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Original Article
ARTICLE IN PRESS
doi:
10.25259/AJC_244_2025

Quantitative analysis of acetic acid and ethanol in aqueous solutions and in fermentation solutions using attenuated total reflection fourier transform infrared (ATR FTIR) spectroscopy

, , , , , , ,
aDepartment of Chemistry, Al-Quds University, Ramallah, 1234, Palestinian Territory, Occupied
bDepartment of Earth and Environment, Al-Quds University, Ramallah, Palestine, Palestinian Territory, Occupied
cDepartment of Physics, Al-Quds University, Ramallah, Palestinian Territory, Occupied

* Corresponding author: E-mail address: falrimawi@staff.alquds.edu (F. Al-Rimawi)

Received: , Accepted: ,
© 2026 Arabian Journal of Chemistry - Published by Scientific Scholar
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This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, transform, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

Abstract

The Palestinian economy relies mainly on the agricultural sector. Palestinian farmers suffer from crop failure, especially dates. One successful way to reduce their losses is to convert poor quality fruits into valuable products such as acetic acid and ethanol through fruit fermentation. It is important for them to analyze the percentage of ethanol and acetic acid in their products in simple, fast, easy, cheap, without sample preparation, robust, accurate and precise method. The objectives of this work are therefore to use an attenuated total reflection fourier transform infrared (ATR FTIR) instrument for quantitative analysis of acetic acid and ethanol in fermentation products. Different solutions of acetic acid in aqueous solutions were prepared in the range of 0.5 to 30% w/w; similarly, different solutions of ethanol in aqueous solutions were prepared in the range of 0.5 to 30% w/w. Infrared (IR) spectra of these solutions were recorded from 4000-400 cm-1. Calibration curves were constructed by plotting mass% % of ethanol or acetic acid vs. absorbance at a certain wavelength and then choosing the best calibration curve with the highest correlation coefficient. The method was validated by conducting precision and accuracy, as well as specificity, linearity, and range of the method. It was found that the mass% % of acetic acid in aqueous solution can be determined from the equation: mass% % acetic acid = 11.902R – 11.763, where R is the ratio of the absorbance of acetic acid in the sample at 1703.0 cm-1 to the absorbance of pure water at 1703.0 cm-1. While the mass% % of ethanol in aqueous solution can be determined from the equation: mass% % ethanol = 56.34R – 15.452, where R is the ratio of absorbance of ethanol in the sample at 1087.8 to that at 1636.0 cm-1. The method has been validated, and results showed that this method for the determination of acetic acid and ethanol is accurate, precise, linear, and selective. FTIR method is a simple, robust, accurate, precise, with no sample preparation step, and fast method for simultaneous determination of acetic acid and ethanol in aqueous solutions, e.g., fermentation products.

Keywords

Absorption
Acetic acid
ATR FTIR
Ethanol
Fermentation products
Wave number

1. Introduction

The fermentation process has a significant historical and cultural importance, leading to the production and preservation of a variety of fermented foods and beverages, such as yogurt, bread, beer, and vinegar [1]. The main types of fermentation include alcoholic fermentation, where yeast converts sugars into ethanol and carbon dioxide; lactic acid fermentation, utilized in dairy products and pickled vegetables; and acetic acid fermentation, which transforms ethanol into acetic acid, the primary component of vinegar [2]. Globally, food spoilage is a significant issue. In 2019, for example, about 17% of all food produced for human consumption was discarded [3]. This waste results not only in economic losses but also contributes to environmental concerns, as decomposing food generates greenhouse gases. Locally, in Palestine, approximately 20% of fresh fruits and vegetables are lost due to spoilage [4]. It was agreed that the fermentation process can be used to transform these spoiled and damaged food items into valuable products, and benefit both the economy and the environment [1].

Fermentation is a crucial tool for biorefining and waste valorization, as well as its historical role in food preservation. Converting low-quality or spoiled agricultural produce (e.g., unsellable dates) into value-added chemicals like ethanol and acetic acid provides an economic incentive for farmers while addressing environmental waste problems. However, for this to be practical at a small or medium scale, producers require accessible, low-cost, and rapid analytical techniques to monitor fermentation processes and assess product quality. This work directly addresses this need by developing a method that can be used outside a specialized laboratory.

