Analysis of Organic Compounds

Overview and Goals of Organic Analysis

Analysis of organic compounds aims to determine:

• Which elements and functional groups are present (qualitative analysis).
• How much of a given compound or functional group is present (quantitative analysis).
• The structure of the compound (connectivity of atoms).

In this chapter, the focus is on classical (wet-chemical) methods for organic substances, i.e. reactions in test tubes, simple separations, and characteristic color or precipitation reactions. Instrumental methods (such as chromatography and spectroscopy) are treated elsewhere.

Organic compounds are usually made of C, H, often O, and sometimes N, halogens (F, Cl, Br, I), S, P, and certain heteroatoms (e.g. B, Si, metals in organometallics). Classical organic analysis therefore typically proceeds in three steps:

1. Preliminary examination (physical properties, simple tests).
2. Elemental (qualitative) analysis – which elements are present?
3. Functional group analysis – which types of bonds and groups are present?

Quantitative (classical) analysis of organic compounds is largely based on combustion and titration methods.

Preliminary Examination of Organic Substances

Before performing specific chemical tests, it is useful to examine:

• Physical state and appearance: solid, liquid, colorless, colored, crystalline or amorphous.
• Odor: often characteristic (e.g. fruity esters, pungent amines, vinegar-like carboxylic acids).
  Caution: sniff cautiously and only if safe.
• Melting and boiling point:
• Sharp melting points suggest relatively pure crystalline substances.
• Range and comparison with literature data assist in identification.
• Solubility behavior:
• In water, dilute acids, dilute bases, organic solvents (e.g. ethanol, ether, hexane).
• Solubility changes on acidification or basification hint at acidic or basic functional groups:
• Soluble in aqueous NaOH but not in water → likely acidic (e.g. carboxylic acids, phenols).
• Soluble in aqueous HCl but not in water → likely basic (e.g. amines).

These simple observations often narrow down the list of possible compound classes.

Elemental Analysis of Organic Compounds (Classical Qualitative Tests)

General Considerations

Elemental analysis in the classical sense means detecting which elements (other than C and H) an organic compound contains. Because C and H are ubiquitous in organic compounds, they are usually not tested qualitatively; their presence is assumed if the substance is organic.

Heteroatoms (N, S, halogens) in organic structures are frequently covalently bound and do not show the typical inorganic reactions directly. Classical methods therefore aim to convert these atoms into simple inorganic ions that can be detected with standard qualitative inorganic tests.

Sodium Fusion (Lassaigne’s Test)

Principle

In Lassaigne’s test, the organic compound is fused with metallic sodium. Under the strongly reducing and high-temperature conditions, heteroatoms are converted to ionic species:

• Nitrogen → cyanide $ \text{CN}^- $
• Sulfur → sulfide $ \text{S}^{2-} $
• Halogens → halides $ \text{X}^- $ (Cl⁻, Br⁻, I⁻)

These ions can then be detected by known inorganic reactions.

Procedure (Conceptual)

1. A small piece of sodium is heated in a fusion tube until molten.
2. The organic sample is added and strongly heated: the mixture glows or chars.
3. The hot tube is plunged into water and broken; the fused mass is crushed and boiled with water.
4. The mixture is filtered to obtain the sodium extract (Lassaigne’s extract), containing $ \text{NaCN} $, $ \text{Na}_2\text{S} $, $ \text{NaX} $, if N, S, or halogens are present.

Different portions of this extract are then used for specific tests.

Detection of Nitrogen

From the sodium extract, nitrogen present in the organic compound has been converted into $ \text{NaCN} $. In solution, $ \text{CN}^- $ can form a complex iron cyanide that gives a characteristic color.

Typical test (Prussian blue test):

1. To a portion of the extract, add a few drops of freshly prepared $ \text{FeSO}_4 $ solution and heat with a little NaOH.
2. Cool and acidify with dilute $ \text{HCl} $.
3. Formation of a deep blue precipitate or coloration (Prussian blue, iron(III) hexacyanoferrate(II)) indicates nitrogen.

Simplified overall reaction sequence (conceptually):

• Formation of complex cyanide:
  $$ \text{Fe}^{2+} + 6 \text{CN}^- \rightarrow [\text{Fe(CN)}_6]^{4-} $$
• Subsequent oxidation and combination yield the Prussian blue complex.

Detection of Sulfur

Sulfur in the sodium extract appears as sulfide $ \text{S}^{2-} $.

