Chem-153L-Organic-Biochem-Prelim-Discussion-1st-Semester-SY2024-2025.pdf

Experiment #1 Separation and Purification of Organic Compounds SEPARATION AND PURIFICATION OF ORGANIC COMPOUNDS ❑Solution and Filtration ❑Extraction ❑Recrystallization ❑Sublimation  Why do we need to separate and/or purify our organic substances? 1.Solution and filtration  1.a. S...

Experiment #1 Separation and Purification of Organic Compounds SEPARATION AND PURIFICATION OF ORGANIC COMPOUNDS ❑Solution and Filtration ❑Extraction ❑Recrystallization ❑Sublimation  Why do we need to separate and/or purify our organic substances? 1.Solution and filtration  1.a. SOLUTION and FILTRATION solution alias: solid-liquid extraction Basis for separation: Relative solubility of components. solvent for 2-component mixture: solute 1: completely soluble solute 2: completely insoluble 1. SOLUTION AND FILTRATION Soluble component can be recovered from filtrate through evaporation of solvent Insoluble component can be filtered and will remain in filter paper 1. SOLUTION AND FILTRATION Benzoic acid sucrose For the sugar-benzoic acid mixture, which component is more soluble: in water? In denatured alcohol? Source: http://www.3dchem.com/imagesofmolecules/Sucrose.gif 2. Extraction A separation technique based on a difference in solubilities of a compound (A) between two immiscible phases (liquid-liquid or liquid-solid) Distribution (Partition) Coefficient solubility of A in O.P.(g/mL) Kp = ---------------------------------------- solubility of A in A.P.(g/mL) Involves transferring a solute from one phase to another The solute is removed from one phase by adding an immiscible solvent in which the solute is more soluble Depending on the phases, the following types of extraction exist: ❏ Solid – liquid extraction ❏ Liquid – liquid extraction Solid-Liquid Extraction ❑Extraction of the analyte from a solid sample matrix into a solvent in which it is soluble. ❑If water is the extracting solvent, the solid sample is simply heated in water. ❑If an organic solvent is an extracting solvent, a Soxhlet extractor is generally employed. Liquid-Liquid Extraction ❑also known as Solvent Extraction ❑separate compounds based on their relative solubilities in two immiscible liquids, usually water and an organic solvent ❑commonly performed after a chemical reaction as part of the work-up Simple Extraction ❑Simple extraction are carried out manually using a separatory funnel. ❑Continuous extraction processes require specially designed equipment, but are generally automated and more efficient. Simple Extraction Principle The procedure involves vigorous shaking of the separatory funnel containing the 2 immiscible liquids. Each liquid is broken up into fine droplets which allows for intimate contact between the phases. The mixture is then allowed to stand to separate it into 2 distinct layers with the denser liquid in the bottom. The lower layer is then drawn off through the stopcock. Simple Extraction Two factors to be considered in the utility of extraction: ❑ Solvent Selection ❑ Extraction Efficiency Solvent Selection Properties of a Good Extracting Solvent 1.It must be practically immiscible with (water) the solution to be extracted; 2. It should have low boiling point for easier removal by distillation or evaporation; 3. It should dissolve the solute better than the other solvent from which it is being extracted (Has a high distribution coefficient for the analyte) 4. Must be chemically inert to the substances present in the sample matrix. Parameters of the Extraction Process: Common Extraction Solvents Solvent Boiling pt. Solubility in Density (oC) water (g/100mL) Diethyl ether 35 6 0.71 Pentane 36 0.04 0.62 Dichloromethane 40 2 1.32 Petroleum ether 40 – 60 Low 0.65 Chloroform 61 0.5 1.48 Hexane 69 0.02 0.66 Toluene 111 0.06 0.87 Extraction Efficiency: Multiple Extraction It is best to perform multiple extraction with a minimum amount of solvent to recover as much of the analyte as possible. Generally, three to four extractions are sufficient.  