N.Z.

Khumalo
213523727
Experiment 8: Gas Chromatography
Use of Internal Standards for quantification of ethanol in beer

Aims
To investigate the use of an internal standard as an aid to analysis. Analysis of the beer sample concerns the
determination of the ethanol weight percentage.

Introduction and Principle

Chromatography is an important laboratory tool which is used for the separation and identification of
compounds (M. Shozi, 2015). There are different types of chromatography including thin layer chromatography
(TLC), liquid chromatography (LC), high performance liquid chromatography (HPLC), ion chromatography
(IC) and gas chromatography (GC). All of these laboratory tools apply the same principle in general. In this
experiment, GC was used for sample analysis and its principle was as follows:

The components of the sample, beer, separated between two phases; the stationary phase and the mobile gas
phase (CHEM340 Practical manual, 2018). During the gas chromatographic separation, the sample was
vaporised and carried by the mobile gas phase through the column. The separation of different components was
achieved based on their relative vapour pressure and affinities for the stationary phase (A.G. Piantanida and
A.R. Barron, 2014). According to A.G. Piantanida and A.R. Barron, they suggested that the affinity of a
substance towards the stationary phase can be described as an equilibrium constant termed the partition
coefficient, which control the migration of different compounds through the column. Therefore differences in
the partition coefficient allow for the chromatographic separation. The equilibrium constant is temperature
dependent and also depends on the chemical nature of the stationary phase. Thus, temperature can be used as
one of the ways to improve the separation of compounds through the column, or a different stationary phase
(A.G. Piantanida and A.R. Barron, 2014).

In this experiment, GC was used for the analysis of beer (Black Label and Castle Larger). Beer consist of
ethanol in its mixture, an alcohol that has short and long-term effects on an individual consuming it. For this
experiment, it was necessary to analyse beer in order to see how much ethanol was there in each sample
provided and to establish the quality of beer. This was achieved by using n-propanol as an internal standard
(IS), which was added in constant amount (10 ml) to samples (F. Nevondo, 2018). The principle of IS was that,
IS must be a compound that is not found in the samples to be analysed. The use of IS for the preparation of
stock solution and sample solution is significant to find the analyte concentration from the calibration curve
generated by plotting concentration against peak area.

Gas chromatography was suitable for this experiment, because it has the ability to analyse the samples both
qualitatively and quantitatively. The qualitative analysis by GC depend on matching the retention times of
unknown and guessed compounds, whereas the quantitative analysis depend on the measurement of the peak –
either its heights or its area (CHEM340 Practical Manual, 2018).
Abstract

Gas chromatography was a laboratory tool used for the analysis of beer using the calibration method called
internal standard. The standard calibration curve had the R2 value of 0.9991. Further calculations from the
experimental data (peak areas) revealed that %volume of ethanol in beer was 13% but the true value was 10%.
This resulted in the experimental error being equivalent to 30%. Related standard deviation percentage for beer
was 15% and 26.7% for Black label and Castle larger, respectively.

Experimental

Using a top-weighing balance, the masses of stock solutions (2.5 g n-propanol and 1.00 g ethanol) were
measured into the vial. Each of the stock solution was transferred into their respective 1 litre volumetric flask
and diluted with distilled water up to the mark. The n-propanol stock solution was used as an IS. Four 100 ml
volumetric flasks (labelled A – D) were used to prepare standard solutions, in which all of them had the same
amount of n-propanol (10 ml) and different amount of ethanol.

Table 1: Amount of n-propanol and ethanol in each flask

Flask A B C D
Final concentration 50 ppm 100 ppm 200 ppm 400 ppm
n-propanol standard 10 ml 10 ml 10 ml 10 ml
Ethanol standard 5 ml 10 ml 20 ml 40 ml

The modification of the experiment was that 10 µl instead of 5 µl portion of standards, were injected three
times in each flask onto the GC column in order to determine the area of each peak on the chromatograms and
plot the standard curve of concentration against ratio of peak area.

