RESULTS AND DISCUSSION
4.2 SILICA GEL COLUMN CHROMATOGRAPHY
4.2.2 Modified Silica Gel Column Chromatography (MSGCC)
Classical column chromatography and TLC are laborious, time consuming (Moreda et al.,
2001), and requiring the use of large amount of organic solvents (Lacaze et al., 2007). In this study, a new silica gel column chromatography (SGCC) was designed with the objective of reducing the amount of solvent as well as operation time required to achieve the same degree of separation as the classical SGCC.
HTGC chromatograms of fractions collected from MSGCC at optimum conditions are shown in Figure 4-2. The initial contents of squalene and FASEs are 6.22 % and 12.28%, respectively (Figure 4-2A). The first fraction collected in the first 4.5 h with hexane as the eluting solvent consists mostly of aliphatic, steroidal and sesquiterpene hydrocarbons and represents about 40 - 50% of the NPLF (Figure 4-2B). The second fraction collected in the next 4.5 h is squalene with very high purity (99 %) as can be seen in Figure 4-2C. The last fraction which contains most FASEs in the NPLF was obtained after eluting the column for 6 h with mixture of hexane and ethyl acetate (99/1, v/v) (Figure 4-2D). Substances that were still adsorbed on silica gel were collected in the washing fraction which consists mostly of FFAs as shown in Figure 4-2E. Results of TLC analyses of each fraction as described in Figure 4-2 are shown in Figure 4-3.
Figure 4-2 HTGC analysis of fractions obtained from modified column chromatography of
NPLF of SODD sample III. Operation conditions for the modified silica gel column chromatography: silica gel to NPLF mass ratio of 10, and elution temperature = 23oC. The GC chromatograms of the NPLF (A), the 1st fraction (B), the 2nd fraction (C), the 3rd fraction (D), and the washing fraction (E) eluted from the modified silica gel column chromatography.4.2.2.1 Isolation of Hydrocarbons
Aliphatic, steroidal and sesquiterpene hydrocarbons represent about 40-50% of the NPLF of SODD. Due to such high content, efficient removal of these hydrocarbons is crucial for obtaining high purity squalene (triterpene hydrocarbon) which is the most polar hydrocarbons in the NPLF.
In this study, we choose squalene as an indicator for successful separation of the first (aliphatic, steroidal and sesquiterpene hydrocarbons) and the second fraction (squalene); parameters such as gradient elution of mobile phase, silica gel to NPLF mass ratio, elution temperature and mobile phase flow rate were adjusted until high purity (> 90 %) squalene with high recovery (> 80%) was obtained.
The major decision required in the design of a satisfactory separation is selection of the mobile phase. Hexane was selected as the eluting solvent for the first and the second fractions because mixture of solvent (hexane and ethyl acetate) did not result in satisfactory separation (data not shown). This is because polarities of aliphatic, steroidal, sesquiterpene, and triterpene hydrocarbons are roughly in the same range.
A lower silica gel to NPLF mass ratio is desirable in column chromatography, because less adsorbent and shorter elution time are required. However, a silica gel to NPLF mass ratio less than 10 did not result in successful separations (data not shown) because it was larger than the linear capacity of the column (Snyder and Kirkland, 1979). Therefore, the mass ratios employed in this study were either 10 or 20.
A B C D E
Aliphatic hydrocarbons Steroidal hydrocarbons Sesquiterpene hydrocarbons Squalene
FASEs
Mainly FFAs A B C D E
Aliphatic hydrocarbons Steroidal hydrocarbons Sesquiterpene hydrocarbons Squalene
FASEs
Mainly FFAs
Figure 4-3 TLC analyses of fractions obtained from MSGCC of NPLF developed in pure
n-hexane. (A) NPLF of SODD sample III; (B) the 1st fraction; (C) the 2nd fraction;(D) the 3rd fraction; (E) the washing fraction.
Table 4-6 shows that a lower silica gel to NPLF mass ratio results in a higher recovery of all
hydrocarbons in the first fraction. The P-value method was applied to determine the significance of these differences. At a higher silica gel to NPLF mass ratio (20), the impurity (mainly squalene) content and recovery in the 1st fraction is significantly lower (p < 0.05) than that obtained if a lower silica gel to NPLF mass ratio of 10 was used.Table 4-6 Effect of (silica gel)/NPLF on squalene content and recovery in the 1
st fraction a (silica gel)/NPLF=20/1 (g/g) (silica gel)/NPLF=10/1 (g/g) CompoundsThe composition of NPLF of SODD sample III is shown in Table 4-3
a Average of three independent experiments. Operation conditions: 150 mL n-hexane as mobile phase, 4.06 mL/min flow rate, 4.5 h elution time and 23 oC elution temperature.
b Recovery = {(1st fraction mass, g x content of the compound in 1st fraction, %) / (NPLF mass, g x content of the compound in NPLF, %)} x 100
c Mainly hydrocarbons such as aliphatic, steroidal, and sesquiterpene hydrocarbons.
d Amount = (1st fraction mass, g / NPLF mass, g) x 100.
