Exploring Solid Phase Extraction Biology Essay

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In solid phase extraction (SPE) compounds that are dissolved in a liquid are separated and isolated by passing the sample through a stationary phase. These compounds can be extracted from the stationary phase by using a different eluting solvent. The effects of column conditioning, pH, eluting solvent strength, column loading rate, and elution speed on solid phase extraction were tested by comparing multiple trials of the extraction of brilliant blue FCF and allura red AC.

In order to submit a sample to solid phase extraction, the sample must be pretreated by diluting the sample, adjusting the pH or adding an internal standard. The SPE column is then conditioned to prepare the stationary phase to retain the desired analyte. When the sample is applied and allowed to flow through to the column, functional groups in the stationary phase attach to the components of the analyte. Undesired components of a mixture are then washed away using a solution of different polarity. An elution is then conducted to extract the analyte from the column and collect it for analysis or further testing. (Reference 2)

Solid phase extraction is often used in forensic toxicology for medical and/or legal investigation of death, poisoning or drug use. Samples of blood and urine are the most used in the determination of alcohol and drug usage. For blood analysis sample pretreatment is used to separate the alcohol and drugs from red blood cells, white blood cells platelets, plasma and proteins. In urine analysis sample pretreatment is used to separate alcohols and drugs from waste products, salts and some proteins. Once the alcohols and drugs are extracted using solid phase extraction, other techniques like HPLC can be used to determine the concentration of each. (Reference 2)

Experimental:

A RapidTrace SPE workstation was used to test the effects of column conditioning, pH, solvent strength, column loading rate and elution speed on the extraction of coloring dyes from solutions. The blue analyte solution was prepared by conducting a 1:4 dilution of the provided brilliant blue FCF (blue # 1 solution). The neutral and acidified allura red solutions (red # 40) were provided. All samples were analyzed using C-18 cartridges by Varian.

The effect of column conditioning on SPE was tested by conditioning a column used to analyze a sample of blue #1 and leaving a second column unconditioned. Parameters used are noted in Table 1 (Appendix).

The effect of pH was tested by comparing a sample of neutral red dye # 40 to a sample of acidified red # 40, while keeping all the columns conditions the same. Parameters used are noted in Table 2 (Appendix)

The effect of the eluting solvent strength was tested by increasing the concentration of the eluting solvent (methanol) to three different columns containing blue analyte samples and comparing the extractions obtained from each column. Parameters used are noted in Table 3 (Appendix)

The effect of the column's loading rate was tested by comparing how three blue #1 samples appeared in a column after being loaded at different rates. Parameters used are noted in Table 4 (Appendix)

The effect of the elution speed of a column was tested by using 70% methanol to elute three columns containing blue analyte at different rates and comparing the extractions obtained from each column. Parameters used are noted in Table 5 (Appendix)

Results:

When comparing the two cartridges obtained from the column conditioning test, the cartridge containing the conditioned column displayed a separation of the blue analyte into a compacted blue strip near the top of the column. The column that was not conditioned, displayed the blue analyzed scattered throughout the cartridge. The intensity of the blue analyte was darker on the conditioned sample than in the sample that was not conditioned.

The cartridges of the neutral and acidified red, displayed no significant changes after being introduced into the column. In both cartridges, the high intensity red analyte strip was located in the upper half of the column.

The columns that were tested at different concentrations of methanol (MeOH) extracted the most amount of blue analyte from the column when the methanol was the most concentrated. The columns in which 5% and 20% methanol were used as eluting solvents made the blue analyte strip appear dark blue and to be located close to the top of the column. The column in which the concentrated methanol was used contained the lightest blue colored strip located close to the top of the column.

The columns in which the blue analyte was loaded at 1 mL/min and 10 mL/min showed almost no difference in the isolation of the blue dye. For these two columns the blue strip was located in the upper half of the column. In contrast, the column that was loaded at a rate of 30 mL/min showed a lighter blue analyte strip undefined and located at a lower position than the other two rates.

The columns eluted with 70 % methanol at a rate of 1 mL/min and 10 mL/min extracted similar intensities of blue colored samples from the column. Their strips were located close to the surface of the column and displayed a light blue color. The column that was eluted at a rate of 30 mL/min extracted a lighter blue colored sample and displayed a darker strip of blue analyte than the previous elution rates.

