Rapid detection of acids and esters in Baijiu by Fourier transform infrared spectroscopy with difference spectroscopy

Rapid detection of acids and esters in Baijiu by Fourier transform infrared spectroscopy with difference spectroscopy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Rapid detection of acids and esters in Baijiu by Fourier transform infrared spectroscopy with difference spectroscopy Yixuan Guo, Zhiqiang Wang, Ruiting Zhang, Lin Ma, Ke Lin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4481737/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Acids and esters are the main aromatic compounds in Baijiu. These compounds affect primarily the flavor and quality of Baijiu. The detection of these compounds is important for the control of Baijiu. Although FTIR spectroscopy has gradually been used to detect Baijiu in recent years, this technology has not been directly employed to measure the infrared spectra of acids and esters in Baijiu. In this paper, a novel FTIR difference spectroscopy is proposed to extract the infrared spectra of acids and esters in Baijiu. This difference spectrum is mainly obtained by subtracting the FTIR spectra of aqueous ethanol from that of Baijiu. The FTIR spectra of some kinds of Baijiu were measured, and the infrared spectra of acids and esters in Baijiu were obtained. The flavor of Baijiu can be distinguished through the difference spectra. Since the acquisition of FTIR spectra only takes less than one minute, this FTIR difference spectrum can be developed as a quick control method for Baijiu. Baijiu Ester Acid Fourier transform infrared spectroscopy Difference spectrum Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Chinese liquor (Baijiu) has a history of more than 3,000 years. One of the reasons why Baijiu is enduring in China is that it contains diverse trace substances.(Shu, Zhang, Zhang, Yue, Liu, & Mu, 2007) Acids and esters are the most content in the trace substances of Baijiu, and they are the main aroma compounds in Baijiu.(Fan, Shen, & Xu, 2011 ; Niu, Yao, Xiao, Xiao, Ma, & Zhu, 2017; Song et al., 2020 ) Because various acids and esters impart the unique flavor and taste to Baijiu, the national standards for Baijiu require the determination of acid and ester content. Different brands, flavors and grades of Baijiu have different acid and ester content. For example, compared to light aroma-type Baijiu, strong aroma-type Baijiu has higher ethyl acetate content. For Baijiu with the same brand, the content of acetic acid and ethyl acetate in Baijiu with premium grade is higher than that in the Baijiu with first-class grade. The content and ratio of acid and ester can also reflect the age of Baijiu. While the storage time of Baijiu becomes longer, the acid in Baijiu will increase, however the ester will decrease.(S. Gu et al., 2020 ) These above properties demonstrate that detection of the concentration of acids and esters is closely related to control the quality of Baijiu. Thus the quantitative detection of acids and esters is particularly important. In order to detect acids and esters in Baijiu, various methods have been developed. For example, in the Chinese national standard GBT 10345 − 2007, the acid-base neutralization titration method is used to measure the acid content in Baijiu, and the gas chromatography method is used to determine the ester content in Baijiu. The chromatographic signal can be easily distinguished, which is why chromatography can be used to quantitatively measure the concentration of various substances in Baijiu. When combined with mass spectrometry, it can also record the concentration of acid and ester with higher accuracy of ng/L. (Buck, Goblirsch, Beauchamp, & Ortner, 2020; Okaru, Abuga, Kibwage, & Lachenmeier, 2017; Sun, Yin, Zhao, Sun, & Zheng, 2018) The chromatography experimental steps are relatively cumbersome, and it generally require several hours. For example, in order to study the chromatographic signal of liquor, the chromatogram of pure possible chemical compounds should be record before the detection of liquor. These methods are not suitable for rapid detection in the market or enterprise automated assembly line. Compared with the gas chromatography, the liquid chromatography has maintained higher accuracy and improved the efficiency, but it still cannot meet the needs of rapid detection. (Charapitsa et al., 2021 ) In addition to the above methods, various spectroscopy methods have also been used to detect the liquor. For example, Raman spectroscopy, infrared spectroscopy, fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy and other spectroscopic methods are often employed. The main advantage of spectroscopy is that the liquor can be detected quickly, and sample pretreatment is not required. And the disadvantage is that the concentration of trace components in the liquor cannot be accurately measured. This is because the spectra of various substances often overlap with each other, and the spectra can hardly be distinguished well. Thus various chemometric methods are applied to analyze these spectra to detect liquor. The principal component analysis, the partial least squares, the support vector machine, and other big data analysis method are usually applied to analyze these spectra. These methods can usually be used to classify liquor, such as source of origin, etc.(Anjos, Santos, Estevinho, & Caldeira, 2016; Joshi, Vi Khanh, Elbourne, Chapman, & Cozzolino, 2019; Li, Tsuta, Tanaka, Tsukahara, & Tsukahara, 2020) Although the spectra of liquor contains the spectral signals of all the chemical components in the liquor, these spectra mainly reflect the most abundant chemical components. For example, in Raman spectra, mid-infrared or near-infrared spectra, the largest spectral peaks are the signal of ethanol and water.(Giannetti, Mariani, Marini, Torrelli, & Biancolillo, 2020; J. Gu et al., 2019 ; Urickova & Sadecka, 2015 ) None of previous spectra showed directly the spectral bands of the trace substances in liquor. In order to enhance the signal of trace components, surface enhanced Raman spectroscopy was used.(de Almeida, Leopold, Franco, & Pereiral, 2019; Qi, Chen, Wang, & Jiang, 2018; Xiao & He, 2019 ) So far, there has not been seen that these spectroscopy methods are used to measure the concentration of acids and esters in liquor. In Baijiu, the content of trace substances is about 1%-2%. The acids and esters are the main components in Baijiu, and their concentration can reach 1-2g/L.(Fan et al., 2011 ) That is to say, in the spectra, especially in Raman or infrared spectra, the signal of acids and esters can account for about 1%-2% of the total spectral signal. Hence the spectra of acids and esters can be detected through infrared spectroscopy or Raman spectroscopy. However the spectra of acids and esters did not present in the previous infrared spectra and Raman spectra of Baijiu, which is maybe that the spectra of these trace substances are overlapped with the large spectral bands of ethanol and water. Therefore, in order to obtain the spectra of acids and esters in Baijiu, the spectra of ethanol and water should be subtracted from the spectra of Baijiu. Here we present a special difference spectroscopic method to subtract the spectral bands of ethanol and water. Using this novel analysis method, we obtained the spectra of acetic acid and ethyl acetate in Baijiu. These spectra were used to detect the quality of Baijiu. These results show that this difference spectroscopic method is a quick detection method, and the method can be used to quantitatively measure the content of acids and esters in Baijiu. 2. Materials and methods 2.1 Materials Ethanol (CH 3 CH 2 OH,99.7%) is purchased from Shanghai Wokai Biotechnology Co., Ltd., acetic acid(CH 3 COOH, 99.5%)is purchased from Tianjin Tianli Chemical Reagent Co., Ltd., ethyl acetate༈CH 3 COOC 2 H 5 , 99.5%༉is purchased from Tianjin Fuyu Fine Chemical Co., Ltd.. Water is Millipore ultrapure water. These chemical compounds were used without further purification. Seven kinds of Baijiu and two kinds of Vodka were all purchased from the supermarket. Their brand names are Yongfeng Erguotou, Xifeng Jiu, Yanghe King, Xiaolang Jiu, Wuliang Chun, Niulanshan, Luzhou Laojiao, Absolute Vodka and Siminuo Vodka. In order to remove the spectral bands of ethanol and water in Baijiu, we prepared aqueous ethanol solutions with the same ethanol concentration of Baijiu. The content of ethanol in Baijiu is present in the volume percentage. The volume percentage can be converted into the mass percentage according to its density-volume relationship. Thus the aqueous ethanol solutions were prepared by weighing. The accuracy of the electronic balance used here is 0.0001g. In order to measure the spectra of acetic acid and ethyl acetate in ethanol aqueous solution, we added a small amount of acetic acid or ethyl acetate into the aqueous ethanol. The concentration of acetic acid or ethyl acetate is 0.67mmol/g. 2.2 Methods The Fourier transform infrared (FTIR) spectra were record with a Nicolet iS20 FTIR spectrometer, which was equipped a triglyceride titanium deuterated sulfate detector and a KBr beam splitter. Instead of the transmission mode, the total reflection mode with ZnSe crystal was used because of the strong absorption of water. The spectra were record from 32 scans, 2 levels of zero filling, Happ Genzel apodization, and Mertz phase correction. The FTIR spectral resolution is 4 cm − 1 . Before each spectrum is collected, the background spectrum is collected independently to eliminate the spectral signals of water vapor and carbon dioxide. The total reflection crystal was cleaned with ultrapure water and was dried between two measurements. 