Extraction Efficiency: Distribution Coefficient Analyte (aqueous phase) Analyte (organic phase) The analyte partitions itself between the two phases based on its distribution coefficient. 3. Recrystallization Products from an organic reaction are seldom obtained in a pure state directly from the reaction mixture. If the product is a solid, it may be purified by recrystallization from a suitable solvent.  Fact: Most organic compounds are more soluble in hot solvents than in cold. The impurities present will have different solubilities from the desired compounds  3. Recrystallization The procedure involves: (a) Selection of a solvent A good recrystallization solvent should: have a relatively low boiling point (evaporates readily) so that crystals can be quite easily dried have a boiling point below the melting point of the crystals have a low flammability if possible not chemically react with the crystals  3. Recrystallization (b) Dissolution of the impure (crude) product Use minimum amount of solvent (addition of more solvent will make your product solution more dilute and result in recovery of fewer pure crystals).  3. Recrystallization (c) Decolorizing add decolorizing carbon (activated carbon) or charcoal to remove colored impurities and other finely dispersed or colloidal matter that is too small to be trapped by filter paper Decolorizing carbon removes materials by adsorption, the attachment of materials to the surface of a solid by intermolecular interactions  3. Recrystallization (d) Hot gravity filtration (not suction filtration because reduced pressure of the latter will evaporate the hot solvent of the filtrate Biggest problem to arise is premature recrystallization To help avoid this problem: a. use a 10-20% excess of the solvent b. preheat the funnel in an oven c. keep the filter and funnel hot during filtration d. use stemless funnel  3. Recrystallization (e) Cooling of the solution deposit crystals of the compound Failure to recrystallize - Remedies: a. Scratching - scratch the inside of the flask vigorously to produce rough surface area where crystals can start to grow b. Seeding - add a tiny amount of pure crystal to the cold, supersaturated solution (the crystal provides sites for larger crystals to grow) c. Cooling - cool the mixture at very low temperatures, e.g. in an ice-salt bath to about -100C or add a few small chunks of dry ice (solid CO2) to the solution to produce a cold spot Problems with Recrystallization If, still crystals do not appear, the solution is unsaturated. - Evaporate the excess solvent. Loss of crystals during filtration. Remedy: Use vacuum filtration. 3. Recrystallization (f) Cold filtration - filtering the crystals from the solution (called the mother liquor) (g) Washing - pour fresh cold solvent; use a minimum amount of solvent if the crystals exhibit much solubility (h) Drying - allow crystals to air dry (to remove the last traces of solvent) It is important to note that: To obtain a good recovery of purified material, the use of unnecessarily large volumes of solvent must be avoided. The amount of pure material lost by retention in the mother liquor will be minimized if the substance is dissolved in the smallest amount of hot solvent. Recrystallization Impurities Compound + Impurities filter Impurities dissolve in hot Compound solvent Cool  4. Sublimation The process of changing the physical state of a substance from solid directly to gas state without passing through the liquid state As a method of purification: It is required that either the desired substance or the impurities sublime, but not both, unless their sublimation temp. are reasonably far apart.  