Determination of Alcohol Content Using Gas Chromatography

1.0 TITLE

Determination of alcohol content in Tapai by using gas chromatography technique

2.0 INTRODUCTION

Alcohol is an organic compound which contain hydroxyl functional group (-OH) that bonded to saturated carbon (C) atom. For centuries, alcohol has been used for many purposes for examples as fuels, to make drinks, as a solvent and as an industrial feedstock. Alcohol can give us many benefits if we use it in the right way but it also can give bad effects to us if we misuse it.

Alcohol can be produced normally by using fermentation process. Fermentation process is a process which uses yeast as the catalyst to break down sugar or glucose into alcohol and carbon dioxide. Ethanol is the most common type of alcohol present from the process of fermentation.

Yeast + glucose alcohol + carbon dioxide

Tapai is one of the most general dessert or fermented food that can be found easily in East- and Southeast Asia such as Malaysia, Indonesia, Singapore and Brunei. Tapai can be made from different main ingredient such as glutinous rice, white rice and cassava. During preparation of Tapai, the ingredients are cleaned and washed before cooked. Cooked glutinous rice is then left to cool down at temperature 30^oC. To produce a matured and good quality Tapai, it has to undergo fermentation for 3-4 days.

Gas chromatography technique has been selected in this study to determine the amount of ethanol content in Tapai. A few aspects have been considered in choosing the right technique to be used. Since Tapai contains alcohol, the Halal issue has been rise in Muslim community whether it is allowed to be consumed or prohibited because of some of the similarities between Tapai and alcoholic beverages.

3.0 LITERATURE REVIEW

3.1 Fermentation

Fermentation is a process of conversion of carbohydrates into alcohols and carbon dioxide or organic acid by using yeast or bacteria as the catalysts. The yeast works to break down the carbohydrates or sugar. Fermentation refers to the chemical conversion of sugars to ethanol. It is a process which is commonly used in order to produce alcoholic beverages such as beer and wine. Besides that, fermentation process is very useful for preservation of food. It produces lactic acids which can make the food can be used in a long period of time. For an example, it is found in such sour foods and pickled cucumber, yogurt and kimchi.

Yeast + glucose alcohol + carbon dioxide

In Malaysia, fermented glutinous rice also known as Tapai is one of the most popular dessert among the Malaysian consumers. Tapai is a sweet or sour alcoholic taste and can be used as a dessert or traditional recipes. Tapai can be made from various types of carbohydrate sources, but commonly from cassava, glutinous rice and white rice. Yeast and sugar are used in the procedure to make Tapai.

Fermented foods generally produce pleasant aroma, texture and have a good keeping quality under ambient environment. Fermented foods are prepared from raw materials. Yeast fermentation generally is an alcoholic fermentation which involves the production of ethanol. Southeast Asia produces 150 million tones of paddy annually. Rice wine is one of the most popular sources to make alcoholic beverages in Asian country. In Malaysia and Indonesia, cassava tubers commonly used as fermented foods because of the high carbohydrates content in it.

To make Tapai, the first step is soaked the rice in a gallon of water and left overnight. Then, the soaked rice is steamed for 45 minutes. Cool the steamed rice to body temperature. After that, the Tape Starter is sprinkled on the rice and mixed for a minute to distribute uniformly. The starter and rice have to be mixed well to prevent the spoilage. Half cup of sugar is mixed with water. The rice is packed in layers into a jar and every layer is sprinkle with some sugar water. This step is taken to help the fermentation process to start. The rice is left for fermentation process to take place for 2-4 days. When the rice already produces a bit of liquid at the bottom of the container or jar and distinctive smell is produced, the fermentation process is complete.

There are 3 stages of fermentation which are Initial Stage (Lag Phase), Middle Stage (Log to Stationary Phases) and Latter Stage (Death Phase). Initial stage is important to make natto bacilli grow sufficiently. The temperature is set at body temperature 37-40^oC. This temperature is an optimum temperature for the bacteria to grow. During the log phase, natto bacilli release a great deal of heat produced by the fermentation process. Sufficient oxygen must be supplied during this phase to make the process runs smoothly. After the stationary phase, the natto bacilli reach the death phase. It undergoes autolysis gradually. 11% of glutamic acid is deliberated 16 hours after the fermentation process started. Organic acids are synthesized in this phase.

