**2. Early Fe+2/H2O2 investigations**

Following the initial discovery, Fenton tested the range of his new reagent. Fenton & Jackson (1899) oxidized aliphatic alcohols, polyalcohols, and benzoic, then FeSO4 followed by H2O2, added stepwise, in molar ratios of (1: 0.1–0.25: 1), adding the peroxide gradually in small amounts.

The aliphatic alcohols: (CH3OH, CH2CH5OH, *n*-C3H7OH, *i*-C3H7OH, and n-C5H11OH), did not produce any visible changes in temperature or precipitates with phenylhydrazine, therefore Fenton assumed that these molecules were non-reactive [5]. Merz & Waters (1947) commented that the 1:1 alcohol: H2O2 ratio used in the experiments by Fenton & coworkers was so high that the alcohols were oxidized directly to organic acids. Fenton & coworkers would have discovered this result if: a) they had assayed their samples for acids, and/or b) assayed the alcohols with different concentrations of H2O2] [6].

On the other hand, the polyalcohols (C2H6O2, C3H8O3, C4H10O4, C5H12O5, and C6H14O6) showed temperature increases and release of gases, whereas the H2O2 only controls showed no reaction for either group. When the oxidized polyalcohols were

*A History of the Fenton Reactions (Fenton Chemistry for Beginners) DOI: http://dx.doi.org/10.5772/intechopen.99846*

reacted with phenylhydrazine forming osazones, indicating that hydroxyl groups of the polyols were oxidized to carbonyls, forming aldoses and/or ketoses. The oxidation of hydroxyl groups in the polyalcohols to aldehydes or ketones required a loss of two hydrogen atoms (H•) from the molecular formula.

Benzoic acid (C7H6O2) was oxidized to salicylic acid (*o*-C7H6O3) as determined by a robust violet color when ferric ion was added to the solution indicating that an exchange of a -H atom with an -OH group adjacent to the -COOH on the benzene ring [5].

Fenton & Jones (1900) repeated testing the oxidizing abilities of Fe+2/H2O2 on a larger set of aliphatic and polyhydroxy acids. Their method was to prepare a 1 M solution of reagent in H2O at 0°C, add FeSO4 to 0.125 M, then add H2O2 to 0.25 M (-FeSO4 was the control). The authors again reported that aliphatic acids appeared non-reactive while polyhydroxy acids showed vigorous and energetic reactions. The oxidized acids reacted with phenylhydrazine, and the precipitates were purified by crystallization, confirming that hydroxyl groups were oxidized to ketones or aldehydes and the molecules identified by melting point determinations. The oxidation of benzoic acid to salicylic acid was also confirmed [7].

Collectively, Fenton (1896) and Fenton et al. (1899, 1900) presented evidence for three different reactions: 1) carbon–carbon double bond formation with loss of 2 H-; 2) carbon–oxygen double bond formation with loss of 2 H- (both aldehydes and ketones; and 3) oxygen addition to phenol.

#### **2.1 Isolation of glucosones**

Following Fenton's lead, Cross, Bevan, & Smith (1898) oxidized glucose with FeSO4 / H2O2 and isolated "glucosone" (2-keto-, glucose). The experiment consisted of: 1) in 100 mL H2O, 4% or 10% glucose; 2) FeSO4 added to a concentration of 1/104 ratio with glucose, and 3) H2O2 was gradually added to a final 1:1 molar glucose/ H2O2 with stirring on ice [8]. Theorizing that 2-keto-, glucose would be indigestible, the authors used yeast to scavenge unoxidized glucose. After filtering out the yeast, the authors found that the solution still contained carbonyl molecules, as indicated by reduction of CuO. The solution retained 20% of the reducing power of the original glucose solution. The oxidized glucose solution also increased in acidity. After drying (105°F / 40.6°C), the dried residue comprised 88% of the weight of glucose including 3.8% furfural.

The solids were resuspended in chilled water and reacted with phenylhydrazine (PHZ), a reversible carbonyl-reactive label). The rationale was that while glucosone and glucose are likely to be equally soluble in most solvents, the double substituted glucosazone was expected to have a different solubility profile from the glucohydrazone. The glucosone reacted with 2 moles of PHZ, whereas glucose reacts with only one mole of PHZ, therefore their solubility in differences in organic solvents would be greater than the unlabeled molecules. After purification, PHZ-labeling was reversed with H2SO4, allowing analysis of purified glucosone. The authors repeated their method with fructose and sucrose recovering only glucosone from all three sugars implying: 1) fructose oxidized to glucosone; and 2) sucrose hydrolyzed to glucose and fructose.

