Gas Chromatography Lab Report

Objective: To determine the concentration of the three analytes ( EtAc, MeBuOH , PrOH, BuOH) or impurities in the whiskey sample by gas chromatography.

Background:

An alcoholic beverage such as whiskey is a source of ethanol, but with different brands there will be a complex blend of trace impurities. Gas Chromatography is an inexpensive apparatus separation is based on partition between mobile phase and stationary phase. Major components used in gas chromatography are gas inlets, injector, column, detector and amplifier. Detector used in FID, flame ionization detector that uses a flame in the column for the compounds exiting gas chromatography.2 The combination of two methods is called GC/ FID, used for the separation of many organic compounds,

hydrocarbons with good sensitivity. This method is useful to separate organic and hydrocarbons and FID is proportional to number of carbon atoms in hydrocarbons, if number of hydrocarbons is more, it has more sensitivity but with hetero atoms the sensitivity is less.
A response factor is used in GC/ FID, which is ratio between response of detector to the compound and the concentration of compound to be analysed it appears as a peak from the detector.3
R.F = Peak area / Concentration (mg/ mL) (1)
It is used to calculate unknown amount in a sample after the measurement of signal strength but it requires high reproducibility to compensate this an internal standard is used called a RRF (relative response factor) which is used to measure signal strength in an unknown sample and compare it with internal standard of known concentration added in to the unknown sample and it is used to calculate the amount in unknown sample.1 FID consists of a fuel and a makeup gas it is best for hydrocarbons, ions moves across the potential difference creating an increase in current then amplified and a peak is recorded.
CH + O CHO+ + e-
Makeup gas can be a nitrogen / helium and it is used to get optimum sensitivity flow rate of gas at 30 ml/ min minimum and for good sensitivity makeup gas such as nitrogen, helium is used. FID cracks the ions as signals and burns completely to smallest ion particles.
Materials required:
Gas chromatography instrument
column (supelco SPB -5)
phase ( 5% phenol 95% dimethylpolysiloxane )
water
ethanol
ethylacetate (EtAc)
1- propanol ( PrOH)
3-methyl-1- butanol( MeBuOH)
1-butanol (BuOH)
Whiskey sample
Procedure:
The whiskey sample matrix (40%) ABV solution of ethanol in water was prepared at 200 c the ABV standard is used worldwide, so the term proof is multiplied by ABV twice thus, alcohol product labelled as 80 proof is 40% ABV.

It is usually 40 %, the contents of EtAc, PrOH, MeBuOH were tested in the whiskey sample and this will be simulated a whiskey sample by using 40% ABV solution of ethanol in water. The mass of all three analytes were weighed into a 50 ml bottle and 40% ABV of ethanol in water was added to 50 ml bottle, followed by preparation of whiskey simulated sample with (IS) its concentration is targeted as 1000 ppm (w/w) so 80µL is transferred to 10 ml bottle and its mass was weighed as (0.0127 g) and total mass of solution was calculated as 12.7 g after adding whiskey to butanol. Next the individual standard solutions were prepared the concentration of each individual standard is calibrated to determine retention time. For each 8µL of all three solutions were transferred to 10 ml bottle then 40% ABV solution of 10 ml of ethanol in water was added to 10 ml bottle. Finally mixed standard solution was prepared so butanol of 1000 ppm was transferred to 50 ml bottle and its mass was recorded and its total mass was calculated, 40µL of EtAc, PrOH, MeBuOH was transferred to 50 ml bottle and weight was recorded then finally 40% ABV solution of ethanol in water was added to 50 ml bottle till mass of solution was

noted.
Then from the results the RF and RRF for each impurity or analyte was calculated followed by RRF the concentration of each analyte in the unknown simulated whiskey sample was calculated with average, standard deviations for each analyte also then calculated.
Results:
Calculations for Simulated Whiskey Sample preparation with Internal Standard:
8µL butanol = wt/ 1000µg/g x 106
Weight of butanol = 0.0127 g
So to find weight of whiskey to be added is,
=0.0127 x 106/ 1000 g
Total mass of whiskey sample only for Butanol =12.7 g