Conclusion:

In this experiment, the effects of column conditioning, pH, solvent strength, column loading rate, and elution speed were tested on the extraction of dyes from water by solid phase extraction. Column conditioning proved to be the most critical step in order to obtain a proper extraction of an analyte. No significant differences were found when analyzing the extractions of neutral and acidified red dye analytes. During the elution solvent strength test, it was found that the most analyte could be extracted when the column was eluted with the concentrated methanol. At rates between 1 mL/ min and 10 mL / min, the loading rate of the sample into the column, and the elution rate of the analyte from the column provided the most favorable results.

Discussion:

Some advantages of performing solid phase extraction over other types of extraction include the low quantities of solvents used and waste produced, less sample handling and preparation making it relatively simple to perform and the availability to make solid phase extraction an automated process. (Reference 1)

There are two types of conducting solid phase extraction. Normal phase SPE involves a non-polar mobile phase and a polar stationary phase located inside a column. In normal phase SPE, retention of the analyte is caused by polar functional groups in the analyte and polar groups of the stationary phase in the column. Silica is the main material used as the packing material in the columns. Some groups bonded to silica used in normal phase SPE include cyanopropyl (LC-CN), diol (LC-Diol) and aminopropyl (LC-N). (Reference 6)

Reversed phase solid phase extraction involves a polar mobile phase and a non polar stationary phase located inside a column. In reverse phase SPE, retention of the analyte is caused by attractive forces between the non-polar groups in the analyte and non-polar groups found in the stationary phase in the column. In reversed phase SPE, some groups bonded to the silica found inside the columns include octadecyl (LC-18), octyl (LC-8), butyldimethyl (LC-4) and phenyl (LC-Ph). (Reference 6)

In both types of SPE, extractions are carried out inside columns containing the stationary phase. Frits are placed at the top columns in order to ensure that the column stays compact and prevent material leakage. Frits are made of polyethylene or copolymers with average pore sizes of 5-50 microns. (Reference 5)

Solid phase extraction can be performed using a vacuum or positive pressure from air or nitrogen to force the solutions through the column. An advantage of performing SPE using vacuum manifolds is the ability for a high number of samples to be processed simultaneously saving time and effort. Positive pressure from air or nitrogen provides a faster and more precise rate of flow for sample loading and solvent elution than vacuum manifolds. A disadvantage on using the vacuum manifold is that if the vacuum is opened too quickly, the sample could cause channeling in the column and the sample won't be able to interact as much as it should. Positive pressure manifolds are not automated and it would take time to conduct a large number of samples. ((Reference 4 and 2)

The column conditioning test results illustrate that in order for SPE to properly separate an analyte, the column requires conditioning to activate the functional groups in the column and prepare them for accepting the sample. The column was first exposed to methanol (MeOH), an organic solvent to "stretch out" the functional groups in the C-18 column and allow them to grab analytes passing by. After the organic the methanol was used, the column was conditioned with water to displace any excess organic solvent and create an acceptable environment for analytes to be retained.

The pH test of the neutral and acidified red analyte did not displayed significant differences and indicates the presence of error in making the sample or in setting up the parameters for the conditioning of the column.

The results obtained by using different solvent strengths to elute the column show that the nature and concentration of the elution solvent must provide an environment such that the analyte will rather be dissolved in the eluting solvent than stay in the solid phase in the column. In the experiment, the highest concentration of methanol resulted in a darker shade of blue analyte (high concentration) obtained in the receiving test tube and a lighter blue strip left behind on the column. The 5 % and 20 % methanol resulted in a light colored blue analyte obtained in the receiving test tubes and dark blue strips left in the column, indicating that some analyte was left behind.

When a sample is loaded into a column, the analyte will react with the stationary phase inside the column, if the sample is introduced too fast, then all of analyte might not stay in the column. The results of loading the columns at different rates indicate that the 1 mL/ min and 10 mL / min were good rates for the sample to be introduced to the column. For both cases a well compacted dark blue strip was observed to be formed near the top of the column. The 30 mL/min rate prove to be too fast because the blue analyte strip was lighter in color and positioned at a lower location when compared to the strips in the other two rates. Given that the 1 mL/ min and 10 mL / min rates separated the blue analyte effectively, a 5 mL/ min will provide the desired results but be faster than the 1 mL /min.

When the samples were eluted using 70 % methanol, the speed in which the elution was carried out affected the amount of blue dye analyte obtained from the samples. Although the blue analyte strips were located around the same position in all the columns for this part of the experiment, the intensity of the blue analyte in the receiving test tubes was greater for the 1 mL / min and the 10 mL / min than the intensity of the blue analyte obtained from 30 mL/min. The results indicate that if the elution solvent speed is too high, then the analyte in the column won't be dissolved by the eluent entirely and some analyte will remain inside the column. Given that the 1 mL/ min and 10 mL / min rates dissolved the analyte effectively, a 5 mL/ min will provide the desired results but will require less time than the 1 mL /min.