Theoretically, fermented food is not considered fully safe since they are prepared under uncontrolled conditions. Nevertheless, natural processes have been related with a few documented cases of food induced disease. Natural processed food has been known to cause serious food poisoning compared to prepared packed food because the production of prepared packed food is been done in a factory with a regular monitoring from the quality control unit.

Figure 1:Process of making Tapai

3.2 Comparison between Tapai and alcoholic beverages

The process of Tapai production is more likely to alcoholic beverages production where both of these productions have to go through the fermentation process. Both processes use yeast in order to produce alcohol.

There might be some differences for alcohol content in wine and Tapai since the other processes except fermentation which are used for alcoholic beverages but not in Tapai production. For Tapai production, the process takes place naturally while for alcoholic beverages production requires temperature control where there are hot fermentation and cool fermentation.

Cronk et al. Went and Geerligs were the first person reported to segregate and identify the essential microorganisms for the fermentation of Tapai in 1895. In order to produce a good Tapai, a mixture of Candida oryzae and Endomycopsis burtonii with 17% of reducing sugar and 2.7% of ethanol are needed. For wine and grapes, the healthy properties can be achieved due to the presence of antioxidants like polyphenols.

From a chapter entitled “Tapai processing in Malaysia: A technology in Transition” by Zahara Marican and Yeoh Quee Lan in a book edited by KH Steinkraus entitle “Industrialization of indigenous fermented foods”, the authors write about the making of Tapai in Malaysia. There are some points that can be considered as useful information from the quote on page 252 “Tapai fermentation is not an anaerobic process and this partly accounts for the low alcohol content. Furthermore the fermentation is normally arrested before all of the sugars are fermented, as the desired end product should not be alcoholic if it is to be consumed by those opposed to alcohol consumption for religious or other reasons”. There is some information gained where Tapai fermentation is not an anaerobic. In the beginning, the enzymes are release to breakdown the sugars by the process of enzyme hydrolysis. This explained why Tapai tasted sweet.

Besides that, Tapai production might be a little different compared to the wine or beer fermentation even though alcohol is produced and sugar is used. For Tapai fermentation, the theory of solid substrate fermentation is involved. Each of the white rice or glutinous rice is coated with a thin layer of sticky liquid to make it difficult for oxygen to diffuse or penetrate. The reaction of the sugar molecules and the liquid caused the Tapai sticky or viscous by the interaction of hydrogen bond between the water molecules and sugar molecules. Furthermore, the low alcohol content in Tapai is caused by the slightly open or not an air tight packing and high room temperature which resulting in the vaporization of alcohol and the alcohols are escaped to the environment. The alcohols as the fermentation product have a short chain and gave a lower evaporation point and boiling point.

Alcoholic beverages contain high amount of ethanol compared to Tapai. Beer contains approximately 6% of ethanol, wine contains 7 to 24% v/v of ethanol, whisky contains 35% of ethanol and Tapai contains only about 5% of ethanol. Besides ethanol, alcoholic beverages contain another chemical composition such as water, glycerine, pestins, acids, polyphenols and some flavours. The least amount of ethanol contained in Tapai is caused by the volatilities of ethanol from the fermented rice due to non-air tight container.

Figure 2:Production of alcoholic beverages

3.3 Analysis of ethanol in fragmented rice using many techniques

Recently, many researches have been made in order to determine ethanol content in fragmented food such as wine and Tapai. Various types of techniques can be used for ethanol determination such as enzymatic method, biosensor, high liquid performance liquid chromatography (HPLC), near-infrared (NIR) spectroscopy and flow injection analysis.

3.3.1 High-performance liquid chromatography (HPLC)

High-performance liquid chromatography (HPLC) analysis was used to quantify the presence of individual phenolic compounds using an HPLC system consisting of a Shimadzu Prominence LC – 20A, with an LC-20AT quaternary pump, a SIL 20 auto sampler– A, a DGU-20A5 on-line degasser, a CBM-20A integrator and a SPD-20AV DAD detector.

