Self-Colorimetric Determination of Bio-Ethanol Using Permanganate in Fermentation Samples

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 in strong acid medium by measuring band absorbance decrease at 267 nm or the unreacted Cr(VI) determination iodometrically after alcohol oxidation. Nevertheless, these titre methods arise difficulties experience analysts from multiple solutions preparation, standardization that should be carried out every day and 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 difficult practical procedures and multiple reagents employed. In this laboratory a self colorimetric method was 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. 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-Lampert law provides limit of detection, limit of quantification and correlation coefficient equal 0.17%, 0.56% and 0.999 respectively.

density, a consumed Cr(VI) determination in strong acid medium by measuring band absorbance decrease at 267 nm or the unreacted Cr(VI) determination iodometrically after alcohol oxidation. Nevertheless, these titre methods arise difficulties experience analysts from multiple solutions preparation, standardization that should be carried out every day and 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 difficult practical procedures and multiple reagents employed.
In this laboratory a self colorimetric method was developed in neutral medium as alcoholspecific oxidizing agent precludes both of its undesirable high oxidizing properties and difficult titrimetric methodologies for bio-ethanol quantification.
It is based on unreacted permanganate optical density difference between a non ethanolcontaining 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-Lampert law provides limit of detection, limit of quantification and correlation coefficient equal 0.17%, 0.56% and 0.999 respectively.

Introduction:
Regardless the chromatographic and enzymatic techniques for ethanol determination that may not be available in many of chemical laboratories, ethyl alcohol determination by titre acidic dichromate is everywhere in literature and applicable in most of winery and biofuel laboratories, noteworthy it comes back since the earlier pioneer work of Widmark [1] and Nicloux [2,3], iodometry enables to determine the unreacted dichromate amount by titrating the amount of iodine librated upon the addition of potassium iodide to the reaction mixture against a standard solution of sodium thiosulfate. Despite of the best repeatability and accuracy of this titre method but it experiences a lot of difficulties arise in end point color change successful detection especially in presence of the produced green color of Cr(III) repeatedly noticed especially with who not well-experienced in the art and this observation agreed to literature [4]. On the other hand, the bad repeatability of the direct spectrophotometric measurement of the resulting Cr(III) green optical density is not be recommended [5,6] or by determining the consumed Cr(VI) by directly measuring the absorbance decrease of a band assigned at 267 nm [7]. Furthermore, the extensive study of Theodore and Rosalind using alkaline permanganate in ethanol determination after its oxidation into oxalic acid [8] as well as Evans and Day work regarding oxidation of ethyl alcohol by means of potassium permanganate reported the oxidized product in neutral medium is acetic acid as an only product [9] inspired us in this laboratory to utilize neutral aqueous permanganate as a specific-alcohol oxidizing agent in ethyl alcohol determination spectrophotometrically precludes its undesirable high oxidation power and eliminates the difficulty involved in titre methods especially at high ethanol levels as closer as fermentation broth-ethanol [10,11] content and to avoid dichromate well known high toxicity.
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 a non ethanol-containing sample 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:

C B -C S α C Ethanol
Where C B is permanganate concentration in the blank and C S is the permanganate concentration after redox reaction and C Ethanol is the ethanol concentration, from Beer-Lambert's law and at specific dilution.

ƐbA B -ƐbA S α C Ethanol
Where A B is absorbance assigned for non ethanol-containing sample and A S assigned for absorbance of ethanol-containing sample after specific dilution, Ɛ 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

Ɛb(A B -A S ) α C Ethanol
Ɛb is a constant value so absorbance difference between blank sample and ethanol-containing sample, ΔA, and ethanol concentration plot, figure (1), gives a linear relationship obeys Beer-Lambert's law.

Results and Discussion:
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.
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.      Table (2), data from absorbance difference ΔA and ethanol concentration plot M is the mean value calculated by the average of five runs SD is the standard deviation of five runs 1 is calculated by (standard deviation/mean value) 100 2 is calculated by mean value/true value * is chosen to predict high acceptable confidence level so far than limit of quantification The limit of detection, LoD, is calculated using the following equation  Proposed method details, the present study, spectrophotometry.

Conclusion:
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 the optical density decrease percent between a non ethanol-containing sample and ethanol-containing sample was used to determine ethyl alcohol content conveniently.