3. Results and discussions 3.1 FTIR spectra of Baijiu Liquor mainly contains ethanol and water, thus the FTIR spectra of liquor is dominated by the spectra of ethanol and water. The Fourier transform total reflection infrared spectrum of a Baijiu (Xifeng Jiu) was measured, and shown in Fig. 1 . Lots of sharper peaks were observed in the spectra. These peaks were also observed in the FTIR spectrum of pure ethanol shown in Fig. 1 , thus these sharper peaks were all assigned to various vibration modes of ethanol. Based on previous spectroscopic studies of ethanol (Malloum, Fifen, & Conradie, 2020 ; P, Hee, & Haiyan, 2016), the peak at ~ 877 cm − 1 and ~ 1043 cm − 1 is assigned to the symmetric and asymmetrical stretching vibration of C-C-O, respectively. The spectral peaks at ~ 1085 cm − 1 and ~ 1273 cm − 1 are assigned to CH 3 swing vibration. The peak at ~ 1325 cm − 1 is considered to the torsional vibration of CH 2 . The peak at ~ 1383cm − 1 is assigned to the bending vibration of CH 3 and CH 2 . The peaks at ~ 1417 cm − 1 , ~ 1453 cm − 1 and ~ 1482 cm − 1 are all attributed to the bending vibration of CH 3 . The peak at ~ 2978 cm − 1 is assigned to CH 3 or CH 2 stretching vibration. The positions of these peaks in pure ethanol and Baijiu are not exactly the same. For example, the strongest peak is located at 1043 cm − 1 in the spectrum of the Baijiu, but at 1045 cm − 1 in the spectrum of pure ethanol. This noncoincidence demonstrated the different intermolecular interactions(Amo & Tominaga, 2000 ) of ethanol molecules in pure ethanol and Baijiu. In pure ethanol, ethanol molecules interact with ethanol molecules through hydrogen bonds and hydrophobic interactions, however ethanol molecules interact with water molecules through hydrogen bonds in Baijiu. In addition to the above sharp peaks, some broad peaks were also observed at ~ 3200 cm − 1 , ~ 1633 cm − 1 and below ~ 1000 cm − 1 in the FTIR spectrum of Baijiu. Compared with the FTIR spectra of water and pure ethanol in Fig. 1 , the broad spectrum peak below 1000 cm − 1 is mainly attributed to the intermolecular vibration of ethanol or water. The peak at ~ 1633 cm − 1 is assigned to the HOH bending vibration of water molecule. The peak at ~ 3200 cm − 1 is assigned to the O-H stretching vibration of water and ethanol molecules.(Malloum et al., 2020 ) These peaks are very broad, mainly because of the strong hydrogen bonds between the molecules in the solutions. Using the peak intensity of C-C-O asymmetric stretching vibration (~ 1043 cm − 1 ), the signal-to-noise ratio of the spectra of Baijiu (Xifeng Jiu) is estimated to be about ~ 1000. Since the concentration of acid or ester in Baijiu is close to 1%, if the infrared absorption coefficient of acid or ester is not much different from that of ethanol, the spectral intensity of acid and ester should be greater than the noise of the spectrum. However the spectral bands of acids and esters are not directly observed in the spectra of Baijiu, which is probably because their peaks are overlapped by the spectral bands of water and ethanol. In order to obtain the spectral peaks of these acids or esters, the spectral bands of ethanol and water should be removed from the spectra of Baijiu. Thus the difference spectra could be employed. Difference spectra were often used in spectral analysis previously. This method is usually used to reflect some small changes in the spectrum to reveal the microstructure of complex small molecule solutions, (Panuszko, Stangret, Nowosielski, & Bruzdziak, 2020) as well as the interaction between cells and drugs in biological solutions, etc.(Ali, Miraz, & Andrew, 2020 ) The most direct method to remove the spectral peaks of ethanol and water is to subtract the spectrum of pure ethanol and the spectrum of pure water. The difference spectra can be obtained from the following equation. In this equation, △I , I Baijiu , I ethanol and I water are the FTIR difference spectrum, the FTIR spectrum of Baijiu, pure ethanol, and pure water, respectively. The coefficients a and b can be calculated according to the concentration of ethanol in Baijiu. $$△I={I}_{Baijiu}-\left(a*{I}_{ethanol}+b{*I}_{water}\right)$$ 1 Using the Eq. ( 1 ), the difference spectrum of the Baijiu (Xifeng Jiu) was obtained, and shown in Fig. 2 a. No matter how the coefficients a and b were adjusted, the spectral bands of ethanol or water could not completely removed. For example, using the coefficients a and b that are according to the concentration of ethanol in Baijiu, the positive and negative spectral peaks are observed in the difference spectrum (Fig. 2 a). This demonstrated the spectral peaks of ethanol and water in Baijiu were inconsistent with those in pure ethanol and pure water. These is because of the different intermolecular interactions in these solutions.(Lin, Hu, Zhou, Liu, & Luo, 2012) In pure water, water molecules interact with water molecules through hydrogen bonds. In pure ethanol, ethanol molecules interact with ethanol molecules through hydrogen bonds and hydrophobic interactions. However, in Baijiu, ethanol molecules not only interact with ethanol molecules, but also interact with water molecules. Therefore, the spectral signals of ethanol and water in Baijiu cannot be totally removed through Eq. ( 1 ). Thus, it is impossible to observe the spectral peaks of only 1%-2% trace compounds in these difference spectra. In order to simulate the influence of the molecular interaction between ethanol and water on the spectrum, we prepared an aqueous ethanol solution with the same ethanol concentration as that of Baijiu. In this solution, the molecular interactions are similar with those of Baijiu, thus the spectral peaks of ethanol or water are very close to those of Baijiu. Using these spectra and Eq. 2 , the new kind of difference spectrum can be obtained. In this equation, △I , I Baijiu and I solution are the FTIR difference spectrum, the FTIR spectrum of the Baijiu and the aqueous ethanol solution. $$△I={I}_{Baijiu}-{I}_{solution}$$ 2 Based on this method, the FTIR difference spectrum of Baijiu (Xifeng) was obtained, and shown in Fig. 2 (a). This difference spectrum is obviously different from the previous difference spectrum by Eq. 1 . Using Eq. 2 , the sharp bands of ethanol is almost completely removed. A weak band at ~ 3300 cm − 1 are still residual, which may be due to the small difference between the concentration of the Baijiu and the concentration of the aqueous ethanol solution. More importantly, some new peaks in this difference spectrum were observed, they were located near ~ 1260 cm − 1 , ~ 1377 cm − 1 , and ~ 1710 cm − 1 , which were shown in the difference spectra of the fingerprint region in Fig. 2 (b). These spectral bands are inconsistent with the peaks of ethanol and water, and they should be assigned to the vibration modes of trace compounds in Baijiu. While the ethanol concentration of Baijiu is exactly the same as that of aqueous ethanol solution, the difference spectrum should be completely free of water and ethanol signals. In the experiment, it is hard to accurately prepare an aqueous ethanol solution with the same alcohol content of Baijiu, which will cause the weak spectrum of ethanol and water to remain in the difference spectrum. Through preparing carefully these aqueous solutions, the vibrational peaks of ethanol and water could be removed as much as possible. This kind of difference spectra method was used to analyze seven different kind of Baijiu to verify the universality of this method. The FTIR spectra of these Baijiu and the corresponding aqueous alcohol solutions were measured. Using Eq. ( 2 ), the difference spectra were obtained, and shown in Fig. 3 . In these spectra, three new vibrational peaks were observed obviously. All the spectra were all similar with each other, which demonstrated the similar substances in these Baijiu. The intensity of the same vibrational bands in these spectra is different with each other, thus the contet of the trace substances were different in the seven kinds of Baijiu. 3.2 Assignments of the FTIR difference spectra Previously, acids and esters were found to be the dominant trace components through chromatographic analysis in Baijiu. Usually, ethyl acetate and ethyl lactate account for more than 96% of the total esters in Baijiu, of which ethyl acetate is the majority.(Wang, Fan, & Xu, 2014 ) Acetic acid is the most representative acids in Baijiu. Thus the vibrational peaks in the difference spectra of Baijiu are likely to be assigned to acetic acid or ethyl acetate. To verify this assignment, the difference spectra of Baijiu should be compared with the Fourier transform infrared spectra of acetic acid and ethyl acetate. However, the spectra of acetic acid and ethyl acetate in aqueous ethanol will be different from those in the pure liquid, which is because of the different intermolecular interactions in these solutions. Thus, in order to compare the difference spectra of Baijiu with the spectra of acetic acid and ethyl acetate, the infrared spectrum of acetic acid or ethyl acetate in aqueous ethanol were measured. Here an aqueous ethanol solution was prepared with the volume concentration 45%, and small amount of acetic acid and ethyl acetate were added in to this aqueous solution, respectively. Then the FTIR spectra were measured for these solutions. Using Eq. 2 , the spectral bands of ethanol and water were removed from the spectra of mixtures. Thus the FTIR spectra of acetic acid or ethyl acetate in the mixtures were obtained, and shown in Fig. 4 . The FTIR spectra are similar with the FTIR difference spectra of Baijiu. Hence then, the vibrational bands in the difference spectra in Baijiu could be assigned to be the vibrational modes of acetic acid or ethyl acetate. In both FTIR spectra of acetic acid and ethyl acetate, the vibrational peaks are all similar, and they peaks were located at ~ 1260 cm − 1 , ~ 1377 cm − 1 , and ~ 1710 cm − 1 . The vibrational peaks at ~ 1260 cm − 1 and ~ 1710 cm − 1 of acetic acid are similar with those of ethyl acetate, but the vibrational bands at ~ 1377 cm − 1 are different. For acetic acid, the vibrational peak at ~ 1377 cm − 1 is very broad, and its full width at half maximum is ~ 60 cm − 1 . This peak is so broad, which is mainly because two sub-bands overlapped with each other. For ethyl acetate, the vibrational peak at ~ 1377 cm − 1 is very narrow, and its full width at half maximum is ~ 10 cm − 1 . Although there is little difference between the FTIR spectra of acetic acid and ethyl acetate, the FTIR spectrum of acetic acid and ethyl acetate in Baijiu is hardly distinguished because of the strong overlapping. In order to assign these peaks, the theoretical infrared spectra of ethyl acetate and acetic acid were calculated through using density functional theory. For acetic acid, there are trans and cis structure. As the trans structure of acetic acid is much more stable than cis structure of acetic acid,(Giubertoni, Sofronov, & Bakker, 2019 ) thus only the theoretical spectra of trans acetic acid was calculated. In Baijiu, the ethyl acetate and acetic acid can interact with water molecules or ethanol molecules through hydrogen bonds. Thus, the polarizable continuum model (PCM) with water or ethanol solvents was employed to simulate the effect of the intermolecular interactions to the IR spectra of acetic acid and ethyl acetate. The scaled factor 0.9614 was used to adjust the theoretical IR spectra. The theoretical spectra were shown in Fig. 4 . The theoretical spectrum of acetic acid or ethyl acetate in water is same with the theoretical spectrum in ethanol. The theoretical spectra agreed with the experimental spectra, hence then the assignment could be obtained from the theoretical calculations. The vibrational peak at ~ 1260 cm − 1 in the difference spectrum is assigned to the C-C stretching vibration. The peak at ~ 1377 cm − 1 is attributed to the bending vibration of the methyl or methylene group and the C-O-H. The peak at ~ 1710 cm − 1 is assigned to the C = O stretching vibration. These assignments were agreed with previous studies.