Download Caffeine Content in Beverages: Lab Experiment on Chromatography and more Lab Reports Organic Chemistry in PDF only on Docsity! 1 CHEM 333: Lab Experiment 5: INTRODUCTION TO CHROMATOGRAPHY; Thin Layer and High Performance Liquid Chromatography: PRE-LAB ASSIGNMENT!!! : The purpose of the first part of today’s experiment is to determine the caffeine content of a beverage sample. Here is your chance to get involved and find out how much caffeine is in that stuff you drink. Which soda has the most caffeine? Red Bull really have more caffeine of normal cola? Tea have more caffeine than coffee? Which of your favorite coffee shops makes the most potent cup? Now is your chance to answer these questions and more that have plagued student-kind for centuries! (After all this information might come in handy as you study for your finals!) YOU ARE ALL TO BRING SAMPLE OF THE CAFINATED BEVERAGE OF YOUR CHOICE TO THIS WEEKS LAB. Pre-lab Reading Chapters 7, 8 and 9 “This lab can actually be kind of fun (in a geeky sort of way). The techniques you will use this week are “analytical”and you will be able to make actual determinations of caffeine levels in beverages. Next week you will learn to perform chromatography on a preparative (though still small) scale.” Liquid Chromatography Liquid Chromatography is a powerful technique to separate and purify organic compounds. This week’s laboratory deals with Thin Layer Chromatography (TLC) and High Pressure Liquid Chromatography (HPLC), which are used as analytical tools to analyze samples. In next week’s laboratory you will use column chromatography. Column chromatography is a preparative method used to isolate or purify larger amounts of material (from milligrams to kilograms). After reading the introductory information in the text, it is very important that you understand chromatography’s relationship to the polarity of organic compounds. Be able to recognize polar functional groups, especially those that can form hydrogen bonds with the solid phase (i.e. 1° and 2° amines, alcohols, and especially carboxylic acids). Make note of Table 7.1 for its ordering of solvent polarities. 2 High Performance Liquid Chromatography (HPLC) High Performance Liquid Chromatography (or High Pressure Liquid Chromatography) is a very sensitive analytical technique. It has the capacity to be employed as both an analytical (milligrams) and preparative (hundreds of milligrams) tool for separating mixtures of closely eluting compounds. The chromatogram of materials separated can also be integrated to give quantitative data on the amounts present in the original mixture (given proper calibration). Two types of detectors are widely used for monitoring separation. An absorbance detector measures the absorbance of a particular wavelength of ultraviolet light, usually tuned for aromatic compounds. The alternative, is a detector that monitors changes in the refractive index of the solvent being eluted. This allows for detection of compounds that may be transparent to UV radiation. A variation of standard chromatography frequently appears with HPLC: the use of “reverse- phase” columns. The solid support in a reverse-phase column is coated with a greasy, non- polar substance (usually a long-chain hydrocarbon mix tethered to the support through a silicon linkage). In this way, the polarity rules are more or less reversed: non-polar compounds get bound to the column and move slowly, while polar compounds move more quickly with the solvent. More polar solvent mixtures (often aqueous) are usually employed. Chromatography Part I. Extraction of Caffeine from Caffeinated Beverages N N N N O CH3 H3C CH3 O Caffeine The purpose of this experiment is to determine the caffeine content of a beverage sample. You must bring your favorite beverage sample with you to lab. This lab uses reverse-phase chromatography: the stationary phase is non-polar and the eluting solvent is polar. There are three steps: 1. Extraction, 2. Sample Preparation, 3. analysis by HPLC.
