The method describes how to determine the content of oxidizable organic substances in water using an oxidizing agent, potassium dichromate.
Potassium dichromate oxidizes many organic and certain inorganic substances to various extents in an acidic solution. Since the level of oxidation depends upon the kinds of substances, the concentration of potassium dichromate, the pH of the solution, and the temperature and reaction time, the procedure described below must be followed precisely. The volume of potassium dichromate required in the analysis is determined potentiometrically. In an acidic solution, the dichromate ions are reduced to chromium(III) ions:
Cr2O72- + 6 e- + 14 H3O+ → 2 Cr3+ + 21 H2O
Dichromate ions in excess of those required are determined through titration with an ammonium iron(II) sulfate solution:
Cr2O72- + 6 Fe2+ + 14 H3O+ →2 Cr3+ + 6 Fe3+ + 21 H2O
Potassium permanganate oxidizes many organic and certain inorganic substances more or less completely in acidic, neutral or alkaline solutions. The volume of potassium permanganate required in the analysis is determined potentiometrically. Since oxidation depends on the type of solution, on its temperature and on the reaction time, the procedure described below must be followed precisely.
In acidic solutions, permanganate ions are typically reduced to manganese(II) ions:
MnO4- + 5 e- + 8 H3O+ → Mn2+ + 12 H2O
In alkaline solutions, the reduction results in tetravalent manganese only:
MnO4- + 3 e- + 4 H3O+ → MnO2 + 6 H2O
Since in both cases the titration takes place in an acidic solution, this is irrelevant for the calculation. By adding oxalic acid, both the excess permanganate ions as well as the tetravalent manganese are reduced to manganese(II) ions:
2 MnO4- + 5 C2O42- + 16 H3O+ → 2 Mn2+ + 24 H2O + 10 CO2
MnO2 + C2O42- + 4 H3O+ → Mn2+ + 6 H2O + 2 CO2
Determination of D-gluconic acid by enzymatic means
This analysis is suitable for non-alcoholic beverages and for those containing alcohol.
Fruit juices
The positive effect of fermented beverages on the human body has been known for centuries. Current beverage trends, like kvass (Russia) and kombucha (Asia), stem from traditions with roots deep in the past. They have always been consumed as healing beverages. Non-alcoholic forms of fermentation employ microorganisms, such as lactic and acetic acid bacteria. They produce organic acids like lactic acid and gluconic acid, which promote digestion and metabolism. Due for the most part to their slightly acidic flavor, these kinds of fermented beverages are popular with consumers as a healthy natural refreshment.
Malt, fruit juice and tea serve as a base for fermented beverages.
As a rule, fermented beverages contain 0.5 – 15 g/l D-gluconic acid.
D-gluconic acid is phosphorylated by adenosine 5'-triphosphate (ATP) in the presence of gluconate kinase to gluconate-6-phosphate
D-Gluconate + ATP \(^{\underrightarrow{\text{gluconate kinase}}}\) D-gluconate-6-P + ADP
The enzyme 6-phosphogluconate dehydrogenase (6-PGDH) catalyzes the oxidation of gluconate-6-phosphate to ribulose-5-phosphate with nicotinamide adenine dinucleotide phosphate (NADP):
D-Gluconate-6-phosphate + NADP+ \(^{\underrightarrow{6-PGDH}}\) ribulose-5-phosphate + NADPH + H+ + CO2
The amount of NADPH formed during the reaction is proportional to the amount of D-gluconic acid.
Determination of oxalic acid by enzymatic means
Suitable for malt, wort, beer, beer-based beverages and soft drinks
Oxalic acid is primarily derived from malt. By reacting with the calcium ions in the brewing liquor, haze caused by calcium oxalate can form. These crystals also serve as nucleation sites for the spontaneous and rapid release of carbon dioxide (gushing). The precise determination of oxalic acid is therefore of great importance in brewing technology.
Oxalic acid (oxalate) is oxidized to carbon dioxide and hydrogen peroxide by the enzyme oxalate oxidase.
\(\text{ Oxalate} \hspace{0.5em}^{\underrightarrow{oxalatoxidase}}\hspace{0.5em} H_2O_2\hspace{0.3em}{+}\hspace{0.3em}CO_2\)
In the presence of the enzyme peroxidase (POD), hydrogen peroxide reacts with MTBH (3-methyl-2-benzo thiazolinone hydrazone) and DMAB (3-dimethyl amino benzoic acid to form a blue quinone complex.
\(H_2O_2+MTBH+DMAB\hspace{0.8em}^{\underrightarrow{POD}} \hspace{0.8em} \text{quinone complex} \space + \space H_2O\)
The intensity of the color is proportional to the concentration of the oxalate in the sample and is measured at 590 nm.
Determination of ascorbic acid by enzymatic means
This analysis is suitable for wort, beer, beer-based beverages and NAB
L-Ascorbic acid (ascorbate) as well as the reducing substances (X-H2) reduce the tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] in the presence of PMS (5-methylphenazinium methosulfate), which mediates electron transfer, at a pH of 3.5 to a formazan:
L-Ascorbate (X-H2) + MTT+ \(^\underrightarrow{PMS}\) dehydroascorbate (X) + MTT formazan +H+
Of all the reducing substances present in the blank, only the ascorbic acid portion of the sample is oxidatively removed by ascorbate oxidase (AAO) in the presence of oxygen. Dehydroascorbate is produced in the reaction and does not react with MTT/PMS:
L-Ascorbate (X-H2) + ½ O2 \(^\underrightarrow{AAO}\) dehydroascorbate (X) + H2O
The absorbance of the sample minus the absorbance of the blank is equivalent to the quantity of ascorbic acid in the sample. MTT formazan serves as the measured variable, and its absorption can be determined photometrically in the visible part of the spectrum at 578 nm.
Determination of acetic acid by enzymatic means
This analysis is suitable for malt, wort, beer, beer-based beverages and soft drinks
Acetic acid (acetate) is converted to acetyl-CoA in the presence of the enzyme acetyl-CoA synthetase (ACS) by adenosine-5'-triphosphate (ATP) and coenzyme A (CoA).
Acetate + ATP + CoA \(^{\underrightarrow{ACS}}\) Acetyl-CoA + AMP + pyrophosphate
Acetyl-CoA reacts with oxaloacetate in the presence of citrate synthase (CS) to form citrate.
Acetyl-CoA + oxaloacetate + H2O \(^{\underrightarrow{CS}}\) citrate + CoA
The oxaloacetic acid required for reaction (2) is produced from malic acid and nicotinamide adenine dinucleotide (NAD) in the presence of malate dehydrogenase (MDH). In doing so, NAD is reduced to NADH:
Malate + NAD+ \(^{\underleftrightarrow{L-MDH}}\) oxaloacetate + NADH + H+
The formation of NADH forms the basis of this analysis, which is measured as an increase in the absorbance at 340, 334 or 365 nm. Since this concerns a previous indicator reaction, the quantity of NADH is not linearly proportional to the acetic acid concentration.