(Matthias, Frank, & Stefan, 2014 ; Nishi, Nakabayashi, & Kosugi, 1999 ; Śmialek et al., 2016 ; Zhang et al., 2020 ) Since the spectra of acetic acid and ethyl acetate in aqueous ethanol are very similar, and their spectra are seriously overlapped, thus the FTIR spectra of acetic acid and ethyl acetate could not be distinguished directly. Hence then, we can only obtain the total spectra of acetic acid and ethyl acetate in the difference spectrum of Baijiu. Using the C = O stretching peak of acid or ester at ~ 1710 cm − 1 , the total content of acid and ester can be estimated, and there is no way to give the content of each acid or ester by the this peak. It is noticed that the FWHM is much different for the peak at ~ 1377 cm − 1 , thus the FWHM of this vibrational peak can be used to qualitatively estimate whether there is more acid or more ester in Baijiu. These band is important to determine the age of Baijiu, which because previous studies by HPLC demonstrated that the content of acid increased with increasing the age of Baijiu(Vanbeneden, Delvaux, & Delvaux, 2006). It can be predicted that the FWHM of the vibrational peak at ~ 1377 cm − 1 would decrease with increasing the age of Baijiu. Hence then, it is possible to employ the FTIR difference spectra to measure the age of Baijiu. 3.3 FTIR Difference Spectra of Baijiu and Vodka Further, the FTIR difference spectra of Baijiu and Vodka are compared to check the characteristic of Baijiu. Different with Baijiu, Vodka is produced through filtering activated carbon and diatomaceous earth.(Abramova, E, G, A, & V, 2020) Thus Vodka contains almost no fusel and acids, esters, aldehydes and other trace compounds. The content of these compounds is generally 8–10 times or more lower than that of Baijiu.(Shu et al., 2007 ; Siristova, Prinosilova, Riddellova, Hajslova, & Melzoch, 2012) FTIR spectra of two kinds of Vodka and Baijiu were measured, and the FTIR difference spectra were obtained through Eq. ( 2 ), the difference spectra of the Vodka and Baijiu (Xifeng Jiu) were plotted in Fig. 5 . Three spectral peaks of acid and ester are observed obviously in the difference spectrum of Baijiu, but these peaks are not observed in the difference spectrum of vodka. There are lots of sharp peaks in the FTIR difference spectrum of vodka. These peaks are assigned to the rotating spectral lines of water vapor, which are usually residual during the background subtraction in the FTIR measurement. Since no vibrational peaks were observed in the difference spectrum of vodka, which demonstrated that the trace components in vodka could not be detected through FTIR. In contrast to the difference spectrum of vodka, the difference spectrum of Baijiu has obvious acid and ester spectrum signals. This demonstrates that it is indeed reasonable to use Fourier Infrared Difference Spectroscopy to detect the acids and esters in Baijiu. 3.4 FTIR Difference Spectra of Baijiu with different flavour Since the FTIR difference spectra can be used to obtain the acid and ester signal in Baijiu, the spectra can be employed to distinguish Baijiu with different flavour. In China, the acid and ester content is different for Baijiu with different flavour. For example, according to the standards of Baijiu, strong aroma-type Baijiu usually has higher acid and ester content, while light aroma-type Baijiu has lower acid and ester content. In order to verify whether the FTIR difference spectra can be used to distinguish the flavor types of Baijiu, the FTIR spectra of two types of light aroma-type Baijiu (Niulanshan zhenbao and Yongfeng Erguotou) and two types of strong aroma-type Baijiu (Luzhou Laojiao and Wuliang Chun) were measured. The FTIR difference spectra are shown in Fig. 6 . It was clearly observed that the vibrational peaks of strong aroma-type Baijiu were generally stronger than those of light aroma-type Baijiu. Take the C = O stretching vibration band as an example, the spectral intensity of strong aroma-type Baijiu is 2 to 3 times that of light aroma-type Baijiu. Because the C = O group is a unique chemical group for acids and esters, stronger C = O stretching band demonstrated higher content of acids and esters in strong aroma-type Baijiu. Thus, the C = O stretching band can be employed to check whether the flavor of Baijiu is strong or light. In addition, combined with other vibrations bands, more complete information about the flavor can be obtained. For example, Niulanshan zhenbao and Yongfeng Erguotou are baijiu with light flavor, however the peaks at ~ 1377 cm − 1 are much different with each other. The FWHM of this peak is smaller for Niulanshan zhenbao than that for Yongfeng Erguotou. As the FWHM of this vibrational band for ester is smaller than that for acid, the concent of ester in Niulanshan zhenbao is more than that in Yongfeng Erguotou. Similarly, although Luzhou Laojiao and Wuliang Chun are baijiu with strong flavor, the content of ester in Wuliang Chun is more than that in Luzhou Laojiao because of the narrower band at ~ 1377 cm − 1 . Only less than one minute is acquired to measure the FTIR spectra of Baijiu. The time for FTIR method is much shorter than that for previous methods such as gas chromatography or liquid chromatography. This advantage indicates that this FTIR method may be developed as a quick inspection method for Baijiu. In this method, the novel difference spectra can show the vibrational bands of the trace compounds in Baijiu, hence then it is necessary to make a FTIR database of aqueous ethanol solutions with various concentrations. In this database, the smaller the concentration interval of aqueous ethanol is, the more accurate the difference spectrum will be. 4. Conclusion A novel FTIR difference spectra analysis method is proposed to detect the acids and esters in Baijiu. Using the spectrum of Baijiu to subtract the spectrum of aqueous ethanol with the same ethanol concentration, the FTIR difference spectra can be obtained. In this difference spectrum, the spectral signals of ethanol and water are almost completely removed, and the remaining spectral peaks come from trace substances in Baijiu. In the difference spectrum, three new peaks at ~ 1260 cm − 1 , ~ 1377 cm − 1 , and ~ 1710 cm − 1 were observed obviously. Through comparing the FTIR difference spectra of baijiu with the experimental and theoretical IR spectra of acetic acid and ethyl acetate, it is demonstrated that these three peaks are assigned to the vibrational modes of acetic acid or ethyl acetate. Different form the FTIR difference spectra of Baijiu, these three peaks are not observed in the FTIR difference spectra of vodka. This is because the trace substances in vodka are filtered out during the manufacturing process of vodka. Based on the intensity and the FWHM of the three peaks in the difference spectrum, the flavor of Baijiu can be detected. The novel FTIR difference spectra we proposed here is expected to become a method for the control of Baijiu. Declarations Acknowledgements This work was supported by the Key Research Project of Shaanxi Provincial Science and Technology Department (2023-YBNY-158), the Xi’an Science and Technology Project (22NYYF016) the Natural Science Foundation of Shaanxi Province (2022JM-087), the Open Fund of the State Key Laboratory of Molecular Reaction Dynamics in DICP, CAS, (SKLMRD-K202413), the Fundamental Research Funds for the Central Universities (QTZX23007) and the 111 Project. Author contribution Guo: Investigation, Writing - original draft, Wang: Formal analysis, Methodology, Funding acquisition Zhang: Formal analysis, Writing - Review & Editing, Funding acquisition Ma: Formal analysis, Funding acquisition Lin: Conceptualization, Methodology, Writing - Review & Editing, Funding acquisition Conflict of Interest: The authors declare no competing interests References Abramova IM, Medrish ME, Romanovs AG et al (2020) Chemical characterization of ethanol and vodka in Russia. IOP Conference Series: Earth and Environmental Science 548: 082059. Ali A, Miraz RK, Andrew CKL (2020) Identifying the Responses from the Estrogen Receptor-Expressed MCF7 Cells Treated in Anticancer Drugs of Different Modes of Action Using Live-Cell FTIR Spectroscopy. ACS omega, 5: 12698-12706. Amo Y, Tominaga Y (2000) Low-frequency Raman study of ethanol-water mixture. Chem Phys Lett 320: 703-706. Anjos O, Santos AJA, Estevinho LM et al (2016) FTIR-ATR spectroscopy applied to quality control of grape-derived spirits. Food Chem 205: 28-35. Buck N, Goblirsch T, Beauchamp J et al (2020) Key Aroma Compounds in Two Bavarian Gins. Appl Sci-Basel, 10: 7269. Charapitsa S, Sytova S, Kavalenka A et al (2021) The study of the matrix effect on the method of direct determination of volatile compounds in a wide range of alcoholic beverages. Food Control, 120: 107528. Almeida MP, Leopold N, Franco R, et al (2019) Expedite SERS Fingerprinting of Portuguese White Wines Using Plasmonic Silver Nanostars. Front Chem 7: 368. Fan W, Shen H, Xu Y (2011) Quantification of volatile compounds in Chinese soy sauce aroma type liquor by stir bar sorptive extraction and gas chromatography-mass spectrometry. J Sci Food Agric 91: 1187-1198. Giannetti V, Mariani MB, Marini F et al (2020) Grappa and Italian spirits: Multi-platform investigation based on GC-MS, MIR and NIR spectroscopies for the authentication of the Geographical Indication. Microchem J 157: 104896. Giubertoni G, Sofronov OO, Bakker HJ (2019) Observation of Distinct Carboxylic Acid Conformers in Aqueous Solution. J Phys Chem Lett 10: 3217-3222. Gu J, Liu H, Ma C et al (2019) Conformal Prediction Based on Raman Spectra for the Classification of Chinese Liquors. Appl Spectrosc 73: 759-766. Gu S, Ren F, Wei Z et al (2020) Rapid Identification of Age and Brand of Chinese Rice Wine Based on Gas-ion Mobility Spectrometry. J Chin Inst Food Sci Technol 20: 248-255. Joshi I, Khanh T, Elbourne A et al (2019) Influence of the Scanning Temperature on the Classification of Whisky Samples Analysed by UV-VIS Spectroscopy. Appl Sci-Basel 9: 3254. Li X, Tsuta M, Tanaka F, et al (2020) Assessment of Japanese Awamori Spirits Using UV-VIS Spectroscopy. Food Anal Methods 13: 726-734. Lin K, Hu NY, Zhou XG, et al (2012) Reorientation dynamics in liquid alcohols from Raman spectroscopy. J Raman Spectrosc 43: 82-88. Malloum A, Fifen JJ, Conradie J (2020) Theoretical infrared spectrum of the ethanol hexamer. Int J Quantum Chem 120: 1-11. Matthias L, Frank F, Stefan L (2014) Direct observation of the cyclic dimer in liquid acetic acid by probing the C=O vibration with