Conventionally, quantitative analysis of fermentation products was determined by chromatographic methods. High-performance liquid chromatography (HPLC) and gas chromatography (GC) are accurate and precise methods for quantitative analysis of fermentation products. However, both mentioned methods need sample preparation and consume a relatively long time for performing analysis [5]. HPLC was used to separate and analyze fermentation products quantitatively as ethanol and acetic acid [6-7]. GC connected with a flame ionization detector was used to analyze the concentrations of ethanol and volatile organic acids quantitatively in the fermentation products [8-9].

Infrared spectroscopy (IR) is mainly used for qualitative analysis of organic compounds by the identification of functional groups. Fourier transform (FT) IR spectrometers are faster, more accurate, and more sensitive when compared to older IR spectrometers. In FTIR, frequencies are measured simultaneously. Data in FTIR were digitized and stored in the computer; data can be easily manipulated, transported, and displayed. IR radiation falls into three regions: Far IR ranges from 670-10 cm-1, mid IR ranges from 400-4000 cm-1, and near IR ranges from 4000-25000 cm-1. IR spectra contain different types of absorption for the same bond, in which the bonding electrons vibrate by stretching (symmetrical and asymmetrical) and by bending (symmetrical and asymmetrical in and out-of-plane). The mid-IR spectrum can be divided into two major regions: The diagnostic region 4000-1500 cm-1, which results from the stretching vibrations of the bonding electrons, and the fingerprint region below 1500 cm-1, which mainly results from the bending vibrations of the bonding electrons [10]. Attenuated total reflection fourier transform infrared (ATR FTIR) spectroscopy is a non-destructive analytical technique that can be used widely to study compounds of different sizes and different conditions, with almost no need for sample preparation [11]. ATR FTIR has a wide range of applications in different fields, including: Quantitative analysis of minerals [12-14], quantitative analysis of pharmaceuticals [15-16], protein analysis [17], food analysis [18-19]. And quantitative determination of olive oil adulteration [20]. Near-infrared spectroscopy (NIR) was used for the simultaneous quantitative determination of the concentrations of ethanol and acetic acid in the fermentation of a rice vinegar. Quantitative analysis is usually carried out with the aid of statistical and mathematical software programs [21]. Quantitative determination of acetic acid and ethanol in human blood serum was determined by NIR with the aid of partial least-squares (PLS) analysis [22].

A large part of the Palestinian economy depends on agriculture. To compensate for their losses, farmers especially date farms (are trying to convert the low-quality product into valuable materials such as acetic acid and ethanol through fermentation. In this context, farmers need an accurate, simple, cheap, and fast method for quantitative analysis of acetic acid and ethanol in the fermentation products. In this work, we are developing a direct method, without sample preparation, simple, cheap, fast, and without complicated mathematical and statistical operations, to determine quantitatively acetic acid and ethanol in aqueous solutions using ATR-FTIR.

2. Materials and Methods

2.1. Chemicals, reagents, and instruments

Ethanol ≥99.9% (GC), gradient grade, suitable for HPLC [STBL4841]. Glacial acetic acid (AA) with ACS specifications [MKCV7157]. Sodium hydroxide [20231205] and potassium hydrogen phthalate [MKCL8595]. All chemicals were purchased from Sigma-Aldrich. A Palestinian natural apple vinegar, from the Shams company. All solutions were prepared in Milli-Q water, using an analytical balance.

The instruments used in this study are IR and GC. A Bruker Tensor II spectrometer with a Platinum ATR accessory (Bruker Daltonics, Billerica, MA, USA) was used, in addition to a Shimadzu GC Nexis GC-2030 system (Shimadzu Corporation, Kyoto, Japan) connected to a flame ionization detector.

2.2. Solutions for calibration curve

Solutions for constructing the calibration curves for quantitative analysis of acetic acid and ethanol have been shown in Table 1 and 2, respectively. The concentration of acetic acid and ethanol in these solutions ranges from 0.5 to 30% w/w, since acetic acid and ethanol concentrations in fermentation solutions will not exceed 30% w/w [23-24].

