Fischer used the results of three characteristic reactions of aldoses to demonstrate the configuration of glucose.

Oxidation by HNO₃

When an aldose is heated in aqueous nitric acid, both the aldehyde and the primary alcohol group are oxidized to carboxylic acids. The product is called a glycaric or aldaric acid (glucaric acid in the case of glucose):

Kiliani synthesis of aldonic acids

When an aldose is treated with HCN at a moderately basic pH, both pairs of diastereomeric cyanohydrins are formed. Hydrolysis then gives the carboxylic acid, one carbon longer than the original aldose. A new stereocenter is formed at what was the carbonyl carbon. This stereocenter could be of either configuration, so a pair of diastereomers is formed. These diastereomers, which differ at only one stereocenter, are called epimers.

 
Osazone formation

Fischer had developed a new hydrazine derivative, phenylhydrazine, that formed solid compounds upon addition to carbonyl compounds. When applied to sugars, this reaction gave crystalline products. However, the reaction was more complicated than the simple nucleophilic addition to an aldehyde. The -OH group at C-2 was also oxidized and derivatized to the phenylhydrazone. Since C-2 is no longer a stereocenter, aldoses that differ in configuration only at C-2 will give identical osazones.
 

 
Fischer's demonstration of the configuration of glucose

Only a few of the pentoses and hexoses were known at the time. Among these were glucose and mannose, hexoses, and arabinose, a pentose. Reaction of glucose and mannose with phenylhydrazine produced the same osazone; thus, glucose and mannose differ only at C-2. They are identical in configuration at C-3, C-4 and C-5.

The same conclusion was obtained by subjecting arabinose to the Kiliani synthesis, followed by nitric acid oxidation. This produced two isomeric glycaric acids which were identical to the glycaric acids produced from glucose and mannose by nitric acid oxidation. Since C-3, C-4 and C-5 are all derived directly from arabinose, they must be identical in the glycaric acids. Thus, mannose and glucose can differ only at C-2.

Fischer recognized that he could only specify the relative configurations of the stereocenters in glucose. His final determination, then, would narrow the possible structures for glucose to a pair of enantiomers. To simplify his reasoning he decided to arbitrarily choose one of this pair in advance by specifying the configuration at C-5. The rest of the stereocenters would then be specified by relation to C-5. The configuration he chose was with the -OH to the right in the standard Fischer projection. He called this the D configuration. We call it R today.

Now going back to arabinose and oxidizing it with nitric acid, Fischer obtained an optically active glycaric acid. This immediately gave him the configuration of C-3 relative to C-5. In the standard Fischer projection, the -OH groups at these two carbons must be on opposite sides. If they were on the same side, the glycaric acid from arabinose would have been a meso compound, regardless of the configuration about C-4.

Returning to the glycaric acids produced from glucose and mannose, he found that both were optically active. Since both ends of the molecules are carboxyl groups, it is possible to have meso glycaric acids. The only way, given the relative configurations at C-3 and C-5, for both acids to be chiral is for the -OH at C-4 to project to the right in the Fischer projection (this prevents it from forming an internal mirror symmetry with the -OH at C-3 in the structure on the left).

Fischer now had two structures, differing only at C-2, of which one was glucose and one mannose. He recognized that the glycaric acids derived from these two structures differed in an important property. One of them could only be derived from the sugar Fischer had; the other could be derived from the other sugar Fischer had, and from another one he didn't have. He set about the task of making that other sugar, and found that, when oxidized with nitric acid it produced the same glycaric acid that could be derived from glucose, thus specifying the configuration of glucose at C-2: the -OH must be to the right.

After Fischer's proof, all papers and textbooks gave the structure of glucose as the enantiomer Fischer had arbitrarily chosen. The final chapter wasn't written until 60 years later, when X-ray diffraction showed that Fischer's choice had been correct. The textbooks wouldn't have to be rewritten.