Solid compounds that sublime are rare, but several can be found in organic chemistry The common characteristic for such compounds is a high volatility due to relatively weak intermolecular forces In order to purify organic molecules in this fashion, it is imperative that the contaminants do not have the same properties.  The typical procedure involves heating the solid in the presence of a clean cold surface. Upon sublimation from the impure mixture, the molecules enter the gas phase at high temperatures and upon contact with the cold surface return to the solid phase as a pure compound.  Advantage of a sublimation versus crystallization: often very clean products are obtained and the method is suitable already for very small amounts of sample. Experiment #2 Thin Layer Chromatography: Analysis of Analgesic Drugs Chromatography - A separation technique - The separation is accomplished by the distribution of the mixture between two phases: one that is stationary and one that is moving (mobile). Principle of Chromatography Different compounds will have different solubilities and adsorption to the two phases between which they are to be partitioned. ▪ different abilities of substances to adsorb, or stick, to surfaces Principle of Chromatography Chromatography Can be used to: - separate mixtures - identify compounds - determine how many components are present in a mixture - determine the purity of a sample Thin Layer Chromatography Stationary phase Thin layer of an adsorbent on a glass or plastic plate Adsorbents ⮚ Finely ground silica (SiO2 nH2O) - a.k.a. silica gel - added with ∼10% by wt Gypsum (CaSO4 2H2O) as binder ⮚ Alumina (Al2O3) Silica gel and alumina are both polar adsorbents but silica gel is less polar than alumina. Thin Layer Chromatography Developer (mobile phase) Closed vessel containing a shallow pool of liquid - Ascends the slide by capillary action within the adsorbent Function: To selectively desorb (remove from the adsorbent) and transport some compounds farther than others.  Developer (mobile phase) Polarity is a major consideration. High polarity Low polarity (decreasing) (decreasing) If too polar: it will carry all Acetic acid Toluene of the spotted materials about the same distance Methanol CCl4 giving a poor separation Ethanol Pentane If too nonpolar: none of anh. Acetone Hexane the spotted materials will Ethyl acetate Pet. ether be moved anh. Ether In general: desorbing and transporting capability Chloroform increases with increasing CH2Cl2 polarity 41 Thin Layer Chromatography Strength of binding of compounds to silica: ⮚ Acids & bases > amides > carboxylic acids > alcohols > ketones ∼ aldehydes > halides > esters > ethers > unsat’d hydrocarbons > sat’d hydrocarbons ⮚ The stronger the binding, the shorter distance travelled The rate at which a compound ascends the thin layer plate depends on: a) polarity of the adsorbent b) polarity of the developing solvent c) polarity of the compound Development: interactions among adsorbent, developer and spotted compounds Molecule is bound to adsorbent. Developer attracts and desorbs it. Molecule is continually adsorbed and desorbed (equilibrium) as solvent moves up the plate. 43 Development: interactions among adsorbent, developer and spotted compounds If molecule is more soluble in solvent, it will easily be desorbed If tightly bound to silica, it will spend more time there A good solvent in chromatography should give a good resolution (separation) ability to have different interactions with the different constituents no matter how similar the constituents are. - usually a mixture of liquids 44 Developing the chromatogram Keep covered to keep chamber saturated with solvent vapor: to maintain equilibrium as solvent moves upward - Adsorbent wetted with solvent will look like wet snow (wet ice) When about 5 mm from opposite unsoaked edge, remove plate - Forgetting to remove the plate when solvent has reached the top edge will enable the spot to continually ascend - Rf values will be higher Mark solvent front while still visible Allow solvent to evaporate If spots already visible, outline the spots by scoring them 45 Visualizing the spots Iodine chamber: Iodine forms a reversible complex with most organic compounds except sat. HCs and alkyl halides ✔ Spots fade rapidly after removal from chamber – must be marked at once. ✔ Prolonged exposure to iodine chamber – entire plate may turn brown, allow to evaporate. ✔ Work in hood – iodine vapors are toxic. 