Tandem Solid-Phase Extraction Columns for Simultaneous Aroma Extraction and Fractionation of Wuliangye and Other Baijiu

Wuliangye baijiu is one of the most famous baijiu in China, with a rich, harmonic aroma profile highly appreciated by consumers. Thousands of volatiles have been identified for the unique aroma profile. Among them, fatty acid esters have been identified as the main contributors to the aroma profile. In addition, many non-ester minor compounds, many of which are more polar than the esters, have been identified to contribute to the characteristic aroma unique to Wuliangye baijiu. The analysis of these minor compounds has been challenging due to the dominance of esters in the sample. Thus, it is desirable to fractionate the aroma extract into subgroups based on functional group or polarity to simplify the analysis. This study attempts a new approach to achieve simultaneous volatile extraction and fractionation using tandem LiChrolut EN and silica gel solid-phase extraction (SPE) columns. A baijiu sample (10 mL, diluted in 40 mL of water) was first passed through the LiChrolut EN (1.0 g) column. The loaded LiChrolut EN column was then dried with air and coupled with a silica gel (5.0 g) SPE column with anhydrous Na2SO4 (10.0 g) in between. The volatile compounds were eluted from the LiChrolut EN column and simultaneously fractionated on the silica gel column based on polarity. The simultaneous extraction and fractionation technique enabled the fractionations of all fatty acid esters into less polar fractions. Fatty acids, alcohols, pyrazines, furans, phenols, hydroxy esters, and other polar compounds were collected in more polar fractions. This technique was used to study the volatile compounds in Wuliangye, Moutai, and Fengjiu baijiu. In addition to fatty acid esters, many minor polar compounds, including 2,6-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-3,5-dimethylpyrazine, p-cresol, and 2-acetylpyrrole, were unequivocally identified in the samples. The procedure is fast and straightforward, with low solvent consumption.


Introduction
Baijiu is one of the oldest distilled spirits globally, with its ethanol content typically 40-65% by volume. The composition of baijiu is very complex due to spontaneous solidstate fermentation. Thousands of volatile compounds have been identified in different types of baijiu, and alcohols, fatty acids, and fatty acid esters dominate the composition [1,2].
Gas chromatography-olfactometry (GC-O) was first used to study the aroma-active compounds in baijiu in 2005 [3]. Among all the aroma-active compounds identified, fatty acid esters have been proven to be the main contributors to baijiu aroma [3][4][5][6][7], exhibiting fruity and floral notes [3][4][5][6]. Free fatty acids and alcohols also make important aroma contributions. In addition to these major compounds, other minor compounds may contribute to the unique aroma of the baijiu. Identifying these minor aroma compounds with unique aroma quality has been challenging due to the high concentration of these In addition, LiChrolut EN resin had low extract efficiency for fatty acids, especially short-chain acids such as acetic acid (6.2%) and propanoic acid (19.0%). The carboxy acid contents in baijiu are very high [32], and these acids would cause significant interference in separating and identifying other volatile compounds in routine analysis. The low extraction efficiency for acids is beneficial because it simplifies the chromatogram to be more conducive to identifying other minor compounds in the extract. The high recoveries for aroma compounds and low recoveries for interfering carboxy acids reveal that LiChrolut EN is an excellent resin for isolating most aroma compounds from baijiu. It needs to be pointed out, however, that LiChrolut EN resin is not ideal for carboxy acid analysis.

Simulated Baijiu Sample
Esters, alcohols, and carboxy acids are the main aroma compounds in baijiu. However, their concentrations vary widely depending on the aroma type and manufacturers, ranging from several ppb to ppm [9,33,34]. A simulated baijiu sample was used to study volatile compounds' extraction and fractionation using tandem SPE columns based on volatile compounds in strong-aroma-type baijiu. However, the concentrations of some trace substances were increased for easy analysis. This simulated sample was used to evaluate the performance of the tandem SPE columns and the feasibility of this technique for separation.