Table 1. Absorption of acetic acid in water as a function of weight % at significant wave numbers for acetic acid, with absorption ratios relative to pure water at each wave number.
% AA in aqueous solution Absorbance at wave number 1703.0 cm-1 Absorption ratio at 1703.0 cm-1 relative to pure water Absorbance at wave number 1287.6 cm-1 Absorption ratio at 1287.6 cm-1 relative to pure water Absorbance at wave number 1233.4 cm-1 Absorption ratio at 1233.4 cm-1 relative to pure water
1 2 3 4 5 6 7
0.0 0.0591 1.0000 0.0467 1.0000 0.0462 1.0000
0.5 0.0615 1.0397 0.0491 1.0508 0.0474 1.0241
1.0 0.0637 1.0770 0.0512 1.0968 0.0473 1.0230
2.0 0.0685 1.1591 0.0564 1.2062 0.0492 1.0640
3.0 0.0731 1.2369 0.0615 1.3158 0.0512 1.1072
4.0 0.0778 1.3155 0.0664 1.4203 0.0530 1.1453
5.0 0.0824 1.3932 0.0712 1.5237 0.0544 1.1772
6.0 0.0880 1.4889 0.0759 1.6239 0.0552 1.1932
7.0 0.0930 1.5730 0.0806 1.7252 0.0568 1.2284
8.0 0.0979 1.6553 0.0853 1.8265 0.0585 1.2660
9.0 0.1027 1.7368 0.0896 1.9174 0.0601 1.3004
10.0 0.1084 1.8323 0.0943 2.0185 0.0619 1.3393
15.0 0.1334 2.2561 0.1165 2.4923 0.0716 1.5474
20.0 0.1586 2.6816 0.1371 2.9341 0.0825 1.7852
30.0 0.2072 3.5036 0.1741 3.7252 0.1104 2.3879

Note: column 3 = column 2/0.0591, column 5 = column 4/0.0467, column 7 = column 6/0.0462.

Table 2. Absorption of ethanol in water as a function of weight % at 1087.8 and 1046.4 cm-1, with absorption ratios relative to the absorbance of each sample at 1636.0 cm-1, which corresponds to water absorption.
% ethanol in aqueous solution Absorbance at wave number 1636.0 cm-1 Absorbance at wave number 1087.8 cm-1 Absorption ratio of 1087.8/1636.0 cm-1 Absorbance at wave number 1046.4 cm-1 Absorption ratio of 1046.4/1636.0 cm-1
1 2 3 4 5 6
0.0 0.1607 0.0465 0.2892 0.0485 0.3016
0.5 0.1596 0.0471 0.2948 0.0516 0.3231
1.0 0.1590 0.0481 0.3028 0.0548 0.3446
2.0 0.1570 0.0501 0.3189 0.0613 0.3906
3.0 0.1551 0.0515 0.3320 0.0674 0.4344
4.0 0.1529 0.0534 0.3489 0.0734 0.4800
5.0 0.1516 0.0552 0.3640 0.0799 0.5270
6.0 0.1523 0.0572 0.3759 0.0868 0.5697
7.0 0.1517 0.0586 0.3861 0.0913 0.6017
8.0 0.1487 0.0608 0.4091 0.0991 0.6668
9.0 0.1478 0.0627 0.4245 0.1050 0.7104
10.0 0.1464 0.0643 0.4391 0.1106 0.7552
15.0 0.1395 0.0732 0.5251 0.1395 1.0004
20.0 0.1337 0.0812 0.6074 0.1636 1.2242
30.0 0.1202 0.1003 0.8347 0.2173 1.8077

Note: Values in column 4 = column 3/ column 2 for each sample solution, and values in column 6 = column 5/ column 2 for each sample solution.

2.3. FTIR instrument and method specifications

The instrument used in this study is Bruker Tensor II with Platinum ATR. The method specifications applied were: resolution 1 cm-1, 120 scans per run in 120 s, and data points of absorption were saved from 4000-400 cm-1. Each sample was measured three times while it was located on the ATR. The data points collected for each sample from the three runs are completely identical.

2.4. Validation of the method

A vinegar sample from a domestic company in Palestine was analyzed for acetic acid and ethanol concentrations using the developed method by FTIR. This sample was measured 12 different times by ATR-FTIR. The % of ethanol was also determined by a reference standard method (GC with flame ionization detector (FID) detector) for assessing the accuracy of the method. The % of acetic acid was also determined by a reference standard method (titration with sodium hydroxide standard solution) for assessing the accuracy of the method.

2.5. GC/FID method for determination of ethanol

For method validation, a Shimadzu GC Nexis GC-2030 connected with a flame ionization detector was used to analyze the concentrations of ethanol in aqueous samples quantitatively. GC is equipped with a split injection port with a split ratio of 5-1, a flame-ionization detector, and a 0.53 mm × 30 m capillary column coated with a 3.0 μm film of phase G43. Helium is used as the carrier gas at a linear velocity of 34.0 cm s-1. The chromatograph is programmed to maintain the column temperature at 50°C for 5 min, then to increase the temperature at a rate of 10° per min to 200°C, and maintain at this temperature for 4 min. The injection port temperature is maintained at 210°C and the detector temperature at 280°C.

