G a s C h r o m a t o g r a p h y Determination of Ethanol in Wine by Head-Space Gas Chromatography

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1 G a s C h r o m a t o g r a p h y Determination of Ethanol in Wine by Head-Space Gas Chromatography Pibulsongkram Rajabhat University Department of Agro-Industry Faculty of Food and Agricultural Technology Mario Jekle Phitsanulok,

2 Principles The characteristic of gas chromatography is that samples can move very rapidly to the stationary phase (and out). You can get narrower bands and a sharper separation. Gas chromatography can only be used for aerial samples. The instrument consists in general of the carrier gas supplier, coarse pressure regulator, a fine pressure gage, an injection port (multi sampler), column, detector and amplifier with analog-digital converter (Pomeranz, Meloan 1994). Figure 1 Schematic of a gas chromatograph. (Pomeranz, Meloan 1994) The carrier gas respectively mobile phase is mostly helium, nitrogen or argon. The pressure differential over the column is responsible for the gas velocity down the column. If the pressure ratio is too low, molecular diffusion remixes the separated components and efficiency decreases. If the ratio is too high, the resistance to mass transfer increases and the efficiency of separation would decrease as well (Pomeranz, Meloan 1994). Two systems for the column are normally used: preparative columns (stationary phase is solid material) and capillary columns (stationary phase is a thin film of not aerial, viscose fluid). It is necessary that the columns are heated to about two-thirds of the boiling point f the highest boiling material in the mixture to be separated. The heating has to be very constant so that the procedure can be reproducible. When the low boiling material is separated, the column temperature can be raised for shortening the time of the separation of the remaining material. The advantage of the capillary column is the rapid and efficient analysis. However in this procedure you need a splitter. Because of the small capacity of the capillary column, only a sample of a few tenths of a micro liter is required. A splitter splits your injected sample into two parts. One small part is used for analysis and the rest is mostly discarded. The stationary phases can divided into fife types: Nonpolar, polar, intermediate, hydrogen bonding and specific (Pomeranz, Meloan 1994). There is a wide range of detectors available, for example thermal conductivity detectors, cross-section detectors, argon ionization detectors or electron capture detector. 2

3 In this experiment a hydrogen flame ionization detector (FID) was used. When the carrier gas and the sample emerge from the column, hydrogen and air (synthetic) are added to the carrier gas to produce a flame of about 2100 C. The pike of the flame serves as cathode and a collector electrode as anode. The carrier gas and the sample substances are ionized so that the conductivity of the flam increases. The electric current between flame and electrode is proportional to the absolute mass of the substance. The evaluation of the substances is possible with the use of a standard: external standard (calibration curve), addition of the detectable substance or internal standard. In the experiment the concentration of ethanol in two fruit wine samples should be examined. As detector FID and for the evaluation an external standard and an internal standard are used. Materials and Methods Standard preparation For the construction of a standard curve it was necessary to examine at least three standards. Three standards with 4 % v/v, 8 % v/v and 12 % v/v ethanol were produced. For that 10 ml of 10 % v/v n-propanol (A), 8 % v/v ethanol (B1), 16 % v/v ethanol (B2) and 24 % v/v ethanol (B3) were made. For the ready standards approximately 0.2 g NaCl, 200 µl of mixture A and 200 µl of mixture B1, B2 or B3 were blended (1:1 mixture A and Bx). NaCl was added to decrease the temperature of the boiling point of the standard. N-propanol served as an internal standard. With the use of an internal standard inaccuracy of the calculation of ethanol in the standards and samples could be avoided. The standards were heated at 70 C for 6 minutes and 0.5 ml of the gas head space of the mixture was injected to the Gas Chromatography instrument. Settings of the Gas Chromatograph: Temperature of Column Oven: 60 C Temperature of Detector: 150 C Temperature of SPL: 150 C Carrier: Helium Detector: FID Method: isocratic The chromatograms showed two peaks: ethanol and n-propanol. The peak of ethanol occurred around the retention time of 4.9 min and of n-propanol around the retention time of 5.4 min. 3

4 To construct a standard curve the peak ratio area of ethanol / n-propanol versus the concentration of ethanol was plotted. Sample preparation Approximately 0.2 g NaCl, 200 µl of mixture A and 200 µl of the sample were mixed. After heating at 70 C for 6 min 0.5 ml of gas head were injected to the Gas Chromatograph. Flow production chart A: 10 % v/v n-propanol, 10 ml B1: 8 % v/v ethanol, 10 ml B2: 16 % v/v ethanol, 10 ml B3: 24 % v/v ethanol, 10 ml + ~ 0.2 g NaCl µl A µl B1 => standard 4 % + ~ 0.2 g NaCl µl A µl B2 => standard 8 % + ~ 0.2 g NaCl µl A µl B3 => standard 12 % Heat with water bath, 70 C, 6 min Inject head space in Gas Chromatograph, 0.5 ml 4

5 Results and discussion Table 1 The results of the Gas Chromatograph: standards standards area ethanol area n-propanol ratio area ethanol/ n-propanol 4 % v/v ethanol ,445 8 % v/v ethanol , % v/v ethanol ,352 Table 1 shows the results of the chromatograms of the gas chromatograph. To avoid inaccuracy in the injection or heating time etc, the ration of the areas ethanol und n-propanol were consulted for the calculation. standard curve 1,6 1,4 area ratio ethanol/ n-propanol 1,2 1 0,8 0,6 0,4 y = 0,1133x - 0,0188 R 2 = 0,9983 standards Linear (standards) 0, c (ethanol) Figure 2 Standard curve: c (ethnanol) vs. ratio area ethanol / n-propanol The plotting of the concentration of ethanol versus the ratio area ethanol / n-propanol resulted in the equation y = x with R 2 = (1) Table 2 The results of the Gas Chromatograph: samples samples area ethanol area n-propanol ratio area ethanol/ n-propanol fruit wine pineapple , fruit wine ,

6 The results of the measurement of the samples are shown in table 2. The sample fruit wine pineapple was produced at another project (alcoholic beverage). The sample fruit wine 2 is a fruit wine produced by the students of the Faculty of Food and Agricultural Technology. For the calculation of the concentration of ethanol it was necessary to convert the equation (1) to: x = y (2) with x = c (ethanol) and y = ratio area ethanol/ n-propanol The received results had to bee multiplied with two because of the dilution in the sample preparation (1:1). The concentration of ethanol in pineapple fruit wine was calculated as 12.6 % and in fruit wine of the students 8.1 %. References Pomeranz, Yeshajahu. Meloan, Clifton E Food Analysis Theory and Practice. Third Edition. Chapman & Hall. 6

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