Objective Statement: The purpose of this lab is to learn the new technique of Solid Phase Extraction to isolate lycopene. You will learn about the technique of SPE and the importance of the resin used in your cartridge for extraction. Where you will also compare your results with your classmates to determine which cartridge was the most effective for lycopene extraction.

Lycopene

Polarity: non-polar

Mass:536.78 g/mol

MF: C40H56

Density: 0.889 g/cm3

BP: 660.9 °C

MP: 177°C

Solubility: Soluble in benzene and ethyl ether. Insoluble in water and ethanol

Tryptamine

Polarity: polarpolar

Mass: 160 g/mol

MF: C10H12N2

Density: 1.2 g/cm3

BP: 137 °C

MP: 118°C

Solubility: Soluble in water, ethanol, and methanol. Insoluble in chloroform.

Acetic acid

Polarity: polar

Mass: 60.052 g/mol

MF: C2H4O2

Density: 1.05 g/cm3

BP: 117.9 °C

MP: 16.7 °C

Solubility: Soluble in wate, alchol and ether. Insoluble in carbon disulfide.

Pre-Lab Questions:

What are the two objectives of this lab?

1. Learn the technique of SPE. 2. Determine the best cartridge for extraction of lycopene.

Give 3 reasons why in society determining amounts of lycopene is important.

Lycopene can be used for sun protection, improve heart health, and lower the risk of certain types of cancer.

Conduct additional research on one amino acid and one organic acid that you find interesting. Report on the structure, and properties and give two interesting pieces of information about each one of the chemicals that you researched.

Tryptamine: an indole—a fused benzene and pyrrole ring, and a 2-aminoethyl group at the second carbon. It is a naturally occurring alkaloid found in a variety of plants and life forms around the world and exists in more than 1500 natural varieties.
Acetic Acid: Acetic acid is a simple monocarboxylic acid containing two carbons. It has a role as a protic solvent, a food acidity regulator, an antimicrobial food preservative, and a Daphnia magna metabolite. It is a conjugate acid of an acetate.

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 acidic or basic? Briefly explain.

The water should be more acidic. This is the way for the organic acids in the tomato to be able to dissolve and be diluted in the water. "Like" dissolves "like".

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.
  - Condition the resin with non-polar methanol.
- 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?

About 3-5 mL of solvent.

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, and water.
- Polar to least polar: water, methanol, isopropanol, acetonitrile, ethyl acetate, and tetrahydrofuran.
Research the SPE cartridge type that you have signed up for using the signup 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) it is best used with. You must state if you would predict it would work well considering the objectives of this laboratory activity.
- The cartridge that my group is doing is the amino propyl cartridge.
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.
- SPE is very similar to the technique of using a separatory funnel. Certain solvents are added to your compound of interest to extract certain polarities or types of compounds out of a solution. In the case of SPE however, solvents are pushed through a solid phase of a mixture, extracting out desired polarities of compounds in a resin by each wash/run-through. A separatory funnel uses solvents that are drilled around in the solution to grab onto and separate desired compounds into a certain type of solution (organic or aqueous). But, it is the same concept of adding a certain polarity/type of solvent to extract a desired compared that shares those properties.

Procedure:

Activity 1 (Setup of SPE):

Weigh approximately about 0.3 g (measured exactly) of tomato in the cleaned beaker.
Dilute the tomato with exactly 100 times the amount of water and adjust to the proper pH. For instance, If you measured out 0.35 grams of paste then you need to dilute it with 35 mL of water. After diluting, mix very well so that the paste is completely dispersed, and then vacuum filter with a Buchner funnel to remove all of the solid plant material that doesn’t dissolve.
Into separate test tubes collect 3 mL of methanol, acetonitrile, isopropanol, ethyl acetate, and ether. You need to collect two 3 mL portions of each solvent. Be sure to label the tubes. You will use these solvents to selectively extract materials from the SPE cartridge in order of polar to non-polar.
In the other beaker collect approximately 20 mL of DI water.
using the steam bath apparatus, make a sand bath and begin heating. The temperature of the sand needs to be warm, not hot.