2.6. Titration method for the determination of acetic acid

0.1 M NaOH aqueous solution, which was prepared and standardized against potassium hydrogen phthalate, was used for quantitative analysis of acetic acid by titration using phenolphthalein indicator. The titration was conducted by a micro titration apparatus, Metrohm 665 Dosimat, equipped with a titration vessel of 7 cm. For method validation, two solutions were used: vinegar from Shams company and a quarterly prepared aqueous solution containing 4.00% w/w AA and 5.1% w/w ethanol.

3. Results and Discussion

3.1. IR spectra of acetic acid, ethanol and water

Figures 1-3 show the IR transmittance spectrums for acetic acid, ethanol, and water, respectively, from 400-4000 cm-1. The data was collected as data points. The significant wavelengths for acetic acid are: a broad peak from about 3700-3000 cm-1 with maximum absorption at 3036.4 cm-1 due to H–O bond stretching. Ethanol has the following absorption peaks: a broad peak from about 3500-3050 cm-1 with maximum absorption at 3316.2 cm-1 due to H–O bond stretching.

IR spectrum of acetic acid.
Figure 1.
IR spectrum of acetic acid.
IR spectrum of ethanol.
Figure 2.
IR spectrum of ethanol.
IR spectrum of water.
Figure 3.
IR spectrum of water.

3.2. Constructing equations for simultaneous quantitative analysis of ethanol and acetic acid in aqueous solutions

For acetic acid, the absorption at 3036.4 cm-1 due to H–O bond stretching strongly overlaps with H–O bond stretching for both ethanol and water. For that, it cannot be useful for the quantitative analysis of any component in the fermentation products. A broad peak from about 3000–2400 cm-1 with maximum absorption at 2933.6 cm-1 due to H–C bond stretching. Also, it is not a useful peak for quantitative analysis due to overlapping with that of H–C bond stretching of ethanol. A sharp and strong peak at 1703.0 cm-1 due to C double bond O bond stretching. Acetic acid absorbs about 80 times more than ethanol and 6 times more than water at this wave number. So absorption at 1703.0 cm-1 is the best choice for quantitative analysis of acetic acid in fermentation product solutions. A medium sharp peak at 1407.5 cm-1 due to O–H bending, however, it strongly overlaps with that of ethanol. It cannot be used for the analysis of any component. The following two wavenumbers 1287.6 and 1233.4 cm-1 (due to C–O bond stretching overlap slightly with ethanol and therefore cannot be used for quantitative determination of acetic acid. Acetic acid absorbs about 7 times at 1287.6 cm-1, more than ethanol, and at 1233.4 cm-1 acetic acid absorbs about 11 times more than ethanol. These two wave numbers can be used for quantitative analysis of acetic acid in fermentation products; however, they will give less accurate results than using 1703.0 cm-1, as we will see in the validation part of the method.

Ethanol has the following absorption peaks: a broad peak from about 3500-3050 cm-1 with maximum absorption at 3316.2 cm-1 due to H-O bond stretching. It is narrower than that corresponding to acid, because hydrogen bonding in acid is stronger [10]. Sharp weak peaks at 2972.1, 2926.4, and 2880.8 cm-1, all due to C-H bond stretching and overlap strongly with that corresponding to acetic acid. Weak broad peak from about 1530-1210 cm-1 corresponding to O–H bending and C–O stretching vibrations of the ethanol molecule, which strongly overlaps with that of acetic acid. A significant characteristic peaks, which are specific for ethanol, are at 1087.8 and 1046.4 cm-1. At 1087.8 cm-1 ethanol absorbs about 5 times more than acetic acid, while at 1046.4 cm-1 ethanol absorbs about 4 times more than acetic acid. We will see in the method validation that using 1087.8 cm-1 for quantitative analysis of ethanol will give more accurate results. Water has a broad peak from about 3700-2800 cm-1 with maximum absorption at 3264.8 cm-1 due to H–O bond stretching. It overlaps strongly with O–H and C–H bond stretching vibrations of both acetic acid and ethanol. Also, it has a sharp medium peak at 1636.0 cm-1 due to O–H bending vibrations. It slightly overlaps with the C double bond O of acetic acid at 1703 cm-1. All fingerprint peaks for acetic acid and ethanol below 1000 cm-1overlap strongly with water absorption peaks.

