Chromatography Forum

I wish you good luck in using ethanol as the internal standard, except for a very narrow set of analytical conditions and sample compositions you are going to need it.

Peter

With the aim of testing the method using Ethanol as an Internal Standard in quantification of volatile components in the alcohol-containing products on the Internet a simple calculator is sited http://www.inpnet.net/el/vcalc
Researcher may input only value of the areas under the peaks of the analyzed component and ethanol. Calculator immediately gives the concentration of the analyzed component in mg per liter of absolute alcohol (AA).

All suggestions and comments are welcome

There are several reasons why using the ethanol in a beverage as an internal standard is a bad idea.

In alcoholic beverages the ethanol is the most abundant or second most abundant component. Let's, for arguments sake take a middle of the range volume fraction of 30%. The minor components are at much lower concentrations, to keep the numbers simple lets say 30 mg/l, an approximate volume fraction of 30e-6. So the ethanol concentration is 10 000 times higher than the concentration of a minor component.

In a routine GC-FID setup a peak needs to have about 1 ng of compound in it to give a comfortable signal:noise ratio. You can go down to 0.1ng but adsorption is likely to become a problem. If you have 1 ng of minor component there will be 10 000 ng = 10 ug of ethanol.

On a non-polar column the ethanol peak will be hopelessly overloaded, on a polar column there will be so much ethanol that the retentions of other compounds will be shifted.

The lower LOD for an FID is around 0.01ng in a peak, its linear range is no more than six orders of magnitude (which is a hundred times better than any other GC detector) which means that the ethanol peak in our generic beverage is dangerously close to the top end of the detector's linear range.

If the ethanol in the beverage is to be used as an internal standard its volume fraction (or mass fraction, or mass/volume) has to be accurately known. How will it be determined ?

Peter

To demonstrate the reliability/robustness of the proposed method “Ethanol-as-IS” the standard ethanol-water (96:4) solution with initial volatile compounds concentration about 4000 mg/L (AA) was analyzed after dilution with water in the ratios 1:1, 1:9, 1:99, 1:1999 and 1:9999. Experimental results are presented in Table 2.







Chromatograms of standard solutions A-F from Table 2. 1 - acetaldehyde, 2 - methyl acetate, 3 - ethyl acetate, 4 - methanol, 5 - 2-propanol, 6 - ethanol, 7 - 1-propanol, 8 - isobutyl alcohol, 9 - n-butanol, 10 - isoamyl alcohol

Even after dilution with water in the ratio 1:999, the difference between the measured concentrations of all compounds and their values calculated using the gravimetric method does not exceed 7.8 %. With the dilution 1:9999 there are peaks of methanol and ethanol only. Other compounds significantly are less than level of detection. But the relative discrepancy of measured concentrations of methanol does not exceed 6.6%.

No bad !

Back again to the simple calculator is sited http://www.inpnet.net/el/vcalc
Researcher may input only value of the areas under the peaks of the analyzed component and ethanol. Calculator immediately gives the concentration of the analyzed component in mg per liter of absolute alcohol (AA).

The following data are unknown in calculation procedure:
- name(kind/type) of sample,
- injection probe value,
- split ratio,
- values of air and hydrogen flows for FID,
- name, technical characteristics and methodic parameters of used GC,
- volume concentration (v/v) of ethanol (strength) in sample,
.- name of Internal Standard (IS),
- quantity of injected IS.
Nevertheless you can rapidly calculate the quantities of analyzed volatile compounds with help the method “Ethanol-as-IS”.

In strong(strict) accordance with the international regulation documents for quality and safety control of alcohol production /International Organization of Vine and Wine (OIV). Compendium of international methods of wine and must analysis, 2009; Vols. 1 and 2; Commission Regulation (EC) No 2870/2000 of 19 December 2000 laying down Community reference methods for the analysis of spirits drinks, 2000/ which prescribe determination of the volatile compounds acetaldehyde, methyl acetate, ethyl acetate, methanol, 2-propanol, 1-propanol, isobutyl alcohol, n-butanol, isoamyl alcohol results of the analysis should be expressed in milligrams per litre (mg/L) of absolute alcohol (AA).

Best Regards,
Siarhei Charapitsa
(Сергей Черепица)

...
Nevertheless you can rapidly calculate the quantities of analyzed volatile compounds with help the method “Ethanol-as-IS”.

