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Experiment 6 Gas Chromatography

Margaret Schnell maschnell 00784786

Partner: Sanja Tresnjic

March 22-24, 2016 April 7, 2016

Analytical Chemistry CHEM:3430:0A01 Prof. Scott Shaw

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Introduction:

Gas chromatography is an analytical separation technique that analyzes compounds

eluted from a column, which carried through the column by a gas mobile phase. The carrier gas

in our experiment was helium. We used helium gas because it has a thermal conductivity that is

higher than any of the analytes we used. A detector is located at the end of the column in gas

chromatography to measure the solutes that are eluted from it. The detector used in our

experiment was a thermal conductivity detector (TCD). TCDs work by passing the eluted analyte

over a heated filament. The thermal conductivity of the gas stream decreases as the temperature

of the filament increases. These changes also change the resistance of the filament; this change

causes a change in the voltage, which is the measured response for this detector. This signal is

proportional to the sample concentrations.

To obtain an ideal chromatogram, there are many parameters that need to be adjusted.

Ideal parameters give chromatograms with the high resolutions in the short amounts of time.

There are many parameters that can affect both the elution time and the resolution, but for this

experiment we focused on the effects of temperature and pressure. In gas chromatography, an

increase in temperature is only favored up to a certain point. The increased temperature

decreases the time of elution for all of the analytes, but after a certain point, it causes a decrease

in the resolution. Pressure also needs to be controlled in order to obtain a favorable

chromatogram. Higher pressures cause backflow which causes elution time to increase. Since the

temperature and pressure are dependent on each other, and it would take too long to figure out

mathematically, these parameters were optimized in our experiment with the Simplex

Optimization software. The Simplex Optimization software can only be used if the

parameters being optimized are dependent on each other. This software used data from test runs

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to calculate what it thought to be a good set of parameters. The results from those parameters

were then reentered into the system to make a better measurement. In our experiment, we

performed as many trials as possible in the time allotted. In the end, we used the parameters that

gave a chromatogram that looked to have the highest resolution in the shortest amount of time.

Experimental Methods:

The CRF values were only recorded for the first three parameters plugged into the

Simplex Optimization. We did this because were not given CRF values for the optimized trials.

We choose the parameters based on looking at the chromatograms from the GC, and not on the

CRF values obtained. Manipulations and calculations were also made to the refractive indices

obtained experimentally. We did this because we needed to correct for the temperature difference

of 23 °C (experimental) and 20 °C (literature values).

The average peak area of ethanol and the internal standard, 1-propanol, were used to

obtain the ratio of ethanol peak area to 1-propanol peak area for each ethanol (v/v) %. A

regression line was included in the calibration graph to show that the results were linear and

reliable to help determine the ethanol (v/v) % in our vodka sample.

Data and Results:

The first part of this experiment was done to determine the optimal conditions for the GC

to run the samples at. These

conditions were determined using

Simplex Optimization. The first

three sets of conditions were set to 30

°C with 30 psi, 50 °C with 20 psi,

and 80 °C with 10 psi. The results from these runs and their respective CRF values are displayed

 Table 1 This table shows the results from the Simplex Optimization of the original three sets of parameters. These were used to give the optimization program sets of data to start choosing the values that would give the optimal results.

Original Parameters

Trial Temperature

(°C) Pressure

(psi) CRF 1 30 30 1.1273 2 50 20 0.8344 3 80 10 0.9747

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in Table 1. From the table, we can see that the pair in the mid-range had the lowest CRF value.

The points that the Simplex Optimization suggested were closest to the mid-range values for

both the temperature and

pressure. The final set of

parameters we decided to

run the samples at was 64

°C and 15.6 psi. The

entire set of parameters

and their times to elute

are recorded in Appendix

B, Calculation 6. Figure 1

is a graphical

representation of the

progression for the measurements made at the different parameters that the Simplex

Optimization program suggested. The points closer to the center of the graph belong to better

sets of parameters. Trials 4-9 are all optimization parameters chosen by the Simplex program.

Trial 5 was chosen as the set of parameters with the best resolution in the shortest amount of

time.  