3.3. Calibration curves for acetic acid and ethanol

Plotting mass% of either acetic acid or ethanol versus absorption at the appropriate wave number in Tables 1 and 2 will give a linear relationship, as shown in Figures 4 and 5. However, absorbance at a certain wavelength for the same sample will change slightly from one time of measurement to another. It will also definitely change depending on the method used for measuring the IR spectrum (ATR, liquid film, CCl4 solution, KBr, etc). When other components of the solution are absorbed, even slightly, with the concerned compound, plotting absorbance directly with the mass% % of the desired compound to be determined will adversely affect the accuracy of the method. This will be seen in the method validation. To solve these problems, we normalize the absorbance at a certain wavenumber relative to pure water at that wave number when dealing with acetic acid, as shown in Figure 4, and as an absorbance ratio for each sample at 1087.8 and 1046.4 cm-1 relative to the absorbance at 1636.0 cm-1 when calculating ethanol concentration, as shown in Figure 5. Since at the later wave numbers, acetic acid is slightly absorbed, as shown in Tables 1 and 2.

(a-c and a’-c’) Calibration curve equations, by which the quantitative determination of acetic acid in aqueous solutions and fermentation products can be potentially calculated. Data from these curves are obtained from Table 1.
Figure 4.
(a-c and a’-c’) Calibration curve equations, by which the quantitative determination of acetic acid in aqueous solutions and fermentation products can be potentially calculated. Data from these curves are obtained from Table 1.
(d,e and d’,e’) Calibration curve equations, by which the quantitative determination of ethanol in aqueous solutions and fermentation products can be potentially calculated. Data from these curves are obtained from Table 2.
Figure 5.
(d,e and d’,e’) Calibration curve equations, by which the quantitative determination of ethanol in aqueous solutions and fermentation products can be potentially calculated. Data from these curves are obtained from Table 2.

Figure 4 and Table 3 summarize the calibration curve equations, by which quantitative determination of acetic acid in aqueous solutions and fermentation products can be potentially calculated. Figure 5 and Table 4 summarize the calibration curve equations, by which quantitative determination of ethanol in aqueous solutions and fermentation products can be potentially calculated, and fermentation products can be potentially calculated. Data from these curves are obtained from Table 1.

Table 3. Calibration curve equations, by which the quantitative determination of acetic acid in aqueous solutions and fermentation products can be potentially calculated.
Equation no. Equation Details of the equation
1

mass % acetic acid = 11.902R - 11.763

R2 = 0.9999, P = 2.20E-26

 Obtained by plotting absorbance ratio R (absorbance of the sample at wave number 1703.0 cm-1/absorbance of pure water at 1703.0 cm-1) versus mass% of acetic acid in the solution, as shown in Figure 4(a)ʹ.
2

mass % acetic acid= 201.22x - 11.758

R2 = 0.9999, P = 2.31E-26

 Obtained by plotting absorbance at wave number 1703.0 cm-1 (x) versus mass% of acetic acid in the solution, as shown in Figure 4(a).
3

mass % acetic acid = 10.785R - 11.291

R2 = 0.9966, P = 1.94E-17

 Obtained by plotting absorbance ratio R (absorbance of the sample at wave number 1287.6 cm-1/absorbance of pure water at 1287.6 cm-1) versus mass % of acetic acid in the solution, as shown in Figure 4(b)ʹ.
4

mass % acetic acid = 230.82x - 11.291

R2 = 0.9966, P = 1.94E-17

 Obtained by plotting absorbance at wave number 1287.6 cm-1 (x) versus mass% of acetic acid in the solution, as shown in Figure 4(b).
5

