Amino acids are dipolar amphoteric molecules (zwitterions) bearing both acidic carboxyl ($-text{COOH}$) and basic amino ($-text{NH}_3^+$) groups. In strongly acidic solution ($text{pH} < 2$), glycine exists as a fully protonated monocation ($^+text{H}_3text{N}–text{CH}_2–text{COOH}$, net charge $+1$).

As strong base ($text{NaOH}$) is incrementally titrated, the $alpha$-carboxyl group dissociates first with an apparent ionization constant $K_1$ ($text{p}K_1 = 2.34$). When exactly $0.5$ equivalent of base is added, $[^+text{H}_3text{N}–text{CH}_2–text{COOH}] = [^+text{H}_3text{N}–text{CH}_2–text{COO}^-]$, and according to the Henderson-Hasselbalch equation, $text{pH} = text{p}K_1$:

At $1.0$ equivalent of $text{NaOH}$, the carboxyl deprotonation is complete, yielding the dipolar zwitterion ($^+text{H}_3text{N}–text{CH}_2–text{COO}^-$, net charge $0$). The pH at which the net electrical charge is zero is the isoelectric point ($text{pI}$), where the molecule displays minimal electrophoretic mobility and lowest solubility:

Further addition of base titrates the $alpha$-amino group with $text{p}K_2 = 9.60$, yielding the fully deprotonated anion ($text{H}_2text{N}–text{CH}_2–text{COO}^-$, net charge $-1$).

Carbohydrates are polyhydroxy aldehydes or ketones classified by degree of polymerization and reducing capacity:

• Molisch Test (General Carbohydrate Test): Concentrated sulfuric acid dehydrates pentoses to furfural and hexoses to 5-hydroxymethylfurfural (5-HMF). These cyclic aldehydes condense with $alpha$-naphthol to form a characteristic purple/violet ring at the liquid-liquid interface.
• Iodine Test (Polysaccharides): Triiodide ions ($text{I}_3^-$) slip into the interior helices of amylose to form an intense deep-blue charge-transfer inclusion complex. Highly branched glycogen and amylopectin yield reddish-brown to purple complexes.
• Benedict’s Test (Reducing Sugars): Sugars with a free hemiacetal or hemiketal group tautomerize into enediols in mild alkaline medium ($text{Na}_2text{CO}_3/text{sodium citrate}$). Enediols reduce cupric ions ($text{Cu}^{2+}$) to insoluble cuprous oxide ($text{Cu}_2text{O}$), creating a green $to$ yellow $to$ brick-red precipitate.
• Barfoed’s Test (Monosaccharide vs Disaccharide): Copper acetate in weak acetic acid ($text{pH} approx 4.6$). Because of the acidic conditions, only rapidly reducing monosaccharides reduce $text{Cu}^{2+}$ within 2–3 minutes of boiling, whereas reducing disaccharides require prolonged boiling (>8–10 min).
• Seliwanoff’s Test (Aldose vs Ketose): Resorcinol in $3,text{M} text{HCl}$. Ketohexoses (fructose) dehydrate much more rapidly than aldohexoses to 5-HMF, which condenses with resorcinol to form a cherry-red complex within 60 seconds.

Amino acids and proteins can be identified by reactions targeting either their shared backbone components ($alpha$-amino, carboxyl, peptide bond) or specific functional R-groups:

• Biuret Reaction: Requires a minimum of two peptide bonds (tripeptides and above). In alkaline solution ($text{NaOH}$), cupric ions ($text{Cu}^{2+}$) form a tetradentate coordination complex with four peptide nitrogen atoms, yielding an intense violet/purple chromophore ($lambda_{max} = 540,text{nm}$).
• Ninhydrin Reaction: Ninhydrin (triketohydrindene hydrate) oxidatively deaminates and decarboxylates free $alpha$-amino acids, releasing $text{NH}_3$, $text{CO}_2$, and an aldehyde. The reduced hydrindantin condenses with another ninhydrin and ammonia to form Ruhemann’s purple ($lambda_{max} = 570,text{nm}$). Secondary amino acids like proline and hydroxyproline yield a characteristic yellow derivative ($lambda_{max} = 440,text{nm}$).
• Xanthoproteic Reaction: Concentrated nitric acid undergoes electrophilic aromatic substitution on benzene rings of aromatic amino acids (Tyrosine and Tryptophan; Phenylalanine reacts slowly), producing yellow nitro derivatives that turn dark orange in alkali.
• Millon’s Reaction: Mercuric nitrate in nitric acid reacts specifically with the phenolic hydroxyl group of Tyrosine, yielding a brick-red mercurated adduct.
• Sakaguchi Reaction: Under alkaline conditions, $alpha$-naphthol and sodium hypobromite react specifically with the guanidino group of Arginine to form a brilliant red dye complex.
• Lead Acetate Test (Sulfur-Containing AAs): Boiling cysteine or methionine with concentrated $text{NaOH}$ hydrolyzes organic sulfur to inorganic sulfide ($text{S}^{2-}$), which precipitates with lead acetate as black lead sulfide ($text{PbS}$).

