CHM 343 Lab Report 3 - CHM343 Lab Report 3 Choon Liang Tan...

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CHM343 Lab Report # 3 02.11.08 Choon Liang Tan CHM 343 Separations Lab Report # 3 Determination of Abscorbic Acid, Benzoic and Caffeine Acid In Unknown Mixture by Reversed-phase HPLC Name: Choon Liang Tan Bronco ID: 007156760 Class Session: CHM343 Lab Lecturer: Dr. Gutnikov & Dr. Dr. Hossein Ahmadzadeh Date: 02.11.08 1
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CHM343 Lab Report # 3 02.11.08 Choon Liang Tan Abstract: Through studies with the reversed-phase HPLC, three standard mixtures of ascorbic and benzoic acid, and caffeine were analyzed. These three components exist in soda drinks and coffee. The first discovery was that ascorbic acid was detected first, thus is the most polar compound. Benzoic acid was the most non-polar compound as it was detected last. The more non-polar the compound, the more it would be retained by the non-polar column, since this is reversed phase. The unknown mixture contained 10.8592ppm of caffeine and 6.0141ppm of benzoic acid. No major uncertainties or errors occurred during this experiment. Benzoic acid can become benzoate ions due to pH values, thus decreasing the retention time with increasing ions. pH is there to help adjust the conditions of the separations in case normal separations are inadequate. Introduction: The structures of large majority of naturally occurring substances encompass non- polar hydrocarbon backbones or chains along with (multiple) polar functional groups (such as –COOH, -OH, -NH2, etc) that render them soluble in water or other polar solvents. Such substances are readily amenable to separation on reversed-phase HPLC. A reversed-phase HPLC system consists of a non-polar stationary phase (such as alkyl chains of various lengths bonded onto silica) and polar mobile phase (commonly water with various proportions of methanol or acetonitrile). Retention, which is based on the interaction of the alkyl carbons of the sample with those of the stationary phase, increases linearly with the carbon number for homologous series. Reversed phase HPLC (RP-HPLC) consists of a non-polar stationary phase and an aqueous, moderately polar mobile phase. One common stationary phase is a silica which has been treated with RMe 2 SiCl, where R is a straight chain alkyl group such as C 18 H 37 or C 8 H 17 . The retention time is therefore longer for molecules which are more non- polar in nature, allowing polar molecules to elute more readily. Retention Time (RT) is increased by the addition of polar solvent to the mobile phase and decreased by the addition of more hydrophobic solvent. Reversed phase chromatography is so commonly used that it is not uncommon for it to be incorrectly referred to as "HPLC" without further specification. The pharmaceutical industry regularly employs RPC to qualify drugs before their release. RPC operates on the principle of hydrophobic interactions, which result from repulsive forces between a polar eluent, the relatively non-polar analyte, and the non- polar stationary phase. The binding of the analyte to the stationary phase is proportional to the contact surface area around the non-polar segment of the analyte molecule upon association with the ligand in the aqueous eluent. This solvophobic effect is dominated by
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  • Spring '08
  • GUTNIKOV
  • Chromatography, pH, High performance liquid chromatography, Choon Liang Tan

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