Common tests:

• Lead acetate test:
• To a portion of the extract, add acetic acid and lead(II) acetate solution.
• A black precipitate of lead(II) sulfide indicates sulfur:
  $$ \text{Pb}^{2+} + \text{S}^{2-} \rightarrow \text{PbS} \downarrow $$
• Sodium nitroprusside test:
• Addition of sodium nitroprusside solution to an alkaline extract containing $ \text{S}^{2-} $ gives a violet coloration.

Detection of Halogens

Halogens in the sodium extract appear as halide ions $ \text{X}^- $.

First, cyanide and sulfide are typically destroyed (e.g. by boiling with concentrated nitric acid) to avoid interference, then the solution is tested for halides:

• Silver nitrate test:
• To the acidified solution, add $ \text{AgNO}_3 $ solution.
• Appearance and behavior of the precipitate suggest which halogen is present:
• White, soluble in dilute ammonia → $ \text{AgCl} $, indicates chloride.
• Cream, partially soluble in concentrated ammonia → $ \text{AgBr} $, indicates bromide.
• Yellow, insoluble in ammonia → $ \text{AgI} $, indicates iodide.

Reactions:
$$ \text{Ag}^+ + \text{Cl}^- \rightarrow \text{AgCl} \downarrow $$
$$ \text{Ag}^+ + \text{Br}^- \rightarrow \text{AgBr} \downarrow $$
$$ \text{Ag}^+ + \text{I}^- \rightarrow \text{AgI} \downarrow $$

Detection of Phosphorus (Conceptually)

Phosphorus in organic compounds can also be converted to an inorganic form (e.g. phosphate) by fusion with sodium peroxide or by oxidative digestion. The resulting $ \text{PO}_4^{3-} $ ions can be detected using classical phosphate tests (e.g. with ammonium molybdate, forming a yellow precipitate). This is less commonly required in basic organic analysis than N, S, and halogens, but follows the same overall idea: conversion to a simple anion and classical inorganic detection.

Functional Group Analysis (Classical Qualitative Tests)

After determining which elements are present, classical organic analysis proceeds to identify functional groups. The emphasis is on characteristic color changes, precipitates, or derivative formation, which signal a particular functional group.

Only selected key tests are outlined here; full coverage of organic functional groups and their reactions is treated in the corresponding organic chemistry chapters.

Tests for Unsaturation (Double and Triple Bonds)

Unsaturation (C=C, C≡C) can be detected by addition reactions that consume characteristic reagents.

Bromine Test

• Principle: Carbon–carbon double and triple bonds add bromine, decolorizing bromine solution.
• Procedure:
• Add a few drops of a bromine solution (often in CCl₄ or another inert solvent) to the sample solution.
• If bromine color disappears rapidly without formation of HBr fumes or other side effects, this suggests addition to a C=C or C≡C bond.
• Note: Aromatic compounds may decolorize bromine more slowly, usually with substitution and formation of HBr, not simple addition; this often requires a catalyst.

Potassium Permanganate Test (Baeyer Test)

• Principle: Unsaturated compounds are oxidized by $ \text{KMnO}_4 $, leading to decolorization of the violet permanganate solution and formation of brown MnO₂.
• Procedure:
• Add dilute, neutral or slightly alkaline $ \text{KMnO}_4 $ solution to the compound in water or another suitable solvent.
• Loss of purple color and appearance of a brown precipitate indicate unsaturation:
  $$ \text{C=C} + \text{KMnO}_4 \rightarrow \text{diols} + \text{MnO}_2 \downarrow $$

Tests for Alcohols

Different classes of alcohols (primary, secondary, tertiary) and polyols can be distinguished by their behavior in oxidation and dehydration reactions.

Lucas Test (for Classification of Alcohols)

• Reagent: Concentrated HCl and anhydrous $ \text{ZnCl}_2 $ (Lucas reagent).
• Principle: Alcohols react with $ \text{HCl} $ to form alkyl chlorides, which are immiscible with water and form a turbidity or separate layer. Reaction rates differ:
• Tertiary alcohols: react quickly at room temperature → immediate turbidity.
• Secondary alcohols: turbidity forms within a few minutes.
• Primary alcohols: react very slowly, often no change at room temperature.

This test helps classify an unknown alcohol but does not unambiguously identify it.

Oxidation Tests (Conceptual)

Mild oxidizing agents (e.g. acidified dichromate) differentiate:

• Primary alcohols → aldehydes → acids.
• Secondary alcohols → ketones.
• Tertiary alcohols → usually no oxidation under mild conditions.

