Without the aid of chromatographic techniques, quantification of bio-ethanol in fermentation-broth distillate becomes inconvenient. Potassium permanganate is preferable over potassium dichromate because of the latter well-known toxic properties, it is common used in ethyl alcohol determination either by visible determination of Cr(III) green optical density, a consumed Cr(VI) determination or the unreacted Cr(VI) determination iodometrically after alcohol oxidation. Nevertheless, these titre methods arise difficulties in experience analysts from multiple solutions preparation, standardization that should be carried out every day to successful end point detection in the presence of Cr(III) green color which leads to a significant ethanol quantification error. Noteworthy permanganate-iodometry drawbacks as same as titre dichromate of difficult practical procedures and multiple reagents employed. In this laboratory, a self colorimetric method is developed in neutral medium as alcohol-specific oxidizing agent precludes both of its undesirable high oxidizing properties and difficult titrimetric methodologies for bio-ethanol quantification and application of Beer-lambert law at high substrate concentrations. It is based on unreacted permanganate optical density difference between a non-ethanol-containing sample as a blank and ethanol-containing sample is directly proportional to the consumed permanganate amount in ethanol red-ox reaction and consequently directly proportional to ethanol content. This optical density difference versus ethanol concentration 1% - 6% v/v obeys Beer-Lambert law which provides limit of detection, limit of quantification and correlation coefficient equal 0.17%, 0.56% and 0.999 respectively.
Regardless of the chromatographic and enzymatic techniques for ethanol determination, due to its high operative cost, in addition to, might be not available in many of biofuel laboratories, ethyl alcohol determination by titre acidic dichromate is everywhere and applicable in most of winery and biofuel laboratories, noteworthy it comes back since the earlier pioneer work of Widmark [
Furthermore, the extensive study of Theodore and Rosalind using alkaline permanganate in ethanol determination after its oxidation into oxalic acid [
The present work depends on the oxidation of ethyl alcohol into acetic acid and reduction of permanganate (VII) into brown precipitate Mn(IV), the principle is based on the purple unreacted Mn(VII) optical density difference between non ethanol-containing samples as a blank and ethanol-containing sample is directly proportional to the amount of consumed permanganate in redox reaction and consequently directly proportional to the amount of ethanol present according to the following equations:
CB ? CS α CEthanol
where CB is permanganate concentration in the blank and CS is the permanganate concentration after sample red-ox reaction and CEthanol is the ethanol concentration, from Beer-Lambert law and at a specific dilution.
εbAB ? εbAS α CEthanol
where AB is absorbance assigned for non-ethanol-containing sample and AS is assigned for absorbance of ethanol-containing sample after a specific dilution, ε is the permanganate relative absorbitivity and it is the same for both of blank and ethanol-containing sample and b is light path length, so
εb(AB ? AS) α CEthanol
εb is a constant value so absorbance difference between blank sample and ethanol-containing sample, ΔA, and ethanol concentration plot,
According to this assay, the ideal dilution giving the highest absorbance difference ΔA between the blank and within ethanol concentrations interval, in addition to, gives blank sample absorbance closer to 2.5 before heating step and absorbance value closer to 2.1 after heating step due to water-permanganate oxidation was 0.375 ml (375 µl):100 ml and the identified optimized reaction environment was incubation in water bath at 60˚C for 30 min, it should be mentioned that no significant increase in absorbance difference between the blank and ethanol containing sample after this mentioned incubation time.
The equation describes oxidation reaction of ethanol in aqueous neutral permanganate is shown below.
3CH3CH2OH + 4KMnO4 = 3CH3COOK + 4MnO2 + KOH + 4H2O
The color decrease percentage absolute equation for ethanol determination as shown in the method details section derived by plotting both terms of absorbance difference and ethanol concentration.
Warming and adjusting the spectrophotometer at 525 nm then zeroing using distilled water.
A 5 ml of permanganate solution is mixed well with 1 ml of distilled water as a blank sample as well as six test tubes contain 5 ml of the same permanganate solution and 1 ml of ethanol concentration ranging 1% to 6% v/v (for ethanol dilutions, ethanol HPLC grade > 99.8% was considered as 100%) and incubate them in water bath altogether at 60˚C for 30 min then cooling and centrifuged to down Mn(IV) precipitate or filtering.
