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Other Discussion & Conclusion
Other discussion:
Vitamin
E is non-polar because when we look at the size of the molecule, and the size of the part
that is hydrocarbon only (i.e. most of it), the polarity of the -OH group will
be overwhelmed by the non-polarity of the remaining molecule. Therefore, these
molecules will be non-polar as a whole, even though they have a polar group
attached. Therefore,
reversed phase HPLC is used in this experiment. A C18 (non-polar) guard and
analytical column is used.
-For the
composition of mobile phase for the linear gradient phase, water is added to
acetonitrile to reduce the eluotropic strength of the mobile phase to the
stationary phase (because is reversed phase HPLC, water is polar). So that the
peaks will not be co-eluted due to short retention times. Short retention times will cause peaks
overlap, and give us less sharp and symmetrical peaks.
-However, in this case, the concentration for linear gradient was only from 95% to 100% acetonitrile. This was too high a concentration for elution to be be done properly and clearly to give define peaks. Therefore, we suggest that in future experiments, we can start from a lower concentration (for example 10%-100% acetonitrile) to prevent peak overlap and give define peaks for analysis.
Thereafter, an isocratic mode is used because the the analysis time is not very long, and band broadening does not occur. Thus it is better to use the isocratic mode because the initial retention time is good enough to produce a sharp and symmetrical peak. So there is not a need to use linear gradient.
-δ- tocopherol is used as internal
standard because it is present in low amounts in the human breast milk.
Moreover, δ- tocopherol has similar physiochemical properties as α and γ tocopherol. Thus, if there
are any changes in the conditions of the HPLC, then the components will be affected in the same way. This way,
the results obtained are not affected greatly.
Lastly, the UV detector is used because the detection limit for the UV range is 180-380nm, and the absorption region of Vitamin E is 275nm-350nm. Since Vitamin E is able to absorb in the UV region, then the UV detector is used.
Conclusion:
Two extraction methods for the evaluation of vitamin E in human milk were compared - With saponification & without saponification.
Direct extraction of tocopherols with hexane resulted in 60 ± 15% recovery of the internal standard, δ-tocopherol. Application of the improved method, which included a simple saponification step, resulted in 99.6 ± 4.0% recovery for the same internal standard.
There was a significant relationship between tocopherol concentrations in milk specimens measured using the two extraction procedures. This implies that both methods of extraction can be used for determination of vitamin E in human milk, although the evidence presented herein suggests that extraction after saponification may significantly improve the reliability of such data.
In
conclusion, human breast milk contains sufficient amount of Vitamin E needed
for infants because the results above showed good indication of sufficient Vitamin E needed for infants. Thus, infants rarely need external supplements of Vitamin E to aid them in their growth.
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