1. Column Chromatographic
Determination of Vitamin A
Isolated from Different Oleaginous
Food Products
PhCh 136–4
Caadan|Carrido|Erscanuela|Fernandez|Guico
Iringan|Lu|Morano|Pineda
Tabilin|Taguinod|Tiosejo|Tiu|Triumfante|Venida
Catabay|Mata

2. Outline
I. Introduction
II. Objectives
III. Significance of Study
IV. Methodology
V. Results and Discussion
VI. Conclusion

3. INTRODUCTION

4. Introduction
Vitamin A
• plays an important
role in vision, bone
growth,
reproduction, cell
division, and cell
differentiation
• helps in the
regulation of the
immune system
• fat-soluble vitamin (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethyl-
cyclohex-1-en-l-yl)nona-2,4,6,8-tetraen-1-ol

5. Introduction
Vitamin A

6. Introduction
Vitamin A Sources

7. Introduction
Vitamin A
Table 1. Recommended Daily Allowance of Vitamin A
Age
Children Male Female Pregnancy Lactation
(years)
1-3 1000 IU
4-8 1320 IU
9-13 2000 IU
14-18 - 3000 IU 2310 IU 2500 IU 4000 IU
19+ - 3000 IU 2310 IU 2565 IU 4300 IU
IU (International Unit)
One IU is equivalent to 0.3 μg retinol.
Reference: US National Institutes of Health

8. Significance of Study
• Vitamin A deficiency is common
among Filipino families and is the
world’s leading preventable cause of
childhood blindness *
• The use of column chromatography as
an inexpensive method for the analysis
of vitamin A
*Reference: Alexandra K. 2009, Malnutrition in the Philippines – perhaps a Double
Burden?

9. Objectives
• General Objective
To evaluate the effectiveness of column
chromatography in the separation of vitamin A
from other nonsaponifiable contents in the sample.
• Specific Objectives
– To determine the proportion of vitamin A eluted
from the column;
– To compare the proportion of vitamin A eluted in
each column per oleaginous product; and
– To compare the percent of vitamin A recovered
among the samples

10. METHODOLOGY

11. Methodology
• Samples
– Margarine (Buttercup ®)
– Butter (Anchor ®)
– Cod Liver Oil Emulsion
(Scott’s Emulsion ®)
• Apparatus
– Laboratory-assembled
column
chromatography set up
(Stationary phase: Basic
aluminum oxide)
– Thermo specific Genesys
10S Series UV-Visible
Spectrophotometer

12. Methodology
Column Chromatography
UVSolvent Extraction
Spectrophotometry
Saponification
Blank: 95% ethanol Stationary Phase: water
Wash with distilled in
Solvent: 50% KOH
basic aluminum oxide
Absorbance was read at 325 nm in 1cm-path
Ethanol
↓
length quartz cells Extract with
Mobile diethyl ether
Phase:
Solvent Column UV
Saponification
Extraction
petroleum ether
Chromatography Spectro
Data
Analysis

13. Methodology
Data Analysis

14. RESULTS AND DISCUSSION

15. Results and Discussion

16. Results and Discussion
Comparison of the Percent of Vitamin A
Recovered Among the Samples
Table 2. Amount of Vitamin A content (in μg per gram) of the oleaginous
product for three different trials.
μg Vitamin A per gram of product
Product Label
Trial 1 Trial 2 Trial 3 Average
Claim
Margarine 9.9 1.27 1.81 0.32 1.13
(Buttercup®)
Butter
5.4 0.90 0.66 0.33 0.63
(Anchor®)
Cod Liver Oil
Emulsion 5.0 2.49 1.25 2.01 1.91
(Scott’s Emulsion®)

17. Results and Discussion
Comparison of the Percent Label Claim of
Vitamin A Among the Samples

18. Results and Discussion
Proportion of Vitamin A Eluted from the Column
Table 3. Percent eluted vitamin A from the column (% Eluted) and percent
retained vitamin A in the column(% Retained) with computed standard
deviations
% Standard
Sample Trial % Eluted
Retained Deviation
1 48.85 51.15
Margarine 2 35.81 64.19 9.46
(Buttercup®)
3 30.46 69.54
1 96.78 3.22
Butter 2 98.37 1.63 21.33
(Anchor®)
3 60.66 39.34
Cod Liver Oil 1 81.58 18.42
Emulsion 2 45.91 54.09
18.84
(Scott’s
3 53.21 46.79
Emulsion®)