46  UV RAYS UV exposure in dark room: a nondestructive way to visualize spots UV causes some chemicals to fluoresce (“glow in the dark”) If silica gel used contains phosphorescent additive, entire plate fluoresces, leaving dark spots where compounds are Avoid looking at the UV source! 47 Thin Layer Chromatography Calculation of Rf (Retention factors) Measure distance traveled by each migrated spot and the solvent front from the center of original spot (origin) For tailing (spot becomes elongated), measure to the point that seems to be the center of area Thin Layer Chromatography Rf is very dependent on: 1. Moisture content (humidity) 2. Thickness of adsorbent 3. Purity of solvent 4. Other factors Identities of compounds established by spotting known compounds with unknown sample. Same Rf values could indicate same compounds. Thin Layer Chromatography Ibuprofen Acetaminophen (Paracetamol) 2-(4-isobutylphenyl)propanoic acid N-(4-hydroxyphenyl)acetamide Thin Layer Chromatography Mefenamic acid Aspirin (acetylsalicylic acid) 2-[(2,3-dimethylphenyl)amino]benzoic acid 2-acetoxybenzoic acid  Experiment #3 Qualitative Analysis of Elements in Organic Compounds Test for Carbon and Hydrogen Oxidizing agent Test for Carbon and Hydrogen The moisture formed (H2O) indicates the presence of H in the compound. This is seen as droplets of water in the tube. The presence of C is detected by the lime water test where the produced CO2 reacts to form a white precipitate. CO2↑ + Ca(OH)2 🡺 CaCO3↓ + H2O Lime water White precipitate Test for Oxygen Ferrox paper - absorbed with thiocyanate (SCN-) and ferric ions (Fe3+) - Fe3+ forms a blood red complex with SCN- Fe3+(aq) + SCN-(aq) 🡺 Fe(SCN)2+ (aq) Blood red complex Oxygen in the compound forms a deep red to purple complex with Fe(SCN)2+ Fe(SCN)2+ --------- O --------- Fe(SCN)2+ Test for Oxygen Comparing you result with a control sample Test tube A. Hexane C6H14 B. Ethanol C2H5OH C. Test compound D. Test compound Sodium fusion Na metal and an organic compound melt together (fusion) during heating. They undergo a redox reaction in which Na is oxidized and hetero elements are reduced Na, heat RX, R3N, R2S NaX, NaCN, Na2S R = alkyl group The fused compounds are dissolved in water, and the ions X- , CN- and S2- are tested. Test for Nitrogen: Lassaigne’s Test 2 NaCN + FeSO4 🡺 Fe(CN)2 + Na2SO4 Fe(CN)2 + 4 NaCN 🡺 Na4Fe(CN)6 Na4Fe(CN)6 + 4 FeCl3 🡺 Fe4[Fe(CN)6]3 Prussian blue ppt (blue green) Test for Sulfur Na2S + Pb(CH3COO)2 🡺 PbS↓ + NaCH3COO Black ppt Test for Halogens Ag+ + NaX 🡺 AgX↓ + Na+(aq) X = Cl (white ppt) X = Br, I (yellow ppt) Test for Halogens Yellow ppt indicates the presence of Br or I but may not imply the absence of Cl White ppt of AgCl may be obscured by the intense yellow color of AgBr or AgI 60 Experiment #4 Qualitative Analysis of Functional Groups Functional Groups Functional Groups ALCOHOL AMINE CARBOXYLIC ACID KETONE ESTER ALDEHYDE Test for Hydrocarbons (Unsaturated) uses dilute alkaline potassium permanganate (KMnO4) to oxidize the carbon-carbon double or triple bond. In this test KMnO4 (purple) reacts with alkenes or alkynes but not alkanes or aromatics. Alkene or Alkyne (positive test) alkene alkyne Test for Hydrocarbons (Unsaturated) In this test KMnO4 (purple) reacts with alkenes or alkynes but not alkanes or aromatics. Test for Hydrocarbons (Unsaturated) Baeyer’s Test for Multiple Bonds Test for Hydrocarbons (Unsaturated) Baeyer’s Test for Multiple Bonds Test