ultrafast coherent Raman spectroscopy. Phys Chem Chem Phys 16: 18010-18016. Nishi N, Nakabayashi T, Kosugi K (1999) Raman spectroscopic study on acetic acid clusters in aqueous solutions: Dominance of acid-acid association producing microphases. J Phys Chem A 103: 10851-10858. Niu Y, Yao Z, Xiao Q et al (2017) Characterization of the key aroma compounds in different light aroma type Chinese liquors by GC-olfactometry, GC-FPD, quantitative measurements, and aroma recombination. Food Chem 233: 204-215. Okaru AO, Abuga KO, Kibwage IO et al (2017) High Ethanol Contents of Spirit Drinks in Kibera Slums, Kenya: Implications for Public Health. Foods 6, 89. Mannix PB, Dong HK, Haiyan F (2016) Revisiting the formation of cyclic clusters in liquid ethanol. J Chem Phys 144: 154302. Panuszko A, Stangret J, Nowosielski B et al (2020) Interactions between hydration spheres of two different solutes in solution: The least squares fitting with constraints as a tool to determine water properties in ternary systems. J Mol Liq 310: 113181. Qi H, Chen H, Wang Y et al (2018) Detection of ethyl carbamate in liquors using surface-enhanced Raman spectroscopy. R Soc Open Sci 5: 181539. Shu D, Zhang L, Zhang W et al (2007) Variation of Flavor Components in Zaopei of Luzhou-flavor Liquor during Fermentation. Food Sci 28: 89-92. Siristova L, Prinosilova S, Riddellova K et al (2012) Changes in Quality Parameters of Vodka Filtered through Activated Charcoal. Czech J Food Sci 30: 474-482. Śmialek MA, Łabuda M, Guthmuller J et al (2016) Electronic state spectroscopy by high-resolution vacuum ultraviolet photoabsorption, He(I) photoelectron spectroscopy and ab initio calculations of ethyl acetate. Eur Phys J D, 70: 1-9. Song XB, Jing S, Zhu L et al (2020) Untargeted and targeted metabolomics strategy for the classification of strong aroma-type baijiu (liquor) according to geographical origin using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry. Food Chem 314: 126098. Sun JY, Yin ZT, Zhao DR et al (2018) Qualitative and quantitative research of propyl lactate in brewed alcoholic beverages. Int J Food Prop 21: 1351-1361. Urickova V, Sadecka J (2015) Determination of geographical origin of alcoholic beverages using ultraviolet, visible and infrared spectroscopy: A review. Spectrochim Acta Part A Mol Biomol Spectrosc 148: 131-137. Vanbeneden N, Delvaux F, Delvau, FR (2006) Determination of hydroxycinnamic acids and volatile phenols in wort and beer by isocratic high-performance liquid chromatography using electrochemical detection. J Chromatogr A 1136: 237-242. Wang XX, Fan WL, Xu Y (2014) Comparison on aroma compounds in Chinese soy sauce and strong aroma type liquors by gas chromatography-olfactometry, chemical quantitative and odor activity values analysis. Eur Food Res Technol 239: 813-825. Xiao S, He Y (2019) Analysis of Sildenafil in Liquor and Health Wine Using Surface Enhanced Raman Spectroscopy. Int J Mol Sci 20: 2722. Zhang Y, Chen GQ, Gu J et al (2020) A theoretical study on dimerization and dissociation of acetic acid in ethanol solvent. Comput Theor Chem 1191: 113029. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 20 Jun, 2024 Reviews received at journal 19 Jun, 2024 Reviews received at journal 14 Jun, 2024 Reviewers agreed at journal 11 Jun, 2024 Reviewers agreed at journal 10 Jun, 2024 Reviewers agreed at journal 07 Jun, 2024 Reviewers invited by journal 07 Jun, 2024 Submission checks completed at journal 28 May, 2024 Editor assigned by journal 28 May, 2024 First submitted to journal 26 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4481737","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":312347291,"identity":"3b67c1ce-f974-4db8-b301-d602e05706c2","order_by":0,"name":"Yixuan Guo","email":"","orcid":"","institution":"Xidian University","correspondingAuthor":false,"prefix":"","firstName":"Yixuan","middleName":"","lastName":"Guo","suffix":""},{"id":312347292,"identity":"5aadb901-88b3-4c4e-9212-ba5a42612a0d","order_by":1,"name":"Zhiqiang Wang","email":"","orcid":"","institution":"Xidian University","correspondingAuthor":false,"prefix":"","firstName":"Zhiqiang","middleName":"","lastName":"Wang","suffix":""},{"id":312347293,"identity":"1a602744-dd3b-4095-a853-efb39d298dbf","order_by":2,"name":"Ruiting Zhang","email":"","orcid":"","institution":"Xidian University","correspondingAuthor":false,"prefix":"","firstName":"Ruiting","middleName":"","lastName":"Zhang","suffix":""},{"id":312347294,"identity":"33ad9c0a-5499-4b7f-bca9-2cf4c5cde0d3","order_by":3,"name":"Lin Ma","email":"","orcid":"","institution":"Xidian University","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Ma","suffix":""},{"id":312347295,"identity":"60ae4ec4-7d79-4dee-85b0-a324fc7ff78a","order_by":4,"name":"Ke Lin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYBACCXbGBoYPDBKMbWAuGzFamBkbGGeQqIWBgZmHgQFoF7FaJJuZ2x7btlnI9rGfPcDwoewwA//sBvxapJkZ241zzkgYt/HkJTDOOHeYQeLOAfxa5JgZ26RzKiQS2xhyDJh52w4zGEgkEKHFwgCohf+NAfNfYrRIg7QwgGyRANrCSIwWyWbGNskekF8k3hgc7DmXziNxg4AWiePtzyR+ttXJzu/PMXzwo8xajn8GAS0o4AAQ85CgfhSMglEwCkYBLgAADjw4k1z2BcUAAAAASUVORK5CYII=","orcid":"","institution":"Xidian University","correspondingAuthor":true,"prefix":"","firstName":"Ke","middleName":"","lastName":"Lin","suffix":""}],"badges":[],"createdAt":"2024-05-27 01:54:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4481737/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4481737/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58116509,"identity":"12e86f4b-db11-44e4-bb31-04cdd4962bf4","added_by":"auto","created_at":"2024-06-11 10:57:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":749190,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of water, ethanol and Baijiu (Xifeng Jiu). The spectra are shifted vertically in order to distinguish these spectra well.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4481737/v1/c73f711171891e3878ea60be.png"},{"id":58117455,"identity":"647d4426-2b98-4904-9327-1a4fc3ec8854","added_by":"auto","created_at":"2024-06-11 11:13:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1031789,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The FTIR difference spectra of Baijiu (Brand name: Xifeng Jiu) through Eq. 1 (black solid line) and Eq. 2 (blue dashed line), (b) the FTIR difference spectrum of Baijiu (Brand name: Xifeng Jiu) in the fingerprint region through Eq. 2.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4481737/v1/e8f61defa282cfffef2d7ec0.png"},{"id":58116503,"identity":"101cceb2-bc0c-4c27-a2eb-c59c9b04bfc1","added_by":"auto","created_at":"2024-06-11 10:57:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":913779,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR difference spectrum of Baijiu. These spectra are shifted vertically in order to present these spectra clearly.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4481737/v1/774a284be2700f879f96cdcb.png"},{"id":58117059,"identity":"2509d542-018b-4ba6-9e79-d6619a2d0fea","added_by":"auto","created_at":"2024-06-11 11:05:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1352786,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR difference spectra of acetic acid and ethyl acetate in aqueous ethanol solution, and the theoretical IR spectra of ethyl acetate and acetic acid in PCM model. The spectra are shifted vertically in order to distinguish these spectra well.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4481737/v1/abceb8ecbbf5f49b08f2a65c.png"},{"id":58116507,"identity":"6d70aa1a-a076-4144-9809-34efa342b824","added_by":"auto","created_at":"2024-06-11 10:57:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":743269,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR difference spectra of Vodka (Brand name: Absolute Vodka and Siminuo Vodka) and Baijiu (Brand name: Xifeng Jiu). The spectra are shifted in order to distinguish these spectra well.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4481737/v1/ed9d72651c070e3e93639892.png"},{"id":58116506,"identity":"fb1baace-67da-4b1a-95b2-ed957cfe42de","added_by":"auto","created_at":"2024-06-11 10:57:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1925285,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR difference spectra of the Baijiu, (a) Luzhou Laojiao, (b) Wuliang Chun, (c) Niulanshan zhenbao and (d) Yongfeng Erguotou\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4481737/v1/ca8ac596ee7846de95d16640.png"},{"id":58117776,"identity":"c2c737d8-2ef3-42a0-b2a8-ae625bd15eda","added_by":"auto","created_at":"2024-06-11 11:21:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6815658,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4481737/v1/f9246c29-7d78-48f1-88a4-1be30b6a56e7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Rapid detection of acids and esters in Baijiu by Fourier transform infrared spectroscopy with difference spectroscopy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eChinese liquor (Baijiu) has a history of more than 3,000 years. One of the reasons why Baijiu is enduring in China is that it contains diverse trace substances.(Shu, Zhang, Zhang, Yue, Liu, \u0026amp; Mu, 2007) Acids and esters are the most content in the trace substances of Baijiu, and they are the main aroma compounds in Baijiu.(Fan, Shen, \u0026amp; Xu, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Niu, Yao, Xiao, Xiao, Ma, \u0026amp; Zhu, 2017; Song et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) Because various acids and esters impart the unique flavor and taste to Baijiu, the national standards for Baijiu require the determination of acid and ester content. Different brands, flavors and grades of Baijiu have different acid and ester content. For example, compared to light aroma-type Baijiu, strong aroma-type Baijiu has higher ethyl acetate content. For Baijiu with the same brand, the content of acetic acid and ethyl acetate in Baijiu with premium grade is higher than that in the Baijiu with first-class grade. The content and ratio of acid and ester can also reflect the age of Baijiu. While the storage time of Baijiu becomes longer, the acid in Baijiu will increase, however the ester will decrease.(S. Gu et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) These above properties demonstrate that detection of the concentration of acids and esters is closely related to control the quality of Baijiu. Thus the quantitative detection of acids and esters is particularly important.