A 0.375 ml (375 µl) of the clear filtrate is diluted to 100 ml for both of the blank and the other tubes set and measure the purple optical density at 525 nm.
Afterthen, calculating the difference in optical density between the blank absorbance and the other set absorbances followed by plotting ethanol concentrations on horizontal axis versus their absorbance difference on vertical axis.
| Reference | Ethanol concentration v/v | Mixing, heating at 60˚C for 30 min, cooling and centrifuge or filtering to down Mn(IV) precipitate. | Absorbance 375 µl:100 ml | Absorbance difference (Ablank − Asample) | ||
|---|---|---|---|---|---|---|
| A | Ref. | ΔA | Ref. | |||
| Blank | 0% | 1 ml H2O + 5 ml of 5% permanganate | 2.103 | AB | - | - |
| S1 | 1% | 1 ml of 1% Alcohol + 5 ml of 5% permanganate | 1.812 | AS1 | 0.291 | AB − AS1 |
| S2 | 2% | 1 ml of 2% Alcohol + 5 ml of 5% permanganate | 1.502 | AS2 | 0.601 | AB − AS2 |
| S3 | 3% | 1 ml of 3% Alcohol + 5 ml of 5% permanganate | 1.211 | AS3 | 0.892 | AB − AS3 |
| S4 | 4% | 1 ml of 4% Alcohol + 5 ml of 5% permanganate | 0.934 | AS4 | 1.169 | AB − AS4 |
| S5 | 5% | 1 ml of 5% Alcohol + 5 ml of 5% permanganate | 0.631 | AS5 | 1.472 | AB − AS5 |
| S6 | 6% | 1 ml of 6% Alcohol + 5 ml of 5% permanganate | 0.363 | AS6 | 1.797 | AB − AS6 |
The equation shown in
The limit of detection, LoD, is calculated using the following equation:
LoD = 3 SD/slope of the linear equation
where SD is the standard deviation of the lowest analyte concentration that can be detected in comparison to the blank, and the slope value of the linear relationship equals 0.297.
The Limit of quantification, LoQ, is calculated using the following equation:
LoQ = 10 SD/slope of the linear equation.
| Parameter | Neutral permanganate oxidation followed by photometry. (Proposed study) | Alkaline permanganate oxidation followed by iodometry. (Theodore and Rosalind 1936) |
|---|---|---|
| Specificity | Alcohol-specific oxidation | Non alcohol-specific oxidation |
| Reagents used | Potassium permanganate | -Potassium permanganate -Sodium thiosulfate -Sulfuric acid -Sodium hydroxide -Potassium iodide -Starch -Sodium tungstate and mercuric sulfate (in case of protein and volatile substances rich samples) |
| Assay procedures testing standard ethanol sample 5% v/v. | 5 ml of permanganate solution plus 1 ml of sample besides blank then heating at 60˚C for 30 min and calculating ΔA followed by ethanol concentration equation from method details section. | (1 ml of 5% ethanol diluted to 25 ml) is mixed with 25 ml of 1 N permanganate and 10 ml of 5 N sodium hydroxide then heating for 20 min in boiling water then cooling and neutralizing with 10 N sulfuric and titration with 1 N sodium thiosulfate using starch indicator. |
| Technique used | Photometrical determination of absorbance difference between a blank and a sample. | Iodometrical determination of unreacted permanganate. |
| Correlation coefficient | 0.999 | Not applicable |
| Intercept | −0.005 | Not applicable |
| Limit of detection | 0.17% | Not applicable |
| Limit of quantification | 0.56% | Not applicable |
| working range | 1% - 6% | Not applicable |
| *Standard 5% v/v ethanol. | M4.88 + _SD0.091 v/v | M3.91 + _SD0.012 wt/v |
| Precision, RSD% | 11.86% | 10.306% |
| Accuracy | 297.6% | 2100.2% |
Mis the mean value calculated by the average of five runs; SDis the standard deviation of five runs; 1is calculated by (standard deviation/mean value) 100; 2is calculated by mean value/true value; *is chosen to predict high acceptable confidence level so far than limit of quantification.
