Pipets, Melting Points, and Organic Synthesis Laboratory Guide

Laboratory Notebook and Preparatory Techniques

• The Why and How of the Laboratory Notebook (MtC Chapter 1):     * Before entering the lab, all procedures for the day must be described in a pre-lab entry in the laboratory notebook.     * The pre-lab should enable completion of the experiment without referring to the lab manual or the Making the Connections (MtC) book.     * During the experiment, all observations and data must be recorded directly into the lab notebook.     * Post-lab write-ups and reports are completed after the session and turned in to the TA.     * Proper Technique for Handling Pipets:         * The most frequent lab injury is cuts from Pasteur pipets sticking up in lockers.         * Pasteur pipets must be kept in a vertical position to avoid losing drops or contaminating the latex bulb.

Experiment 0: Pipets and Melting Points

• Approximating Volume by Drop Count:     * It is useful to determine how many drops from a specific pipet equal $1\,mL$.     * Example: If a Pasteur pipet delivers $1\,mL$ in $28\,drops$, adding $7\,drops$ equals roughly $0.25\,mL$.

• Calibration Procedure:     * Draw water into a $5\,3/4\,inch$ Pasteur pipet using a rubber bulb.     * Add water dropwise into a graduated cylinder until the volume reaches $2\,mL$.     * Record the number of drops to calculate the average volume per drop.     * Pasteur pipets can be permanently marked with a Sharpie at the $0.5\,mL$ and $1.0\,mL$ levels using water from a graduated cylinder as a reference.

• Filter Pipets Preparation:     * Standard Filter Pipet: Created by pushing a pea-sized amount of loose cotton into the constriction of the pipet using a metal wire. It is used to remove unwanted solids from a liquid. The plug must be snug enough not to float but loose enough to allow solvent flow.     * Filter-tip Pipet: Prepared by rolling a tiny cylinder of cotton and pushing it into the very tip of the pipet. This is used to remove small amounts of liquid from a solid, leaving the solid behind.

• Melting Point Theory:     * Melting point is an intensive property used for the qualitative identification and purity assessment of organic compounds.     * Purity Indicators: A pure sample typically has a very distinct and narrow melting point range (approximately $2-3\,^\circ C$).     * Impurities: The presence of an impurity lowers the melting point and broadens the melting point range (analogous to freezing point depression).

• Melting Point Procedure:     * Equipment: DigiMelt SRS apparatus ($Figure\,0.2$).     * Heating Rate: Use a rate of $1-2\,^\circ C/min$ during the final $10\,degrees$ prior to melting.     * Reporting: Always report melting point as a range ($T_{start}-T_{finish}$).

• Mixed Melting Points Technique:     * Used to confirm the identity of an unknown.     * If an unknown is mixed with a known identical compound, the melting point range will not change.     * If the unknown is mixed with a different known compound, the melting point will be lower and the range broader.

Experiment 1: Recrystallization

• Recrystallization Principles:     * Process involves four steps: dissolution of a solid in hot solvent, filtration of the heated solution, crystal formation upon cooling, and isolation of crystals.     * Successful recrystallization depends on solubility factors and temperature gradients.

• Hot Plate Safety:     * Never dial a hot plate above half-way or $300\,^\circ C$.     * Keep surfaces clean to avoid toxic fumes from spills.     * Be aware of the autoignition temperature (e.g., diethyl ether is $160\,^\circ C$).

• Macroscale Procedure:     * Weigh $0.5\,g$ of impure unknown.     * Dissolve in the minimum amount of hot deionized water ($DI\,H_2O$), not exceeding $25\,mL$.     * Add boiling chips to prevent bumping.     * Decolorization: Use a pea-sized amount of charcoal pellets or powder to remove colored impurities (chromophores) via adsorption. Never add charcoal to a boiling solution.     * Hot Gravity Filtration: Use a pre-warmed funnel and fluted $11\,cm$ Whatman #4 filter paper to remove insoluble impurities and charcoal without inducing premature crystallization.     * Crystallization: Allow to cool slowly to room temperature, then place in an ice bath ($0\,^\circ C$). Slow growth produces purer crystals. Scratching the glass or adding a seed crystal can induce nucleation.     * Isolation: Use suction filtration with a B\u00fcchner funnel and Whatman #50 filter paper. Wash with cold water.     * Desiccator: Dry overnight using Drierite ($CaSO_4$). Blue indicator turns pink when hydrated.

• Microscale Procedure (Craig Tube):     * Used for solids in the $10-100\,mg$ range.     * Weigh approximately $75\,mg$ of unknown.     * Heat in a $10 \times 75\,mm$ test tube with $0.5-1.5\,mL$ of hot water.     * Filter using a pre-warmed filter pipet into a $2\,mL$ Craig tube.     * Isolation: Use centrifugation instead of vacuum filtration. The Craig tube assembly is inverted in a centrifuge tube ($Figure\,1.3$); rapid rotation forces solvent past the glass interface, leaving crystals behind.