\u003c/p\u003e \u003cp\u003eIn order to detect acids and esters in Baijiu, various methods have been developed. For example, in the Chinese national standard GBT 10345\u0026thinsp;\u0026minus;\u0026thinsp;2007, the acid-base neutralization titration method is used to measure the acid content in Baijiu, and the gas chromatography method is used to determine the ester content in Baijiu. The chromatographic signal can be easily distinguished, which is why chromatography can be used to quantitatively measure the concentration of various substances in Baijiu. When combined with mass spectrometry, it can also record the concentration of acid and ester with higher accuracy of ng/L. (Buck, Goblirsch, Beauchamp, \u0026amp; Ortner, 2020; Okaru, Abuga, Kibwage, \u0026amp; Lachenmeier, 2017; Sun, Yin, Zhao, Sun, \u0026amp; Zheng, 2018) The chromatography experimental steps are relatively cumbersome, and it generally require several hours. For example, in order to study the chromatographic signal of liquor, the chromatogram of pure possible chemical compounds should be record before the detection of liquor. These methods are not suitable for rapid detection in the market or enterprise automated assembly line. Compared with the gas chromatography, the liquid chromatography has maintained higher accuracy and improved the efficiency, but it still cannot meet the needs of rapid detection. (Charapitsa et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eIn addition to the above methods, various spectroscopy methods have also been used to detect the liquor. For example, Raman spectroscopy, infrared spectroscopy, fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy and other spectroscopic methods are often employed. The main advantage of spectroscopy is that the liquor can be detected quickly, and sample pretreatment is not required. And the disadvantage is that the concentration of trace components in the liquor cannot be accurately measured. This is because the spectra of various substances often overlap with each other, and the spectra can hardly be distinguished well. Thus various chemometric methods are applied to analyze these spectra to detect liquor. The principal component analysis, the partial least squares, the support vector machine, and other big data analysis method are usually applied to analyze these spectra. These methods can usually be used to classify liquor, such as source of origin, etc.(Anjos, Santos, Estevinho, \u0026amp; Caldeira, 2016; Joshi, Vi Khanh, Elbourne, Chapman, \u0026amp; Cozzolino, 2019; Li, Tsuta, Tanaka, Tsukahara, \u0026amp; Tsukahara, 2020) Although the spectra of liquor contains the spectral signals of all the chemical components in the liquor, these spectra mainly reflect the most abundant chemical components. For example, in Raman spectra, mid-infrared or near-infrared spectra, the largest spectral peaks are the signal of ethanol and water.(Giannetti, Mariani, Marini, Torrelli, \u0026amp; Biancolillo, 2020; J. Gu et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Urickova \u0026amp; Sadecka, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) None of previous spectra showed directly the spectral bands of the trace substances in liquor. In order to enhance the signal of trace components, surface enhanced Raman spectroscopy was used.(de Almeida, Leopold, Franco, \u0026amp; Pereiral, 2019; Qi, Chen, Wang, \u0026amp; Jiang, 2018; Xiao \u0026amp; He, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) So far, there has not been seen that these spectroscopy methods are used to measure the concentration of acids and esters in liquor.\u003c/p\u003e \u003cp\u003eIn Baijiu, the content of trace substances is about 1%-2%. The acids and esters are the main components in Baijiu, and their concentration can reach 1-2g/L.(Fan et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) That is to say, in the spectra, especially in Raman or infrared spectra, the signal of acids and esters can account for about 1%-2% of the total spectral signal. Hence the spectra of acids and esters can be detected through infrared spectroscopy or Raman spectroscopy. However the spectra of acids and esters did not present in the previous infrared spectra and Raman spectra of Baijiu, which is maybe that the spectra of these trace substances are overlapped with the large spectral bands of ethanol and water. Therefore, in order to obtain the spectra of acids and esters in Baijiu, the spectra of ethanol and water should be subtracted from the spectra of Baijiu. Here we present a special difference spectroscopic method to subtract the spectral bands of ethanol and water. Using this novel analysis method, we obtained the spectra of acetic acid and ethyl acetate in Baijiu. These spectra were used to detect the quality of Baijiu. These results show that this difference spectroscopic method is a quick detection method, and the method can be used to quantitatively measure the content of acids and esters in Baijiu.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eEthanol (CH\u003csub\u003e3\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eOH,99.7%) is purchased from Shanghai Wokai Biotechnology Co., Ltd., acetic acid(CH\u003csub\u003e3\u003c/sub\u003eCOOH, 99.5%)is purchased from Tianjin Tianli Chemical Reagent Co., Ltd., ethyl acetate༈CH\u003csub\u003e3\u003c/sub\u003eCOOC\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003e, 99.5%༉is purchased from Tianjin Fuyu Fine Chemical Co., Ltd.. Water is Millipore ultrapure water. These chemical compounds were used without further purification. Seven kinds of Baijiu and two kinds of Vodka were all purchased from the supermarket. Their brand names are Yongfeng Erguotou, Xifeng Jiu, Yanghe King, Xiaolang Jiu, Wuliang Chun, Niulanshan, Luzhou Laojiao, Absolute Vodka and Siminuo Vodka.\u003c/p\u003e \u003cp\u003eIn order to remove the spectral bands of ethanol and water in Baijiu, we prepared aqueous ethanol solutions with the same ethanol concentration of Baijiu. The content of ethanol in Baijiu is present in the volume percentage. The volume percentage can be converted into the mass percentage according to its density-volume relationship. Thus the aqueous ethanol solutions were prepared by weighing. The accuracy of the electronic balance used here is 0.0001g. In order to measure the spectra of acetic acid and ethyl acetate in ethanol aqueous solution, we added a small amount of acetic acid or ethyl acetate into the aqueous ethanol. The concentration of acetic acid or ethyl acetate is 0.67mmol/g.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.2\u003c/b\u003e Methods\u003c/h2\u003e \u003cp\u003eThe Fourier transform infrared (FTIR) spectra were record with a Nicolet iS20 FTIR spectrometer, which was equipped a triglyceride titanium deuterated sulfate detector and a KBr beam splitter. Instead of the transmission mode, the total reflection mode with ZnSe crystal was used because of the strong absorption of water. The spectra were record from 32 scans, 2 levels of zero filling, Happ Genzel apodization, and Mertz phase correction. The FTIR spectral resolution is 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Before each spectrum is collected, the background spectrum is collected independently to eliminate the spectral signals of water vapor and carbon dioxide. The total reflection crystal was cleaned with ultrapure water and was dried between two measurements.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussions","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.1 FTIR spectra of Baijiu\u003c/h2\u003e \u003cp\u003eLiquor mainly contains ethanol and water, thus the FTIR spectra of liquor is dominated by the spectra of ethanol and water. The Fourier transform total reflection infrared spectrum of a Baijiu (Xifeng Jiu) was measured, and shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Lots of sharper peaks were observed in the spectra. These peaks were also observed in the FTIR spectrum of pure ethanol shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, thus these sharper peaks were all assigned to various vibration modes of ethanol. Based on previous spectroscopic studies of ethanol (Malloum, Fifen, \u0026amp; Conradie, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; P, Hee, \u0026amp; Haiyan, 2016), the peak at ~\u0026thinsp;877 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and ~\u0026thinsp;1043 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is assigned to the symmetric and asymmetrical stretching vibration of C-C-O, respectively. The spectral peaks at ~\u0026thinsp;1085 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and ~\u0026thinsp;1273 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are assigned to CH\u003csub\u003e3\u003c/sub\u003e swing vibration. The peak at ~\u0026thinsp;1325 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is considered to the torsional vibration of CH\u003csub\u003e2\u003c/sub\u003e. The peak at ~\u0026thinsp;1383cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is assigned to the bending vibration of CH\u003csub\u003e3\u003c/sub\u003e and CH\u003csub\u003e2\u003c/sub\u003e. The peaks at ~\u0026thinsp;1417 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, ~\u0026thinsp;1453 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and ~\u0026thinsp;1482 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are all attributed to the bending vibration of CH\u003csub\u003e3\u003c/sub\u003e. The peak at ~\u0026thinsp;2978 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is assigned to CH\u003csub\u003e3\u003c/sub\u003e or CH\u003csub\u003e2\u003c/sub\u003e stretching vibration. The positions of these peaks in pure ethanol and Baijiu are not exactly the same. For example, the strongest peak is located at 1043 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the spectrum of the Baijiu, but at 1045 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the spectrum of pure ethanol. This noncoincidence demonstrated the different intermolecular interactions(Amo \u0026amp; Tominaga, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) of ethanol molecules in pure ethanol and Baijiu. In pure ethanol, ethanol molecules interact with ethanol molecules through hydrogen bonds and hydrophobic interactions, however ethanol molecules interact with water molecules through hydrogen bonds in Baijiu.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition to the above sharp peaks, some broad peaks were also observed at ~\u0026thinsp;3200 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, ~\u0026thinsp;1633 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and below ~\u0026thinsp;1000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the FTIR spectrum of Baijiu. Compared with the FTIR spectra of water and pure ethanol in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the broad spectrum peak below 1000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is mainly attributed to the intermolecular vibration of ethanol or water. The peak at ~\u0026thinsp;1633 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is assigned to the HOH bending vibration of water molecule. The peak at ~\u0026thinsp;3200 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is assigned to the O-H stretching vibration of water and ethanol molecules.