compounds: 1. Cyclohexane (C6H12) 2. Bromobenzene (C6H5Br) 3. linoleic acid (C18H32O2) Test for Hydrocarbons (Unsaturated) Ignition Test for High Degrees of Unsaturation Unsaturated bonds form luminous (yellow) flame and sooty deposits. Aromatic hydrocarbons burn with a yellow, sooty flame due to their high carbon content. Aliphatic hydrocarbons burn with flames that are yellow, but less sooty. Test for Hydrocarbons (Unsaturated) Ignition Test for High Degrees of Unsaturation Test compounds: 1. Benzophenone (C13H10O) 2. Cyclohexane (C6H12) (C6H5)2CO + O2 → CO2 + H2O (+C(s)) Jones Oxidation for 1o and 2o Alcohols Jones Reagent - a solution of chromium trioxide (CrO3) in aqueous sulfuric acid (H2SO4). As an alternative, potassium dichromate (K2Cr2O7) can be used. - Using acetone as a reaction solvent, the reagent is used for the oxidation of primary and secondary alcohols to carboxylic acids and ketones, respectively. - The oxidation is very rapid and exothermic. Jones Oxidation for 1o and 2o Alcohols Test Compounds: 1. 1-Butanol, CH3CH2CH2CH2OH 1. 2-Butanol, CH3CH(OH)CH2CH3 1. t-Butyl alcohol, (CH3)3COH Jones Oxidation for 1o and 2o Alcohols A positive test is marked by the formation of a green color within 15 seconds upon addition of the yellow-orange reagent to a primary or secondary alcohol. Tertiary alcohols do not give visible reaction within 2 seconds, the solution remaining orange in color. 1-BUTANOL > 2-BUTANOL >>> t-BUTANOL Jones Oxidation for 1o and 2o Alcohols Aldehydes and Ketones Many, but not all, aldehydes and ketones have pleasant fruity odors. Formaldehyde has a sharp, unpleasant odor. Benzaldehyde has the characteristic odor of almonds. Acetone (a key ingredient in nail polish remover) has a fruity smell. Tests for Aldehydes and Ketones 2,4-DNPH (2,4-Dinitrophenylhydrazine) TEST General test for Carbonyl compounds TOLLENS’ TEST General test for Aldehydes FEHLING’S TEST General test for Aliphatic aldehydes Tests for Aldehydes and Ketones Test compounds: 1. Formaldehyde 1. Benzaldehyde 1. Acetone Tests for Aldehydes and Ketones 2,4-DNPH TEST - a general test for aldehydes and ketones - Aldehydes and ketones react with 2,4-DNPH at room temperature to give a yellow-orange to red precipitate of 2,4-dinitrophenylhydrazone. Tests for Aldehydes and Ketones 2,4-DNPH TEST Aromatic Simple aldehydes and aldehydes and ketones give a yellow ketones give an precipitate. orange precipitate. Positive Tests for Aldehydes and Ketones 2,4-DNPH TEST Tests for Aldehydes and Ketones Tollens’ Test (a general test for aldehydes) Tollen’s reagent is a solution of aqueous silver nitrate (AgNO3) with aqueous ammonia (NH3) All aldehydes give a positive Tollen’s test. In general, ketones don’t react with the Tollen’s reagent. Tests for Aldehydes and Ketones OXIDATION AND REDUCTION Tests for Aldehydes and Ketones Tollen’s Test -an oxidation reaction in which aldehydes reacts w/ ammoniacal silver nitrate to yield a ppt of silver metal, which appears as a mirror. Tests for Aldehydes and Ketones Fehling’s Test (a general test for aliphatic aldehydes) In this test the presence of aldehydes but not ketones is detected by reduction of the deep blue solution of copper(II) to a red precipitate of insoluble copper oxide (Cu2O). Tests for Aldehydes and Ketones Fehling’s Test (a general test for aliphatic aldehydes) OXIDATION AND REDUCTION Carboxylic Acids The smallest carboxylic acids have sharp odors, whereas some of the larger ones are suggestive of body odors. Acetic acid has strong acidic odor. In more dilute solution, the odor can take on a smell of overripe fruit. Succinic acid is odorless. Tests for Carboxylic Acid Sodium bicarbonate test Carboxylic acids react with NaHCO3 solution to form the carboxylate anion and carbon dioxide gas. Tests for Carboxylic Acid Sodium bicarbonate test Test compounds: 1. Acetic acid (CH3COOH) 2. Succinic acid (HOOC-CH2CH2-COOH) + H2O