4.2.2.2 Isolation and Purification of Squalene
The effect of silica gel to NPLF mass ratio on the isolation of squalene is shown in Table
4-7. It can be seen that the squalene content is significantly lower (p < 0.05) while the recovery of
squalene is significantly higher (p < 0.05) at (silica gel)/NPLF=20/1 (g/g) than those obtained at (silica gel)/NPLF=10/1 (g/g). At (silica gel)/NPLF=10/1 (g/g), squalene with high purity and recovery can be obtained in the 2nd fraction in 2.5 h by using large mobile phase flow rate of 33.32 mL/min (Table 4-8). The time required is much shorter than that required to achieve comparable results (in terms of squalene purity and recovery) when classical silica gel column chromatography was used.Table 4-7 Effect of (silica gel)/NPLF on purity and recovery of squalene in the 2
nd fraction a (silica gel)/NPLF=20/1 (g/g) (silica gel)/NPLF=10/1 (g/g) CompoundsThe composition of NPLF of SODD sample III is shown in Table 4-3
a Average of three independent experiments. Operation conditions: 150 mL n-hexane as mobile phase, 17.32 mL/min flow rate, 4.5 h elution time and 23 oC elution temperature.
b Recovery = {(2nd fraction mass, g x content of the compound in 2nd fraction, %) / (NPLF mass, g x content of the compound in NPLF, %)} x 100
c Mainly hydrocarbons such as aliphatic, steroidal, and sesquiterpene hydrocarbons.
d Amount = (2nd fraction mass, g / NPLF mass, g) x 100.
The identification of compounds in the second fraction was confirmed by HTGC, TLC and GCMS analyses. The GCMS fragmentation of the peak showed a molecular ion (M+) at 410
(relative intensity: 1.09 %) and base peak at 69.05. Peaks were also observed at m/z at 149, 137, 121, 109, 95, 81, 55, and 41. These fragmentation patterns were quite similar to those for squalene reported in U.S. National Bureau of Standards library (2005).
The fractionations obtained in this study give better separation than previous study of Lanzόn et al. (1994) in which the unsaponifiable matter of both virgin and refined olive oil was fractioned by classical silica gel column chromatography with a silica gel to unsaponifiable matter mass ratio of 75, using hexane as eluent. The fraction containing squalene was eluted after aliphatic, steroidal and sesquiterpene hydrocarbons, the purity of squalene was not reported in their study. However, judging from the chromatogram in their study, low squalene purity was obtained.
4.2.2.3 Isolation of FASEs
Using single solvent as mobile phase is more desirable than using mixture of solvents in column chromatography because of easier recovery and reuse of solvent. When pure hexane was employed as the eluent, it was impossible to recovery most FASEs in reasonable short time in the third fraction. It was found that it required 83 h to isolate FASEs with a purity of 83.06% and a recovery of 97.84% at 23oC and 17.32 mL/min using hexane as the mobile phase. The elution time can be reduced to 20 h by increasing the elution temperature (temperature in the packing region) to 64oC due to increasing solubility of sample in mobile phase. However, the purity of FASEs obtained was much lower (39.01% purity, 96.15% recovery). These results agree with previous observation that once squalene was completely eluted, polarity of the mobile phase must be increased to elute other more polar compounds (Lanzόn et al. 1994). Thus, mixture of hexane and ethyl acetate was employed as the eluent in order to successfully recover most FASEs in the third fraction in shorter time.
Table 4-8 Effect of mobile phase flow rate on the purity and recovery of squalene in the 2
nd fractionThe composition of NPLF of SODD sample III is shown in Table 4-4
a Average of three independent experiments. Operation conditions: 150 mL n-hexane as mobile phase, (silica gel)/NPLF = 10/1 (g/g), and 23oC elution temperature.
b Recovery = {(2nd fraction mass, g x content of the compound in 2nd fraction, %) / (NPLF mass, g x content of the compound in NPLF, %)} x 100
c Mainly hydrocarbons such as aliphatic, steroidal, and sesquiterpene hydrocarbons.
d Amount = (2nd fraction mass, g / NPLF mass, g) x 100.
