Experiment 3

Introduction to Solid Phase Extraction

Jessica Nicholson and Madi Kreitz

9/28/2017

Mike Miller
Introduction:

Solid Phase Extraction, or SPE, is a process that can be used to isolate the compound of

interest from the aqueous matrix allowing for further analysis. It separates solutes (mobile

phases) based on their affinities for a solid stationary phase. Our mobile phase for this

experiment will be methanol, which is a polar molecule. Our stationary phase is C18 – OH which

is highly nonpolar. Our goal in this experiment is to separate Blue Dye #1, a relatively

polar/ionic molecule, and separate Red Dye #40 in both neutral, which is slightly nonpolar, and

acidic, which is polar/ionic, forms. When red dye is acidified it will become more ionic and

move through the stationary phase and it will separate more efficiently than the neutral red dye

that is used previously with the same stationary phase due to the acidified version having a

higher affinity for the mobile phase which is moving out of the column and the neutral version

having a higher affinity for the stationary phase and wanting to remain in it.

Experimental Procedure:

Part 1: The four steps of SPE

1. Obtain a new SPE column and label it “100% methanol”

2. Obtain a clean and dry test tube and label it “100% methanol”

3. Condition: Add 2 mL of methanol to the top of the SPE column and push the liquid

through with a plunger into the waste beaker

4. Condition: Add 2 mL od DI water to the top of the SPE column and push the liquid

through with a plunger into a waste beaker

5. Load: Add 2 mL of Blue Dye #1 to solution to the SPE column and push the liquid with a

plunger into the waste beaker

 6. Wash: Add 2 mL of DI water to the SPE column and push the liquid through with a

plunger into the waste beaker

7. Elute: Add 2 mL of methanol to the SPE column and push the liquid through with a

plunger into a clean dry test tube labeled “100% methanol”

8. Keep your SPE column labeled “100% methanol” and test tube labeled “100% methanol”

to compare with the results in part three

Part 2: The effect of column conditioning

1. Label one new SPE column “conditioned” and label another new SPE column “NOT

conditioned”

2. Using the column labeled “conditioned”, repeat steps 3-4 from part 1 to condition the

SPE column

3. Load: Add 2 mL of Blue Dye #1 solution to the SPE column and push the liquid through

with a plunger into the waste beaker

4. Using the column labeled “NOT conditioned” Load: Add 2 mL of Blue Dye #1 solution

to the SPE column and push the liquid through with a plunger into the waste beaker

Part 3: The effect of solvent strength

1. Label two new SPE columns 5% methanol and 20% methanol

2. Label two clean and dry test tubes 5% methanol and 20% methanol

3. Prepare 5% methanol in a 10 mL graduated cylinder. Stir the solution carefully with a

clean glass stir rod to be sure the solution is uniformly mixed

4. Condition the SPE column labeled “5% methanol”

 5. Load: Add 2 mL of Blue Dye #1 solution to the SPE column and push the liquid through

with a plunger into the waste beaker

6. Elute: Add 2 mL of 5% methanol to the SPE column and push the liquid through with a

plunger into a clean dry test tube labeled 5% methanol

7. Prepare 20% methanol in a 10 mL graduated cylinder. Stir the solution carefully with a

clean glass stir rod to be sure the solution is uniformly mixed

8. Condition the SPE column labeled “20% methanol”

9. Load: Add 2 mL of Blue Dye #1 solution to the SPE column and push the liquid through

with a plunger into the waste beaker

10. Elute: Add 2 mL of 20% methanol to the SPE column and push the liquid through with a

plunger into a clean dry test tube labeled 20% methanol

11. Use the SPE column and test tube from Par 1 for the 100% methanol results

Part 4: The effect of pH

1. Label two new SPE columns Red Dye and Acidified Red Dye

2. Condition the SPE column labeled “Red Dye”

3. Load: Add 2 mL of Red Dye #40 solution to the SPE column and push the liquid through

with a plunger into the waste beaker

4. Condition the SPE column labeled “Red Dye”

5. Load: Add 2 mL of Acidified Red Dye #40 solution to the SPE column and push the

liquid through with a plunger into the waste beaker

Chemical Hazards:

 Hydrochloric acid is corrosive and should be handled with extreme caution

  Blue and /red Dye contact with skin or eyes must be avoided for it can irritate

 Methanol is slightly hazardous in the case of contact with skin, and contact with eyes

 Materials should be kept in the fume hood at all times

 Organic waste should be placed in the proper hazardous waste containers

Results:

Part 1:

Summarize your observations for all four steps of SPE. Be sure to clearly state what the

stationary and mobile phase look like in each step

In the conditioning step, all the solution went through and the stationary and mobile

phases remained clean, during the loading step blue dye was added, the stationary phase had a

visible line of blue and the mobile phase remained clear. Next, we wash with water the mobile

phase stays clear and the dye remains in the stationary phase. Last, we elute the dye, the mobile

phase is sent through and it turns blue on its exit and the stationary phase should be back to its

normal color.