Accurate quantification of protein concentration is fundamental to all enzyme kinetics, molecular biology, and biochemical purification protocols:

• Lowry Method (Folin-Ciocalteu Assay): Combines the Biuret reaction with the reduction of the Folin-Ciocalteu phenol reagent (phosphomolybdate and phosphotungstate). In alkaline copper solution, peptide bonds chelate $text{Cu}^{2+}$ to form $text{Cu}^+$. Monovalent copper, along with tyrosine, tryptophan, and cysteine side chains, reduces the yellow phosphomolybdic-phosphotungstic acid complex to an intense molybdenum/tungsten blue chromophore measured at $lambda = 660,text{nm}$. Sensitivity: $5–100,mutext{g/mL}$.
• Bradford Method (Dye-Binding Assay): Employs Coomassie Brilliant Blue G-250. In acidic medium, the dye exists in a doubly protonated red/brown cationic state ($lambda_{max} = 465,text{nm}$). Upon binding to basic (primarily arginine, lysine, histidine) and aromatic amino acid residues of proteins, the dye is converted to an unprotonated blue anionic sulfonate form ($lambda_{max} = 595,text{nm}$). The absorbance at $595,text{nm}$ is directly proportional to protein concentration via the Beer-Lambert law ($A = varepsilon cdot c cdot l$). Sensitivity: $1–50,mutext{g/mL}$.

The GOD-POD method is the gold-standard enzymatic photometric assay for blood glucose quantification due to its absolute specificity for $beta$-D-glucose over other circulating aldohexoses or reducing sugars:

In the first step, GOD catalyzes the oxidation of glucose to D-glucono-$delta$-lactone with the equimolar generation of hydrogen peroxide ($text{H}_2text{O}_2$). In the second coupled step, POD uses $text{H}_2text{O}_2$ to oxidatively couple 4-aminoantipyrine with phenol, synthesizing a bright pink quinoneimine chromophore. The intensity of color measured at $lambda_{max} = 505,text{nm}$ is directly proportional to glucose concentration.

Salivary $alpha$-amylase ($alpha$-1,4-glucan 4-glucanohydrolase, EC 3.2.1.1) is a calcium-dependent metalloenzyme that endo-cleaves internal $alpha(1to 4)$ glycosidic linkages in amylose and amylopectin, releasing maltose, maltotriose, and $alpha$-limit dextrins. The rate of maltose formation follows the Michaelis-Menten equation:

Where $v_0$ is initial velocity, $V_{max}$ is maximal velocity at enzyme saturation, and $K_m$ (Michaelis constant) represents the substrate concentration at which velocity is half-maximal ($v_0 = V_{max}/2$). Taking the reciprocal yields the linear Lineweaver-Burk double reciprocal equation:

Maltose generated by enzymatic hydrolysis is quantified using the 3,5-dinitrosalicylic acid (DNSA) assay. Under alkaline boiling conditions, maltose reduces DNSA (yellow) to 3-amino-5-nitrosalicylic acid (ANSA, dark reddish-brown), measured spectrophotometrically at $lambda = 540,text{nm}$.

Thin Layer Chromatography (TLC) operates on differential partition and adsorption chromatography between two phases:

• Stationary Phase: A thin ($0.25,text{mm}$) layer of microcrystalline silica gel ($text{SiO}_2cdot xtext{H}_2text{O}$) bound to an inert aluminum or glass plate with calcium sulfate binder (Gypsum, silica gel G). Highly polar silanol groups ($-text{Si}-text{OH}$) form hydrogen bonds with polar solutes.
• Mobile Phase: A non-polar/moderately polar solvent mixture, typically BAW (n-Butanol : Glacial Acetic Acid : Water in $4:1:1 text{v/v/v}$).

As the solvent ascends the plate by capillary action, amino acids with non-polar hydrophobic side chains (e.g., Leucine, Valine) partition preferentially into the organic mobile phase and migrate rapidly (high $R_f$). Highly polar or charged amino acids (e.g., Aspartic acid, Lysine) interact strongly with the stationary phase and migrate slowly (low $R_f$).

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) in the Laemmli buffer system resolves proteins purely on the basis of polypeptide chain length (molecular mass):

• Denaturation & Charge Uniformity: The anionic detergent SDS binds cooperatively to denatured polypeptide backbones at a constant stoichiometric ratio of approximately 1.4 g SDS per 1.0 g of protein (~one SDS molecule per two amino acids). SDS imparts an overwhelming, uniform negative charge that masks intrinsic charges, creating an identical charge-to-mass ratio for all proteins. Reducing agents ($beta$-mercaptoethanol or DTT) break covalent disulfide bonds.
• Discontinuous Gel & Isotachophoretic Stacking:
  • Stacking Gel ($4% text{Acrylamide, pH } 6.8$): Operates between trailing glycine zwitterions (net charge $approx 0$) and fast leading chloride ions ($text{Cl}^-$). The sharp voltage gradient between $text{Cl}^-$ and glycine compresses all protein samples into an ultra-thin starting zone ($< 1,mutext{m}$) before entering the sieving gel.
  • Resolving Gel ($12% text{Acrylamide, pH } 8.8$): At higher pH, glycine ionizes to a fully charged anion ($text{glycine}^-$), outrunning the proteins. The concentrated polyacrylamide molecular mesh then separates proteins strictly by size through sieving.