(Malloum et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) These peaks are very broad, mainly because of the strong hydrogen bonds between the molecules in the solutions.\u003c/p\u003e \u003cp\u003eUsing the peak intensity of C-C-O asymmetric stretching vibration (~\u0026thinsp;1043 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), the signal-to-noise ratio of the spectra of Baijiu (Xifeng Jiu) is estimated to be about\u0026thinsp;~\u0026thinsp;1000. Since the concentration of acid or ester in Baijiu is close to 1%, if the infrared absorption coefficient of acid or ester is not much different from that of ethanol, the spectral intensity of acid and ester should be greater than the noise of the spectrum. However the spectral bands of acids and esters are not directly observed in the spectra of Baijiu, which is probably because their peaks are overlapped by the spectral bands of water and ethanol. In order to obtain the spectral peaks of these acids or esters, the spectral bands of ethanol and water should be removed from the spectra of Baijiu. Thus the difference spectra could be employed. Difference spectra were often used in spectral analysis previously. This method is usually used to reflect some small changes in the spectrum to reveal the microstructure of complex small molecule solutions, (Panuszko, Stangret, Nowosielski, \u0026amp; Bruzdziak, 2020) as well as the interaction between cells and drugs in biological solutions, etc.(Ali, Miraz, \u0026amp; Andrew, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe most direct method to remove the spectral peaks of ethanol and water is to subtract the spectrum of pure ethanol and the spectrum of pure water. The difference spectra can be obtained from the following equation. In this equation,\u003cem\u003e△I\u003c/em\u003e, \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eBaijiu\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eethanol\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003ewater\u003c/em\u003e\u003c/sub\u003e are the FTIR difference spectrum, the FTIR spectrum of Baijiu, pure ethanol, and pure water, respectively. The coefficients \u003cem\u003ea\u003c/em\u003e and \u003cem\u003eb\u003c/em\u003e can be calculated according to the concentration of ethanol in Baijiu.\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$△I={I}_{Baijiu}-\\left(a*{I}_{ethanol}+b{*I}_{water}\\right)$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eUsing the Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), the difference spectrum of the Baijiu (Xifeng Jiu) was obtained, and shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. No matter how the coefficients \u003cem\u003ea\u003c/em\u003e and \u003cem\u003eb\u003c/em\u003e were adjusted, the spectral bands of ethanol or water could not completely removed. For example, using the coefficients \u003cem\u003ea\u003c/em\u003e and \u003cem\u003eb\u003c/em\u003e that are according to the concentration of ethanol in Baijiu, the positive and negative spectral peaks are observed in the difference spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). This demonstrated the spectral peaks of ethanol and water in Baijiu were inconsistent with those in pure ethanol and pure water. These is because of the different intermolecular interactions in these solutions.(Lin, Hu, Zhou, Liu, \u0026amp; Luo, 2012) In pure water, water molecules interact with water molecules through hydrogen bonds. In pure ethanol, ethanol molecules interact with ethanol molecules through hydrogen bonds and hydrophobic interactions. However, in Baijiu, ethanol molecules not only interact with ethanol molecules, but also interact with water molecules. Therefore, the spectral signals of ethanol and water in Baijiu cannot be totally removed through Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Thus, it is impossible to observe the spectral peaks of only 1%-2% trace compounds in these difference spectra.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to simulate the influence of the molecular interaction between ethanol and water on the spectrum, we prepared an aqueous ethanol solution with the same ethanol concentration as that of Baijiu. In this solution, the molecular interactions are similar with those of Baijiu, thus the spectral peaks of ethanol or water are very close to those of Baijiu. Using these spectra and Eq.\u0026nbsp;\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the new kind of difference spectrum can be obtained. In this equation, \u003cem\u003e△I\u003c/em\u003e, \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eBaijiu\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003esolution\u003c/em\u003e\u003c/sub\u003e are the FTIR difference spectrum, the FTIR spectrum of the Baijiu and the aqueous ethanol solution.\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$△I={I}_{Baijiu}-{I}_{solution}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eBased on this method, the FTIR difference spectrum of Baijiu (Xifeng) was obtained, and shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(a). This difference spectrum is obviously different from the previous difference spectrum by Eq.\u0026nbsp;\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Using Eq.\u0026nbsp;\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the sharp bands of ethanol is almost completely removed. A weak band at ~\u0026thinsp;3300 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are still residual, which may be due to the small difference between the concentration of the Baijiu and the concentration of the aqueous ethanol solution. More importantly, some new peaks in this difference spectrum were observed, they were located near ~\u0026thinsp;1260 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, ~\u0026thinsp;1377 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and ~\u0026thinsp;1710 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which were shown in the difference spectra of the fingerprint region in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(b). These spectral bands are inconsistent with the peaks of ethanol and water, and they should be assigned to the vibration modes of trace compounds in Baijiu. While the ethanol concentration of Baijiu is exactly the same as that of aqueous ethanol solution, the difference spectrum should be completely free of water and ethanol signals. In the experiment, it is hard to accurately prepare an aqueous ethanol solution with the same alcohol content of Baijiu, which will cause the weak spectrum of ethanol and water to remain in the difference spectrum. Through preparing carefully these aqueous solutions, the vibrational peaks of ethanol and water could be removed as much as possible.\u003c/p\u003e \u003cp\u003eThis kind of difference spectra method was used to analyze seven different kind of Baijiu to verify the universality of this method. The FTIR spectra of these Baijiu and the corresponding aqueous alcohol solutions were measured. Using Eq.\u0026nbsp;(\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), the difference spectra were obtained, and shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. In these spectra, three new vibrational peaks were observed obviously. All the spectra were all similar with each other, which demonstrated the similar substances in these Baijiu. The intensity of the same vibrational bands in these spectra is different with each other, thus the contet of the trace substances were different in the seven kinds of Baijiu.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Assignments of the FTIR difference spectra\u003c/h2\u003e \u003cp\u003ePreviously, acids and esters were found to be the dominant trace components through chromatographic analysis in Baijiu. Usually, ethyl acetate and ethyl lactate account for more than 96% of the total esters in Baijiu, of which ethyl acetate is the majority.(Wang, Fan, \u0026amp; Xu, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) Acetic acid is the most representative acids in Baijiu. Thus the vibrational peaks in the difference spectra of Baijiu are likely to be assigned to acetic acid or ethyl acetate.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo verify this assignment, the difference spectra of Baijiu should be compared with the Fourier transform infrared spectra of acetic acid and ethyl acetate. However, the spectra of acetic acid and ethyl acetate in aqueous ethanol will be different from those in the pure liquid, which is because of the different intermolecular interactions in these solutions. Thus, in order to compare the difference spectra of Baijiu with the spectra of acetic acid and ethyl acetate, the infrared spectrum of acetic acid or ethyl acetate in aqueous ethanol were measured. Here an aqueous ethanol solution was prepared with the volume concentration 45%, and small amount of acetic acid and ethyl acetate were added in to this aqueous solution, respectively. Then the FTIR spectra were measured for these solutions. Using Eq.\u0026nbsp;\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the spectral bands of ethanol and water were removed from the spectra of mixtures. Thus the FTIR spectra of acetic acid or ethyl acetate in the mixtures were obtained, and shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The FTIR spectra are similar with the FTIR difference spectra of Baijiu. Hence then, the vibrational bands in the difference spectra in Baijiu could be assigned to be the vibrational modes of acetic acid or ethyl acetate.\u003c/p\u003e \u003cp\u003eIn both FTIR spectra of acetic acid and ethyl acetate, the vibrational peaks are all similar, and they peaks were located at ~\u0026thinsp;1260 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, ~\u0026thinsp;1377 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and ~\u0026thinsp;1710 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The vibrational peaks at ~\u0026thinsp;1260 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and ~\u0026thinsp;1710 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of acetic acid are similar with those of ethyl acetate, but the vibrational bands at ~\u0026thinsp;1377 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are different. For acetic acid, the vibrational peak at ~\u0026thinsp;1377 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is very broad, and its full width at half maximum is ~\u0026thinsp;60 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. This peak is so broad, which is mainly because two sub-bands overlapped with each other. For ethyl acetate, the vibrational peak at ~\u0026thinsp;1377 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is very narrow, and its full width at half maximum is ~\u0026thinsp;10 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Although there is little difference between the FTIR spectra of acetic acid and ethyl acetate, the FTIR spectrum of acetic acid and ethyl acetate in Baijiu is hardly distinguished because of the strong overlapping.