Lab 11: Solid Phase Extraction 

Pre-Lab:

1.  After reading the introduction above, what are two objectives that you see for this second part of the lab?  Include this in your  “objectives” section along with the objective of the normal extraction lab.

The purpose of this lab is to combine column chromatography and extraction to extract lycopene, polar sugars, organic acids, and amino acids found within tomato paste that are normally difficult to extract through other methods. In this lab, we will be using cartilage packed with resins that will allow us to extract the desired products. Each laboratory group will be using a different cartridge to allow for the comparison of results based on the different cartridge types. 

(Also pasted above in "objective" section of lab report)

2. 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

   • Lycopene has been shown to inhibit tumor growth in different parts of the body, including the colon, liver, lungs, prostate, and breasts.

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

   • Lycopene is a known antioxidant and can remove free radicals from the body.

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

   • Lycopene gives tomatoes their red appearance. A greater presence of lycopene causes a tomato to appear more red, making it more likely to be purchased by consumers and helping to increase profit within the tomato industry. 

3. 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.

Malic Acid - 

Malic acid is a dicarboxylic acid and has the chemical formula C4H6O5. 

Facts:

•  Malic acid has two known stereoisomers but only one form, the (L) form exists naturally. The (R) stereoisomer must be manufactured in a lab.

• Malic acid helps to make foods taste sour and contributes to the sour taste of green apples.

Tyramine - 

Tyramine is an amino acid and has the chemical formula C8H11NO.

Facts:

• Tyramine has been known to help regulate blood pressure. 

• Tyramine is unable to cross the blood-brain barrier.

4. 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.

The water should be made more acidic. The organic acids that we are attempting to isolate through this experiment are acidic in nature, therefore if we added a basic solution it would neutralize the acidic components, causing the product to not be isolated in the experiment.

5. 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.

A polar solvent should be used to catch the nonpolar lycopene that will move through the system. 

• • 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 is about 0.5 mL so how much should we use for preconditioning?

Approximately 3-5 mL should be used. (0.5*6=3 and 0.5*10=5)

6. 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.

From most to least polar: Water, Methanol, Acetonitrile, Isopropanol, Ethyl Acetate, and then Tetrahydrofuran. 

7. 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.  

The SPE cartridge type that my lab partner and I have signed up for is a Si cartridge. Its chemical structure has two methyl groups with two Si atoms bonded to two hydroxy groups and one additional oxygen. The Si cartridge is a polar sorbent that is used to extract polar analytes from non-polar matrices. Due to lycopene being a non-polar chemical, I feel that the Si cartridge will not be effective at isolating lycopene but it may be effective at isolating another more polar organic compound found withing the tomato paste. 

8. 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.

It seems that liquid column chromatography has a similar process to solid-phase extraction. Both processes require the use of a column, a resin for the chemicals to move through, and a solvent to extract the desired product. A good understanding of the nature of the chemicals used in both columns as well as the nature of the chemicals you are seeking to obtain as a product is also required for both procedures to ensure you are selecting the proper chemicals throughout the experimental process. 

Results:

Solvent: 3:1 hexane/ethyl acetate solvent.

The  TLC  resulted in very faint spots.

Height of the Solvent Front Plate 1 (Far Left):

Height of the Solvent Front Plate 2 (Middle): 

Height of the Solvent Front Plate 3 (Far Right):

Conclusion: This lab was effective at eluting compounds from tomato paste. However, due to poor evaporation techniques, the spots present on the TLC plates were faint. With more time, it is possible that more compounds would have been present upon analysis.