Mass % acetic acid= 22.216R - 20.978

R2 = 0.982, P = 1.21E-12

 Obtained by plotting absorbance ratio R (absorbance of the sample at wave number 1233.4cm-1/absorbance of pure water at 1233.4 cm-1) versus mass% of acetic acid in the solution, as shown in Figure 4(c)ʹ.
6

mass % acetic acid = 480.45x - 20.978

R2 = 0.982, P = 1.21E-12

 Obtained by plotting absorbance at wave number 1233.4 cm-1 (x) versus mass% of acetic acid in the solution, as shown in Figure 4(c).
Table 4. Calibration curve equations, by which the quantitative determination of ethanol in aqueous solutions and fermentation products can be potentially calculated.
Equation no. Equation Details of the equation
7

mass % ethanol = 56.34R - 15.452

R2 = 0.991, P = 8.48E-15

 Obtained by plotting absorbance ratio R (absorbance of the sample at wave number 1087.8 cm-1/absorbance of the sample at 1636.0 cm-1) versus mass% of ethanol in the solution, as shown in Figure 5(d)ʹ.
8

mass % ethanol = 559.32x - 25.907

R2 = 0.9995, P = 3.88E-23

 Obtained by plotting absorbance at wave number 1087.8 cm-1 (x) versus mass% of ethanol in the solution, as shown in Figure 5(d).
9

mass % ethanol = 20.272R - 5.6673

R2 = 0.997, P = 8.55E-18

 Obtained by plotting absorbance ratio R (absorbance of the sample at wave number 1046.4 cm-1/absorbance of the sample at 1636.0 cm-1) versus mass% of ethanol in the solution, as shown in Figure 5(e)ʹ.
10

mass % ethanol = 176.1x - 8.990

R2 = 0.998, P = 1.88E-18

 Obtained by plotting absorbance at wave number 1046.4 cm-1 (x) versus mass% of ethanol in the solution, as shown in Figure 5(e).

3.4. Validation of the method

3.4.1. Validation parameters

Analytical method validation is a critical process in ensuring the reliability, accuracy, and reproducibility of a method used to determine the composition, structure, or quality of a sample. When employing FTIR spectroscopy as an analytical tool, method validation ensures the technique’s suitability for its intended purpose, whether for qualitative or quantitative analysis. Validation of an FTIR method follows internationally recognized guidelines such as those by the International council for harmonization. The main validation parameters include specificity, linearity and range, precision, and accuracy.

3.4.2. Validation results and application to real samples

Specificity is the ability of the method to measure the analyte without interference from other components in the matrix. In FTIR, this involves confirming that the absorption peaks are unique to the analyte of interest. This is achieved by selecting a specific/characteristics wavelength (at a specific wave number) for both ethanol and acetic acid in aqueous solutions, and specific equations were constructed to quantitatively determine the concentration of acetic acid and ethanol in aqueous solutions, e.g., fermented sample solutions.

Linearity, which demonstrates a direct relationship between absorbance (at a specific wavelength) and the mass% % of ethanol /acetic acid in aqueous solutions. Calibration curves are typically used for this purpose. As shown in Figures 4 and 5, a direct relationship was achieved between absorbance (at a specific wavenumber) and the mass% of ethanol /acetic acid in aqueous solutions with a high correlation coefficient (R2), confirming the linearity of the proposed method. The range of the current method was selected to be from 0-30% (w/w) for both ethanol and acetic acid, as shown in Figures 4 and 5. The highest limit of linearity (30%) was chosen as the highest mass% of acetic acid in fermented solutions is 30%.

Accuracy, which measures the closeness of the measured value to the true value, is often assessed by recovery studies using known standards. Precision, on the other hand, measures the reproducibility of results under the same conditions. Precision is evaluated through repeatability (intra-day) and intermediate precision (inter-day or between operators). To this end and to assess both accuracy and precision of the method, an aqueous solution containing accurately 4.00% w/w acetic acid and 5.10% w/w ethanol was prepared. This solution was used to examine the accuracy and precision (repeatability/intra-day precision) of the method using equations in Tables 3 and 4, for simultaneous quantitative analysis of acetic acid and ethanol in aqueous solutions. This sample was measured 12 different times by ATR-FTIR. Absorbance at the significant wave numbers has been shown in Table 5. The calculated average value, percent error, standard deviation (SD), and relative standard deviation (RSD) for acetic acid and ethanol are shown in Tables 6 and 7, respectively. It can be seen that equations 1-6 are both precise and accurate for quantitative analysis of acetic acid in aqueous solutions. The most accurate and precise equations of these are 1 and 2, which depend on the absorption at the wave number 1703 cm-1. This wave number has the least matrix effect. Equation. 1 will be adopted to calculate the concentration of acetic acid in fermentation solutions. Table 7 shows that calculating mass% of ethanol in aqueous solution, based on plotting absorbance versus mass% (equations 8 and 10), is precise but not accurate. This is due to the matrix effect. Since acetic acid slightly absorbs IR radiation at the wave numbers 1087.6 and 1046.4 cm-1, and is strongly absorbed by ethanol. On the other hand, equations 7 and 9, which are based on plotting the ratio of absorbance for the sample (1087.6/1636.0 cm-1 and 1046.4/1636.0 cm-1) versus mass% % ethanol, are both precise and accurate. However, equation 7 is more accurate than equation 9, because the extent of acetic acid absorption at 1046.4 cm-1 is larger. Equation. 7 will be adopted to calculate the concentration of ethanol in fermentation solutions.