\u003c/p\u003e \u003cp\u003eIn order to assign these peaks, the theoretical infrared spectra of ethyl acetate and acetic acid were calculated through using density functional theory. For acetic acid, there are trans and cis structure. As the trans structure of acetic acid is much more stable than cis structure of acetic acid,(Giubertoni, Sofronov, \u0026amp; Bakker, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) thus only the theoretical spectra of trans acetic acid was calculated. In Baijiu, the ethyl acetate and acetic acid can interact with water molecules or ethanol molecules through hydrogen bonds. Thus, the polarizable continuum model (PCM) with water or ethanol solvents was employed to simulate the effect of the intermolecular interactions to the IR spectra of acetic acid and ethyl acetate. The scaled factor 0.9614 was used to adjust the theoretical IR spectra. The theoretical spectra were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The theoretical spectrum of acetic acid or ethyl acetate in water is same with the theoretical spectrum in ethanol. The theoretical spectra agreed with the experimental spectra, hence then the assignment could be obtained from the theoretical calculations. The vibrational peak at ~\u0026thinsp;1260 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the difference spectrum is assigned to the C-C stretching vibration. The peak at ~\u0026thinsp;1377 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is attributed to the bending vibration of the methyl or methylene group and the C-O-H. The peak at ~\u0026thinsp;1710 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is assigned to the C\u0026thinsp;=\u0026thinsp;O stretching vibration. These assignments were agreed with previous studies.(Matthias, Frank, \u0026amp; Stefan, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Nishi, Nakabayashi, \u0026amp; Kosugi, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Śmialek et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eSince the spectra of acetic acid and ethyl acetate in aqueous ethanol are very similar, and their spectra are seriously overlapped, thus the FTIR spectra of acetic acid and ethyl acetate could not be distinguished directly. Hence then, we can only obtain the total spectra of acetic acid and ethyl acetate in the difference spectrum of Baijiu. Using the C\u0026thinsp;=\u0026thinsp;O stretching peak of acid or ester at ~\u0026thinsp;1710 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the total content of acid and ester can be estimated, and there is no way to give the content of each acid or ester by the this peak. It is noticed that the FWHM is much different for the peak at ~\u0026thinsp;1377 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, thus the FWHM of this vibrational peak can be used to qualitatively estimate whether there is more acid or more ester in Baijiu. These band is important to determine the age of Baijiu, which because previous studies by HPLC demonstrated that the content of acid increased with increasing the age of Baijiu(Vanbeneden, Delvaux, \u0026amp; Delvaux, 2006). It can be predicted that the FWHM of the vibrational peak at ~\u0026thinsp;1377 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e would decrease with increasing the age of Baijiu. Hence then, it is possible to employ the FTIR difference spectra to measure the age of Baijiu.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.3 FTIR Difference Spectra of Baijiu and Vodka\u003c/h2\u003e \u003cp\u003eFurther, the FTIR difference spectra of Baijiu and Vodka are compared to check the characteristic of Baijiu. Different with Baijiu, Vodka is produced through filtering activated carbon and diatomaceous earth.(Abramova, E, G, A, \u0026amp; V, 2020) Thus Vodka contains almost no fusel and acids, esters, aldehydes and other trace compounds. The content of these compounds is generally 8\u0026ndash;10 times or more lower than that of Baijiu.(Shu et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Siristova, Prinosilova, Riddellova, Hajslova, \u0026amp; Melzoch, 2012)\u003c/p\u003e \u003cp\u003eFTIR spectra of two kinds of Vodka and Baijiu were measured, and the FTIR difference spectra were obtained through Eq.\u0026nbsp;(\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), the difference spectra of the Vodka and Baijiu (Xifeng Jiu) were plotted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Three spectral peaks of acid and ester are observed obviously in the difference spectrum of Baijiu, but these peaks are not observed in the difference spectrum of vodka. There are lots of sharp peaks in the FTIR difference spectrum of vodka. These peaks are assigned to the rotating spectral lines of water vapor, which are usually residual during the background subtraction in the FTIR measurement. Since no vibrational peaks were observed in the difference spectrum of vodka, which demonstrated that the trace components in vodka could not be detected through FTIR. In contrast to the difference spectrum of vodka, the difference spectrum of Baijiu has obvious acid and ester spectrum signals. This demonstrates that it is indeed reasonable to use Fourier Infrared Difference Spectroscopy to detect the acids and esters in Baijiu.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.4 FTIR Difference Spectra of Baijiu with different flavour\u003c/h2\u003e \u003cp\u003eSince the FTIR difference spectra can be used to obtain the acid and ester signal in Baijiu, the spectra can be employed to distinguish Baijiu with different flavour. In China, the acid and ester content is different for Baijiu with different flavour. For example, according to the standards of Baijiu, strong aroma-type Baijiu usually has higher acid and ester content, while light aroma-type Baijiu has lower acid and ester content. In order to verify whether the FTIR difference spectra can be used to distinguish the flavor types of Baijiu, the FTIR spectra of two types of light aroma-type Baijiu (Niulanshan zhenbao and Yongfeng Erguotou) and two types of strong aroma-type Baijiu (Luzhou Laojiao and Wuliang Chun) were measured. The FTIR difference spectra are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. It was clearly observed that the vibrational peaks of strong aroma-type Baijiu were generally stronger than those of light aroma-type Baijiu. Take the C\u0026thinsp;=\u0026thinsp;O stretching vibration band as an example, the spectral intensity of strong aroma-type Baijiu is 2 to 3 times that of light aroma-type Baijiu. Because the C\u0026thinsp;=\u0026thinsp;O group is a unique chemical group for acids and esters, stronger C\u0026thinsp;=\u0026thinsp;O stretching band demonstrated higher content of acids and esters in strong aroma-type Baijiu. Thus, the C\u0026thinsp;=\u0026thinsp;O stretching band can be employed to check whether the flavor of Baijiu is strong or light.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, combined with other vibrations bands, more complete information about the flavor can be obtained. For example, Niulanshan zhenbao and Yongfeng Erguotou are baijiu with light flavor, however the peaks at ~\u0026thinsp;1377 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are much different with each other. The FWHM of this peak is smaller for Niulanshan zhenbao than that for Yongfeng Erguotou. As the FWHM of this vibrational band for ester is smaller than that for acid, the concent of ester in Niulanshan zhenbao is more than that in Yongfeng Erguotou. Similarly, although Luzhou Laojiao and Wuliang Chun are baijiu with strong flavor, the content of ester in Wuliang Chun is more than that in Luzhou Laojiao because of the narrower band at ~\u0026thinsp;1377 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOnly less than one minute is acquired to measure the FTIR spectra of Baijiu. The time for FTIR method is much shorter than that for previous methods such as gas chromatography or liquid chromatography. This advantage indicates that this FTIR method may be developed as a quick inspection method for Baijiu. In this method, the novel difference spectra can show the vibrational bands of the trace compounds in Baijiu, hence then it is necessary to make a FTIR database of aqueous ethanol solutions with various concentrations. In this database, the smaller the concentration interval of aqueous ethanol is, the more accurate the difference spectrum will be.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eA novel FTIR difference spectra analysis method is proposed to detect the acids and esters in Baijiu. Using the spectrum of Baijiu to subtract the spectrum of aqueous ethanol with the same ethanol concentration, the FTIR difference spectra can be obtained. In this difference spectrum, the spectral signals of ethanol and water are almost completely removed, and the remaining spectral peaks come from trace substances in Baijiu. In the difference spectrum, three new peaks at ~\u0026thinsp;1260 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, ~\u0026thinsp;1377 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and ~\u0026thinsp;1710 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were observed obviously. Through comparing the FTIR difference spectra of baijiu with the experimental and theoretical IR spectra of acetic acid and ethyl acetate, it is demonstrated that these three peaks are assigned to the vibrational modes of acetic acid or ethyl acetate. Different form the FTIR difference spectra of Baijiu, these three peaks are not observed in the FTIR difference spectra of vodka. This is because the trace substances in vodka are filtered out during the manufacturing process of vodka. Based on the intensity and the FWHM of the three peaks in the difference spectrum, the flavor of Baijiu can be detected. The novel FTIR difference spectra we proposed here is expected to become a method for the control of Baijiu.