Experiment 2: Extraction

• Liquid-Liquid Extraction Theory:     * Separation based on the partitioning of a compound between two immiscible solvents (usually an organic solvent and water).     * Partition Coefficient ($K_D$): $K_D = \frac{C_2}{C_1}$, where $C$ is concentration in each solvent.     * The less dense solvent forms the upper layer (e.g., MtBE), while the more dense solvent forms the lower layer (e.g., water or chlorinated solvents).

• Acid-Base Extraction:     * Carboxylic Acids ($RCO_2H$): React with aqueous bases (NaOH or NaHCO3) to form water-soluble sodium carboxylates ($RCO_2^-Na^+$).     * Phenols ($ArOH$): React with NaOH but not with the weaker base NaHCO3 ($pK_a$ difference allows separation).     * Amines ($RNH_2$): React with aqueous HCl to form water-soluble organo-ammonium ions ($RNH_3^+Cl^-$).

• Drying Agents:     * Used to remove trace water from organic layers.     * Examples: Anhydrous Sodium Sulfate ($Na_2SO_4$). Blue Drierite indicator.

• Part A: $K_D$ of Benzoic Acid:     * Dissolve $100\,mg$ of benzoic acid in $2\,mL$ of methyl tert-butyl ether (MtBE) in a centrifuge tube.     * Add $3\,mL$ of $DI\,H_2O$, shake vigorously for $30\,seconds$, and separate.     * Dry the MtBE layer with $50\,mg$ $Na_2SO_4$, transfer to a tared vial, and evaporate solvent to determine recovery.

• Part B: Multi-Component Separation:     * Mixture of a carboxylic acid, a phenol, and a neutral compound.     * Step 1: Extract carboxylic acid into aqueous phase using $3 \times 1.5\,mL$ of $5\%\,NaHCO_3$. Acidify aqueous layer with $10\%\,HCl$ to precipitate and isolate via Hirsch funnel.     * Step 2: Extract phenol into aqueous phase using $3 \times 1.5\,mL$ of $5\%\,NaOH$. Acidify and isolate precipitate.     * Step 3: Neutral component remains in MtBE. Wash with $DI\,H_2O$, dry over $Na_2SO_4$, and evaporate.

Experiment 3a: Thin Layer Chromatography (TLC)

• Chromatography Fundamentals:     * Separation based on competitive interaction between a mobile phase (eluent) and a stationary phase (adsorbent).     * Stationary Phase: Usually silica gel ($SiO_2$) or alumina ($Al_2O_3$). Polar compounds adsorb more strongly to these polar stationary phases.     * Mobile Phase Polarity (Elutropic Series): Water > Methanol > Ethanol > Acetone > Ethyl Acetate > Diethyl Ether > Dichloromethane > Toluene > Cyclohexane > Hexanes.

• Retention Factor ($R_f$):     * $R_f = \frac{\text{distance from origin to center of spot}}{\text{distance from origin to solvent front}}$     * Values range from $0$ to $1$. Polar compounds have smaller $R_f$ values.

• Spotting and Developing:     * Origin marked with a pencil $1\,cm$ from the bottom.     * Developed in a chamber with a filter paper wick to saturate the air with eluent.     * Visualization: UV light ($254\,nm$) makes spots appear dark on a fluorescent background. Iodine ($I_2$) vapors adhere to compounds for temporary visualization.

• Analgesic Analysis:     * Active ingredients: Acetaminophen, Aspirin (acetylsalicylic acid), Caffeine, Ibuprofen.     * Crushed tablet dissolved in ethyl acetate and compared against standards via TLC.

Experiment 3b: Acetylation of Ferrocene and Column Chromatography

• Friedel-Crafts Acylation:     * Ferrocene is "superaromatic," $10^5$ times more reactive than benzene.     * Reaction: Ferrocene + Acetic Anhydride + $85\%\,H_3PO_4$ (catalyst) + Heat ($95\,^\circ C$ for $30-45\,min$).     * Yields: Acetylferrocene (orange-red) and potentially diacetylferrocene (red).

• Column Chromatography Procedure:     * Stationary Phase: Alumina powder ($Al_2O_3$).     * Packing: Standard slurry method (alumina/ligroin). Sequence: cotton/frit \u2192 sand ($1/4\,inch$) \u2192 alumina slurry \u2192 sand ($1/4\,inch$).     * Elution Order: (Least polar first): 1. Ferrocene (ligroin), 2. Acetylferrocene ($70:30\,ligroin:ethyl\,acetate$), 3. Diacetylferrocene (pure ethyl acetate).

Experiment 4a: Simple and Fractional Distillation

• Theory:     * Simple Distillation: Used for liquids with large boiling point differences.     * Fractional Distillation: Used for liquids with close boiling points (<\,25\,^\circ C difference). Incorporates a fractionating column (e.g., copper mesh) to provide more "theoretical plates" for heat exchange between vapor and condensate.

