Micro-extraction of urine and/or stomach contents and analysis by TLC

5) Run the TLC. Let the solvent go about 90% of the way up the plate. 6) Remove the plate from the chamber and mark the solvent front immediately with a pencil - you will use this to calculate the Rf. 7) Let the solvent dry off of the

Other information that can be obtained from a TLC analysis is the progress of a chemical reaction, which can be done by taking samples from the reaction mixture at various intervals and spotting them on TLC plates and taking down the Rf factor. Also TLC analysis can be used to determine the optimum conditions required to obtain a high yield of the desired product with little to no side products. Yield can be ascertained via TLC analysis in that the reactant and product intensity on the TLC plate will have varying degrees of intensity. At the end of the reaction, the product should have the most intensity, while the reactant has the least intensity. At the beginning of the reaction, it would be the other way around. With this, you can determine the percent yield of the

baseline (see Figure 9.4 on page 288 for help). If the solvent traveled 57 mm

Table 1: This table shows the position that the solution was at inside the graduated tube it was held in at each time interval it was measured.

In the first part, there may have been cross contamination of materials, especially since periods before us had already performed the experiment. During chromatography, our spot sizes were initially very varied, as we didn’t know exactly how much of each substance to place when we began. Thus, the substances in more abundance would have greater opportunity to travel up with the eluent, skewing the results. In the beginning, we even put 6-7 drops, causing some of the substances to mix together. Touching the TLC plate may also have disrupted some of the substances traveling up.

In Table 1, fluorene travels further up the TLC plate versus fluorenone since a more nonpolar 80:20 pentane-ether developing solvent is used. Since fluorine is more nonpolar than 9-fluorenone, it will be more soluble in the highly nonpolar solvent and enter the mobile phase easier than the polar 9-fluorenone. Fraction 2 is expected to have two different spots because this is the collection of the eluted solution between Fraction 1 and Fraction 3, where the fraction contains both the remaining fluorene and 9-fluorenone. Fraction 1, however, should not contain any 9-fluorenone since if correctly performed only fluorene and pentane should have eluted out in the mobile phase. A crack in the column during the elution of Fraction caused one section

In this experiment, separation of fluorene and 9-fluorenone took place using column chromatography. The pureness of the separate compounds was checked using TLC and melting point analysis. Rf values were the determined from the TLC plates and calculated to compare the experimental Rf values to the starting mixture Rf values. TLC is useful to separate tiny samples and is considerably quick, while chromatography is used to separate considerably larger mixtures but takes more time. The melting point of both desired molecules was found and contrasted with the actual values. The melting point is used to assess the purity of each compound recovered. Column chromatograpahy separates compounds based on polarity. Column chromatography contains a mixture dissolved in solvent and put in a column that had solid adsorbent and an eluent. There are different phases in the column: a polar stationary phase (solid adsorbent), the alumina, and mobile phases (eluent), which can flow throughout the column. There are different phases due to different polarities, resulting in dissimilar bands. TLC separates compounds due to polarity. Each sample is dotted on the plate and placed in eluent and the samples quickly separate due to polarities and adsorption. The spots can either move far or short distances. The weakly adsorbed compounds move faster than the stronger adsorbed components. The textbook mentions simple elution stepwise elution to elute each fraction in the column chromatography. Simple

The standard reference mixture did not have a Rf value because it ended up moving off the TLC plate. For part 2 of the experiment, the Rf values for acetaminophen, aspirin caffeine and ibuprofen were .50, .67, .18, and .82. Also for part 2 of the experiment the Rf values for acetaminophen, aspirin and ibuprofen from pills were .42, .54 and .69. The unknown mixture from part 1 is fluorenone. The standard reference mixture from part 1 was composed of all three compounds.

Since the stationary phase is the silica gel (very polar), polar compounds will have a strong affinity for it and travel slower up the TLC plate resulting in a low Rf. Since Compound C is the most polar molecule, it will have a high affinity for the stationary phase and travel the slowest, so it will have the lowest Rf. Compound A is less polar than Compound C, so it will travel faster and further leading to a higher Rf. Compound B is the least polar compound so it will have a higher affinity for the mobile phase meaning it will travel the furthest resulting in the highest Rf.

The bigger the Rf, the further the spot moved and that the Rf should be the same for a component regardless of how far the solvent moves. Results & Discussion: Comparing the efficiency between the plates with the unknown was quite effective, the first plate contained the unknown, Aspirin, Acetaminophen and Ibuprofen. The outcomes came out very clear, Aspirin had a measure of 2.9cm, Acetaminophen had 2.6cm , Ibuprofen had 3.5cm and my unknown had 3cm. It was clear that Ibuprofen had too high of a distance, Acetaminophen was too low of a distance, while Aspirin was right around the length my unknown traveled.

After realizing that the solvent moved up in ¾ the TLC plate was removed and marked where the solvent reached by using a pencil. The next step was spotting under UV light, and draw around different spot appeared. The Rf value obtained, showed a close range between Tylenol and acetaminophen, and between Anacin and Acetylsalicylic acid (Aspirin). This combination were also used in Co- spotting where on TLC plate, only two dots were drawn and placed a sample of acetaminophen overlapping Tylenol and Anacin overlapping aspirin on a second spot, and then used UV light gave two spot as predicated. Throughout this experiment possibly some error could have happened, like not putting the a lid on developing chamber as soon as possible, the solvent dried so fast when removed from glass jar this could have caused error in drawing a line in the wrong place and therefore interfering with our Rf values. TLC experiment are useful in biochemical analysis in separation of biochemical metabolites or constituent from its body fluids, blood plasm, and urine. TLC can be used in pharmaceutical industry for detection of impurity in a pharmacopoeias chemical (TLC

Incorporation of assay controls included setting up a spectrophotomer and running the chart recorder with a full-scale deflection before the start of the assay. The set recorder had a corresponding value of 1 for the change in the absorbance. Therefore, prior testing was done to observe whether a change occurred in the readings. This helped to indicate that the results were valid, as they could have been affected by a fault during the setting up of the spectrophotometer. On the other hand this was considered as one of the controls for the experiment. Nevertheless, a new cuvette had to be used for each assay.

We are testing for the presence of lipids, the amount and how polar they are. The bigger the dot, the higher the concentration and the farther the dot the more nonpolar it is. A positive test for lipid chromatography appears to have phospholipids, fatty acids and triglycerides in that order on the powdered strip while a negative test appears to not have those three in that order or even have all those three show up. We tested chromatography solvent and placed it on the silica gel strip. Our hypothesis was that the nonpolar molecules in the solvent would move up farther from the solvent causing the Triglycerides to be at the top of the paper but the polar molecules would move slower causing the phospholipids to stay at the bottom of the paper.

Once the solvent line has risen, the plate was removed and allowed to dry, and the solvent line marked. Once the TLC plate was dried, it was analyzed under a UV Light and circles placed around visible spots where the solutions had traveled (Figure 3). The Rf values were calculated, Table 3, by using the measured distance the spot

Therefore, a mathematical equation that relates the concentration of analyte absorbed in the PDMS pellets as a function of uptake or release rate constants k, equilibrium concentration of the compounds in the polymer and the t95% can be used (Smith, Oostingh, and Mayer 55). T95% is the time to reach 95% of the maximum measure concentration or steady state (Smith, Oostingh, and Mayer 55), so at t95% the concentration of analyte at time t is 0.95 of the equilibrium concentration (Ct = 0.95Ceq) (Brown and A. 4097).