In strong(strict) accordance with the international regulation documents for quality and safety control of alcohol production /International Organization of Vine and Wine (OIV). Compendium of international methods of wine and must analysis, 2009; Vols. 1 and 2; Commission Regulation (EC) No 2870/2000 of 19 December 2000 laying down Community reference methods for the analysis of spirits drinks, 2000/ which prescribe determination of the volatile compounds acetaldehyde, methyl acetate, ethyl acetate, methanol, 2-propanol, 1-propanol, isobutyl alcohol, n-butanol, isoamyl alcohol results of the analysis should be expressed in milligrams per litre (mg/L) of absolute alcohol (AA).

Best Regards,
Siarhei Charapitsa
(Сергей Черепица)

Sergey, if you mention Regulation 2870/2000 and accordance with it, it must be stressed out that the only acceptable internal standards are pentan-3-ol (CAS 584-02-1), pentan-1-ol (CAS 71-41-0), 4-methylpentan-1-ol (CAS 626-89-1) or methyl nonanoate (CAS 1731-84-6).

The strenght of spirits (for expressing results per 1 hL of absolute ethanol) may be measured by 3 different methods, but none of them is GC.

Let's carefully read the Basic Regulation Documents:
1. International Organization of Vine and Wine (OIV). Compendium of international methods of wine and must analysis, 2009; Vols. 1 and 2;
2. Commission Regulation (EC) No 2870/2000 of 19 December 2000.

In the chapter
III.2. GAS CHROMATOGRAPHIC DETERMINATION OF VOLATILE CONGENERS: ALDEHYDES, HIGHER ALCOHOLS, ETHYL ACETATE AND METHANOL
in the paragraзр
11. Method performance characteristics (precision)
Statistical results of the interlaboratory test: the following tables give
the values for the following compounds: ethanal, ethyl acetate, acetal,
total ethanal, methanol, butan-2-ol, propan-1-ol, butan-1-ol, 2-methylpropan-
1-ol, 2 methyl-butan-1-ol, 3 methyl-butan-1-ol.
The following data were obtained from an international method
performance study carried out to internationally agreed procedures.
Sample types
A Brandy; blind duplicates.
B Kirsch; blind duplicates.
C Grappa; blind duplicates.
D Whisky; split levels (*).
E Rum;
In accordance to above mentions OIV and EC regulation documents the final results should be presented in milligrams per litre (mg/L) of absolute alcohol (AA).
But in all tables experimental results from interlaboratory test are presented in micrograms per grams.

Where did such frivolity ?

I think that participants could not measure the volume content of ethanol in samples by traditional methods. Probably, the volume of samples was insufficient for performing measurements of alcohol strength.

The proposed method "Ethanol-as-IS" avoids this problem.

Thousands of analytical and testing laboratories all over the world carry out gas chromatographic analysis of volatile compounds in spirit drinks. Researchers may validate this new method in actual practice, making sure its simplicity, accessibility and effectiveness in everyday practice. The obtained results show the possibility of developing a new international standard of measurement procedure, which will allow increase the data accuracy and will considerably simplify the measurement procedure.

...
The proposed method "Ethanol-as-IS" avoids this problem.
...

Sergey,

before any further divagation would you please remind us the definition of internal standard ?

I mean that IS is a subtance not present in analysed samples.

There is one correct decision only:
Ethanol should be IS in determination of volatile compounds in spirit products.

What we have today?
In accordance to the above mentions OIV and EC regulation documents the final results should be presented in milligrams per litre (mg/L) of absolute alcohol (AA).
To obtain the most reliable data to use method of IS, no doubt.
As the IS we should use the component whose concentration is known to be maximum accurate.
Concentration of ethanol in spirit product is equal to 789300 milligrams per litre (mg/L) of absolute alcohol (AA).
Why should we abandon this fact of nature?

Theoretical background was presented, for example, here http://pubs.acs.org/doi/abs/10.1021/jf3044956

Best regards,
Siarhei

There is one correct decision only:
Ethanol should be IS in determination of volatile compounds in spirit products.

What we have today?
In accordance to the above mentions OIV and EC regulation documents the final results should be presented in milligrams per litre (mg/L) of absolute alcohol (AA).
To obtain the most reliable data to use method of IS, no doubt.
As the IS we should use the component whose concentration is known to be maximum accurate.
Concentration of ethanol in spirit product is equal to 789300 milligrams per litre (mg/L) of absolute alcohol (AA).
Why should we abandon this fact of nature?

Theoretical background was presented, for example, here http://pubs.acs.org/doi/abs/10.1021/jf3044956

Best regards,
Siarhei

The alcohol content is for straight run raw spirit: 200 degrees proof, 95 % v/v, which is not what most people drink. Depending where you are in the world "spirits" are about 30 - 40 % ethanol v/v. Liqueurs, fortified wines and beers have progressively less ethanol.