For sample analysis, 50 ml of each sample (Black Label and Castle Larger) was diluted to 1 liter volumetric
flask to make a stock solution. From the stock solution, 10 ml was drawn and mixed with 10 ml of the n-
propanol standard into 100 ml volumetric flask. This was diluted to the mark with distilled water. These were
injected in the same way as the standards, but were injected five times in each flask onto the GC column in
order to determine the area of each peak on the chromatograms. The obtained experimental data was used to
determine the peak area ethanol/ peak area of n-propanol for each chromatogram as well as determining the
weight percentage ethanol.

Results / Calculations and Discussion

Results for prepared standard solutions

a) b) Sample A (injection 2).

Sample A (injection 1).
c) Sample A (injection 3). d) Sample B (injection 1).

e) Sample B (injection 2) f) Sample B (injection 3)

g) Sample C (injection 1) h) Sample C (injection 2)

 i) Sample C (injection 3) j) Sample D (injection 1)

k) Sample D (injection 2) l) Sample D (injection 3)

Figure 1: The gas chromatograms of n-propanol and ethanol standards are represented on the plot of peak area
against retention time (in minutes), each injected 3 times.

Calculations

Flask A
Average peak area = (18.2 + 15.9 + 12.4) / 3 = 15.5

Flask B
Average peak area = (33.8 +36.2 +37.5) / 3 = 35.8

Flask C
Average peak area = (67 +75.8 +66.6) / 3 = 69.8
Flask D
Average peak area = (147.8 + 188.5 +111.6) /3 = 149
Table 1: The average peak areas observed from chromatograms of sample A - D

Flask Concentration Peak Peak Peak Average

(ppm) area 1 area 2 area 3 peak
(pA*s) (pA*s) (pA*s) area
(pA*s)

A 50 18,2 15,9 12,4 15,5

B 100 33,8 36,2 37,5 35,8
C 200 67 75,8 66,6 69,8
D 400 147,8 188,5 111,6 149,3

160
y = 0.3803x - 3.7087
140 R² = 0.9991
AVERAGE PEAK AREA [PA*S]

120

100

80

60

40

20

0
0 50 100 150 200 250 300 350 400 450
CONCENTRATION (PPM)

Figure 2: The standard calibration curve of average peak area against concentration

Discussion for standard solutions

From the above chromatograms there are two peaks representing the ethanol and n-propanol components of the
mixture. Each of them had different values of peak area (or height) due to the constituent of the mixture from
each flask. Since each standard solution was injected three times onto the GC column, the mean peak area was
calculated for each mixture in order to draw a standard calibration curve.

The calibration curve obtained was an expected linear plot of peak area versus concentration. The R2 value
being equal to 0.9991 was an indication that the calibration curve was a good linear plot with high sensitivity.
This also suggested that the values on the x and the y-axis related very well to each other. Thus, the results of
analyte concentration determined from this curve was reliable.
Results for analysis of the sample beer

a) Black Label (injection 1) b) Black label (injection 2)

C) Black label (injection 3) d) Black label (injection 4)

e) Black label (injection 5) f) Castle larger (injection 1)

g) Castle larger (injection 2) h) Castle lager (injection 3)

i) Castle larger (injection 4) j) Castle larger (injection 5)

Figure 3: The gas chromatograms of beer with n-propanol internal standard.

Table 2: The retention time and peak area ethanol/peak area of n-propanol for each chromatogram

Injection Sample name Retention time (min) Peak area [pA*s]

1 Black label 2.527 41.81
2 Black label 2.494 28.05
3 Black label 2.530 40.80
4 Black label 2.525 40.37
5 Black label 2.531 39.00
1 Castle larger 2.528 43.75
2 Castle larger 2.533 32.13
3 Castle larger 2.525 45.69
4 Castle larger 2.505 34.31
5 Castle larger 2.500 22.15
 Calculations

Black Label
Average peak area = (41.81 + 28.05 + 40.80 +40.37 + 39.00) / 5 = 38.0
Standard deviation = 5.7