Cretaed a vacuum set-up for the SPE column.
Thoroughly cleaned the vacuum set-up, 20 test tubes, and two small beakers with soapy water and thoroughly rinsed. Working with really small amounts of material so have to make sure all glassware is clean.
Put .30 g of tomato paste into a 150 mL beaker. 30 ml of water that was brought to a pH of 4 by three drops of HCl was added to the beaker and mixed for 5 minutes. Stir until there are no red clumps left and the solution is a murky orangish-brown.
Collected 20 mL of DI water for the next couple of steps.
Made a steam bath apparatuses and brought the sand to a warm, not a hot temperature. Was a 4 for our heating mantles.

Activity 2 (Conditioning of the Column):

Connected a vacuum to the vacuum set-up and turned on the vacuum to a 1/4 pressure.
Filled an aminopropyl chamber with water and then placed the cartridge into the hole on the rubber stopper of the vacuum set-up. Ran the vacuum until there was only a little layer of water left, do not let the cartridge go dry. Removed the cartridge before all the water pulled through.
Repeated the previous step with water three more times. If you let the cartridge dry, must restart the whole conditioning process. A definitive white layer was forming on top of the chamber, probably the result of water reacting with some of the chemicals in the aminopropyl.

Activity 3 (Loading of the Column):

Used the vacuum at 1/4 pressure, and pulled exactly 5 mL of the filtered tomato solution through the cartridge. Can not allow the cartridge to run dry.
Rinsed the cartridge with water by filling the cartridge with water and vacuuming the water out. Do not let the cartridge go dry. A small red layer formed under the first white layer from activity 2.
Repeated the previous step three more times.
After the fourth water rinse, dried the cartridge by leaving it on the vacuum for 1 minute. Turned off the vacuum.

Activity 4 (Solid Phase Extraction):

Took apart the vacuum assembly and cleaned it. Removed all traces of sugar from the inside of the flask. Using a paper towel, remove most of the water from the inside of the flask.
Secured the flask using a ring stand, placed a test tube into the flask, and then fit the rubber stopper over the top with the glass tubing extending into the test tube.
Filled the cartridge with the first solvent, the most polar solvent, methanol. Pulled the remaining ~ 1 mL of solvent into a pipette
Turned on the vacuum only ¼ of a turn, placed the cartridge into the hole in the stopper, and while the loaded solvent pulled through, added the remaining methanol to the top. The cartridge cannot go dry until all the methanol has been passed through.
When all the methanol has been passed through, turned off the vacuum and slowly removed the cartridge so that you only slowly release the pressure. If you release too quickly, the eluent will not stay within the test tube.
Removed the test tube, labeled and placed it standing up in the sand bath with a boiling stick. If any solvent is pulled into the flask, take apart the assembly, put the solvent into the test tube then rinse the assembly with water.
Placed a new test tube into the assembly and with vacuum pressure, extracted the second portion of the same solvent.
Continued the extraction process working through the remaining solvents, one at a time, following all of the same steps. The order of use is acetonitrile then isopropanol then ethyl acetate then finally ether. Collected 10 fractions. Each of the fractions was placed in the warm sand bath for evaporation. Use boiling sticks in test tubes.

Activity 5 (Analysis):

Kept the fractions in the sand bath until all but 0.5 mL has evaporated. When evaporated, spot on a TLC plate and analyze the fractions. Made the spots as dark as possible. Some fractions without material. A hexane/ethyl acetate combination seems to work best for these compounds.
Obtained results from two other groups and compare your results to theirs.

