, 0.95 EA EAIKDC 97:3 0.23, 0.47 EA2KDC 95:5 0.49,0.95 EA3KDC 9:1 0.48, 0.64, 0.95 AC ACKl 95:5 0.37, 0.56, 0.68, 0.78, 0.97 These samples were placed in water bath to evaporate the trace of solvents completely and stored in eppendorf tube for carrying out preparatory thin layer chromatography. 170
Figure 8.1:- Column chromatography of (Petroleum Ether) PE extract of Lasianthus lucidus leaf. Figure 8.2:- Photograph of the fractions collected by eluting column chromatography, (a) Dilute fractions collected in conical flask; (b) Fractions in concentrated form. Figure 8.3:- Photograph showing TLC Plate of various fractions obtained from PE extract. 171
8.2 PREPARATORY TLC The column fractions were further separated using preparatory thin layer chromatographic technique. Using this technique only separation of AlKDC and EA2KDC were possible. The method adopted to run preparatory TLC was similar to that of general TLC and the method was discussed earlier in chapter 4. The solvents used here as developing system were petroleum ether and ethyl acetate at the ratio 9:1. Uniform spots of the same fraction were given over the silica gel plate in series by using capillary tube. After developing the chromatogram, the single spots obtained were collected in a small glass vessel and dipped in methanol. Now silica gel was separated from the sample, methanol was evaporated and the sample was stored for further analysis. It was not possible to separate other fractions because of lack of proper isolation methods. Various developing systems were prepared using different ratios of solvents to separate those fractions, but no signal spot was obtained as such. After performing PTLC of AlKDC and EA2KDC, the samples were stored for HPLC, mass spectral analysis and bioactivity assay. 8.3 HPLC High Performance Liquid Chromatography (HPLC) is considered an instrumental technique of analytical chemistry. HPLC has many uses including medical, legal, research and manufacturing. It is used for identifying, quantifying or purifying individual components from the mixture. HPLC is basically a highly revolutionised form of column chromatography. Here, the solvent is forced under high pressure of up to 400 atmospheres instead of running the solvent through the column under gravity. That makes it much faster (Hung and Parcher, 1988). HPLC was performed on the column fractions AlKDC and EA2KDC after their separation using preparatory TLC. The purpose for performing HPLC is to check the purity of the samples under study. Number of peaks present in a HPLC chromatogram indicates the number of different phytochcmicals present in the fraction. 172
HPLC of the two fractions AlKDC and EA2KDC were performed at Central Instrumental Laboratory (CIL), Assam University, Silchar. The samples were diluted with methanol and 10 il of the diluted sample was injected to the HPLC and the separation was realised into a RP 18 reverse phase HPLC column (250 x 4.6 mm) (stationary phase) with low pressure gradient. The flow rate of the solvent used (methanol) in HPLC was 1 ml/ minute. The results of HPLC spectral analysis of the fractions showed several peaks at the retention time between 0-20 minutes. After 20 minutes, compound within the range of polarity have already been flushed out which indicates the polarity of the solvents have increased by increasing the percentage of the methanol used to 30, 50 and 70% at the 5, 10 and 15 retention time respectively. After 70% methanol were used, there were no peaks appeared any more to indicate that all compounds were being flushed out from the column. The HPLC chromatogram of AlKDC and EA2KDC were presented in figure 8.4 and 8.5 respectively. The peak spectrum of AlKDC showed the highest peak spectrum at 1.57 minutes. The second highest peak spectrum was observed at 1.23 minute. While at 2 to 3, 5.32, 7.81 and 19.19 minutes showed low peak spectrum. The highest peak spectrum in case of EA2KDC was observed at 6.11 minutes. No moderate peak spectrum was found in this sample, but some small peak spectrum was observed at 1 to 4, 5 to 6.9, 8.83, 10.14, 12.39, 13.65 and 16.79 minutes. 173
si r'&^s, -s.tfc-y^sisc'''' *^'*^^''?'^'*^*"' r:i Figure 8.4:- High Performance Liquid Chromatogram of Column Fraction (AlKDC). m^i:. Of = \ - 1 i L Figure 8.5:- High Performance Liquid Chromatogram of Column Fraction (EA2KDC). 174
