CHEM 231: Organic Form and Function
CHEM 231: Organic Form and Function
Laboratory 3 CSI Meadville: Preparative purification of a mixture Specific Practical Goals
to effect physical separation by leveraging chemical properties to master the technique of aqueous extraction using a separatory funnel to separate a solid from liquid using vacuum filtration to analyze samples using TLC and HPLC
The Premise Benzocaine, or ethyl p-aminobenzoate (4), is a widely used inexpensive local anæsthetic. As part of a homicide investigation, your forensics lab receives a 2 gram sample of impure benzocaine seized from a basement laboratory. The sample is believed to contain significant amounts of benzoic acid (1), most probably the result of low-quality starting materials. Your job is to use the chemical properties of benzocaine and benzoic acid to separate the two components in high purity and recovery (yield), to estimate the purity of the isolated compounds using analytical tools (balance, TLC, HPLC), and to determine the percent composition of the original sample.
Scheme 1. Proton transfer reactions of benzoic acid, ethyl p-aminobenzoate, and hydroxide
Week 1 Pre-lab Reading Technique Primers 2 & 3: Filtration & The Separatory Funnel Review of Acid-base Chemistry You will recall from freshman chemistry that the pKa of an acid corresponds to the pH at which it is exactly half ionized. Therefore, if hydrofluoric acid (HF) has a pKa of 3.2, and you make an aqueous solution of HF at pH 3.2, then the solution will consist of exactly 50% HF and 50% F . Also bear in mind that the pH (and pKa) scale is logarithmic, so that by changing the pH by one unit, we change this balance tenfold. In other words. an HF solution at pH 4.2 would consist of 90% F , and one at pH 5.2 would be 99% F . Thus, pH can be a powerful tool for modifying the charge properties of a compound. If this feels a bit rusty, you should brush off the freshman chemistry text and review the section on acid-base chemistry, particularly the passages dealing with the acidity constant. Safety Considerations You must abide by all laboratory safety rules. Benzoic acid [65-85-0]. Harmful by ingestion. Irritant; harmful by inhalation. Sodium benzoate [532-32-1]. May be harmful if inhaled. Causes respiratory tract irritation. May be harmful if absorbed through skin. Causes skin irritation. Causes eye irritation. May be harmful if swallowed. Benzocaine [94-09-7]. May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. May be harmful if swallowed. Dichloromethane [75-09-2]. Harmful if swallowed. Causes skin and eye irritation. Suspected of causing cancer. May be harmful if inhaled. Causes respiratory tract irritation. Harmful if absorbed through skin. Causes skin irritation. Causes eye irritation. Harmful if swallowed. Sodium hydroxide solution [1310-73-2]. Causes severe skin burns and eye damage. May be harmful if inhaled. Material is extremely destructive to the tissue of the mucous membranes and upper respiratory tract. May be harmful if absorbed through the skin. May be harmful if swallowed. Concentrated hydrochloric acid [7647-01-0]. Causes severe skin burns and eye damage. May cause respiratory irritation. Procedural Overview Important Note: You will be evaluated on the amount of compound you recover. Therefore, make sure all transfers are quantitative. If you are unsure how to do this, ask your instructor. Likewise, purity counts, so make sure all glassware is clean before using. As soon as you receive the sample, record its weight and ID number in your notebook. Completely dissolve the 1 solid in 20 mL of methylene chloride, taking a small aliquot of this solution for a TLC sample. Transfer the solution 2 to a separatory funnel and partition using an equal amount of 3M sodium hydroxide. Separate the phases into two Erlenmeyer flasks, taking care to keep track of which flask contains which phase (aqueous or organic). Extract the organic phase with another portion of 3M sodium hydroxide and separate the phases again into two 3 Erlenmeyer flasks, combining this aqueous phase with the last. Dry the organic phase with sodium sulfate and 4 remove the solvent using rotary evaporation. To the aqueous phase carefully add concentrated hydrochloric acid with stirring until the mixture is acid to litmus. Cool in an ice bath and isolate the resulting precipitate by vacuum filtration. Notes: 1
2 3 4
to make the TLC sample, add 2 drops of the solution to a small sample vial. Close the vial tightly, label it clearly, and carefully store it for later analysis. always use a ring stand for a sep funnel—never use a clamp for holding the funnel. make sure the sodium sulfate container is immediately and tightly resealed! remember to tare the round-bottom flask before you transfer the solution to it.