As shown in Table 4-9, as the polarity increases by increasing the percentage of ethyl
acetate in the mobile phase from 1% to 3%, the purity of FASEs in the third fraction decreases from 78.48% to 55.14%, while the corresponding recoveries varies only slightly. One of the important criteria for successful separation in this study is to recover most of the FASEs in the third fraction. FASEs contents are significantly higher (p < 0.05) when mobile phase contained 1% ethyl acetate as compared to those obtained when 2% and 3% ethyl acetate were used.
However, the contents and recoveries of the FFAs and acylglycerols are not significantly different (p > 0.05) among the three studies using different percentages of ethyl acetate. These results agree with previous observation that once the squalene was completely eluted, polarity of the mobile phase must be increased to elute other more polar compounds Lanzόn et al. (1994).
More silica gel yielded a higher adsorption area available per unit NPLF, which resulted in longer elution time with comparable separation due to a higher amount of mobile phase that was used to displace FASEs. For example, at a silica gel to NPLF mass ratio of 20 and with 2% ethyl acetate in the mobile phase, the elution time to obtain the third fraction that contains FASEs with a purity of 69.64 ± 9.99% and a corresponding recovery of 95.18 ± 6.45 % was significantly longer (6 h, p < 0.05) than those obtained while using a silica gel to NPLF mass ratio of 10 (2 h).
Table 4-9 Effect of mobile phase polarity on composition and recovery of 3
rd fraction aMobile phase ( hexane/ethyl acetate, v/v)
99/1 98/2 97/3
Compounds
Purity, wt.% Recovery,% b Purity, wt.% Recovery, % Purity,
wt.% Recovery, %
The composition of NPLF of SODD sample III is shown in Table 4-3
a Average of three independent experiments. Operation conditions: (silica gel)/ NPLF =10/1 (g/g), 150 mL mobile phase volume, 17.32 mL/min flow rate, and 23 oC elution temperature.
b Recovery = {(3rd fraction mass, g x content of the compound in 3rd fraction, %) / (NPLF mass, g x content of the compound in NPLF, %)} x 100
c Mainly ketones and aldehydes.
d Amount = (3rd fraction mass, g / NPLF mass, g) x 100.
Among the mobile phase polarity that tried in this study (100/0, 99/1, 98/2 and 97/3, hexane/ethyl acetate, v/v), only mixture of hexane/ethyl acetate = 99/1 yield satisfactory separation. When packing region temperature was controlled at -10 and 0oC, satisfactory separation was not significantly different (data not shown). The packing region temperature was controlled at 23oC in this isolation of FASEs study.
The effect of mobile phase flow rate on FASEs is shown in Table 4-10. It can be seen that FASEs contents and recoveries are not significantly different (p > 0.05) while elution time is significantly higher (p < 0.05) at 17.32 mL/min than those obtained at 33.33 mL/min.
Table 4-10 Effect of flow rate on the purity and recovery of FASEs in the 3
rd fraction17.32 mL/min a 33.33 mL/min The composition of NPLF of SODD sample III is shown in Table 4-3.
Operation conditions: (silica gel)/NPLF=10/1 (g/g), 150 mL mobile phase of 99/1 (hexane/ethyl acetate, v/v), and 23oC elution temperature.
a Average of three independent experiments.
b Recovery = {(3rd fraction mass, g x content of the compound in 3rd fraction, %) / (NPLF mass, g x content of the compound in NPLF, %)} x 100
c Mainly aldehydes and ketones.
d Amount = (3rd fraction mass, g / NPLF mass, g) x 100.
As shown in Table 4-11, for comparable separation, both elution time and amount of solvent required are much less when using MSGCC as compared to classical SGCC. By employing MSGCC to obtain the squalene-rich fraction, the mobile phase volume and elution time required as fractions of those needed in classical silica gel column chromatography are 1/73 and 1/18, respectively. To obtained the FASEs-rich fraction, the corresponding mobile phase volume and elution time are 1/221 and 1/23, respectively of those needed in classical SGCC.
This separation was confirmed in subsequent experiments, carried out under the same conditions except with a NPLF that contains low percentage of squalene and FASEs (Table 4-12) and with a NPLF obtained from SODD sample IV (Table 4-13). Squalene content in the second fraction and FASEs recoveries in the third fraction in this case were not significantly different (p
> 0.05), compared to those obtained using a NPLF with high squalene and FASEs contents. Also, it was found that the third fraction was richer in FFAs due to higher content of FFAs in NPLF.