In practice, changes in color of the oxidant (e.g. orange Cr(VI) to green Cr(III)) and subsequent detection of the oxidation products (e.g. aldehydes) support alcohol identification.

Tests for Phenols

Phenols are aromatic compounds with hydroxyl groups directly attached to aromatic rings. They show characteristic acidic behavior and distinct color reactions.

Ferric Chloride Test

• Principle: Many phenols form colored complexes with $ \text{Fe}^{3+} $ ions.
• Procedure:
• Dissolve the sample in water or ethanol; add a few drops of neutral or slightly acidic $ \text{FeCl}_3 $ solution.
• Appearance of intense coloration (often violet, blue, green, or red) indicates phenolic OH groups.
• Different phenols may give distinct colors due to different complex structures.

Tests for Aldehydes

Aldehydes are strongly reducing and can be detected by specific oxidation reactions that yield visible deposits.

Tollens’ Test (Silver Mirror Test)

• Reagent: Tollens’ reagent (ammoniacal silver nitrate, containing $ [\text{Ag(NH}_3)_2]^+ $).
• Principle: Aldehydes reduce $ \text{Ag}^+ $ to metallic silver while being oxidized to carboxylic acids:
  $$ \text{R-CHO} + 2 [\text{Ag(NH}_3)_2]^+ + 3 \text{OH}^- \rightarrow \text{R-COO}^- + 2 \text{Ag} \downarrow + 4 \text{NH}_3 + 2 \text{H}_2\text{O} $$
• Observation:
• Formation of a bright silver mirror on the inner surface of a clean test tube or a black/grey precipitate of silver indicates an aldehyde.

Some reducing sugars and other reducing substances may also give a positive Tollens’ test.

Fehling’s/Benedict’s Tests

• Reagent: Cu²⁺ complexes in alkaline solution (Fehling’s solution, Benedict’s reagent).
• Principle: Aldehydes (especially aliphatic) reduce $ \text{Cu}^{2+} $ to $ \text{Cu}_2\text{O} $, a red precipitate:
  $$ \text{R-CHO} + 2 \text{Cu}^{2+} + 5 \text{OH}^- \rightarrow \text{R-COO}^- + \text{Cu}_2\text{O} \downarrow + 3 \text{H}_2\text{O} $$
• Observation:
• Brick-red precipitate of $ \text{Cu}_2\text{O} $ indicates a reducing aldehyde.

Ketones generally do not react under these conditions (with important exceptions among α-hydroxy ketones and sugars).

Schiff’s Test (Color Reaction)

• Reagent: Schiff’s reagent (decolorized fuchsin solution).
• Principle: Aldehydes react with the reagent to restore the dye color, typically giving a red-violet coloration.
• Observation:
• Development of purple/pink color after a short time indicates an aldehyde.

Tests for Ketones

Ketones are less reactive toward mild oxidation than aldehydes. Their identification in simple classical analysis relies mostly on:

• Their failure to give aldehyde-specific tests (e.g. Tollens, Fehling), and
• Formation of derivatives with characteristic melting points (e.g. 2,4-dinitrophenylhydrazones, see below).

Some ketones, particularly methyl ketones, give additional characteristic reactions (iodoform test).

2,4‑Dinitrophenylhydrazine (2,4‑DNPH) Test for Carbonyl Groups

• Reagent: 2,4-dinitrophenylhydrazine in acidic solution (Brady’s reagent).
• Principle: Both aldehydes and ketones (i.e. carbonyl compounds) react with 2,4‑DNPH to form hydrazone derivatives:
  $$ \text{R-CO-R'} + \text{H}_2\text{NNH-}(\text{NO}_2)_2 \rightarrow \text{R-C=N-NH-}(\text{NO}_2)_2 + \text{R'-OH} $$
  (schematic representation)
• Observation:
• Formation of a yellow to orange crystalline precipitate indicates a carbonyl group.
• Application:
• The derivative’s melting point can be measured and compared with literature values to help identify the specific aldehyde or ketone.