HPLC technique has been designed to determine the amount of ethanol produced and amount of unwanted byproducts produced during the fermentation process. HPLC can produce the shortest analysis time and maintaining enough resolution between components for proper quantification and identification. By using this technique, all components can be analyzed less than 10 minutes.

3.3.2 Short wave-near infrared (SW-NIR)

Short wave-near infrared (SW-NIR) is one of the type of NIR. SW-NIR is one of the techniques that can be used to detect alcohol content in beverages and food products. The region of SW-NIR is between 700 to 1100 nm (Fu et al., 2012). The SW-NIR is suitable for non-destructive or non-invasive analysis of biological and biomedical materials (Ozaki et al., 1992; Šašić & Ozaki, 2001; Sato et al., 2000). The SW-NIR can penetrate more intensely into a sample with less heating effect compared than long-wave NIR and the obstruction arising from the intense water bands can be diminished (Reeves III, 1994). Tungsten lamps, LEDs or the sunlight can be utilized as the SW-NIR light source and the light can be transmitted through glass optical fibers (Fu et al., 2012). In order to determine the content of alcohol in aqueous samples, temperature factor needs to be emphasized (Wülfert et al., 1998; Barboza & Poppi, 2003). It is because temperature can cause a change in the vibration spectra (Fu et al., 2012). Figure 2 below shows the schematic diagram of the multichannel SW-NIR spectrometer module.

Figure 2:Schematic diagram of the multichannel SW-NIR spectrometer module

3.3.3 Fourier transform infrared spectroscopy (FTIR)

In determination of alcohol, FTIR had been used for the quality control of alcoholic drinks such as spirit drinks and beer. FTIR with combination of multivariate data analysis can determine the essential compound quantitatively (Lachenmeier, 2007). Apart from that, Vonach et al. (1998) also stated that the combination of HPLC-FTIR can be used to determine the components of wine such as acetic, fructose, succinic, glycerol, ethanol, glucose, citric, lactic, malic and tartaric acid directly when used a real wine as a sample. By using FTIR, to obtain the information about the product, for example the chemical composition, possibly we can make it within in a short time. Liquid Fourier transform-middle infrared spectrometry can be used to analyze wine in a 90 s (Patz et al., 2004). Nowadays FTIR can give us advantages in order to determine and analyze the beverages because it can help us to reduce the time cost, and at the same time, it is also contribute for a good precision and accuracy when dealing with the parameters (Moreira et al., 2004). Furthermore, according to Lachenmeier (2007), a simple sample preparation is needed when dealing with FTIR spectroscopy compared to the other analytical methods.

3.3.4 Other spectroscopy

Raman spectroscopy is not a preferable technique in determining ethanol content in Tapai due to the expensive instrument required. As HPLC method is low sensitivity but accurate weighing process and the sample distillation are required, HPLC is not a good technique in this study. NIR spectroscopy is time consuming in establishing calibration curves and has low accuracy. It can be disturbed by other alcohol in the sample (Wang et al., 2003).

3.4 Determination of ethanol in Tapai by using Gas Chromatography

Gas-liquid chromatography is one of the most sophisticated analytical techniques in spectroscopy. Gas chromatography becomes more preferable because of its utilization of electronic digital data-processing equipment for accurate qualitative and quantitative analysis.

Traditional ethanol analysis methods are distillation and mass determination. These methods include small accuracies because it can be interfere by other component in the sample. In order to evaluate the accuracy and precision of the gas-liquid chromatography, the ethanol standards and typical Tapai samples are necessary to be analyzed (B.Stackler and E.N.Christensen., 1974)

To analyze ethanol contents in alcoholic beverages, Bouthilet et al. had made a packed GC method. Yet, the method needs at least 100mL of sample and distillation as the pretreatment process. These official gas liquid chromatography methods can be adjusted with different sample preparation procedures when dealing with different samples. These samples require distillation or dilution as the pretreatment process. By using packed column, lower resolution and interference by other alcohols in alcoholic beverages still exist. When capillary gas chromatography is used for determining the ethanol contents in alcoholic samples, time-consuming pretreatment procedures are still required. The capillary GC method is one of the most important techniques because of its advantage of high resolution and sensitivity (Wang et al., 2003).