What is the affinity of the Blue Dye for the following phases; aqueous mobile phase, SPE

column (Stationary Phase), methanol mobile phase?

The blue dye has a high affinity for the methanol mobile phase, a low affinity for the SPE

column (stationary phase), and no affinity for the aqueous mobile phase.
Summarize what is happening to Blue Dye based on its location on the column throughout

the procedure.

During the conditioning step, the blue dye is not yet present in the column, once we hit

the loading step the solute gets stuck in the stationary phase because it has a low affinity for it.

This dye is washed out with the mobile phase during the elute step of SPE.

Part 2:

Summarize your observations of the column packing after the Blue Dye had been loaded.

The conditioned column retained more of the dye than the not conditioned column.

What are your conclusions about the importance of column conditioning based on your

results and the information given in the introduction?

Conditioning is important because it prepares the stationary phase to hold the

solute/dye/analyte. Separation without conditioning will not be accurate because the dye is

already run down and weaker than when conditioned.

Part 3:
Summarize your observations of the column packing and the resulting eluent when the

Blue Dye is eluted with the different strengths of methanol, 5%, 20%, 100%.

The mobile phase of 5% methanol caused most of the dye to remain in the stationary

phase, mobile phase 20% methanol the dye is moved further through the stationary phase but still

very little exits with the mobile phase. Last, the 100% methanol fully removes all of the blue

dye.

Using your observations, state which solvent Blue Dye has the strongest affinity for and

which solvent Blue Dye has the weakest affinity for.

Blue dye has the strongest affinity for 100% methanol and the weakest affinity for 5%

methanol.

Using your results and the information from the introduction, explain why Blue Dye has

different affinity for the mobile phase based on the solvent strength.

Blue dye has a different affinity for each of these mobile phases because they each have

different polarities. 100% methanol will have the highest polar qualities, 20% will be watered

down and less polar, and then 5% will be the least polar, the less polar the substance the less

affinity.

Which solvent would be best to elute all the Blue Dye from the SPE column?

The mobile phase of 100% methanol will be the best to elute all of the blue dye from the

SPE column because it has the highest relative affinity.

Part 4:

Summarize your observations of the column packing after each solution of Red Dye has

been applied to the column.

The neutral red dye bled down the stationary phase more than the acidified red dye which

stayed in a straight line in the stationary phase.

Explain what the observations mean in terms of the affinity of each solution of Red Dye for

the stationary phase vs. the aqueous mobile phase.

The neutral red dye does have a strong affinity for the stationary phase, but it also has a

slight affinity for the mobile phase so it is taken down the slightest amount in the stationary

phase but none leaves the container. The acidified red dye has no movement as all which means

it has a very high affinity for the stationary phase and no affinity for the aqueous mobile phase.

To separate Red Dye #40 from an aqueous solution, would it be better load the column with

a neutral solution or an acidified solution?

It would be better to use an acidified solution because the lower pH of the acid will

contrast the neutral pH of the water and draw out the Red Dye.

Conclusion:

In this experiment, we used many solvents to separate and test different dyes. Blue Dye

#1 was tested against three solvents and it was found that 100% ethanol was most effective at

separating the dye. This is because our Blue Dye is a polar molecule which is paralleled by the
polar aspects of methanol. They will have a high affinity for one another. Red Dye Was not

separated because it remained in the stationary phase due to the nonpolar-nonpolar tie which was

mimicked with acidified red dye to a higher degree. Though, neutral red dye did have a slight

affinity for the aqueous mobile phase, as shown by it slightly running down the stationary phase.

The pH of the acidified red dye makes it more difficult to be separated, it would call for a polar

solvent and a nonpolar mobile phase to be able to fully separate it.

Work Cited:

Huynch, L., Henck, C., Saxton, K., & Wang, J. (n.d.). Introduction to Forensic Chemistry:

Laboratory Manual (Vol. 1). University at Albany.