Relative standard deviation = (5.7 / 38) x 100 = 15%

Using calibration curve straight line equation and average peak area from black label, sample concentration of
analyte could be found.
Y = 0.3803x – 3.7087
38.0 = 0.3803x – 3.7087
0.3803x = 41.7087
X = 109.7
Therefore, concentration of analyte = 110 ppm
Response factor = (area of analyte / area of IS) ÷ (concentration of analyte / concentration of IS)
= (38.0 / 35.8) ÷ (110 ppm / 100 ppm)
= 0.96

Castle larger
Average peak area = (43.75 + 32.13 + 45.69 + 34.31 + 22.15) / 5 = 35.6
Standard deviation = 9.5

Relative standard deviation = (9.5 / 35.6) x 100 = 26 7%

Using calibration curve straight line equation and average peak area from castle larger, sample concentration of
analyte could be found.
Y = 0.3803x – 3.7087
35.6 = 0.3803x – 3.7087
0.3803x = 39.3087
X = 103.4 ppm
Therefore, concentration of analyte = 103 ppm

Response factor = (area of analyte / area of IS) ÷ (concentration of analyte / concentration of IS)
= (35.6 / 35.8) ÷ (103 ppm / 100 ppm)
= 0.97

% volume = (volume of solute) ÷ (volume of solvent) x 100

= (10 ml ÷ 80 ml) x 100%
= 13%
% v of ethanol in beer is 10%

Experimental error = [(experimental data - true value) ÷ true value] x 100

= [(13% - 10%) ÷ 10%] x 100
= 30%

Discussion for sample analysis results

The two peaks on the above chromatograms represent two compounds, ethanol and n-propanol. Retention times
observed for ethanol were all 2.5 minutes and those of n- propanol were 2.7 minutes. The role of retention times
was to provide the qualitative aspect of the chromatogram for the two beer samples (T.A. Berger, 1996). For
each of the five sample injections (10 µl), the retention time of a compound was observed to be the same
because the samples were analysed under the same chromatographic conditions.

For quantitative analysis, the chromatographic peak area was related to the quantity of analyte. For
determination of the actual amount of the compound, the average peak areas of the samples were compared
against standards of known concentration. This is where the straight line equation of the calibration curve, Y =
0.3803x – 3.7087, was used to determine the amount of ethanol in each beer sample. The amount of ethanol in
Black label was found to be 110 ppm and 103 ppm in Castle larger.
The analysis of the samples was further studied by making basic stats calculations so that it could be concluded
if the use of internal standard method was suitable for this experiment or not. According to these calculations,
the sensitivity is very low because high values of concentrations were obtained and the slope of the calibration
curve was small (0.3803).

Conclusion
Based on the calculations of basic stats, internal standard was not suitable for this experiment. The reason for
this was that the relative standard deviation percentage (%RSD) obtained for beer was very large (%RSD >
1%), 15% and 26.7% for Black Label and Castle Larger. This suggested that the precision of the analysis was
poor, which resulted in an experimental error being 30%.

The aim of the experiment was achieved but the obtained results were not expected, hence, they were not
satisfying. The percentage volume of ethanol in beer was expected to be 10% but from the calculations using
experimental data, it was found to be 13%. This could be due to errors encountered during the experiment.
Internal standard method can compensate for several types of both random and systematic errors. Random
errors are difficult to eliminate whereas systematic errors can be avoided since they are instrumental/method or
personal errors. One can prevent them from occurring when performing a similar experiment as this one by
appropriately adhering to the experimental procedure.

References

 A.G. Piantanida and A.R. Barron (2014). Principles of Gas Chromatography. OpenStax Journals.
Volume 1 (2), pp 1-12.
 CHEM340 Practical Manual (2018). UKZN WC, Gas Chromatography, pp 37 - 41.
 F. Nevondo (2018). Introduction to Instrumental Analysis. UKZN WC Lecture Notes, page 1-42.
 M.Shozi (2015). Chromatography. UKZN WC Lecture Notes, pp 1-66.
 T.A. Berger (1996). Separation of Gasoline on an open tubular column with 1.3m effective plates.
SpringerLink Journals. Volume 42 (1-2), pp 63-71.