Column Chromatographic    Determination of Vitamin AIsolated from Different Oleaginous           Food Products            ...
OutlineI. IntroductionII. ObjectivesIII. Significance of StudyIV. MethodologyV. Results and DiscussionVI. Conclusion
INTRODUCTION
IntroductionVitamin A  • plays an important    role in vision, bone    growth,    reproduction, cell    division, and cell...
IntroductionVitamin A
IntroductionVitamin A Sources
IntroductionVitamin ATable 1. Recommended Daily Allowance of Vitamin A     Age                 Children          Male     ...
Significance of Study• Vitamin A deficiency is common  among Filipino families and is the  world’s leading preventable cau...
Objectives• General Objective  To evaluate the effectiveness of column  chromatography in the separation of vitamin A  fro...
METHODOLOGY
Methodology• Samples  – Margarine (Buttercup ®)  – Butter (Anchor ®)  – Cod Liver Oil Emulsion    (Scott’s Emulsion ®)• Ap...
Methodology       Column Chromatography        UVSolvent Extraction           Spectrophotometry            Saponification ...
MethodologyData Analysis
RESULTS AND DISCUSSION
Results and Discussion
Results and DiscussionComparison of the Percent of Vitamin ARecovered Among the SamplesTable 2. Amount of Vitamin A conten...
Results and DiscussionComparison of the Percent Label Claim ofVitamin A Among the Samples
Results and DiscussionProportion of Vitamin A Eluted from the ColumnTable 3. Percent eluted vitamin A from the column (% E...
Results and DiscussionProportion of Vitamin A Eluted from the ColumnTable 4. Percent vitamin A recovered among trials per ...
Results and Discussion Proportion of Vitamin A Eluted in Each Column per Oleaginous ProductTable 5. Percent eluted vitamin...
Results and DiscussionComparison of the Percent Label Claim ofVitamin A Among the SamplesTable 2. Amount of Vitamin A cont...
Results and DiscussionComparison of the Percent Label Claim ofVitamin A Among the Samples        Butter           Margarin...
Conclusion“To determine the proportion of vitamin A eluted from the column”       The proportion of vitamin A in the colum...
Conclusion     Column        chromatography        isineffective in the separation of vitamin Afrom other nonsaponifiable ...
Recommendations• Perform flash chromatography instead  • Since collected amount is small• Perform TLC  •   To assess if ot...
ReferencesDam, H. (n.d.). Fat-Soluble Vitamins. Retrieved March 10, 2012, from Annual Reviews: http://www.annualreviews.or...
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Column Chromatographic Determination of Vitamin A Isolated from Different Oleaginous Food Products

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  • Fat-soluble properties are due to its structure…
  • Presence of many carbon and hydrogen which have similar electronegativities making it nonpolar solubility: insoluble in water and soluble in organic solvents such as diethyl etherAnother notable moiety is its extensive conjugated double bond Enables it to absorb light which eventually fluoresce
  • Presence of many carbon and hydrogen which have similar electronegativities making it nonpolar solubility: insoluble in water and soluble in organic solvents such as diethyl etherAnother notable moiety is its extensive conjugated double bond Enables it to absorb light which eventually fluoresce
  • Expressed in IU…The Recommended daily allowance of vitamin A by the national institute of healthRange 1000-4000 IU depending on the age group and consumer typeHowever RDA is not commonly attained by majority of Filipinos, there the significance of the study…
  • Deficiancy (first siginificance)Therefore there is a need to determine the vitamin A content of food products using an inexpensive method such as column chromatography
  • Sample products were procured from Robinsons supermarket ermita
  • SaponificationThe samples saponified using a mixture of 95% ethanol and 50% KOH and subjected to boiling under reflux for 5 mins.Solvent Extraction:The resulting solution was then extracted twice using diethyl ether to separate the saponifiable components, such as triglycerides, from Vitamin A since the they are soluble in aqueous layer while vitamin A is more soluble in the ether layer. Column ChromatographyThe ether extract was then subjected to solid-liquid column chromatography with basic aluminum oxide as the stationary phase and petroleum ether as the mobile phase. The eluates were only collected after the orange bands, composed mainly of carotenes, have been removed from the column. The vitamin A present in the column may be detected via exposure to UV radiation causing it to fluoresce.DetectionThe vitamin A in the collected eluates were then quantified using UV Spectrophotometry
  • The goal of the saponification process is to
  • Per trial the percent eluted and retained are not definite pattern Possible reasons:End point determination, possibility that viatmin A available to fluoresce (remaining) is diffused at the center of the colmn packing Column properties: interaction with the solvent – pet ether most suitable (cannot be pointed out as the cause, also used in the official) Therefore the possible problem is the stationary phase
  • *erase standard deviationAve% vit a recovered +/- stddev
  • Add computation for label claim
  • Butter, margarine, emulsion still have different matrices after saponification. After column chrom, only vitamin A will be eluted.
  • Explanation for 2nd conclusion:This is due to factors such as column packing, uniformity of column properties (eg. Column diameter and length), difficulty in end-point determination during the elution process caused by the diffusion of the analyte in the alumina.
  • Column Chromatographic Determination of Vitamin A Isolated from Different Oleaginous Food Products

