The objective of this lab is to isoalte lycopene from toamto paste using a solid phase extraction technique. 

The two objectives from this part of the lab is to seperate various organic compounds along with the lycopene and introduce us to a type of extraction that's part column chromatography and part extraction while using what we know about the polarity of the subtances of intresta and the lab materials.

1. Isolating and determining amounts of natural products in tomatoes is a very important field of research.  Read the introduction sections of the papers linked below and give 3 reasons why in society determining amounts of lycopene is important.

   • https://drive.google.com/file/d/1N6uECNZzIMRfZ0oztnw-3MmMKd98rXJ4/view?usp=sharing

   • https://drive.google.com/file/d/1N99OXoKpxtkvdygAWa1YlZtjT1l_gpdg/view?usp=sharing

   • https://drive.google.com/file/d/1IbmAA1Rr1i6UeiykKb1ztw7ml9CyNUpL/view?usp=sharing

Lycopene helps to protect tissues from oxidative or photooxidative damage by free radicals.

Lycopene helps inhibit prostate cancer.

Lycopene is useful for processed and healtheir food.

2. The chemical composition of tomatoes is very complex.  Refer to the paper linked at https://drive.google.com/file/d/1IbmAA1Rr1i6UeiykKb1ztw7ml9CyNUpL/view?usp=sharing that has been highlighted on canvas and conduct additional research on one amino acid and one organic acid that you find are interesting.  Report on the structure, properties and give two interesting pieces of information about each one of the chemicals that you researched.  Add these two chemicals to your “compound of study” section along with Lycopene.

Tyramine amino acid:

-Tyramine helps to regulate blood pressure.

-Tyramine is produced in the body and is supplimented through various foods.

Citric acid

-Citric acid is used in various cosmetics, foods, and cleaning products.

-Citric acid is also present in animal tissue.

3. Before working with the SPE cartridge, the tomato has to be diluted with water.  To make sure that the organic acids are in their non-polar acid form, we have to adjust the pH of the water.  Should the water be made acidic or basic? Briefly explain.

4. Before adding the tomato to the cartridge, the cartridge needs to be preconditioned. The cartridges are dry.  Answer the following questions.

   • The cartridges are to be preconditioned in a solvent that is of the opposite polarity of the resin.  In the lab will you condition the resin with a polar or non-polar solvent?  Explain.  To answer, you need to consider what the polarity of the resin will be to capture our compounds of interest.

   • The cartridges are to be preconditioned in 6-10 times the hold volume of the cartridge.  The hold volume of the cartridges we will be using are about 0.5 mL and so how much should we use for preconditioning?

The cartridges must be pre-conditioned with a polar solvent because the resin is non-polar to capture lycopene, a non-polar compound. We will be using about 3-5 mL of solvent.

5. Because there are a wide variety of chemical compounds that we wish to isolate, we are going to extract from the solid into several kinds of liquid solvents.  The hope is that each solvent only extract one or two compounds of interest.  This is a trick often used by natural product chemists to attempt to separate and isolate chemicals from one another.  We have tried the same kind of trick before in an extraction lab by manipulating the pH of the extract.  In this lab, we are manipulating the polarity of the extract.  The rule is to begin with the most polar solvent and end with the most non-polar solvent.  Rank the following solvents in order of most polar to least polar: acetonitrile, ethyl acetate, isopropanol, methanol, tetrahydrofuran, water.

Ranking from most to least polar:

water > methanol>  acetonitrile > isporopanol > ethyl acetate > tetrahydrofuran

6. Research the SPE cartridge type that you have signed up using the sign up sheet on the canvas page and  give a brief report.  You must report on the chemical composition of the resin in the cartridge (use words to describe and don’t upload a picture).  You must state what kinds of compounds (polar or non-polar) that it is best used with.  You must state if you would predict it would work well considering the objectives of this laboratory activity.  

Si is an unbonded silica sorbent. It uses hydrogen bonding to isolate polar compounds. It is best used when trying to isolate polar compounds. Considering the compound of interest is lycopene whihc is non-polar, I don't predict it successfully isolating lycopene but instead isolating a polar organic compund within the tomato paste.

7. After reading the introduction and reviewing the links provided, what technique have you performed in the past that is a lot like solid phase extraction (SPE).  Explain in good detail.