19. Results and Discussion
Proportion of Vitamin A Eluted from the Column
Table 4. Percent vitamin A recovered among trials per oleaginous food
product (Margarine, Butter, and Cod Liver Oil Emulsion.)
% of Vitamin A Average % of Vitamin
Sample
Recovered A Recovered
12.84
Margarine 18.30 11.44 ± 7.65
(Buttercup®)
3.19
16.63
Butter 12.31 11.70 ± 5.26
(Anchor®)
6.16
Cod Liver Oil 49.84
Emulsion 24.91 38.29 ± 12.57
(Scott’s Emulsion®) 40.12

20. Results and Discussion
Proportion of Vitamin A Eluted in Each Column
per Oleaginous Product
Table 5. Percent eluted vitamin A from the column (% Eluted) and percent
retained vitamin A in the column (% Retained) with computed standard
deviations among trials per oleaginous food product (Margarine, Butter, and
Cod Liver Oil Emulsion)
% Standard
Sample Trial % Eluted
Retained Deviation
1 48.85 51.15
Margarine 2 35.81 64.19 9.46
(Buttercup®)
3 30.46 69.54
1 96.78 3.22
Butter 2 98.37 1.63 21.33
(Anchor®)
3 60.66 39.34
Cod Liver Oil 1 81.58 18.42
Emulsion 2 45.91 54.09 18.84
(Scott’s Emulsion®) 3 53.21 46.79

21. Results and Discussion
Comparison of the Percent Label Claim of
Vitamin A Among the Samples
Table 2. Amount of Vitamin A content (in μg per gram) of the oleaginous
product for three different trials.
μg Vitamin A per gram of product
Product Label
Trial 1 Trial 2 Trial 3 Average
Claim
Margarine 9.9 1.27 1.81 0.32 1.13
(Buttercup®)
Butter
5.4 0.90 0.66 0.33 0.63
(Anchor®)
Cod Liver Oil
Emulsion 5.0 2.49 1.25 2.01 1.91
(Scott’s Emulsion®)

22. Results and Discussion
Comparison of the Percent Label Claim of
Vitamin A Among the Samples
Butter Margarine Emulsion

23. Conclusion
“To determine the proportion of vitamin A eluted from the column”
The proportion of vitamin A in the column and in the
eluate are variable – there is no trend observed.
“To compare the proportion of vitamin A eluted in each column per
oleaginous product”
Large standard deviations suggest that the proportion of
vitamin A in the column and in the eluate are variable between
trials, therefore there is low repeatability.
“To compare the percent of vitamin A recovered among the samples”
The percent vitamin A recovered varies among samples.
This may be due to the differences in their matrix. The use of
column chromatography in isolating Vitamin A is not robust.

24. Conclusion
Column chromatography is
ineffective in the separation of vitamin A
from other nonsaponifiable contents in
the sample.

25. Recommendations
• Perform flash chromatography instead
• Since collected amount is small
• Perform TLC
• To assess if other compounds are separated as
well.
• Subject the Vitamin A eluate itself to the
handheld UV lamp
• To assess if it contains vitamin A

26. References
Dam, H. (n.d.). Fat-Soluble Vitamins. Retrieved March 10,
2012, from Annual Reviews:
http://www.annualreviews.org/doi/abs/10.1146/annure
v.bi.20.070151.001405
Eden, E. (1950). A Micro Method for Separating Free and
Esterified Vitamin A. Biochem, 259-261.
Merck & Co. (2001). Vitamin A. The Merck Index.
Whyte, B. (1996). Vitamin A and Retinoids. Retrieved
March 10, 2012, from
http://www.chem.qmul.ac.uk/iubmb/newsletter/1996/
news2.html