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Key Research Project of Shaanxi Provincial Science and Technology Department (2023-YBNY-158), the Xi\u0026rsquo;an Science and Technology Project (22NYYF016) the Natural Science Foundation of Shaanxi Province (2022JM-087), the Open Fund of the State Key Laboratory of Molecular Reaction Dynamics in DICP, CAS, (SKLMRD-K202413), the Fundamental Research Funds for the Central Universities (QTZX23007) and the 111 Project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGuo: Investigation, Writing - original draft,\u003c/p\u003e\n\u003cp\u003eWang: Formal analysis, Methodology, Funding acquisition\u003c/p\u003e\n\u003cp\u003eZhang: Formal analysis, Writing - Review \u0026amp; Editing, Funding acquisition\u003c/p\u003e\n\u003cp\u003eMa: Formal analysis, Funding acquisition\u003c/p\u003e\n\u003cp\u003eLin: Conceptualization, Methodology, Writing - Review \u0026amp; Editing, Funding acquisition\u003c/p\u003e\n\u003cp\u003eConflict of Interest: The authors declare no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbramova IM, Medrish ME, Romanovs AG et al (2020) Chemical characterization of ethanol and vodka in Russia. IOP Conference Series: Earth and Environmental Science 548: 082059. \u003c/li\u003e\n\u003cli\u003eAli A, Miraz RK, Andrew CKL (2020) Identifying the Responses from the Estrogen Receptor-Expressed MCF7 Cells Treated in Anticancer Drugs of Different Modes of Action Using Live-Cell FTIR Spectroscopy. ACS omega, 5: 12698-12706. \u003c/li\u003e\n\u003cli\u003eAmo Y, Tominaga Y (2000) Low-frequency Raman study of ethanol-water mixture. Chem Phys Lett 320: 703-706. \u003c/li\u003e\n\u003cli\u003eAnjos O, Santos AJA, Estevinho LM et al (2016) FTIR-ATR spectroscopy applied to quality control of grape-derived spirits. Food Chem 205: 28-35. \u003c/li\u003e\n\u003cli\u003eBuck N, Goblirsch T, Beauchamp J et al (2020) Key Aroma Compounds in Two Bavarian Gins. Appl Sci-Basel, 10: 7269.\u003c/li\u003e\n\u003cli\u003eCharapitsa S, Sytova S, Kavalenka A et al (2021) The study of the matrix effect on the method of direct determination of volatile compounds in a wide range of alcoholic beverages. Food Control, 120: 107528. \u003c/li\u003e\n\u003cli\u003eAlmeida MP, Leopold N, Franco R, et al (2019) Expedite SERS Fingerprinting of Portuguese White Wines Using Plasmonic Silver Nanostars. Front Chem 7: 368. \u003c/li\u003e\n\u003cli\u003eFan W, Shen H, Xu Y (2011) Quantification of volatile compounds in Chinese soy sauce aroma type liquor by stir bar sorptive extraction and gas chromatography-mass spectrometry. J Sci Food Agric 91: 1187-1198. \u003c/li\u003e\n\u003cli\u003eGiannetti V, Mariani MB, Marini F et al (2020) Grappa and Italian spirits: Multi-platform investigation based on GC-MS, MIR and NIR spectroscopies for the authentication of the Geographical Indication. Microchem J 157: 104896. \u003c/li\u003e\n\u003cli\u003eGiubertoni G, Sofronov OO, Bakker HJ (2019) Observation of Distinct Carboxylic Acid Conformers in Aqueous Solution. J Phys Chem Lett 10: 3217-3222. \u003c/li\u003e\n\u003cli\u003eGu J, Liu H, Ma C et al (2019) Conformal Prediction Based on Raman Spectra for the Classification of Chinese Liquors. Appl Spectrosc 73: 759-766. \u003c/li\u003e\n\u003cli\u003eGu S, Ren F, Wei Z et al (2020) Rapid Identification of Age and Brand of Chinese Rice Wine Based on Gas-ion Mobility Spectrometry. J Chin Inst Food Sci Technol 20: 248-255. \u003c/li\u003e\n\u003cli\u003eJoshi I, Khanh T, Elbourne A et al (2019) Influence of the Scanning Temperature on the Classification of Whisky Samples Analysed by UV-VIS Spectroscopy. Appl Sci-Basel 9: 3254. \u003c/li\u003e\n\u003cli\u003eLi X, Tsuta M, Tanaka F, et al (2020) Assessment of Japanese Awamori Spirits Using UV-VIS Spectroscopy. Food Anal Methods 13: 726-734. \u003c/li\u003e\n\u003cli\u003eLin K, Hu NY, Zhou XG, et al (2012) Reorientation dynamics in liquid alcohols from Raman spectroscopy. J Raman Spectrosc 43: 82-88. \u003c/li\u003e\n\u003cli\u003eMalloum A, Fifen JJ, Conradie J (2020) Theoretical infrared spectrum of the ethanol hexamer. Int J Quantum Chem 120: 1-11. \u003c/li\u003e\n\u003cli\u003eMatthias L, Frank F, Stefan L (2014) Direct observation of the cyclic dimer in liquid acetic acid by probing the C=O vibration with ultrafast coherent Raman spectroscopy. Phys Chem Chem Phys 16: 18010-18016. \u003c/li\u003e\n\u003cli\u003eNishi N, Nakabayashi T, Kosugi K (1999) Raman spectroscopic study on acetic acid clusters in aqueous solutions: Dominance of acid-acid association producing microphases. J Phys Chem A 103: 10851-10858. \u003c/li\u003e\n\u003cli\u003eNiu Y, Yao Z, Xiao Q et al (2017) Characterization of the key aroma compounds in different light aroma type Chinese liquors by GC-olfactometry, GC-FPD, quantitative measurements, and aroma recombination. Food Chem 233: 204-215. \u003c/li\u003e\n\u003cli\u003eOkaru AO, Abuga KO, Kibwage IO et al (2017) High Ethanol Contents of Spirit Drinks in Kibera Slums, Kenya: Implications for Public Health. Foods 6, 89. \u003c/li\u003e\n\u003cli\u003eMannix PB, Dong HK, Haiyan F (2016) Revisiting the formation of cyclic clusters in liquid ethanol. J Chem Phys 144: 154302. \u003c/li\u003e\n\u003cli\u003ePanuszko A, Stangret J, Nowosielski B et al (2020) Interactions between hydration spheres of two different solutes in solution: The least squares fitting with constraints as a tool to determine water properties in ternary systems. J Mol Liq 310: 113181. \u003c/li\u003e\n\u003cli\u003eQi H, Chen H, Wang Y et al (2018) Detection of ethyl carbamate in liquors using surface-enhanced Raman spectroscopy. R Soc Open Sci 5: 181539. \u003c/li\u003e\n\u003cli\u003eShu D, Zhang L, Zhang W et al (2007) Variation of Flavor Components in Zaopei of Luzhou-flavor Liquor during Fermentation. Food Sci 28: 89-92. \u003c/li\u003e\n\u003cli\u003eSiristova L, Prinosilova S, Riddellova K et al (2012) Changes in Quality Parameters of Vodka Filtered through Activated Charcoal. Czech J Food Sci 30: 474-482. \u003c/li\u003e\n\u003cli\u003eŚmialek MA, Łabuda M, Guthmuller J et al (2016) Electronic state spectroscopy by high-resolution vacuum ultraviolet photoabsorption, He(I) photoelectron spectroscopy and ab initio calculations of ethyl acetate. Eur Phys J D, 70: 1-9. \u003c/li\u003e\n\u003cli\u003eSong XB, Jing S, Zhu L et al (2020) Untargeted and targeted metabolomics strategy for the classification of strong aroma-type baijiu (liquor) according to geographical origin using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry. Food Chem 314: 126098. \u003c/li\u003e\n\u003cli\u003eSun JY, Yin ZT, Zhao DR et al (2018) Qualitative and quantitative research of propyl lactate in brewed alcoholic beverages. Int J Food Prop 21: 1351-1361. \u003c/li\u003e\n\u003cli\u003eUrickova V, Sadecka J (2015) Determination of geographical origin of alcoholic beverages using ultraviolet, visible and infrared spectroscopy: A review. Spectrochim Acta Part A Mol Biomol Spectrosc 148: 131-137. \u003c/li\u003e\n\u003cli\u003eVanbeneden N, Delvaux F, Delvau, FR (2006) Determination of hydroxycinnamic acids and volatile phenols in wort and beer by isocratic high-performance liquid chromatography using electrochemical detection. J Chromatogr A 1136: 237-242. \u003c/li\u003e\n\u003cli\u003eWang XX, Fan WL, Xu Y (2014) Comparison on aroma compounds in Chinese soy sauce and strong aroma type liquors by gas chromatography-olfactometry, chemical quantitative and odor activity values analysis.\u003cem\u003e \u003c/em\u003eEur Food Res Technol 239: 813-825. \u003c/li\u003e\n\u003cli\u003eXiao S, He Y (2019) Analysis of Sildenafil in Liquor and Health Wine Using Surface Enhanced Raman Spectroscopy. Int J Mol Sci 20: 2722. \u003c/li\u003e\n\u003cli\u003eZhang Y, Chen GQ, Gu J et al (2020) A theoretical study on dimerization and dissociation of acetic acid in ethanol solvent. Comput Theor Chem 1191: 113029. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-analytical-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Analytical Methods](https://www.springer.com/journal/12161)","snPcode":"12161","submissionUrl":"https://submission.nature.com/new-submission/12161/3","title":"Food Analytical Methods","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Baijiu, Ester, Acid, Fourier transform infrared spectroscopy, Difference spectrum","lastPublishedDoi":"10.21203/rs.3.rs-4481737/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4481737/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAcids and esters are the main aromatic compounds in Baijiu. These compounds affect primarily the flavor and quality of Baijiu. The detection of these compounds is important for the control of Baijiu. Although FTIR spectroscopy has gradually been used to detect Baijiu in recent years, this technology has not been directly employed to measure the infrared spectra of acids and esters in Baijiu. In this paper, a novel FTIR difference spectroscopy is proposed to extract the infrared spectra of acids and esters in Baijiu. This difference spectrum is mainly obtained by subtracting the FTIR spectra of aqueous ethanol from that of Baijiu. The FTIR spectra of some kinds of Baijiu were measured, and the infrared spectra of acids and esters in Baijiu were obtained. The flavor of Baijiu can be distinguished through the difference spectra. Since the acquisition of FTIR spectra only takes less than one minute, this FTIR difference spectrum can be developed as a quick control method for Baijiu.\u003c/p\u003e","manuscriptTitle":"Rapid detection of acids and esters in Baijiu by Fourier transform infrared spectroscopy with difference spectroscopy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-11 10:56:58","doi":"10.21203/rs.3.rs-4481737/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-20T10:51:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-19T13:29:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-14T04:23:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"179779097118107190774264177248914578910","date":"2024-06-12T00:45:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"139194607211660733184869698550368990091","date":"2024-06-11T00:03:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"120900989025529195520427100779170795059","date":"2024-06-07T16:14:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-07T16:12:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-29T03:24:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-29T03:24:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food Analytical Methods","date":"2024-05-27T01:53:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-analytical-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Analytical Methods](https://www.springer.com/journal/12161)","snPcode":"12161","submissionUrl":"https://submission.nature.com/new-submission/12161/3","title":"Food Analytical Methods","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fec05975-00b9-49be-be4d-d0b9a27cfd39","owner":[],"postedDate":"June 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-08-14T11:39:59+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-11 10:56:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4481737","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4481737","identity":"rs-4481737","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}