After we chose our optimal set of parameters, we

ran known standard solutions of pure ethanol, 1-propanol,

and water in the GC. The results obtained are tabulated in

Table 2. These were run to be sure we knew which peaks Table 2 The table shows times at which the three standards of the compounds we are testing are eluted. These are controls so we know which peak corresponds to which compound in our mixture samples.

Known Standards

Standard Retention Time

(min) Pure EtOH 1.658 1-propanol 2.248

Water 2.536

10

14

18

22

26

30

30   40   50   60   70   80  

Pres

sure

(psi

)

Temperature  (Celsius)  

Simplex Optimization Trials Trial  1  

Trial  2  

Trial  3  

Trial  4  

Trial  5  

Trial  6  

Trial  7  

Trial  8  

Trial  9  

Figure 1 This graph follows original parameters run then the parameters chosen by the Simplex Optimization program. The points closer to the center of the graph are the better parameter settings. The parameters shown by the marker for Trial 5 was selected as the best, and the parameters were used for the remainder of the solutions to be tested.

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in the chromatograms corresponded to which compounds in the mixture. It is important to note

that these are just estimates and the actual retention times vary with each solution and each

individual run.

Next, we ran the four mixtures of water, ethanol, and 1-propanol and the vodka sample in

a refractometer. This was done to be sure we obtained the exact ethanol (v/v) % of the solutions.

The data was collected and manipulated, and the results are recorded in Table 3. The refractive

indices were corrected from 23 °C to 20 °C. The corrected ethanol (v/v) % allowed us to make a

more precise and accurate calibration curve for the known samples.

After the refractive indices were

measured, the samples were ready to be run

at the optimal parameters in the GC.

Mixtures of 0.6 uL of each ethanol (v/v) %

standard with 0.6 uL of the 40% (v/v) 1-

propanol standard were tested to make a

calibration curve. The curve obtained is

Sample Refractive

Index Measured

Refractive Index

Corrected

Mass (g)/g Solution Density Density

Pure EtOH

Actual EtOH (v/v)%

20% (v/v) EtOH 1.3428 1.3440 0.150 0.977 0.7893 18.6%

30% (v/v) EtOH 1.3481 1.3493 0.238 0.963 0.7893 29.0%

40% (v/v) EtOH 1.3531 1.3543 0.320 0.950 0.7893 38.5%

50% (v/v) EtOH 1.3569 1.3581 0.383 0.940 0.7893 45.6%

Table 3 The column of refractive indices measured is the data that was obtained from the refractometer. The measurements were made at 23 °C, and so had to be corrected for a temperature of 20 °C. Once this was done, a series of calculations was made to determine the actual ethanol (v/v) %.

y = 0.0252x + 0.0542 R² = 0.98554

0.5

0.75

1

1.25

1.5

15 25 35 45 55

Peak

Are

a Et

OH

: Pea

k A

rea

1-pr

opan

ol

EtOH (v/v) %

Peak Area EtOH:Peak Area 1-propanol vs. EtOH (v/v)%

Figure 2 (Left) This is a graph of the calibration curve of the solutions with known EtOH (v/v) %. The R2 value indicates that the curve is a reliable measurement, and that it is safe to determine the EtOH (v/v) % of the vodka with this curve.

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shown in Figure 2, and was used to determine the exact ethanol (v/v) % in our vodka solution.

The graph was corrected for the new ethanol (v/v) % that were determined by the corrected

refractive indices. The slope of the graph is 0.0252 and an R2 value of 0.98554 was obtained.

The ethanol (v/v) % in the vodka sample was determined to be 42.0% from the calibration curve

with an error of 6%.

Discussion:

The temperature and pressure are dependent on each other and both affect the time of

elution of the sample. The Simplex Optimization program was run to help us in determining the

optimal parameters to get the best resolution between components in the fastest time. The

original parameters (Table 1) were run to give the program starting points to determine optimal

parameters. The lowest CRF value is generally correlated to the optimal conditions, but this is

not always true. The middle to high temperature and the middle to low pressures of the original

parameters gave the lowest CRF values. Because of this, we assume that the optimal parameters

will fall around and between these sets of parameters.