Wang et al. (2004) has studied the gas chromatography coupled with flame ionization detection (GC-FID) to detect the ethanol content in alcoholic beverages. A selected column used is mega-pore capillary column (CP-Wax 58 CB). Acetonitrile was chosen as the internal standard for ethanol and, 2-pentanol is used for methanol. This selection is due to the water solubility of acetonitrile is more rather than 2-pentanol. Direct injection GC coupling with dual internal standards was used to determine ethanol and methanol content can be obtained less than 12 min.

Due to the success in the previous study, Brill and Wagner (2012) have performed the new method using GC-MS in order to detect the alcohol content that has been adapted by Wang et al.’s (2004) experiment that used capillary gas chromatography together with FDI detector. This further study is quite costly, but it is worth since the accuracy is high. The column in this study was replaced by polar capillary column instead of mega-pore column in the previous study. 0.2 µL splitless injections were chosen to directly inject the samples to the GC-MS. Helium set at pressure set at 7.35 psi and 1mL/min of flow rate acted as the carrier gas in GC-MS. The polar VF-WAXms selected was the reliable because the samples could be run in 15 minute intervals.

Capillary gas chromatography is a simple and rapid method that developed to determine ethanol content in alcoholic beverages using megapore polar column (CPWax58 CB, 30 m ¥ 0.53 mm) with direct injection gas chromatography. Contrary to packed GLC method, distillation or stepwise dilution of samples were not necessary by the method developed in this study. In conclusion, gas chromatography method is the most suitable and quick method for determination of ethanol contents in alcoholic beverages with complex alcohol contents and small sample amount.

Solid-phase microextraction (SPME) is a non-harmful environmentally pre-concentration technique which is clean, selective, rapid, efficient, simple and solvent-free. The derivation of polyphenols allowed non-volatile compounds to be converted into volatile derivatives, thus improving the separation, selectivity or sensitivity of SPME–GC determination. This step can be performed in the sample solution prior to sampling, on the SPME fibre coating or during desorption in the GC injection port. For on-fibre derivation, the chemical agent has to be loaded on the fibre prior to or after analyte extraction. Commonly, the analytes are first extracted and then the fiber is exposed to the vapour of the derivatizing agent. The coupling of GC and SPME has been used to determine resveratrol isomers and the total free resveratro was also determined by direct-exposure-probe positive-ion chemical ionization mass spectrometry. However, no references have been found to the use of the hyphenated technique SPME–GC–MS for the determination of polyphenol mixtures (Pilar Vinas et al., 2009).

4.0 PROBLEM STATEMENTS

The purpose of this study is to determine the most suitable and appropriate method for alcohol determination in the sample used which is fermented rice or commonly called as Tapai. Tapai is a traditional dessert or fermented food frequently found in East and Southeast Asia. Nowadays, Tapai has been established and being promoted to many countries. Determination of ethanol perhaps is the most important analysis in the fermentation industry for the quality control. It is necessary for the industry to use the most accurate and easiest method in order to check the alcohol composition in the products manufactured before sending them to the markets.

Besides that, this study is fundamental in order to determine the Halal limits for alcohol content in the sample. The consumption of Tapai had raised the Halal issue to Muslim community since it has similar alcohol content with beer which is 5%. So, the study should offer the method that can give the short period for the detection and the easiest way to handle the experiment or the test include the easy sample preparation with the accurate results.

5.0 OBJECTIVES

These are few objectives that have been identified in order to deal up with the problem statement and carried out the research on determination of alcohol content in Tapai by using gas chromatography spectroscopy.

1. To develop gas chromatography method with the best accuracy compared to other methods for alcohol detection in the sample.
2. To determine the lower limit of detection (LOD) of alcohol.
3. To identify the amount of ethanol content in the sample.