The overall goal of this procedure is to use small molecule flora fours based on the so-called cruciform geometry to analyze and qualitatively discriminate analytes from several compound classes. This is accomplished by first preparing solutions of these cruciform sensors in several different solvents, thus capitalizing on their highly solvent dependent emission. The second step is to expose these solutions to the analytes of interest, including carboxylic acids, onic acids, or organic amines, which can be added either as solids or in solution.
Next, the combined sensor analyte solutions are irradiated under 365 nanometer ultraviolet light, causing them to emit light in the visible region. These emission colors are photographed using a standard digital photography setup. The final step is the generation of emission color panels, which are used to detect qualitative differences in emission of different analytes solutions.
Ultimately, this fluorescence method will be useful for rapid identification of common analytes in the pharmaceutical and food industries. The main advantage of this technique over existing methods like chromatography is that it can directly analyze highly polar analytes without the need for a derivative decision. This method can help answer key questions in a routine quality control field where it could be used to quickly determine whether a well-defined composition is authentic or has been compromised.
Visual demonstration of this method is critical as method itself uses optical detection and because of its ultimate application would involve non-specialized operators. Demonstrating the procedure will be Abel from Buns Laboratory in Heidelberg and Rio Color Lire from my laboratory at the University of Houston. Both the graduate students First prepare fresh stock solutions of Cruciforms one through three with a concentration of 50 milligrams per 10 milliliters, di chloro methane, and set aside.
Weigh out 500 milligrams of the carboxylic acid analyte in six 16 milliliter DR vials and add 16 milliliters of di chloro methane ethyl acetate, acetonitrile nn, dimethylformamide isopropyl alcohol or methanol. Next, add 20 microliters of cruciform stock solution to each vial and shake the vial if heterogeneous allow the corresponding solution to settle. Position the uncapped vials on a glass plate under the UV lamp with a distance between the camera lens and the sample vials of 60 centimeters.
Turn on the UV lamp and turn off the room light. Take pictures varying the exposure times from 0.25 to 15 seconds for each solution to produce images reflecting the color of remission. Following this, prepare a 1.0 times 10 to the negative fourth molar solution of cruciform four in di Chloro methane and set aside.
Next, prepare five individual solutions for each onic acid analyte by dissolving 50 milligrams of the analyte in three milliliters each of Acetonitrile. 1, 2, 4, tri chloro benzine di chloro methane cyclo heine, and chloro benzine. Transfer 1.8 milliliters of each of the analyte solutions into five separate 10 by 10 millimeter quartz cuvettes.
Then add 20 microliters of the previously prepared cruciform solution into each of the five cuvettes and stir the two solutions to homogenize. If any precipitation is observed, allow the corresponding solution to settle. Place all five Q vets onto a glass plate and irradiate them from the top.
Using a handheld 365 nanometer UV lamp, ensure that the room is dark and immediately take a digital photograph of the emission colors of the solutions with a distance between the camera lens and the cuvettes of 45 centimeters and a shutter speed of 0.5 seconds. Next, prepare at least 80 milliliters each of 1.0 times 10 to the negative six molar solutions of cruciform four in Acetonitrile 1 2 4, tri chloro benzene cyclohexane di chloro methane and chloroform dissolve 152.6 milligrams of two six DI chlorophyl onic acid in 40 milliliters of each of the cruciform solutions. Separately dissolve 97.6 milligrams of phenyl onic acid in 40 milliliters of each of the cruciform solutions.
Then dissolve 40 milligrams of the desired amine analyte into two milliliters of each onic acid cruciform solution. For each Amin analyte, prepare five solutions with two six di chlorophyl onic acid and five solutions with phenyl onic acid as additives. Transfer aliquots of the 10 prepared solutions into 10 separate quartz cuvettes.
Place the two five vete sets onto a glass plate irradiate at 365 nanometers by a handheld UV and immediately photograph using the settings described previously using an image processing program. Cut out a representative square segment from the digital photographs of the emission colors of each photographed vial. Then organize these cutouts into panels similar to those shown here.
If quantification of differences in emission color is desired, extract red green blue values from the panels and then statistically treat them to obtain relative standard deviations of emission colors of one analyte relative to another. Use the following equation. Finally, use the equation to identify unknown carboxylic acid analytes and to determine the deviation between the unknown analyte to all substances of the calibration data set using cruciform Fluor one through three in six different solvents, the fluorescent responses were found to be dependent upon the concentration and the structural identity of a carboxylic acid pictured.
Here is the digitally recorded emission color of all flora four solvent carboxylic acid combinations. This array exhibits 18 characteristic emission colors per analyte, which can be used to uniquely characterize and analyte using the red green blue values of the emission color. All analytes can be discerned relative to carboxylic acids, a one through a 10 and identified as shown in the auto correlation plot here using a completely analogous procedure.
Onic acids B one through B nine are readily discriminated from each other using cruciform four as evidenced by the emission color panel and the correlation graph shown here. Analysis of amines is achieved using situ two formed complexes of cruciform four with a large excess of onic acid additives, two six di chlorophyl onic acid and phenyl onic acid. These complexes whose emission colors are different from those of the pure cruciform can respond to the amine analytes in two ways.
A means more basic than purine displace cruciform four from its complexes with onic acid additives, thus regenerating the emission colors of pure uncomplex cruciform four. On the other hand, less basic species appear to bind to the cruciform onic acid complex without destroying it, and this event results in the modulation of the complexes fluorescence emission. Therefore, vicarious sensing methodology is characterized by a leveling effect wherein analytes above a certain threshold of can no longer be discriminated from each other Once mastered, this technique can be done in a couple of hours if it is performed properly.
After watching this video, you should have a good understanding of how to analyze carboxylic acids, baroni acids, and the means using a simple setup employing cruciform flu force in a set of several different solvents. Don't forget that working with organic solvents and UV lamps can be hazardous and precaution such as the use of well ventilated rooms and goggles should always be taken by performing this procedure.