Table 5. Absorption of the aqueous solution containing 4.00% w/w acetic acid and 5.10% w/w ethanol was measured 12 times, at significant wave numbers for both components.
Run No. Absorbance at wave number 1703.0 cm-1 Absorbance at wave number 1636.0 cm-1 Absorbance at wave number 1287.6 cm-1 Absorbance at wave number 1233.4 cm-1 Absorbance at wave number 1087.8 cm-1 Absorbance at wave number 1046.4 cm-1
1 0.0780 0.1567 0.0672 0.0518 0.0573 0.0842
2 0.0782 0.1570 0.0674 0.0518 0.0568 0.0832
3 0.0779 0.1568 0.0672 0.0517 0.0572 0.0840
4 0.0779 0.1568 0.0672 0.0517 0.0571 0.0838
5 0.0782 0.1573 0.0674 0.0519 0.0570 0.0840
6 0.0779 0.1568 0.0673 0.0518 0.0571 0.0843
7 0.0780 0.1569 0.0673 0.0518 0.0572 0.0845
8 0.0780 0.1566 0.0672 0.0518 0.0573 0.0843
9 0.0779 0.1568 0.0672 0.0517 0.0573 0.0844
10 0.0779 0.1563 0.0671 0.0517 0.0569 0.0842
11 0.0778 0.1561 0.0670 0.0517 0.0570 0.0842
12 0.0779 0.1568 0.0672 0.0517 0.0571 0.0844
Table 6. The calculated average value, percent error, standard deviation (SD) and relative standard deviation (RSD) for acetic acid in the sample contains 4.00% w/w acetic acid and 5.10% w/w ethanol, depending on equations 1-6.
Equation no. applied for determining the mass % acetic acid The calculated mean value of mass % acetic acid The percent error SD RSD
1 3.94 1.42% 0.021 0.52%
2 3.93 1.63% 0.020 0.52%
3 4.23 5.83% 0.026 0.61%
4 4.23 5.63% 0.025 0.59%
5 3.92 2.03% 0.028 0.72%
6 3.90 2.57% 0.028 0.72%
Table 7. The calculated average value, percent error, (SD) and (RSD) for ethanol in the sample contains 4.00% w/w acetic acid and 5.10% w/w ethanol, depending on equations 7-10.
Equation no. applied for determining the mass % ethanol The calculated mean value of mass % ethanol The percent error SD RSD
7 5.08 0.31% 0.07 1.38%
8 6.04 18.60% 0.101 1.67%
9 5.21 2.24% 0.054 1.03%
10 5.83 14.24% 0.062 1.07%

To evaluate the accuracy and precision of the current method, a real vinegar sample from a domestic company in Palestine called Shams company was analyzed for acetic acid and ethanol concentrations using equations 1 and 7, respectively. This sample was measured 12 different times by ATR-FTIR. Absorbance at the significant wave numbers has been shown in Table 8. Applying the data in column e of Table 8 in equation 1 to calculate the mass % of acetic acid in the sample, the mean value was 3.80% with SD = 0.079 and RSD = 2.08%. This result is in good agreement with the result obtained from titrating the sample five times against 0.1M standard NaOH aqueous solution using phenolphthalein indicator in a micro titration apparatus. The mass % of acetic acid obtained from the titrations is: 3.85, 3.85, 3.8, 3.69, and 3.76%, with an average value = 3.79%. The % error in acetic acid % in this real sample using the proposed IR method in comparison with the reference method of titration with sodium hydroxide was calculated to be 0.26% demonstrating high accuracy of the method for acetic acid determination. Low RSD of the proposed IR method (2.08%) indicated high precision of the method. Applying the data in column f of Table 8 in equation 7 to calculate the mass % of ethanol in the sample, the mean value was 1.61% with SD = 0.053 and RSD = 3.26%. This result is in good agreement with the result obtained from GC measurement (1.57%). The % error in ethanol % in this real sample using the proposed IR method in comparison with the reference method (GC method) was calculated to be 2.5% demonstrating good accuracy of the method for ethanol determination. Low RSD of the proposed IR method (3.26%) indicated high precision of the method.