Anyway, if ethanol as an internal standard really does work to calculate contaminant content as a fraction of ethanol content, the ethanol content makes no difference.

Peter

Let's carefully read the Basic Regulation Documents:
1. International Organization of Vine and Wine (OIV). Compendium of international methods of wine and must analysis, 2009; Vols. 1 and 2;
2. Commission Regulation (EC) No 2870/2000 of 19 December 2000.
...
In accordance to above mentions OIV and EC regulation documents the final results should be presented in milligrams per litre (mg/L) of absolute alcohol (AA).

But you were not enough dilligent Sergey
According to Regulation No 2870/2000 results should be presented in grams per 100 L of absolute alcohol (equals to milligrams per 100 mL of absolute alcohol). Surely not a problem to convert.

I don't mention OIV documents because they are not the source of law for official control of spirits drinks in EU countries.

With the aim of testing the method using Ethanol as an Internal Standard in quantification of volatile components in the alcohol-containing products on the Internet a simple calculator is sited http://www.inpnet.net/el/vcalc
Researcher may input only value of the areas under the peaks of the analyzed component and ethanol. Calculator immediately gives the concentration of the analyzed component in mg per liter of absolute alcohol (AA).

All suggestions and comments are welcome

O.K. - time for your calculator.
I inputed real peak areas gained when analysing Whisky congeners CRM LGC5100.
http://imgur.com/JKXZ18d
The output of methanol concentration calculated by your tool is 6.68 mg/L AA. And certified value is 5.20 mg/100 mL AA - very huge difference.

Just for testing purposes I have changed the area of ethanol peak by multiplying it by 1000.
It has no impact on methanol concentration.
Seems that this calculator doesn't work properly.

O.K. - time for your calculator.
I inputed real peak areas gained when analysing Whisky congeners CRM LGC5100.
http://imgur.com/JKXZ18d
The output of methanol concentration calculated by your tool is 6.68 mg/L AA. And certified value is 5.20 mg/100 mL AA - very huge difference.

Dear dblux_ ,
Ok,
please, send me (SVCharapitsa@tut.by) or put in the public domain measured chromatogram of your CRM LGS5100, for example, in AIA Chromatography format (*.cdf).

Best Regards,
Siarhei

Sergey, after modifications you made your calculator is working correctly with one exception.
Values in the last column (g/100L) are 100 times higher than should be.
Ethanol should be 78930.00 not 7893000.00 and methanol 5.34313 not 534.313 g/100 L AA.
http://imgur.com/64ktNQP
Not tested other congeners.

Ok,
in accordance with dblux_'s wishes now calculator presents final results in g per 100 L (AA) http://www.inp.bsu.by/english/calculator_en.htm .
The test version http://www.inpnet.net/el/vcalc will be corrected on monday.
Nevertheless we till are waiting for promised file of measured chromatogram of your CRM LGS5100.
No matter in what format: AIA Chromatography format (*.cdf) or HP Chemstation Data file (*.ch).

Best Regards,
Siarhei

Hi,

I have received the measured chromatograms of “Whisky - Congeners CRM LGC 5100” and scans of real Certificate LGC5100, too.
Thanks to Ann and to Bob.


Figure 1. Scan of Certificate LGC5100, the 1st page.


Figure 2. Scan of Certificate LGC5100, the 2nd page.

Final results are presented in the following Figure 3-5.


Figure 3.


Figure 4.


Figure 5.

For simplicity and clarity in the right of the column "C, g/100 L (AA)" inscribed value from Certificate LGC5100.
Fragment of chromatogram with discussed methanol is presented in Figure 3. Separation between peaks of ethyl acetate and methanol wants to be better. But, we work with what we have.

In the case of methanol calculator gives values 5.8 (Ann) and 4.1 (Bob).
According to Certificate LGC5100 concentration of methanol is 8.2.

In the case “2+3-methyl butanol” calculator gives values 73.2 (Ann) and 66.9 (Bob).
According to Certificate LGC5100 concentration of “2+3-methyl butanol” is 70.1

Recently performed experiments from three different laboratories are presented in this publication http://arxiv.org/ftp/arxiv/papers/1406/1406.6358.pdf

I believe that the accumulation of experimental data from the intensive laboratories will be made appropriate adjustments coefficients RRF.

All remarks and comments are welcome.

Best Regards,
Siarhei

Unless there is something odd about the vertical scale that ethanol peak is the classic shape for column overload and detector saturation - therefore its area should not be used as a standard for other components.