Ester Distribution
Extracts were fractionated on the tandem SPE columns using pentane-dichloromethane or methanol-dichloromethane at different proportions with increased polarity. As shown in Table 2, pentane (F1) could not elute any compounds from the tandem columns. However, a small number of esters were eluted by pentane-dichloromethane (98:2) (F2), and most of the ethyl esters were eluted in pentane-dichloromethane (95:5) (F3) and pentanedichloromethane (90:10) (F4). The acetates, however, were primarily eluted in F5 (80:20 pentane-dichloromethane) and some in F6 (50:50 pentane-dichloromethane). This fraction-ation is meaningful because the most abundant esters in baijiu are ethyl esters, grouped from F2 to F5. In addition, some of the acetates, including ethyl acetate, butyl acetate, isopentyl acetate, and hexyl acetate, were eluted out in F6. Except for ethyl acetate, concentrations of acetates are much lower in baijiu. Therefore, their presence in F6 will unlikely impose major problems for analyzing other polar volatile compounds. Fan et al. [5,10] reported that esters of baijiu extracts would mainly be eluted out in the fractions of pentane:diethyl ether = 98:2 and pentane:diethyl ether = 95:5 from silica gel. Laura Culleré [19] indicated that when extracts of wines were fractionated in LiChrolut EN resins, ethyl esters of fatty acids would elute in the first fraction because of low retention factors. However, when tandem LiChrolut EN and silica gel columns were used in this study, a more polar solvent was needed to elute the esters. This may be because ester compounds would go through two chromatographic separations, so more polar solvents were needed to elute all the esters from both columns.

More Polar Fractions
F6 (50:50 pentane-dichloromethane) and F7 (90:10 dichloromethane-methanol) were the more polar fractions. All the acids, alcohols, pyrazines, hydroxy esters, and dibasic esters were eluted in F7 because of their strong polarity. Similarly, lactones, furans, and phenolics were mainly eluted in F7, although some were eluted in F6 (Table 3). It can be seen that these compounds were separated from ethyl esters so that the interference of esters on the identification of these substances can be eliminated. Aromatic esters (except ethyl benzoate) were eluted in F5 and F6, and benzeneacetaldehyde was eluted in F6 and F7, whereas benzyl alcohol and phenylethyl alcohol were eluted in F7 due to being more polar.
It can also be observed that some compounds, such as 2,6-dimethylphenol, nonanal, and ethyl benzoate, were poorly chromatographed and appeared in several fractions. In addition, only a few aldehydes and ketones were included in the simulated sample, so their elution order was not apparent.
It has been reported previously that alcohols, phenols, aldehydes, and ketones can be eluted with pentane:diethyl ether from 95:5 to 50:50, depending on experimental conditions [5,10]. When wine volatiles were fractionated on the LiChrolut EN column, it was reported that most of the volatile compounds could be eluted in less polar fractions (pentane and pentane:dichloromethane = 90:10), whereas fatty acids, phenolics, some lactones, benzyl alcohol, and benzaldehyde are shown in more polar fractions [19]. Compared to the fractionation on silica gel and LiChrolut EN, the elution order was similar on the tandem LiChrolut EN and silica gel columns. This result encouraged further research to develop a simultaneous extraction and fractionation method to separate ester compounds from baijiu extracts.

Simultaneous Extraction and Fractionation of Baijiu by Tandem Lichrolut EN and Silica Gel SPE Columns
The simultaneous extraction and fractionation method was applied in three aroma types of baijiu (Wuliangye, Moutai, and Fenjiu) to separate esters from other compounds ( Table 4). The results showed that the elution order of volatile compounds was quite similar to the simulated baijiu (see Section 2.1, Tables 2 and 3). Under chromatographic conditions, esters were mainly eluted in less polar fractions (F1-F5 combined fractions). Because of their high concentrations, only a small amount of ethyl hexanoate and ethyl heptanoate were in more polar fractions (F6-F7 fractions). In addition, some long-chain ketones (≥C 6 ), short-chain acetals, some aromatic compounds (benzaldehyde and aroma esters), phenol, and furfural were eluted in these fractions. On the other hand, acids, alcohols, pyrazines, furans, phenolics, hydroxy esters, and dibasic esters were eluted in more polar fractions (except furfural and phenol). Therefore, simultaneous extraction and fractionation using tandem SPE columns is an effective method to separate esters from other compounds in different aroma types of baijiu. As shown in Figure 1, very few esters were present in the polar fraction (F7), which allows for the unequivocal identification of minor polar compounds, including 2,6-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-3,5-dimethylpyrazine, p-cresol, and 2-acetylpyrrole, in the samples. Comparing the three types of baijiu, both Wuliangye and Moutai had more polar compounds than Fengjiu.