Iodoform Test (for Methyl Ketones and Certain Alcohols)

• Reagents: Iodine and base (e.g. $ \text{I}_2 $ in NaOH).
• Principle: Compounds containing the $ \text{CO-CH}_3 $ group (methyl ketones) and alcohols that can be oxidized to such ketones (e.g. ethanol, certain secondary alcohols) form iodoform $ \text{CHI}_3 $, a yellow solid with a characteristic odor.
• Overall simplified reaction:
• For a methyl ketone:
  $$ \text{R-CO-CH}_3 + 3 \text{I}_2 + 4 \text{OH}^- \rightarrow \text{R-COO}^- + \text{CHI}_3 \downarrow + 3 \text{I}^- + 3 \text{H}_2\text{O} $$
• Observation:
• Yellow precipitate of $ \text{CHI}_3 $ indicates presence of a methyl ketone or a precursor alcohol.

Tests for Carboxylic Acids

Carboxylic acids are weak Brønsted acids and show typical acid–base behavior, as well as specific reactions forming characteristic salts or derivatives.

Acid–Base Reactions and Solubility

• Solubility in aqueous sodium bicarbonate or sodium carbonate with evolution of carbon dioxide:
  $$ \text{R-COOH} + \text{NaHCO}_3 \rightarrow \text{R-COO}^- \text{Na}^+ + \text{H}_2\text{O} + \text{CO}_2 \uparrow $$
• Observation:
• Effervescence (bubbling) due to CO₂ gas indicates a carboxylic acid.

Phenols (except very strongly acidic ones) usually do not liberate CO₂ from bicarbonate, allowing discrimination between phenols and carboxylic acids.

Formation of Characteristic Derivatives

Carboxylic acids can form:

• Amides (by reaction with ammonia or amines),
• Esters (by reaction with alcohols, often catalyzed by acid),

whose melting points and other properties can support identification. However, in basic introductory qualitative organic analysis, simple solubility and effervescence tests are usually the primary tools.

Tests for Amines

Amines are organic bases and react with acids as proton acceptors. They also show characteristic color reactions and sometimes distinctive odors.

Basicity and Solubility

• Amines react with dilute mineral acids (e.g. HCl) to give water-soluble ammonium salts:
  $$ \text{R-NH}_2 + \text{HCl} \rightarrow [\text{R-NH}_3]^+ \text{Cl}^- $$
• Observation:
• Compounds insoluble in water but soluble in dilute HCl are often amines (or other basic organics).

Hinsberg Test (Classification of Amines)

• Reagent: Benzenesulfonyl chloride in the presence of base (e.g. NaOH).
• Principle: Primary, secondary, and tertiary amines respond differently:
• Primary amines form sulfonamides that are soluble in alkali (due to an acidic N–H).
• Secondary amines form sulfonamides insoluble in alkali.
• Tertiary amines do not react under these conditions and remain insoluble; however, they can form soluble salts with acids in a subsequent step.
• This test allows classification of amines by their substitution pattern.

Nitrous Acid Test (for Aromatic Amines, Conceptual)

• Aromatic primary amines react with nitrous acid (generated in situ from sodium nitrite and mineral acid) to form diazonium salts, which can further couple with aromatic compounds to give azo dyes (intense colors).
• For aliphatic amines, reaction with nitrous acid often leads to evolution of nitrogen gas and formation of alcohols or other products.

Tests for Nitro Compounds (Conceptual)

Nitro compounds can often be identified by reduction to amines (e.g. with tin and HCl or Fe and HCl), followed by the known tests for amines. A positive amine test after reduction suggests that the original compound contained a nitro group.

Tests for Esters

Esters are typically neutral, often pleasant-smelling compounds (fruity odors). Classical tests include:

• Hydrolysis (saponification) in alkaline solution:
  $$ \text{R-COOR'} + \text{OH}^- \rightarrow \text{R-COO}^- + \text{R'-OH} $$
• Identification of the products:
• The resulting carboxylate can be acidified to regenerate the acid.
• The alcohol can be identified by its own characteristic tests.
• Odor:
• Characteristic fruity odors may suggest certain simple esters (e.g. ethyl acetate, isoamyl acetate).

Tests for Halogen-Containing Organic Compounds

Beyond Lassaigne’s test, halogenated organic compounds can often be detected by:

• Beilstein test:
• A clean copper wire is dipped into the organic compound and passed through a flame.
• A green or blue-green flame indicates the presence of halogens (formation of volatile copper halides).
• This test is very sensitive but not entirely specific; certain inorganic halides also give positive results.