Morell *et al.* (1899–1905) conducted a larger and more detailed survey of the oxidation of monosaccharides to corresponding -osones with Fenton's reagent. Following Cross & Bevan's method [9–14]. Morrell et al. (1899a) expanded the study of the osones to include galactose, rhamnose, arabinose, and mannose [9].

Morrell et al. (1899b) increased glucose to glucosone yields by: 1) slow addition of H2O2, 2) controlling temperature with refrigeration, 3) controlling pH and precipitating organic acid with (Pb(OAc)2). This method increased glucosone yield to 10%. Morell et al. purified PHZ-glucosazone and PHZ-mannosazone from their corresponding PHZhydrazones, but were not able to purify PHZ-arabinosazone, PHZ-rhamnosazone, and PHZ-galactosazone from their PHZ-hydrazone contaminants [10].

Morrell *et al*. (1900) oxidized glucose, fructose, arabinose, rhamnose, galactose, maltose, lactose and sucrose, then labeled both aldoses and osones with PHZ. The authors increased purity by precipitating the saccharic acids with Pb(OAc)2, while controlling pH with Ba(OH)2. PHZ-rhamnosazone was also separated from its PHZ-hydrazone, but not galactose or arabinose [11].

Morrell *et al*. (1902) found that reacting a glucosone with bromine severed the C2- C3 bond and oxidized C3 to a carboxylic acid, the final product being erythronic acid (trihydroxy-butyric acid), which was then dehydrated to butyric acid with nitric acid. The identity of erythronic acid was confirmed by calculating formula weights of the lead and barium salts [12].

Morell et al. (1903) oxidized aldoses with Fenton's reagent, precipitate organic acids with Pb(OAc)2 and Ba(OH)2, then label the oxidation mixture for carbonyl groups with methyl-, phenyl-hydrazine (MPHZ). However, galactose and arabinose MPHZ-osazones were not separable from their MPHZ-hydrazones. The authors tested bromo-, phenylhydrazine (BPHZ) and found that BPHZ-arabinosazone was easily separable from BPHZ-arabinose hydrazone using benzene as the solvent [13].

Morrell & Bellars (1905) retested purification of all the aldose osazones with BPHZ. BPHZ-labeling after Fenton oxidation allowed sharp separations of BPHZ -osazones from the corresponding hydrazones in benzene, thus achieving the goal of acquiring pure osazones after Fe+2/H2O2 oxidation [14]. [Considering that both Cross & Bevan (1898) and Morell et al. (1899) stated that the purified osones were tasted, the implication is that both groups were investigating the osones as non-caloric sweeteners].

### **2.2 Isolation of aldonic acids and other byproducts**

Cross & Bevan (1898) surveyed the by-products yielded by Fenton oxidation of aldoses to glucosones. The secondary products included: tartronic acid, ( 8%), acetic acid (5%), formic acid (15%), and furfural(s) ( 4%). Missing carbohydrate mass was assumed to be lost as carbon dioxide (CO2). The authors noted that Fenton oxidation of glucose produced furfural, but fructose did not; on the other hand, glucose produced lower amounts of dicarboxylic acids and pentoses [8]. Cross & Bevan (1899) oxidized a 2% solution of furfural with H2O2 and a catalytic amount of FeSO4. Formic, acetic acid, and a red precipitate identified as pyromucic acid were isolated. The authors also reported that Fe+2/H2O2 oxidized benzene to phenol, followed by additional hydroxylations [15].

Morrell et al. (1903) used Pb(OAc)2 and Ba(OH)2 to precipitate the sugar acid impurities in the quest to purify the osones. From the oxidation of glucose and fructose, the experimenters recovered the several polyhydroxy acids after precipitation with Pb+2 or Ba+2 ions. After removal of Pb+2 and Ba+2 ions with H2SO4, the solubilized acids were identified as glycolic and oxalic. The Pb+2 / Ba+2 soluble acids were separately precipitated as calcium salts and identified as glyoxylic and trihydroxy-butyric acids [13].

In sum, Cross & Bevan (1898, 1899) and Morrell et al. (1899–1903) confirmed that Fenton's reagent was responsible for the following reactions: dehydrations producing C=C bonds, C-C bond cleavages, and oxygen atom additions forming hydroxyl, aldehyde, ketone, and carboxylic acid groups, creating new classes of organic molecules.