8.4 MASS SPECTRAL ANALYSIS Mass spectral analysis of AlKDC and EA2KDC were carried out to find out their molecular masses. For mass spectral analysis the samples were sent to Sophisticated Analytical Instrumentation Facility (SAIF), North Eastern Hill University, Shillong. The mass spectral data of AlKDC and EA2KDC are represented in figure 8.6 and 8.7 respectively. The molecular weights of AlKDC and EA2KDC were found to be 803.63 and 990.26 respectively. The very high molecular weights of the isolated materials are indicative of possible macromolecular structure or very long chain fatty acid system. ;A2 KDC, KAJYAYAN! DUTTA CHOWD^)URV,S>t::Pi- OF Li^E SCif-NCt ANDBIOiNFO^MATJC. AU?1 Af»-SOU 17-04.14 BCB.KOLKATA, v,'t.> Mem KXJ J.JiO iso. K»p,. 360 400 450 WO &S0 SCO «ffip, 700 rep. i^ 8S0 90O. fiso 1iX».-. Figure 8.6:- Mass Spectra of Column Fraction (AlKDC) 175
Figure 8.7:- Mass Spectra of Column Fraction (EA2KDC) 8.5 RESULTS AND DISCUSSION Column chromatography, high performance liquid chromatography (HPLC) and mass spectral analysis were performed as part of phytochemical screening of Lasianthus lucidus Blume. Thin layer chromatographic (TLC) profiling was presented in chapter 4, has provided an idea about the mode of separation of various phytochemicals on silica gel plates. The spots produced by various phytochemicals were detected and their Rf values were calculated. Components showing high Rf value in less polar solvent system have low polarity and with less Rf value have high polarity. Now, on the basis of TLC profiling, various crude extracts of the plant leaves were needed to be separated using column chromatographic techniques. The solvent systems in which the extracts exhibited distinct separation were selected as mobile phase solvent in column chromatography. Column chromatography were carried out on PE, EA and AC extracts, ME extract was discarded because to separate its high polar phytoconstituents more developed solvent systems were required. From PE extract four fractions namely AlKDC, A2KDC, A3KDC and A4KDC were separated. From EA three 176
fractions (EAIKDC, EA2KDC and EA3KDC) and from AC one fraction (ACKl) were separated. All the fractions were semi-solid in appearance, soluble in organic solvents like petrol ether, ethyl acetate, methanol and chloroform and shows slightly alkaline properties. The fractions, AlKDC and EA2KDC were further separated using preparatory TLC and the distinct spots with low Rf value (0.42 in case of AlKDC and 0.49 in case of EA2KDC) were collected. Now these collected samples were screened for HPLC analysis to check the purity of the samples. In the HPLC chromatogram of AlKDC, highest peak spectrum was obtained at 1.57 minutes while in EA2KDC, it was observed at 6.11 minutes. In case of both the fractions, along with a single high peak, 4-5 small peaks were observed which indicates along with one compound in maximum quantity, trace of other materials were also present. Mass spectral analysis of AlKDC and EA2KDC were performed to find out their respective molecular masses. The molecular weights of AlKDC and EA2KDC were found to be 803.63 and 990.26 respectively. Mass spectral data of both the samples only provides the information that the very high molecular weights of the isolated materials are indicative of the possible presence of macromolecular structure or very long chain fatty acid system. 8.6 CONCLUSION In the present study, on the basis of TLC profiling of various leaf extracts of Lasianthus lucidus Blume, column chromatographic separation of the plant extracts, their HPLC and mass spectral analysis were carried out. The future prospect of this study lies in structural elucidation of natural compound(s) from these fractions with active biological principles. REFERENCES Hung, L. B., Parcher, J. F., Shores, J. C. and Ward, E. H. 1988. Theoretical and experimental foundation for surface-coverage programming in gas-solid chromatography with an adsorbable carrier gas. Journal of American Chemical Society. 110(11): 1090. 177
Laurence, M.H. and Christopher, J.M., 1989. Experimental organic chemistry: Principles and Practice. 2" edition. Blackwell Publication, pp; 180-185. http://www.orgchem.colorado.edu/labs/3361.html. (Organic Chemistry at CU Boulder, lecture courses. Original content 2012 University of Colorado at Boulder, Department of Chemistry and Biochemistry.} 178