Recovery of Volatile Compounds Extracted by LiChrolut EN Resin
Some main volatile compounds in baijiu (Table 1) were selected to determine the recovery of LiChrolut EN resin. Each compound standard was dissolved in 52% (v/v) ethanol-water mixture at a concentration of 5 ppm. A single LiChrolut EN SPE column (0.2 g of LiChrolut EN resins packed in a 6 mL standard SPE column) was used to extract the volatile compounds.
The SPE bed was conditioned sequentially by 5 mL of dichloromethane, methanol, and 10% ethanol-water. The sample (2 mL) was diluted to 10% ethanol (v/v) with Milli-Qquality water. Then, the diluted sample was passed through the SPE bed with a flow rate of 1 mL/min. After the sample was loaded, the LiChrolut EN SPE bed was rinsed with 5 mL of water, then dried under vacuum at ambient temperature. The volatile compounds were finally eluted with 5 mL dichloromethane. The extract was dried with anhydrous sodium sulfate, filtered, and slowly concentrated to 500 µL under a gentle stream of nitrogen. The internal standard (4-octanol, 5 ppm) was added, and the extract was analyzed by GC-MS. Triplicate samples were prepared to calculate the standard deviation.
The volatile compound concentrations were determined using calibration graphs built with dichloromethane solutions containing known amounts of volatile compounds and a fixed quantity of the internal standard. The recovery was calculated using the concentration of volatile compounds found in eluted dichloromethane solutions divided by the concentration in the original mixture.

Preparation of Simulated Baijiu
A simulated baijiu sample (52% ethanol by vol) was prepared from pure standards based on the typical concentration range reported in strong-aroma-type baijiu (Table 5).

Simultaneous Extraction and Fractionation Using Tandem SPE Columns
The tandem SPE for fractionation (see Step 3 in Section 3.4.3) process constitutes one LiChrolut EN column, one anhydrous sodium sulfate column, and one silica gel column ( Figure 2). The purpose of the LiChrolut EN, anhydrous sodium sulfate, and silica gel columns is to extract volatile compounds, remove free water, and achieve the final fractionation, respectively.

Simultaneous Extraction and Fractionation Using Tandem SPE Columns
The tandem SPE for fractionation (see Step 3 in Section 3.4.3) process constitutes one LiChrolut EN column, one anhydrous sodium sulfate column, and one silica gel column ( Figure 2). The purpose of the LiChrolut EN, anhydrous sodium sulfate, and silica gel columns is to extract volatile compounds, remove free water, and achieve the final fractionation, respectively.

Figure 2.
Tandem Lichrolut EN and silica gel SPE columns for simultaneous extraction and fractionation.

Step 1: Extraction of Volatile Compounds Using LiChrolut EN SPE Column
Volatile compounds were extracted by a single LiChrolut EN SPE column (1.0 g of LiChrolut EN resins packed in a 12 mL standard SPE column). The SPE bed was conditioned sequentially by 20 mL of dichloromethane, 20 mL of methanol, and 20 mL of 10% ethanol in water (v/v). The sample (10 mL) was diluted to 10% ethanol by volume with Milli-Q water. Then, the diluted sample was passed through the SPE column at a flow rate of 1 mL/min. After the sample loading, the LiChrolut EN SPE column was rinsed with 20 mL of water, then dried for 10 min under vacuum at ambient temperature.

Step 2: Installation of the Anhydrous Sodium Sulfate Column
A SPE bed packed with 10.0 g of anhydrous sodium sulfate in a 20 mL standard SPE column was connected to the volatile-loaded LiChrolut EN column in Step 1 (Figure 2).

Step 1: Extraction of Volatile Compounds Using LiChrolut EN SPE Column
Volatile compounds were extracted by a single LiChrolut EN SPE column (1.0 g of LiChrolut EN resins packed in a 12 mL standard SPE column). The SPE bed was conditioned sequentially by 20 mL of dichloromethane, 20 mL of methanol, and 20 mL of 10% ethanol in water (v/v). The sample (10 mL) was diluted to 10% ethanol by volume with Milli-Q water. Then, the diluted sample was passed through the SPE column at a flow rate of 1 mL/min. After the sample loading, the LiChrolut EN SPE column was rinsed with 20 mL of water, then dried for 10 min under vacuum at ambient temperature.