A technique preformed in the past similar to SPE was colum chromatography. Column chromatography used silica gel, sand, fiber glass, and a slected solvent to isolate two seperate comounds with simialr or the same physical properties from one another. This technique is used again in SPE to provide powerful purification to the compund.

• Two beakers, 15 test tubes, and a finger flask were cleaned with soap anjd water.

• 0.30 grams of tomato paste was measured and put  into one of the clean beakers.

• a flask containing 30 mL of water was measured out and HCl was added until it reached a pH of about 4.5.

• The solution was added to the tomato paste and mixed until it was mostly dissolved. 

• The solution was then vaccum filtered to remove all solids within the mixture.

• 5 solvents were measured into 3 mL fractions into test tubes with two tubes for each solvent resulting in 10 tubes.

• A side arm flask was set up to the vaccum and a tube was placed inside the flask to collect each sample.

• The column was then conditioned with water running which was fully filled and ran through the column 3 different times being careful the coumn didn't completly dry.

• 5 mL of the tomato extract was then ran through the column followed by 3 more full rinses with water letting the water run all the way through and the column dry.

• The column was then ran through with 3 mL of the most polar solvent which was methanol.

• The tube collecting the methanol and extracted compounds was then put into a heated sand bath to evaporate some of the solvent.

• This was repeated with the second fraction of methanol.

• This was repeated in decreasing polarity with acetylnitrile, isoproponyl, ethyl acetate, then ether and they were left to boil to remove the solevnt.

• Once the solvent had evaporated from each of the tubes, they were anlyzed by TLC with a 3:1 hexane ethyl acetate solvent.

• Our TLC plates were analyzed and compared to other groups with other column types.

Using solid phase extraction (SPE), various organic compounds were eluted. Our group used a column with Si cartilage which was a polar compound meant to extract polar compounds. Our group failed to evaporate enough of the solvent to get a clear and dark spot on our TLC analysis but, an analysis of the plates present still indicates the nature of the SPE and the column type chosen. Looking at our group's TLC plates, its obvious polar compounds were eluted as the spots were the most present for methanol, which was the most polar compound. Spots were present for the other compounds but the more non-polar the compound was, the more it moved to the top of the plate with the majority non-polar solvent. As the compound became more non-polar, past methanol, The spots started to look the same. This could be an indication that the same compound was being eluted throughout the experiment and therefore the same spots were present on the TLC plate. The non-polar solvents seem to have lighter spots but no distinct conclusion can be made because lots of solvent was still present within the solution when the analysis was performed. In total, about 7 fairly distinct compounds can be observed from the TLC plate This leads me to believe there are only so many organic substances within the processed tomato paste that also work well with the polar SPE column.

Looking at the other group's TLC plate, they have some very distinct spots that are a result of the NH2 column they used which served best to remove polar substances. Their SPE cartilage doesn't seem to be as polar as ours as its spot for methanol, the most polar compound, is lighter than many other of their spots. The darkest spot is the acetonitrile which speaks to the polarity of the compound. Our TLC plate and the other groups' TLC plate both have spots for acetonitrile which shows the similarities within the policy of the SPE columns. Their spots for isopropanol were also very light/non-existent which leads me to believe that the isopropanol solvent was difficult to evaporate leading to poor TLC spots because one of the TLC analyses at least should've displayed a clear spot due to the polarity of the column and the clear spots for acetonitrile and ethyl acetate that came before and after. As their group moved to more non-polar compounds their spots became more streaky and light similar to our groups. They ere able to identify about the same amount of spots and the same kind of spots which further supports the nature of a polar SPE column and the amount of polar organic compounds within processed tomato paste.

This technique seems to be effective in removing compounds as various spots were present in both groups analysis indicating that many compounds were isolated like predicted.

Overall, this lab was effective in teaching about SPE extraction and the properties of a column for a desired result. I learned about SPE extraction and how to properly pack and utilize its function for small amounts of starting material. It was a new way to pack a column which was unique and cool to perform. I continued to practice TLC analysis which does an excellent job in showcasing results and compounds present. If I were to repeat this lab, I would evaporate more solvent to make clearer TLC plates for a more in-depth analysis. This lab went well and was effective in a general sense of eluting compounds based on their polarity and extracting various compounds from tomato paste but, I think with more time, more compounds could've been present upon analysis.