Title:

Gas Chromatography - Determination of ethanol in water

Objective:

To determine the concentration of ethanol in the unknown sample

Introduction:

Gas chromatography is a widely used technique in analytic chemistry to analyse volatile

substances. Because separation of compound mixtures on the column occurs while they are in
gaseous state, solid and liquid samples must first be vaporized. Its use is limited to the study of
thermostable and sufficiently volatile component.

A gas chromatograph consists of several components within a frame including injector, column
and the detector, associated with a thermostatically controlled that allow the column to attain
high temperatures. For a gas chromatograph, mobile phase and stationary phase are required. The
mobile phase which is a carrier gas transports the analytes through the column. The carrier gases
are usually helium, hydrogen or nitrogen. The carrier gas must be chemically inert and free of
oxygen, all traces of hydrocarbon and water vapour. Therefore, there is a molecular sieve inside
the carrier gas system to remove water and other impurities.

The column is usually a narrow-bore tube which coils around itself with a length that can vary
from 1 to over 100m, depending upon the type and the contents of the stationary phase. There are
two types of column, packed columns and capillary columns. For packed column, the stationary
phase is deposited or bonded by chemical reaction onto a porous support. For capillary columns
a thin layer of stationary phase is deposited onto or bound to the inner surface of the column.

The injection in gas chromatograph is done by microsyringe to inject very small quantity of
sample in the solution. The thermostatted oven is used to control the temperature of the column.
The excessively high temperature of column may have poor separation even though with a short
retention time.

After sample is injected, the sample is vapourised and carried by the carrier gas through the
column. Different components have different interactions with stationary phase. The species
present can be qualitatively identified based on the delay in the sample passing through the
column and it can be presented in a form of chart after the components passing through the
detector.

Reagents and Apparatus:

Reagents Apparatus
1 % standard solution of ethanol Gas chromatograph (GC)
3 % standard solution of ethanol GC injection syringe
5 % standard solution of ethanol
Unknown sample solution

Procedure:

Instrument Set-up

1) The He carrier gas should be on prior to heating the column. The He gas was allowed to flow
through the column for approximately 15 min before turning on the oven.

2) The flow rate of the carrier gas was adjusted to approximately 40cm3/min.

3) After the 15 minute column conditioning period, the injector and detector temperatures were
set to 250° C and the column temperature was set at 60° C.

4) The voltage output from the detector was verified that was connected to the Serial Box
Interface using the Instrumentation Amplifier. The conversion of the analog signal at the detector
to a digital signal was allowed by these components, capable of being stored on the computer.
For basic instruction on using Logger Pro, consult "Data Collection for GC using Logger Pro".

Determination of retention time

1) 1.0 μL of ethanol solution was injected precisely into column and record the retention time.

Determination of ethanol

1) Sample solution for analysis (unknown and ethanol) was prepared similarly as the calibration
solutions.

2) 1.0 μL of each of the calibration solutions was injected precisely, in triplicate, and the
Chromatograms were recorded.

3) Step 2 was repeated for the Unknown sample solution.

Result:

Concentration (%) Retention Time (min) Peak Area

1 3.162 3738.0635
3 3.357 8258.8076
5 3.463 16051.9355
Sample 3.423 15998.3477

Graph of Concentration Vs Peak Area

18000
16051.94
16000
f(x) = 3078.47 x + 114.2
14000 R² = 0.98

12000
Peak Area

10000
8258.81
8000

6000
3738.06
4000

2000

0
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5

Concentration

Calculation of concentration of NaOH in unknown sample

y = 3078.5x + 114.2

y−114.2
x=
3078.5
15998.3477−114.2
x=
3078.5
 ≈ 5.16 (concentration of NaOH in unknown sample)

Percentage error of concentration of NaOH in unknown sample, actual concentration in

unknown sample is 4%

|4−5.16|
Percentage error = × 100 %
4
= 29.00%

Sample Calculation of Percentage error between Group 29 & Group 28

By using concentration of 1%
3738.0635 – 2850.7231
Percentage error = × 100%
3738.0635
= 23.74 %

Concentratio Retention Time Retention Time Peak Area Peak Area Percentage
n (%) (Group 29) (Group 28) (Group 29) (Group 28) error (%)
(min) (min)
1 3.162 3.340 3738.0635 2850.7231 23.74
3 3.357 3.405 8258.8076 6840.6484 17.17
5 3.463 3.444 16051.9355 13825.5703 13.87
Sample 3.423 3.464 15998.3477 15971.5225 0.17

Discussion

Comparing the results of 2 groups, it can be seen that the peak area deviates significantly for
known concentration of 1%, 3% and 5% ethanol solution. The percentage error for 1%, 3% and
5% ethanol solution are 23.74%, 17.17% and 13.87% respectively. However, for sample
solution, the percentage error is 0.17% only. Errors must be made when the experiment is
conducted since both the groups are using same solutions and tested using same machine but
different values with high percentage errors are obtained. However, through calibration curve, it
is found that the percentage error of the sample concentration of ethanol solution is 29%.

There are several reasons to the cause of the difference in values. Firstly, the error
occurred when 1 microliter of solution is drawn up using the microliter syringe. It is hard to read
the value by looking at the white spot in at the end of the syringe. Hence, it will cause inaccuracy
and a precise reading of the volume of solution cannot be obtained. Besides, there might be
bubbles trapped in the syringe. The bubbles will greatly affect the results obtained as the bubbles
occupied space and thus, the volume of solution will be lesser than expected. Lastly, the error
might due to the injection of solution. The needle of the syringe is not fully put into the gas
chromatography or the injection of solution into gas chromatography was not fast enough.