Step 2: Installation of the Anhydrous Sodium Sulfate Column
A SPE bed packed with 10.0 g of anhydrous sodium sulfate in a 20 mL standard SPE column was connected to the volatile-loaded LiChrolut EN column in Step 1 (Figure 2).

Step 3: Connection of the Silica Gel SPE Column and Simultaneous Fractionation on the Tandem SPE Columns
A silica gel SPE column was prepared by packing silica gel (5.0 g) in a 12 mL standard SPE tube. The column was sequentially conditioned with an aliquot of 40 mL of methanol, dichloromethane, and pentane. The prepared silica gel column was installed in tandem with the sample-loaded LiChrolut EN column, with the anhydrous sodium sulfate column in between ( Figure 2). Next, an aliquot of 40 mL of the mixture of pentane:dichloromethane, each at different compositions (F1: 100:0, F2: 98:2, F3: 95:5, F4: 90:10, F5: 80:20, F6:50:50), was sequentially applied to elute the volatile compounds from the tandem columns at a flow rate of 1 mL/min. Finally, 40 mL of dichloromethane:methanol (90:10, F7) was applied. All eluents were slowly concentrated to 2 mL and then to a final volume of 500 µL with a stream of nitrogen. An aliquot of 50 µL of the internal standard (4-octanol, final concentration was 5 mg/L) was added to each fraction for GC-MS analysis. Standard calibration curves were used to estimate volatile concentration in each fraction.

Gas Chromatography-Mass Spectrometry Analysis
Identification and quantitation of volatile compounds in each concentrated fraction were performed on an Agilent 7890B GC equipped with an Agilent 5977B mass selective detector (MSD, Agilent Technologies, Inc., Santa Clara, CA, USA), and a PAL RTC autosampler (CTC Analytics AG, Zwingen, Switzerland). A volume of 1 µL of concentrated fractions was injected into the GC injector and separated on an HP-Innowax column (60 m length, 0.32 mm i.d., 0.25 µm film thickness; Agilent Technologies, Inc.). Helium was used as the carrier gas at a constant flow rate of 1.0 mL/min. The GC injector temperature was set at 230 • C. The oven temperature was programmed at 40 • C for a 5 min holding and ramped up to 230 • C at a rate of 4 • C/min with 15 min holding. The MS transfer line and ion source temperatures were 250 and 230 • C, respectively. Electron ionization mass spectrometric data from m/z 35-350 were collected using a scan rate of 5.2/s, with an ionization voltage of 70 eV. The results were calculated using MassHunter software (version B.08.00, Agilent Technologies, Inc.). A C 7 -C 30 n-alkane mixture was injected in the same conditions to calculate the retention indices (RIs), and RIs were calculated in accordance with the method of van den Dool and Kratz [41].

Volatile Compound Identification
Identification of volatile compounds was based on the following criteria: mass spectra (MS) of unknown compounds were compared with those in the NIST 17 database (Agilent Technologies Inc.), and RIs relative to those of pure reference compounds were compared to the RIs relative to those in the literature (RIL).

Application of Fractionation in Baijiu
The Wuliangye, Moutai, and Fenjiu baijiu samples were extracted and fractionated using the tandem SPE column method described in Section 3.4.

Conclusions
In conclusion, tandem LiChrolut EN-silica gel SPE columns allow fast volatile extraction and fractionation from Wuliangye and other baijiu. It integrates extraction and fractionation steps into a simple procedure. The method is quick and straightforward, with low solvent consumption and minimum extraction bias. It has excellent recoveries for important aroma compounds in baijiu. Furthermore, the technique can successfully eliminate the esters' interference and facilitate other volatile compound identification and analysis. When this technique was used to analyze the minor polar compounds in Wuliangye, Moutai, and Fengjiu baijiu, many minor polar compounds, including 2,6dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-3,5-dimethylpyrazine, p-cresol, and 2-acetylpyrrole, could be unequivocally identified in the samples.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.