    1. 1. Column Chromatographic Determination of Vitamin AIsolated 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. 2. OutlineI. IntroductionII. ObjectivesIII. Significance of StudyIV. MethodologyV. Results and DiscussionVI. Conclusion
    3. 3. INTRODUCTION
    4. 4. IntroductionVitamin 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. 5. IntroductionVitamin A
    6. 6. IntroductionVitamin A Sources
    7. 7. IntroductionVitamin ATable 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 IUIU (International Unit)One IU is equivalent to 0.3 μg retinol.Reference: US National Institutes of Health
    8. 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 DoubleBurden?
    9. 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. 10. METHODOLOGY
    11. 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. 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 UVSaponification Extraction petroleum ether Chromatography Spectro Data Analysis
    13. 13. MethodologyData Analysis
    14. 14. RESULTS AND DISCUSSION
    15. 15. Results and Discussion
    16. 16. Results and DiscussionComparison of the Percent of Vitamin ARecovered Among the SamplesTable 2. Amount of Vitamin A content (in μg per gram) of the oleaginousproduct 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. 17. Results and DiscussionComparison of the Percent Label Claim ofVitamin A Among the Samples
    18. 18. Results and DiscussionProportion of Vitamin A Eluted from the ColumnTable 3. Percent eluted vitamin A from the column (% Eluted) and percentretained vitamin A in the column(% Retained) with computed standarddeviations % 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. 19. Results and DiscussionProportion of Vitamin A Eluted from the ColumnTable 4. Percent vitamin A recovered among trials per oleaginous foodproduct (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. 20. Results and Discussion Proportion of Vitamin A Eluted in Each Column per Oleaginous ProductTable 5. Percent eluted vitamin A from the column (% Eluted) and percentretained vitamin A in the column (% Retained) with computed standarddeviations among trials per oleaginous food product (Margarine, Butter, andCod 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. 21. Results and DiscussionComparison of the Percent Label Claim ofVitamin A Among the SamplesTable 2. Amount of Vitamin A content (in μg per gram) of the oleaginousproduct 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. 22. Results and DiscussionComparison of the Percent Label Claim ofVitamin A Among the Samples Butter Margarine Emulsion
    23. 23. Conclusion“To determine the proportion of vitamin A eluted from the column” The proportion of vitamin A in the column and in theeluate are variable – there is no trend observed.“To compare the proportion of vitamin A eluted in each column peroleaginous product” Large standard deviations suggest that the proportion ofvitamin A in the column and in the eluate are variable betweentrials, 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 ofcolumn chromatography in isolating Vitamin A is not robust.
    24. 24. Conclusion Column chromatography isineffective in the separation of vitamin Afrom other nonsaponifiable contents inthe sample.
    25. 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. 26. ReferencesDam, H. (n.d.). Fat-Soluble Vitamins. Retrieved March 10, 2012, from Annual Reviews: http://www.annualreviews.org/doi/abs/10.1146/annure v.bi.20.070151.001405Eden, 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

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