After plugging in the original values, the program gave us the first set of parameters to

use on the GC. After we ran the GC with the suggested set of parameters we reported the values

obtained back into the Simplex Optimization program and it would give us a new set of

parameters, and the process was repeated again as many times as possible (in our case, six

times). We looked at each chromatogram to find which we thought had the best resolution (and

most narrow bandwidths) in the shortest amount of time. We decided to run the rest of our

samples at 64 °C and pressure of 15.6 psi. These parameters gave us a resolution of 3.06 in 2.83

minutes. We choose the parameters based on how the chromatograms looked and not actually

calculating the resolutions for the sake of time. Due to poor injections, some of the peaks were

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broken up into multiple peaks, and so, did not look as good as they actually were. Our

parameters were good, although they may not have been the best possible, and they still allowed

us to run our samples while collecting good data.

As mentioned in the data and results section, the known standards were then run at the set

parameters. These served as guides for the chromatograms of the mixtures of the compounds for

the remainder of the experiment. The time of elution of each compound in the chromatograms

would change slightly depending on the mixture, but overall, these were good estimates of where

to look for each compound.

The four different ethanol (v/v) % mixtures were first tested in the refractometer. This

step was essential because it enabled us to find the exact ethanol (v/v) % in each of the solutions.

With small changes in temperature, comes a change in the refractive index. Due to these

changes, it is important to correct the refractive index to a temperature of 20 °C, which is the

temperature at which literature values of densities and mass percentages of ethanol are obtained.

All refractive indices were experimentally measured at 23 °C. The corrected refractive indices all

had similar, but slightly lower, ethanol (v/v) % to what we expected them to be.

The utilization of three different calibration curves enabled us to find the exact ethanol

(v/v) % in the vodka sample. The vodka sample could not be measured in the refractometer

because it was unknown if there were any other analytes in the sample that would interfere with

the instrument. The refractometer was used to obtain a refractive index at 23 °C. Because

literature values of ethanol densities are recorded at 20 °C, we needed to correct for the

temperature difference. The exact mass percentages for each corrected refractive index was

found by using the slope equation in Appendix A, Calibration Curve 1. The exact mass

percentages were then converted to exact densities (g/cm) by using the slope equation in

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Appendix A, Calibration Curve 2. The exact densities and mass percentages were then used to

make the calibration curve in Figure 2. These compared the exact mass percentages to the peak

area of ethanol: peak area of 1-propanol. This calibration curve contains a very good R2 value

that was close to one (perfect). Because of the good R2 value, we can use it as a reliable

calibration curve within the calculated error.

The ethanol (v/v) % in the vodka sample was quoted to be 40% on the Hawkeye bottle.

The ethanol (v/v) % we found from our experiments and related calibration curves was 42%

(v/v). The error in our calibration curve was +/- 6%. The value quoted on the bottle is within the

error range, meaning that the value obtained was accurate. The relative standard deviation

between the quoted and the experimental values was 5%. Since the values fall within this range,

they are precise.

Conclusion:

Overall, it is important that parameters that are dependent on each other are optimized to

the best of our ability. Software’s, such as Simplex Optimizations, make finding these

parameters much easier. Finding the corrected refractive index was an important part of this

experiment because, without doing so, our calibration curve in Figure 2 would have been off.

Overall, the final goal was to determine the ethanol (v/v) % in our vodka sample. The value we

obtained experimentally and mathematically was 42%. This value proved to be both accurate and

precise with respect to the errors associated with it.

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References

1. Dorman, F. L., Whiting, J. J., Cochran, J. W., and Gardea-Torresdey, J. (2012) Gas

Chromatography. Analytical Chemistry Anal. Chem. 12, 4775–4785.

2. Mostafa, A., Edwards, M., and Gorecki, T. (2012) Optimization aspects of comprehensive

two-dimensional gas chromatography. Journal of Chromatography A 1255, 38–55.

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Appendix A: Supplemental Information

Calibration Curve 1 was used to determine the actual mass percent with a known corrected value for the refractive index (or x value to be plugged into the slope equation).

