Ethanol as Internal Standard for Determination of Volatile Compounds in Alcohol Drinks by Gas Chromatography

Introduction

The new methodical approach of using ethanol as internal standard in gas chromatographic analysis of volatile compounds in spirit drinks in daily practice of testing laboratories is proposed. This method provides determination of volatile compounds concentrations in spirit drinks directly expressed in milligrams per liter (mg/L) of absolute alcohol according to official methods without measuring of alcohol strength of analyzed sample.

Briefly about this method.

Theoretical background

The main difference of the proposed method "ethanol as ISTD" from classical method of IS in this case is the following. In the classical case calibration of chromatograph includes the measuring of relative detector response factors for every analyzed compound relative to IS. Numeric values of these factors RF_i are calculated from chromatographic data for standard solutions with known concentrations of analyzed compounds and may be expressed by the following equation:

(1)

where A^st_i and A^st_IS are peak areas of i-th compounds and IS respectively; C^st_i (mg/L) and are concentrations of i-th compounds and IS respectively expressed in mg per 1 liter of solution. Concentration of i-th sample compound relative to absolute alcohol C_i [mg/L] is expressed by the following formula [1–3]:

(2)

where A_i and A_IS are the peak areas for i-th compound and IS respectively, C_IS (mg/L) is concentration of IS, Strength is concentration of alcohol in solution expressed in % volume. In the case of "ethanol as ISTD" the formulas (1) and (2) looks as follows:

(3)

where C^st_i is concentration of i-th compounds expressed in mg per 1 liter of absolute alcohol, ρ_Et = 789300 mg/L is the density of ethanol. Concentration of i-th sample compound relative to absolute alcohol C_i [mg/L(AA)] is expressed by the following formula:

(4)

According to (4) we obtain value of i-th sample compound concentration directly expressed in mg per 1 liter of absolute alcohol directly without any additional measurement of strength and without of any procedure of IS adding in an analyzed sample.

Experimental results

To compare proposed methodical approach "ethanol as ISTD" with classical commonly used method of internal standard experimental researches were planned and executed. Seven standard ethanol-water (96:4) solutions VC and VCC were prepared by weight. Concentrations of analyzed volatile compounds in the prepared solutions are presented in Table 1. The standard solutions of volatile compounds VC-1, VC-2 and VC-3 were used to generate calibration curves. The standard solutions VCC-0, VCC-1, VCC-2, VCC-3 and certified reference materials GSO-8404 and GSO-8405 were used to control trueness (ISO 5725-1994. Accuracy (trueness and precision) of measurement methods and results) of the proposed methodical approach. Every standard solution was injected two times.

**Table 1** Concentrations of analyzed volatile compounds are expressed in mg/L (AA), **1-pentanol** was introduces as internal standard
Compound	Concentration, mg/L (AA)							Relative error, % (P=0,95%)
Compound	VC-1	VC-2	VC-3	VCC-0	VCC-1	VCC-2	VCC-3	Relative error, % (P=0,95%)
acetaldehyde	111	11,2	1,13	4275	1096	56,2	2,22	± 3%
methyl acetate	114	11,5	1,17	4397	1128	57,8	2,29	± 3%
ethyl acetate	108	10,9	1,11	4173	1070	54,9	2,17	± 3%
methanol	1092	113,3	14,3	41995	10774	555,5	24,96	± 3%
2-propanol	105	12,1	2,70	3991	1025	54,1	3,69	± 3%
1-propanol	104	10,5	1,06	4012	1029	52,8	2,08	± 3%
isobutyl alcohol	103	10,4	1,05	3975	1020	52,3	2,06	± 3%
n-butanol	106	10,7	1,08	4071	1044	53,5	2,11	± 3%
isoamyl alcohol	106	10,7	1,08	4071	1044	53,5	2,11	± 3%
1-pentanol (IS)	27,13	27,13	27,13	27,13	27,13	27,13	27,13	± 3%

In all cases calibration coefficients were been generated by the considered three methods. At the first case 1-pentanol was used as IS. At the second case the ES method was used. And in the third case the ethanol as IS was used. Analytical characteristics of the obtained calibration coefficients are presented in Table 2.

**Table 2** Analytical characteristics of the calibration coefficients from INP
Compound	1-pentanol as IS		ES		Ethanol as IS		LOD* (mg/L)
Compound	Slope	Correlation c-nt, R²	Slope(mg/L)/(pA*min)	Correlation c-nt, R²	Slope	Correlation c-nt, R²	LOD* (mg/L)
acetaldehyde	2,396	0,9997	266,1	0,9997	1,710	0,9997	0,344
methyl acetate	2,491	0,9997	276,7	0,9996	1,779	0,9999	0,683
ethyl acetate	1,757	0,9997	195,1	0,9997	1,254	0,9999	0,322
methanol	2,133	0,9998	236,9	0,9997	1,523	0,9999	0,046
2-propanol	1,400	0,9998	155,5	0,9997	0,999	0,9999	0,119
ethanol	1,413	N/A	155,5	N/A	1	N/A	N/A
1-propanol	1,179	0,9997	130,9	0,9996	0,841	0,9999	0,222
isobutyl alcohol	1,018	0,9998	113,0	0,9997	0,727	0,9999	0,178
n-butanol	1,117	0,9999	124,1	0,9998	0,798	0,9999	0,189
isoamyl alcohol	1,030	0,9999	114,1	0,9998	0,735	0,9999	0,179
1-pentanol (IS)	1	N/A	110,1	N/A	0,708	N/A	0,271
*limit of detection (LOD)

The main obtained experimental results are presented below in the form of screenshots.