Materials and Methods:
All reagents were of analytical grade.
Original method details, Titrimetry, Theodore and Rosalind 1936.
Alkaline permanganate oxidation followed by Iodometry.
Reagents and Equipments:
1-Potassium permanganate 3.3%.
2-Sulfuric acid 10 N.
3-Potassium iodide.
4-Starch solution.
5-Sodium thiosulfate.
6-Sodium hydroxide 5 N.
7-Water bath.
8-Sodium tungstate and mercuric sulfate are added to ethanol-containing sample prior to distillation to prevent foams caused by presence of proteins and to remove volatile substances may be present and oxidizable other than ethanol under alkaline permanganate.
Procedures:
1-A 25 ml of potassium permanganate solution (diluted if needed according to the expected ethanol percent under determination) and 10 ml of 5 N sodium hydroxide are added to both of 25 ml of non ethanol-containing sample as a blank experiment and 25 ml of ethanol-containing sample with mixing all the contents very well then placed in boiling water bath in an attached cooling closed condenser for 20 min.
2-The solutions are cooled then neutralized by adding 10 ml of 10 N sulfuric acid followed by addition of 10 gm potassium iodide.
3-The iodine liberated is titrated against standard sodium thiosulfate as exactly the same strength as the permanganate solution.
Calculation of ethanol content:
[(volume of sodium thiosulfate for the blank) − (volume of thiosulfate for the sample)] = volume of thiosulfate equivalent to the permanganate used in the oxidation.
1 ml of 1 N of consumed permanganate is equivalent to 4.275 mg alcohol, (1 N permanganate, 3.3 gm permanganate in 100 ml H2O digested in hot water for 3 - 5 h).
Proposed method details, the present study, spectrophotometry.
Neutral permanganate oxidation followed by photometry.
Reagents and Equipments
1-Potassium permanganate, analytical grade.
2-Water bath.
4-Spectrophotometer.
Procedures:
1-Weight accurately 5 gm potassium permanganate and dissolve in 100 ml distilled water stirring on cold using magnetic stirrer for 0.5 h till homogeneity.
2-Mix well 5 ml of permanganate solution to both of 1 ml distilled water as a blank and 1 ml of ethanol solution (maximum ethanol concentration 6% v/v) and incubate them in water bath at 60˚C for 30 min.
3-Centerifuge or filter ethanol-containing sample using glass wool and dilute 0.375 ml from the clear filtrate (375 µl) to 100 ml distilled water for both of blank and ethanol sample and measure both absorbances at 525 nm after zeroing using distilled water and calculate absorbance difference ΔA and color decrease percentage as follows.
ΔA = AB − ASwhere AB absorbance assigned to Blank and AS absorbance assigned to ethanol-containing sample after the previous mentioned 375 µl:100 ml dilution.
Color decrease percentage (CDP) = (ΔA/AB)100
4-Plot absorbance difference and/or color decrease percentage versus ethanol concentrations 1% - 6% v/v.
A facile colorimetric method for ethanol quantification 1% - 6% v/v has been outlined with acceptable accuracy and precision. Fermentation broth-distillate ethanol content is oxidized by alcohol-specific aqueous neutral permanganate into acetic acid by heating in water bath at 60˚C for 30 min and decrease percent of the optical density between a non ethanol-containing sample and ethanol-containing sample is used to determine ethyl alcohol content conveniently and application of Beer-Lamber law at high substrate concentrations.
This paper is dedicated to the soul of Dr. Ahmed Kamal Youssef, Assistant Professor, Chemistry Department, Faculty of Science, Assiut University, Egypt 1976-2021.
The authors declare no conflicts of interest.
Geies, A. and Abdelazim, M. (2021) Self-Colorimetric Determination of Bio-Ethanol Using Permanganate in Fermentation Samples. Open Access Library Journal, 8: e7762. https://doi.org/10.4236/oalib.1107762