Friday, March 23, 2012

Purification of Caffeine and Analysis

Well who would have thunk it last post we started to ponder how do I get chlorophyll out of my extracted sample...this post we solve this issue.....Eh it may go a little sum sum like dis.

How about some free coffee and or tea before we start?

For your entertainment: Purification and Thin-Layer Chromatographic Analysis of Caffeine

Purpose
  The purpose of this experiment was to continue the study and purification of caffeine isolated in a previous experiment via sublimation and Thin Layer Chromatography. Molecules are shown below.

 
Stuff about Stuff
Caffeine: Look at the previous post. If you're too lazy just know that its a white powdery substance in its natural form without odor and a rather bitter taste.

Chlorophyll a: For now just know that it provides a greenish color.
 

Procedure

All procedures were carried out under a hood, all individuals involved wore nitrile gloves and safety goggles. Glassware was cleaned, rinsed with deionzed water and if necessary acetone before usage.

 

Safety like it or not it's important. If you haven't heard about Carol....here she is. She never wore her safety goggles and now she doesn't need them. Don't be Carol. Or even worse meet Bill (Maturity warning....). Don't be Bill.




  Prior to the beginning of this experiment a sample of caffeine extracted from tea leaves and was stored in a 25 mL Erlenmeyer flask that was corked and allowed to sit for a week. From this crude extracted sample a small mass was put into a test tube and about .1 mL dichloromethane was added to it. The test tube was covered with wax paper and allowed to sit. This sample was labeled “Crude solution” and was to be used for the Thin Layer Chromatography later in the experiment.
The remaining crude sample was put into a filter flask with a side arm, and 1.9 mL of dichloromethane was added to it. A filter adapter was then set in the filter flask and a test tube was placed so that it sat about .5 cm off the bottom of the filter flask. The filter flask was placed in a 100 mL sand bath so that the sand covered a few mm of the flask wall. The sand bath was connected to a transformer to allow for control in process of heating. To add stability and decrease movement of the equipment the flask and test tube were each clamped separately to vertical rods in the hood. The side arm of the filter flask was connected via appropriate tubing to the vacuum in the hood. The vacuum was turned on the check for any leakage of air, once it was clear there was none, the sand bath and vacuum were both turned on until it seemed that no more caffeine was collecting on the inner tube. Meanwhile the ice water was placed into the test tube (fitted in the filter flask) kept being replaced every few minutes. After both the sand bath and vacuum had been turned off, the equipment was taken off the sand bath and raised in the air to cool.
            A spatula was used to scrap the caffeine off the test tube and onto the weigh boat. The weigh boat was weighed prior to and after the addition of the caffeine. The sublimed caffeine was put into another test tube where.50 mL dichloromethane was added. The test tube was labeled “Purified solution” and was covered with wax paper and stored.


  For the TLC a developing chamber was created (shown to the left). A 150 mL beaker was fit with filter paper, placed in beaker so that it sat on the sidewalls of the beaker but made contact with the bottom. 5 mL of ethyl acetate were added and the chamber was covered with aluminum foil. While the chamber was being set up the TLC plate was prepared for spotting of different caffeine solutions. The TLC plate was 6.7 cm in length and 4.5 cm in width. A 2 mm line was marked from the bottom edge of the plate and another about 1 cm from the top. The line at the bottom was divided into 3 distinct marked areas.
            Using a micropipette 3 spots of the crude caffeine solution was placed in the center of the first area, 3 spots of the purified caffeine were added to the second and then 1 spot of the standard solution provided to the class was placed onto the plate. The TLC plate was then placed in the chamber and the ethyl acetate mobile phase was allowed to run. Note tweezers were used to handle the TLC plate upon entry and extraction of the plate from the chamber. The plate was then taken out when the ethyl acetate was near the line marking 1 cm from the top.  Immediately after the plate was taken out of the chamber and how far the ethyl acetate traveled was marked with a pencil. The plate was then analyzed with 254 nm UV radiation. During this process each spot of caffeine was outlined with a pencil and the chlorophyll was marked. Rf values were calculated for each caffeine solution by dividing the midpoint of the caffeine spot by the total distance traveled by the ethyl acetate solvent in cm. What we got the Rf value for the “Crude Caffeine Solution” was .121, for the “Purified Caffeine Solution” the Rf value was .144 and for the “Standard Caffeine Solution the Rf value was .124. .

Things that may be good to know.
Rf values = Distance between midpoint of spot from origin in cm
                        Distance traveled by the solvent in cm
 
 Questions? (Feel free to add your own)

So how does one see the results of the thin layer chromatography?
The visualization technique used in the TLC portion of this experiment involves the use of
ultraviolet (UV) light. The caffeine absorbs the UV light and gives off visible light.
This phenomenon is called fluorescence. For more on wiki go here.



Bored? Go here 



Its Question Time:

What is sublimation?And why is it useful in lab settings involving purification?

Sublimation is the process of a phase change between solid and gas. In general, if an experimental
product can sublime, one can use it as a purification technique. This is because in most cases, impurities do not sublime.

Caffeine and sublimation

 Caffeine will normally sublime around 180 degree C (at 1 atm).

In general, in order for a solid to sublime, its triple point pressure must be higher than 1 atm. In order to accelerate the sublimation process for caffeine (see below), external pressure must be reduced to drive the equilibrium towards gas phase. Vaccum filtration helps by lowering the pressure inside the filtering flask  and will promote the caffeine to sublime at a temperature well below 180 degree C.

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