Week 2 Pre-lab Reading Technique Primers 1 & 7: Thin-layer chromatography & HPLC Safety Considerations You must abide by all laboratory safety rules. Exposure routes for methylene chloride are transdermal and inhalation. Avoid contact with skin and always work in a fume hood. Do not look directly into the UV lamp! Protect skin from prolonged exposure. Procedural Overview Important Note: You will need to bring a ruler and pencil to lab. Spot the two TLC samples from last week, along with TLC standards for benzoic acid and benzocaine, onto a precut silica gel plate. Develop the plate in a solvent of methylene chloride containing 1% acetic acid. Visualize the developed plate with a UV lamp and calculate all Rf values. After weighing the isolated solids, prepare HPLC samples of each (1-5 mg in 1 mL acetonitrile) and analyze using the procedure below. HPLC Operating Procedure a.
Make sure you are operating in the proper software window (left window is for left instrument)
b.
Ensure that the system is available—a green "Ready" box will show in the upper left quadrant
c.
On the top menu, go to RunControl Sample Info; enter your sample ID in the “Sample name:” field
d.
With injector in “Load” position (counterclockwise), flush out sample loop with methanol 2 times using a full syringe (handle syringe with care!)
e.
Load sample into injector using 2 full injections, avoiding any air bubbles in the syringe; leave the syringe in the injector port after the last injection
f.
Turn injector to “Inject” position (clockwise), leaving the syringe in the injector; software will set a zero point on the trace and the pump will start
g.
Do not make any hardware adjustments while a run is in progress
h.
When the sample has finished, the pump will stop and your trace will automatically print out
revision 9-17-14
Laboratory 3 CSI Meadville: Preparative purification of a mixture Specific Practical Goals
to effect physical separation by leveraging chemical properties to master the technique of aqueous extraction using a separatory funnel to separate a solid from liquid using vacuum filtration to analyze samples using TLC and HPLC
The Premise Benzocaine, or ethyl p-aminobenzoate (4), is a widely used inexpensive local anæsthetic. As part of a homicide investigation, your forensics lab receives a 2 gram sample of impure benzocaine seized from a basement laboratory. The sample is believed to contain significant amounts of benzoic acid (1), most probably the result of low-quality starting materials. Your job is to use the chemical properties of benzocaine and benzoic acid to separate the two components in high purity and recovery (yield), to estimate the purity of the isolated compounds using analytical tools (balance, TLC, HPLC), and to determine the percent composition of the original sample.