Table 8. Absorption of the vinegar sample at significant wave numbers for each component in the solution.
Run no. Absorbance at wave number 1703.0 cm-1 Absorbance at wave number 1636.0 cm-1 Absorbance at wave number 1087.8 cm-1 Absorption ratio at 1703.0 cm-1 relative to pure water at the same wave number Absorption ratio of 1087.8/ 1636.0 cm-1
a b c d e f
1 0.0769 0.1623 0.0490 1.3005 0.3018
2 0.0770 0.1632 0.0495 1.3031 0.3036
3 0.0777 0.1641 0.0496 1.3145 0.3021
4 0.0772 0.1640 0.0496 1.3066 0.3027
5 0.0772 0.1630 0.0494 1.3058 0.3029
6 0.0775 0.1630 0.0492 1.3119 0.3020
7 0.0773 0.1626 0.0494 1.3084 0.3041
8 0.0763 0.1618 0.0490 1.2917 0.3029
9 0.0774 0.1625 0.0495 1.3093 0.3049
10 0.0778 0.1619 0.0491 1.3163 0.3030
11 0.0775 0.1630 0.0492 1.3110 0.3019
12 0.0773 0.1622 0.0491 1.3084 0.3025

Note: Values of column e = values of column b/ 0.0591, where 0.0591 is the absorbance of pure water at wave number 1703.0 cm-1. Values of column f = values of column d/ values of column c.

3.5. Comparison with previous studies

Compared to previously published methods, the presented method offers distinct advantages. While studies by Yano et al. 1997 [21] and Paprocki et al. 2023 [22] successfully utilized NIR spectroscopy with complex multivariate calibration PLS for similar analyses, our approach uses a simpler mid-IR-based univariate calibration. This eliminates the need for advanced chemometric software and expertise, making it more accessible. Compared to the chromatographic methods (HPLC, GC) that are considered the standard method [6-7,9,25-27], our ATR-FTIR technique requires no sample preparation or consumables, reducing analysis time from hours to minutes and significantly lowering the cost per analysis.

4. Conclusions

ATR FTIR spectroscopy can be used as a robust method for the simultaneous quantitative determination of acetic acid and ethanol in aqueous solutions, especially fermentation products. This method is simple, accurate, precise, fast, with minimum cost, and with no sample preparation. Appropriate equations were selectively constructed for the determination of acetic acid and ethanol in aqueous solutions. The following equation accurately and precisely measures the concentration of acetic acid in aqueous solutions: mass% acetic acid = 11.902R – 11.763, in which R is the absorbance of the sample at 1703.0 cm-1/absorbance of pure water at 1703.0 cm-1. The following equation: mass%ethanol = 56.34R – 15.452, accurately and precisely measures the concentration of ethanol in aqueous solutions, in which R is the absorbance of the sample at 1087.8/1636.0 cm-1.

The practical application of this method is significant for small-scale producers and agricultural cooperatives, particularly in regions like Palestine. It allows for the rapid, in-house quality control of vinegar and other fermentation products without the need for expensive instrumentation, toxic solvents, or highly trained personnel. Real-time process decisions are made possible by the ease with which a producer can place a drop of the fermentation broth on the ATR crystal, obtain a spectrum in two minutes, and use the supplied equations to determine the concentrations of ethanol and acetic acid instantly.

Acknowledgment

Al-Quds center for measurement and analysis at Al-Quds University especially Mr. Zahi Turabi are acknowledged for their help in analyzing samples with GC.

CRediT authorship contribution statement

Mahmoud Alkhatib, Samia Alkhatib, and Suha Zareer: Conceptualization, Methodology, Investigation, Data curation, Writing – original draft. Mohannad Qurie and Mutaz Qutob: Investigation, Resources, Visualization and Validation. Amer Marei, and Musa Abu Teir: Resources, Supervision, Writing – review & editing. Fuad Al-Rimawi: Conceptualization, Supervision, Project administration, Writing – review & editing.

Declaration of competing interest

There are no conflicts of interest.

Data availability

The data supporting the findings of this study are available from the corresponding author upon request.

Declaration of generative AI and AI-assisted technologies in the writing process

The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.

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