Appendix - Table of Contents.

(All instrument parameters used in the tables can be found in Reference 3)

Table # 1: Effect of column conditioning on SPE

Table # 2: Effect of pH on SPE

Table # 3: Effect of solvent strength on SPE

Table # 4: Effect of column loading rate.

Table # 5: Effect of Elution Speed.

Table # 1: Effect of column conditioning on SPE

To 2 test tubes, add 4 mL of blue analyte solution and place them in position 1 and 2 in the sample rack.

SPE steps for 1stcolumn

STEP

Source

Output

Volume

Speed

Condition

MeOH

Org. Waste

3 mL

12 mL/min

Condition

Water

Aq. Waste

3 mL

12 mL/min

Load

Sample 1

Org. Waste

4 mL

5 mL/min

SPE steps for 2ndcolumn

STEP

Source

Output

Volume

Speed

Load

Sample 2

Org. Waste

4 mL

5 mL/min

Table # 2: Effect of pH on SPE

4 mL of red analyte in a test tube placed in position 1 of the sample rack.

4 mL of acidified red analyte in a test tube in position 2 of the sample rack.

SPE steps for 1stcolumn

STEP

Source

Output

Volume

Speed

Condition

MeOH

Org. Waste

3 mL

12 mL/min

Condition

Water

Aq. Waste

3 mL

12 mL/min

Load

Sample 1

Org. Waste

4 mL

5 mL/min

SPE steps for 2ndcolumn

STEP

Source

Output

Volume

Speed

Condition

MeOH

Org. Waste

3 mL

12 mL/min

Condition

Water

Aq. Waste

3 mL

12 mL/min

Load

Sample 2

Org. Waste

4 mL

5 mL/min

Table # 3: Effect of solvent strength on SPE

To 3 test tubes add 4 mL of blue analyte solution and place them in positions 1, 2 and 3 in the sample rack.

These conditions were kept constant for all three columns.

STEP

Source

Output

Volume

Speed

Condition

MeOH

Org. Waste

3 mL

12 mL/min

Condition

Water

Aq. Waste

3 mL

12 mL/min

SPE steps for 1stcolumn

Load

Sample 1

Org. Waste

4 mL

5 mL/min

Elute

5% MeOH

Fraction 1

2 mL

2 mL/min

SPE steps for 2nd column

Load

Sample 2

Org. Waste

4 mL

5 mL/min

Elute

20% MeOH

Fraction 1

2 mL

2 mL/min

SPE steps for 3rd column

Load

Sample 3

Org. Waste

4 mL

5 mL/min

Elute

MeOH

Fraction 1

2 mL

2 mL/min

Table # 4: Effect of column loading rate.

To 3 test tubes add 4 mL of blue analyte solution and place them in positions 1, 2 and 3 in the sample rack.

These conditions were kept constant for all three columns.

STEP

Source

Output

Volume

Speed

Condition

MeOH

Org. Waste

3 mL

12 mL/min

Condition

Water

Aq. Waste

3 mL

12 mL/min

SPE steps for 1stcolumn

Load

Sample 1

Org. Waste

4 mL

1 mL/min

SPE steps for 2nd column

Load

Sample 2

Org. Waste

4 mL

10 mL/min

SPE steps for 3rd column

Load

Sample 3

Org. Waste

4 mL

30 mL/min

Table # 5: Effect of Elution Speed.

To 3 test tubes add 4 mL of blue analyte solution and place them in positions 1, 2 and 3 in the sample rack.

These conditions were kept constant for all three columns.

STEP

Source

Output

Volume

Speed

Condition

MeOH

Org. Waste

3 mL

12 mL/min

Condition

Water

Aq. Waste

3 mL

12 mL/min

SPE steps for 1stcolumn

Load

Sample 1

Org. Waste

4 mL

5 mL/min

Elute

70% MeOH

Fraction 1

3 mL

1 mL/min

SPE steps for 2nd column

Load

Sample 2

Org. Waste

4 mL

5 mL/min

Elute

70% MeOH

Fraction 1

3 mL

10 mL/min

SPE steps for 3rd column

Load

Sample 3

Org. Waste

4 mL

5 mL/min

Elute

70% MeOH

Fraction 1

3 mL

30 mL/min

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