Tests for Carbohydrates (Basic Classical Reactions)

Classical organic analysis often includes simple carbohydrate tests:

• Molisch test (general test for carbohydrates):
• Reagent: α‑naphthol in ethanol (Molisch reagent), followed by concentrated sulfuric acid.
• Principle: Carbohydrates dehydrate to furfural derivatives, which form colored products with α‑naphthol.
• Observation: A violet ring at the interface of the two layers indicates carbohydrates.
• Specific tests (e.g. Barfoed, Seliwanoff) distinguish between monosaccharides and disaccharides, or aldoses and ketoses, by their different reaction rates and color changes.

Classical Quantitative Organic Analysis (Combustion and Elemental Composition)

While qualitative tests identify which elements and functional groups are present, classical quantitative organic analysis determines the elemental composition – that is, the mass percentages of C, H, N, etc. This allows the empirical formula to be calculated.

Combustion Analysis for C and H

Principle

An accurately weighed sample of the organic compound is completely burned in an oxygen stream. The resulting $ \text{CO}_2 $ and $ \text{H}_2\text{O} $ are absorbed in suitable absorbents and weighed. From the mass of $ \text{CO}_2 $ and $ \text{H}_2\text{O} $ formed, the amounts of C and H in the original sample are calculated.

Example (conceptual):

• Organic compound (unknown formula) is burned:
  $$ \text{C}_x\text{H}_y\text{O}_z + \text{O}_2 \rightarrow x \text{CO}_2 + \frac{y}{2}\text{H}_2\text{O} $$

If a mass $m_{\text{CO}_2}$ of $ \text{CO}_2 $ and a mass $m_{\text{H}_2\text{O}}$ of $ \text{H}_2\text{O} $ are produced, then:

• Moles of CO₂:
  $$ n_{\text{CO}_2} = \frac{m_{\text{CO}_2}}{M(\text{CO}_2)} $$
  which equals moles of C in the sample.
• Moles of H₂O:
  $$ n_{\text{H}_2\text{O}} = \frac{m_{\text{H}_2\text{O}}}{M(\text{H}_2\text{O})} $$
  which contains twice as many moles of H:
  $$ n_{\text{H}} = 2 \, n_{\text{H}_2\text{O}} $$

From these and the sample mass, mass percentages of C and H can be computed.

Determination of Nitrogen (Kjeldahl Method, Classical Concept)

The Kjeldahl method is a classical procedure for determining nitrogen in organic compounds (especially in foods and biological materials).

• The sample is digested with concentrated sulfuric acid (and catalysts), converting nitrogen to ammonium sulfate.
• The solution is then made alkaline, and the released ammonia is distilled into a known excess of standard acid.
• The remaining acid is back-titrated with base.
• From the amount of acid neutralized by ammonia, the nitrogen content is calculated.

This method is central in classical organic elemental analysis but does not differentiate between different nitrogen-containing functional groups.

Empirical Formula Determination

Combining data from combustion analysis (C, H, and sometimes O by difference) and nitrogen/sulfur/halogen determinations allows calculation of the empirical formula.

Outline:

1. Convert mass percentages into moles of each element.
2. Divide all mole values by the smallest to obtain a simple whole-number ratio.
3. These integers give the empirical formula (smallest integer ratio of atoms).

Structural information (arrangement of atoms) still requires additional data (e.g. qualitative functional group tests and, in modern practice, instrumental methods).

Systematic Approach to Classical Analysis of Organic Compounds

In practical qualitative organic analysis, the different tests are applied in a systematic order to identify an unknown substance:

1. Preliminary tests:
• Physical state, melting/boiling point, odor, elemental composition by sodium fusion.
2. Solubility and acid–base behavior:
• Solubility in water, dilute acids, dilute bases, organic solvents.
• Gas evolution (e.g. with NaHCO₃) to test for carboxylic acids.
3. Tests for specific functional groups:
• Unsaturation (bromine, $ \text{KMnO}_4 $).
• Alcohols and phenols (Lucas test, FeCl₃).
• Carbonyl groups (2,4‑DNPH).
• Aldehydes (Tollens, Fehling, Schiff).
• Ketones (e.g. iodoform test for methyl ketones).
• Amines (basicity, Hinsberg test).
• Esters (saponification, odor).
4. Preparation of derivatives:
• Crystalline derivatives (e.g. hydrazones, oximes, amides, esters) with known melting points can confirm identity by comparison with reference data.
5. Consistency check:
• All observations (solubility, tests, derivatives, melting point) are combined to propose a structure consistent with the empirical formula from elemental analysis.

Although modern instrumental techniques have largely replaced many classical wet-chemical procedures, these methods remain important for understanding the chemical behavior of organic functional groups and for simple laboratory identifications.