• Gas Chromatography (GC) Analysis:     * Used to determine percent composition of distillate fractions.     * Triangulation: $Area = \text{height (h)} \times \text{width at half-height (w}<em>{1/2})$     * Correction Factors: Toluene used as standard ($C</em>{factor}=1$). $C_{factor\,Cyclohexane} = A_c/A_t$ from a $1:1$ standard mixture.     * $Corrected\,Area = Area / C_{factor}$. Volume percent: $V_c\% = \frac{Corrected\,Area_c}{Total\,Corrected\,Area} \times 100$.

• Operating Parameters:     * Distillation rate: $1\,drop$ every $2-3\,seconds$.     * Fractions: Collected in $5\,mL$ increments.

Experiment 4b: Steam Distillation

• Theory of Immiscible Liquids:     * $P_{total} = P^\circ_A + P^\circ_B$. The system boils when the sum of individual vapor pressures equals atmospheric pressure, which always occurs below $100\,^\circ C$.     * Useful for isolating volatile oils from plants that would decompose at their normal high boiling points.

• Isolation of Eugenol from Cloves:     * Grind $1.0\,g$ of cloves buds.     * Mix with $15\,mL$ $DI\,H_2O$ in a $25\,mL$ round-bottom flask.     * Apparatus: Hickman distillation head for microscale steam distillation ($Figure\,4.2$).     * Aluminum block must reach at least $130\,^\circ C$ for distillation to start.     * Extract distillate with $CH_2Cl_2$, dry over $Na_2SO_4$, and evaporate solvent to isolate eugenol.     * Identification via Infrared (IR) spectrum ($3525\,cm^{-1}$ for OH, $1639-1612\,cm^{-1}$ for aromatic C=C).

Experiment 5: The E1 Reaction - Synthesis of Cyclohexene

• Dehydration Mechanism:     * E1 pathway: Protonation of alcohol OH \u2192 Loss of water to form carbocation \u2192 Loss of beta-proton to form alkene.     * Reagents: Cyclohexanol + Mixed acid ($H_2SO_4 + H_3PO_4$).

• Procedure Specifics:     * Add $0.074\,moles$ cyclohexanol to $1.5\,mL\,85\%\,H_3PO_4$ and $0.5\,mL\,conc.\,H_2SO_4$.     * Fractionating column used to separate cyclohexene (bp $82.8\,^\circ C$) from unreacted cyclohexanol (bp $161\,^\circ C$) during the reaction.     * Odor Control: A drying tube containing calcium chloride ($CaCl_2$) is attached to the vacuum adapter.     * Workup: Wash distillate with water, $10\%\,sodium\,carbonate$, and brine. Dry over $Na_2SO_4$.

Experiment 6: Oxidative Cleavage - Green Synthesis of Adipic Acid

• Project Overview:     * Alternative to industrial nitric acid oxidation of cyclohexanol (which releases $N_2O$ greenhouse gas).     * Utilizes "Green Chemistry" principles: safer oxidants, atom economy, and catalysis.

• Reaction Components:     * Oxidant: $30\%\,Hydrogen\,Peroxide\, (H_2O_2)$. By-product is water.     * Catalyst: Sodium Tungstate Dihydrate ($Na_2WO_4 \cdot 2H_2O$).     * Phase Transfer Catalyst (PTC): Aliquat 336 ($CH_3N[(CH_2)_7CH_3]_3Cl$). Transfers tungstate ions from the aqueous phase to the organic cyclohexene phase.

• Procedure:     * Mix $2.00\,g$ cyclohexene, $11.98\,g\,30\%\,H_2O_2$, $0.5\,g$ tungstate, $0.5\,g\,PTC$, and $0.37\,g\,KH_SO_4$.     * Reflux with vigorous stirring for $1\,hour$. Stirring is critical for PTC efficiency.     * Isolation: Pipet off the aqueous layer below the unreacted cyclohexene and above the oily PTC layer. Cool in ice to precipitate adipic acid; isolate by vacuum filtration (Hirsch funnel).

• Characterization: Melting point ($151-153\,^\circ C$), IR (broad OH peak), and NMR in DMSO solvent.

Questions & Discussion (Post-Lab Review)

• Mixed Melting Point: If adipic acid and citric acid (similar mp) are compared, a mixture will show a depressed and broadened range if they are different.

• Heating Rate: Exceeding $1-2\,^\circ C/min$ causes the observed melting point to be inaccurate (usually higher) due to the thermometer lag.

• Sample Behavior:     * Turning white to brown indicates decomposition.     * Disappearing from the tube indicates sublimation.

• Recrystallization Solvent Selection: Acetone is a poor choice because many solids are too soluble in it at all temperatures, preventing crystal formation.

• Extraction Layer Identification: Add a drop of water to the centrifuge tube; the layer the drop enters and mixes with is the aqueous layer.

• TLC Troubleshooting: If spots are on the baseline and the solvent front is at the top, the solvent is too polar. If the eluent level is above the origin spots, the spots will wash off into the solvent pool.

• Steam Distillation Mass Balance: Mass of oil co-distilling depends on the ratio: $\frac{mass\,oil}{mass\,water} = \frac{(P^\circ_{oil} \times MW_{oil})}{(P^\circ_{water} \times MW_{water})}$.