Fig.1 - Obtained calibration coefficients for the case "ethanol as ISTD".

Fig.2 - The final report of analysis of control sample VCC-1 (2 mg/L). 1-pentanol was used as IS.

Fig.3 - The final report of analysis of control sample VCC-1. Preview.

Fig.4 - The final report of analysis of control sample VCC-1. ES method was used.

Fig.5 - The final report of analysis of control sample VCC-1. ES method was used. Preview.

Fig.6 - The final report of analysis of control sample VCC-1. Ethanol was used as IS.

Fig.7 - The final report of analysis of control sample VCC-1. Ethanol was used as IS. Preview.

Fig.8 - All necessary calculations were performed directly in Unichrom. Ethanol was used as IS.

Fig.9 - Chromatogram of the control sample VC-1 (1000 mg/L).

Fig.10 - The final report of analysis of control sample VCC-1. 1-pentanol was used as IS.

Fig.11 - The final report of analysis of control sample VCC-1, ES method.

Fig.12 - The final report of analysis of control sample VCC-1. Ethanol was used as IS.

The main obtained experimental results are presented below in the form of screenshots.

Conclusion

Relative detector response factors RF for analyzed compounds relative to ethanol are sufficiently stable in wide regions of impurities concentrations. These relative factors can be tabulated for every certain type device. It follows that the proposed method "ethanol as IS" allows make measurements without often periodic calibration of gas chromatograph. Thousands of testing laboratories all over the world carry out gas chromatographic analysis of volatile compounds in spirit drinks day-and-night. They may test this approach in their real practice. It is important to note that there is no need to perform any additional measurements. This method could be tested while performing current measurements with existing instrumentation. Calculations could be done in parallel by three different methods: using traditional IS method [1–3], ES method [4–8] and using "ethanol as IS".

Literature cited

Commission Regulation (EC) No 2870/2000 of 19 December 2000 laying down Community reference methods for the analysis of spirits drinks, 2000.
AOAC Official Methods 972.10. Alcohol (higher) and ethyl acetate in distilled liquors. Alternative gas chromatographic method, 1975.
AOAC Official Methods 972.11. Methanol in distilled liquors. Gas chromatographic method, 1973.
GOST R 51698-2000. Vodka and ethanol. Gas-chromatographic express-method for determination of toxic microadmixtures content, 2000.
GOST R 51762-2002. Vodka and ethanol from food raw materials. Gas-chromatographic method for determination of carbonic acids and furfural content, 2002.
GOST R 52363-2005. Ethanol containing waste of food alcohol and liqueur-vodka production. Gas-chromatographic method for determination of volatile organic admixtures content, 2005.
GOST R 52788-2007. Ethanol and ethanol containing production. Gas-chromatographic method for determination of crotonaldehyde (denaturant additive), 2007.
GOST R 53419-2009. Raw ethanol from food raw materials. Gas-chromatographic method for determination of volatile organic admixtures, 2009.

There are the following prepared templates ReportVCADEIS-eng.uwb and ReportVCADEIS-eng.xls for illustration how this method is working.

Fig.13 - GC control panel. The carrier gas was hydrogen.

Fig.14 - Split ratio was 1 : 6,6.

Fig.15 - The sitting parameters of liquid autosampler (LAS.)

Fig.16 - Layout of the screen for visualization of the basic metrological parameters of measured data.

Fig.17 - The following layout presents all parameters of graduation curves. Ethanol as ISTD.

Fig.18 - Graduation curves for methyl acetate. Method of external standard.

Fig.19 - Graduation curves for methanol. Ethanol as ISTD.

Fig.20 - Graduation curves for methanol. The value of RRMS for methanol is 7,9812 and 9,4101 for ethanol, respectively, in the case of ES. The value of RRMS for methanol is 1,2776 in the case when we use ethanol as IS (see Fig. 5). There is essential reduction of RRMS more then six times from value 7,9812 to value 1,2776.

Fig.21 - Layout with presentation of scenario (macros) treatment of measurement data.

Fig.22 - Preview of final report form before printing.

Fig.23 - Construction of a final report form with the help of MS Excel.

Fig.24 - Screenshot of the report form generated with the help of Excel. ReportVCADEIS-eng.xls.

Fig.25 - Printed report generated by ReportVCADEIS-eng.xls.

Fig.26 - Analysis of the sample «А310». Control of accuracy was not been done and so function "Control of accuracy" was turned off. There are results of reproducibility only.

Fig.27 - Function "Control of accuracy" was turned on. There are results of reproducibility and accuracy.

Fig.28 - Preview of final report. Function "Control of accuracy" was turned on.

Fig.29 - Analysis of sample "РВ-2".

Fig.30 - Final report. Analysis of sample "РВ-2".

Fig.31 - Preview of final report with analysis of sample "РВ-2".

Head of Laboratory:

Charapitsa Siarhei	e-mail: chere@inp.bsu.by
Ph.D	Tel.: +(375 17) 212 17 26
	Mob.: +(375 29) 651 33 91

Laboratory of Analytical Research