Scheme 1. Proton transfer reactions of benzoic acid, ethyl p-aminobenzoate, and hydroxide
Week 1 Pre-lab Reading Technique Primers 2 & 3: Filtration & The Separatory Funnel Review of Acid-base Chemistry You will recall from freshman chemistry that the pKa of an acid corresponds to the pH at which it is exactly half ionized. Therefore, if hydrofluoric acid (HF) has a pKa of 3.2, and you make an aqueous solution of HF at pH 3.2, then the solution will consist of exactly 50% HF and 50% F . Also bear in mind that the pH (and pKa) scale is logarithmic, so that by changing the pH by one unit, we change this balance tenfold. In other words. an HF solution at pH 4.2 would consist of 90% F , and one at pH 5.2 would be 99% F . Thus, pH can be a powerful tool for modifying the charge properties of a compound. If this feels a bit rusty, you should brush off the freshman chemistry text and review the section on acid-base chemistry, particularly the passages dealing with the acidity constant. Safety Considerations You must abide by all laboratory safety rules. Benzoic acid [65-85-0]. Harmful by ingestion. Irritant; harmful by inhalation. Sodium benzoate [532-32-1]. May be harmful if inhaled. Causes respiratory tract irritation. May be harmful if absorbed through skin. Causes skin irritation. Causes eye irritation. May be harmful if swallowed. Benzocaine [94-09-7]. May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. May be harmful if swallowed. Dichloromethane [75-09-2]. Harmful if swallowed. Causes skin and eye irritation. Suspected of causing cancer. May be harmful if inhaled. Causes respiratory tract irritation. Harmful if absorbed through skin. Causes skin irritation. Causes eye irritation. Harmful if swallowed. Sodium hydroxide solution [1310-73-2]. Causes severe skin burns and eye damage. May be harmful if inhaled. Material is extremely destructive to the tissue of the mucous membranes and upper respiratory tract. May be harmful if absorbed through the skin. May be harmful if swallowed. Concentrated hydrochloric acid [7647-01-0]. Causes severe skin burns and eye damage. May cause respiratory irritation. Procedural Overview Important Note: You will be evaluated on the amount of compound you recover. Therefore, make sure all transfers are quantitative. If you are unsure how to do this, ask your instructor. Likewise, purity counts, so make sure all glassware is clean before using. As soon as you receive the sample, record its weight and ID number in your notebook. Completely dissolve the 1 solid in 20 mL of methylene chloride, taking a small aliquot of this solution for a TLC sample. Transfer the solution 2 to a separatory funnel and partition using an equal amount of 3M sodium hydroxide. Separate the phases into two Erlenmeyer flasks, taking care to keep track of which flask contains which phase (aqueous or organic). Extract the organic phase with another portion of 3M sodium hydroxide and separate the phases again into two 3 Erlenmeyer flasks, combining this aqueous phase with the last. Dry the organic phase with sodium sulfate and 4 remove the solvent using rotary evaporation. To the aqueous phase carefully add concentrated hydrochloric acid with stirring until the mixture is acid to litmus. Cool in an ice bath and isolate the resulting precipitate by vacuum filtration. Notes: 1
2 3 4
to make the TLC sample, add 2 drops of the solution to a small sample vial. Close the vial tightly, label it clearly, and carefully store it for later analysis. always use a ring stand for a sep funnel—never use a clamp for holding the funnel. make sure the sodium sulfate container is immediately and tightly resealed! remember to tare the round-bottom flask before you transfer the solution to it.
Week 2 Pre-lab Reading Technique Primers 1 & 7: Thin-layer chromatography & HPLC Safety Considerations You must abide by all laboratory safety rules. Exposure routes for methylene chloride are transdermal and inhalation. Avoid contact with skin and always work in a fume hood. Do not look directly into the UV lamp! Protect skin from prolonged exposure. Procedural Overview Important Note: You will need to bring a ruler and pencil to lab. Spot the two TLC samples from last week, along with TLC standards for benzoic acid and benzocaine, onto a precut silica gel plate. Develop the plate in a solvent of methylene chloride containing 1% acetic acid. Visualize the developed plate with a UV lamp and calculate all Rf values. After weighing the isolated solids, prepare HPLC samples of each (1-5 mg in 1 mL acetonitrile) and analyze using the procedure below. HPLC Operating Procedure a.
Make sure you are operating in the proper software window (left window is for left instrument)
b.
Ensure that the system is available—a green "Ready" box will show in the upper left quadrant
c.
On the top menu, go to RunControl Sample Info; enter your sample ID in the “Sample name:” field
d.
With injector in “Load” position (counterclockwise), flush out sample loop with methanol 2 times using a full syringe (handle syringe with care!)
e.
Load sample into injector using 2 full injections, avoiding any air bubbles in the syringe; leave the syringe in the injector port after the last injection
f.
Turn injector to “Inject” position (clockwise), leaving the syringe in the injector; software will set a zero point on the trace and the pump will start
g.
Do not make any hardware adjustments while a run is in progress
h.
When the sample has finished, the pump will stop and your trace will automatically print out
revision 9-17-14