Some precaution steps can be done before conducting the experiment in order to improve
the accuracy of results. The bubbles in the microliter syringe must be removed as it will affect
the reading in the column. Next, the needle of the syringe must be cleaned every time before the
injection of solution to ensure that there is no dust deposit on the surface on the needle. The
introduction of solution into gas chromatography must be fast enough to avoid zone broadening.

There are a few improvements that can be considered to obtain a more consistent and
perfect results. The solutions should be prepared freshly before conducting the experiment as
degradation and precipitation will occur is the solutions have not been kept properly.
Additionally, more microliter syringe should be provided. Using the same microliter syringe in
drawing up different solutions will cause contamination in other solutions. To avoid this
situation, the microliter syringe can also be rinsed with distilled water and the solution of each
samples before drawing up the solution.

Internal standard are often used in gas chromatography. This method of internal standards
is used to improve the precision of quantitative analysis. An internal standard is a known
concentration of a substance that is present in every sample that is analyzed. Internal standard
calibration used in this experiment involves the comparison of the instrument responses from the
target compounds in the sample to the responses of reference standard added to the sample or
sample extract before injection. They can also be used to correct for variability due to analytic
loss in sample storage and treatment. (Tutorial: Internal Standards, n.d.)

The terminology of internal standard is very simple. A constant amount of internal

standard is added to all samples or extracts. That same internal standards is also included in each
of the calibration standards. Instead of keeping track of absolute peak area or height for
calibration purposes, you use the ratio of the area or height of the analytic and internal standard.
For example, consider the blue chromatogram inset in the figure 1.1. The normal sample is
shown as the peaks comprising lines. To this sample, a known amount of another compound
(represented by the solid peak) is added as an internal standard. The same process is used to
prepare the calibration samples: calibrators at different concentrations each have the same
amount of internal standard added to them. When the chromatogram is integrated, the peak areas
of the analytic and the internal standard are measured. The ratio of these areas is then used in the
calibration process. (Separation Science HPLC Solutions., n.d)

Figure 1.1 Peak Area Ratio vs. Concentration ratio

The ratio is termed the response factor (RF) or relative response factor (RRF),
indicating that the target compound response is calculated relative to that of the internal standard.

Response Factor Equation

RF = (( A x ¿(Cis ) ¿ ×(( Ais )(C x ))

 Where

A x = Area of the compound

C x = Concentration of the compound

Ais = Area of the internal standard

C is = Concentration of the internal standard

Internal standards selected that are similar in analytical behavior to the compound
of interest and expected to be found in the samples. The analyst needs to demonstrate that the
measurements of the internal standard is not affected by target analysts, surrogates or matrix
interference. However, this is not as useful as GC and HPLC method with non – MS detectors,
unless the internal standards could be separated from target compounds chromatographically.

The internal standard is choose because it accounts for routine variation in the response
of the chromatographic system. Besides that, account for the variations in the exact volume of
sample or sample extract introduced into the chromatographic system. The next benefit is the
retention times of the target compound and the internal standard may be used to calculate the
relative retention time (RRT) of the target compound and can be used to compensate for small
retention time shifts. Although there are so many benefit of internal standard, the main
advantages of internal standard calibration is that the internal standards must be compounds that
are not fond in the samples to be analyzed and they must produce an unambiguous response on
the chromatographic detector system. (Internal Standard Calibration, n.d.). 

An internal standard is usually used when performing mass spectrometer quantitation. An

appropraiate internal standard is vital in controlling for extraction, HPLC injection and ionization
variability. In a comples matrix, it is common for two different standard levels to give identical
response. Two points can only be differentiated when an internal standard is used. ("The Internal
Standard", 2016)

It is very important to choose the appropriate internal standards in order to improve accuracy
and precision of the method. The proper internal standard should be chemically similar to the
analyte, but it should not be presented in the sample during the experiment. It is best to choose
the compounds which have the same functional groups, boiling points and activity as the target
compounds. The best internal standard is an isotopically labeled version of the analyte.
Validation of the internal standard is needed to confirm it does correct for small amounts of
variation within the analytical process. (Kelly, 2014)

Conclusion

The concentration of ethanol in the unknown sample is determined as 5.16% from the
experiment. The concentration is different from the concentration determined by the other group
with a percentage error of 0.17% when the results is compared to the other group. The slight
different may be due to the injection of the sample into GC by different people.

References

Kelly, K. (2014). Choosing an Internal Standard. Retrieved from https://www.

Phenomenex.com/Info/Page/internalstandard

Separation Science HPLC Solutions. (n.d). Internal Standards #1: How Does It Work? Retrieved

from http://blog.sepscience.com/separationscience/hplc-solutions-134-internal-standards-
part-1-how-does-it-work
The Internal Standard. (2016). Retrieved from http://www.ionsource.com/tutorial/msquan/is.htm

Internal Standard Calibration. (n.d.). Internal Standard Calibration. Retrieved from

https://www.azdhs.gov/documents/preparedness/state-laboratory/lab-licensure-
certification/technical-resources/calibration-training/04-internal-standard-calib.pdf

Tutorial: Internal Standards (n.d.). Tutorial: Internal Standards. Retrieved from

https://facultystaff.richmond.edu/~cstevens/301/is_general.html