Calibration Curve 2 was used to determine the actual densities of the solutions with mass percentages found from Calibration Curve 2 (or x value to be plugged into the slope equation).

y = 1651.5x - 2204.6 R² = 0.99135

16

20

24

28

32

36

40

1.344 1.346 1.348 1.35 1.352 1.354 1.356 1.358

Mas

s %

Refractive Index

Calibration Plot to Determine Mass Percent

y = -0.0016x + 1.001 R² = 0.99454

0.935

0.94

0.945

0.95

0.955

0.96

0.965

0.97

0.975

16 20 24 28 32 36 40

Den

sity

(g/c

m)

Mass %

Calibration for Determination of Ethanol Solutions

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Appendix B: Calculations

1. The table below shows the measured refractive indices and the corrected refractive indices

based on the temperature they were measured at. These were corrected by using the equation:

[Corrected RI= Measured RI - (0.0004/°C)  x  (temperature  of  measurement  -­‐  20°C)]

Refractive  Indices  of  Samples  (@  23  °C)  

Sample  Refractive  Index  

Measured  Refractive  Index  

Corrected  2%  v/v  EtOH   1.3428   1.344  3%  v/v  EtOH   1.3481   1.3493  4%  v/v  EtOH   1.3531   1.3543  5%  v/v  EtOH   1.3569   1.3581  Vodka   1.3536   1.3548  

2. and 3. These are both included under the discussion section in Figure 2.

4. The percentage obtained from the calibration curve was:

slope of the calibration= (peak area ratio)/(EtOH (v/v)%)

0.0252= (1.11)/(EtOH (v/v)%)

EtOH (v/v)% = (1.11)/(0.0252) = 44%

The error in this measurement was:

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The RSD% = [(42-40)/40] = 5%

5. Figure 1 under discussion section contains the plot that traces the progress of the optimization.

6.

Simplex Optimization Trials

Trial Temperature (°C)

Pressure (psi)

Elution Time (min)

1 48 22.5 2.818 2 64 15.6 2.5 3 62 18.1 2.218 4 68 17.2 2.043 5 57 19.5 2.381 6 60 21 2.056

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7. The number of plates and the plate heights calculated for each sample are recorded below.

The length of the column in this experiment was 25 meters, but 25,000 millimeters is used to

find plate height.

The plate number was found with: (16*Retention Time EtOH2)/(Width of EtOH Peak2)

The plate height was found with: (Length of Column)/(Plate Number)

Sample Average

Time (min)

Average Width (min.)

Average Plate

Number

Average Plate

Height (mm)

Vodka 1.623 0.16 1645 15 20 % EtOH

(v/v) 1.614 0.17 1443 17

30 % EtOH (v/v) 1.614 0.17 1442 17

40% EtOH (v/v) 1.619 0.18 1369 18

50% EtOH (v/v) 1.619 0.19 1161 22

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Appendix C: Questions

1. Thermal Conductivity Detectors measure how well a substance can transfer heat from a

hot region to a cold region. Helium is the carrier gas used in our experiment because its

thermal conductivity is very high; this helps to lower the conductivity of the gas stream

of our ethanol. In turn, the helium gives the lowest limit of detection for the TCD. When

our ethanol leaves the column, it flows into the TCD and over the hot tungsten-rhenium

filament. This causes the conductivity of the gas stream to decrease, the filament to get

hotter, the electrical resistance to increase, and this then causes the voltage across the

filament to change. The change in voltage is what the detector measures. Some TCD split

the carrier gas into the analytical column and the reference column. The reference column

minimizes the change in flow as the temperature is changed. The resistance of the

analytical column is then measured with respect to the reference column.

2. An internal standard is usually used in gas chromatographic analysis because the quantity

of sample analyzed and the instrument response varies slightly from run to run. Since gas

chromatography flow rates can change with each injection (if not all injections are

performed exactly the same way), the internal standard helps to correct for this.

3. UV-Vis is another experiment that would benefit from using Simplex Optimization to

help choose parameters. The resolution and signal to noise ratio are two ways to judge

how good the absorption is. These need to be balanced, just like resolution and time in

this experiment, to obtain good data. These two are balanced by adjusting slit width and

radiant power. Radiant power is related to the square of slit width.