Results:

Set Up

Post packing and washing of cartridge

TLC of polar solvents

Solvent System: 3:1 Hexan : Ethyl acetate

4A & 4B: Ethyl acetate

-No data

5A: Tetrahydrofuran

-Spot 1 (bottom): Rf = 2.2 cm / 4.0 cm = .55

-Spot 2 (darkest): Rf = 2.9cm / 4.0 cm = .725

-Spot 3 (top): Rf= 3.5 cm / 4.0 cm = .875

TLC of non-polar to slightly-polar solvents

Solvent System: 3:1 Hexan : Ethyl acetate

1A: Methanol first run

-Spot 1(bottom): Rf = 2.1 cm / 4.0 cm = .525

-Spot 2(darkest): Rf = 2.9 cm / 4.0 cm = .725

1B-3B: Methanol second run, isopropanol, and acetonitrile

-No data

TLC of group #2:

Si (unbonded silica) cartridge

Solvent System: 3:1 Hexan : Ethyl acetate

Methanol A:

-Spot 1: Rf = 3.9 cm / 4.2 cm = .93

Methanol A:

-Spot 1: Rf = 3.9 cm / 4.2 cm = .93

Isopropanol A:

-Spot 1: Rf = 4.0 cm / 4.2 cm = .95

Conclusion/Discussion:

My least polar and most polar fractions have spots, all other fractions had no indication of material in them. This indicates the compounds in tomatoes are either pretty polar or not polar at all. It was interesting to see though that even despite the major difference in polarities, my first and ninth fractions had a spot with the same Rf value. This was most likely due to my solvent system being a mixture of non-polar and polar solvents. this means that the SPE extraction process is very selective. With using a variety of solvents, and proper packing of the cartridge, I was able to effectively select and extract different compounds on the opposite ends of the polarity spectrum. Both compounds dilute at the same rate in my solvent system, indicated by the same RF value between the two compounds. In total, I observed five spots between those two fractions. Indicating five major organic compounds in tomatoes. 3 compounds being non-polar and 2 compounds being very polar. However, I do not see high concentrations of these compounds. My spots were not very dark. Only two of the spots, the ones with the same Rf values, are dark. The other three, two non-polar and one very-polar, were very dull and hard to see. When comparing to my Lab9A, I still see the same Rf value of lycopene in the non-polar plan, indicating that lycopene was extracted, but this lab proved the presence of four other compounds. Lycopene was still present, but there are two more non-polar, and two more polar compounds found in tomatoes that were not accounted for in the previous lab. Indicating that normal extraction was more effective than extracting Lycopene on its own. however, SPE was way more educational and effective in extracting a variety of compounds with opposite polarities. The comparison, the other group above, however, had much different results. They only extracted non-polar compounds. Whereas the methanol and isopropanol solvents were only effective in extracting compounds. This is most likely due to their starting different cartridges that most likely did not allow for polar compounds to pass through. They, however, were more effective in extracting Lycopene, or compounds like lycopene. Without multiple spots, and only extracting non-polar compounds, what they did extract was more effective in only getting lycopene. the silica cartridge was a lot more effective in only extracting one variety of chemical compounds.

So overall, SPE was more effective in extracting a variety of compounds. Normal extraction was a lot more effective in extracting a polarity-specific targeted compound. The type of cartridge used also plays a major role in either allowing or determining certain polarities to be extracted through SPE.

Reflection:

In this lab, I learned about the importance of using the right extraction technique for what you are trying to achieve in a lab. Many techniques have varieties that yield different products. In this case, when comparing Lab #10 and Lab #11, the difference was using SPE to extract and analyze multiple compounds from one product instead of isolating and extracting a certain compound. I do believe I could have done this lab a little more effectively as there was a lot to work through and understand that we left some of our fractions in the sand bath for too long. Some of the fractions boiled instantly and loss more than .5 mL of solution. this definitely caused some compounds to be evaporated out of the fractions and could cause some compounds that were extracted through the cartridge to not be able to analyze. So next time, I will make sure to research, understand, and better maintain the purification of the fractions for the most effective analysis of the whole lab procedure.