Table 1. Mass of the impurities in the whiskey sample.
Mass Concentration ppm, (w/w)
BuOH 0.0127g / 12.7 g only for butanol 999.89 approx. = 1000 ppm
EtAc 0.0159g 919 ppm
PrOH 0.0155g 895.86 ppm
MeBuOH 0.0163g 942.098 ppm
Solution Total 17.3018g

Graph of PrOH:

Concentration = mass of impurity g / total mass of solution g x 106
For BuOH = 0.0127 g / 12.7 g x 106
= 1000 ppm (w/w) , but for others EtAc = 0.0159g / 17.301 g x 106 = 919 ppm
Graph of MeBuOH:

Graph of EtAc:

Table 2. Simulated Whiskey sample M1:
Peak # Peak area compound
1 776140 Ethanol and water
2 24370999 Ethanol and water
3 1381715 Ethanol and water
4 101594 PrOH
5 56486 EtAc
6 96556 BuOH
7 105979 MeBuOH

Table 3. Mixed samples with concentration and peak area.
Mixed sample BuOH
ppm Peak area BuOH EtAc
ppm Peak area EtAc
MeBuOH
ppm Peak area MeBuOH PrOH
ppm Peak area PrOH
1 1000 81784 919 41349 942.09 78466 895.86 69239
2 1000 92826 919 46419 942.09 88976 895.86 78677
3 1000 106122 919 52878 942.09 103631 895.86 90650

Calculation of RF value for BuOH in mixed standard solution:
RF = peak area / concentration of BuOH
Peak area = 81784 / 1000
= 81.784
Table 4. RF values for the mixed standard solutions.
Mixed solution RF BuOH RF PrOH RF EtAc RF MeBuOH
1 81.784 77.28 45 83.28
2 92.82 87.82 51 94.44
3 106.12 101.18 58 110.00

Calculations for average RF for BuOH:
Here N = 3,
Avg RF = 81.784 + 92.82 + 106.12 / 3
= 280.72 / 3
= 93.57
Graph of whiskey 1:

Graph of whiskey 2:

Graph of whiskey 3:

Table 5. Average RF values for mixed standard solutions:
Standard solution Avg RF value
BuOH 93.57
PrOH 89
EtAc 51
MeBuOH 95.90 = 96

Calculation of unknown PrOH by RF value:
Concentration = Peak area / RF of PrOH
Peak area of PrOH is 101594
RF is 89
Concentration = 101594 / 89
= 1141.5 ppm
For the other standard solutions it was calculated as the above concentration with peak areas and RF values of respective values.
Table 6.

Simulated whiskey samples with concentrations.

sample Peak area PrOH Peak area

EtAc Peak area

MeBuOH Ppm of PrOH Ppm of EtAc Ppm of MeBuOH

1 101594 56486 105979 1141.5 1107.5 1103.94

2 99241 55083 103861 115.06 1080.05 1081.88

3 98822 54002 102856 1110.35 1058.86 1071.41

Calculation of Average concentration of PrOH:

Avg conc. = 1141.5 + 1115.06+ 1110.35 / 3

= 1122.30 ppm

Table 7. Average ppm and Standard deviation values.

Standard solution Avg ppm Standard deviation

PrOH 1122.30 16.78

EtAc 1082.13 24.38

MeBuOH 1085.46 16.60

Standard deviation for PrOH:

S.D = ? (1141.5 – 1122.30)2 + (1115.06 – 1122.30)2 + (1110.35 – 1122.30)2 / 3-1

= ? 368.25 + 52.70 + 142.80 / 2

= ? 563.75 / 2

= ?281.64 = 16.78

Calculation for RRF of PrOH in Mixed Solutions:

RRF = RF of PrOH / RF of BuOH

RRF = 77.28 / 81.78

RRF = 0.94497

Table 8. RRF values of the three impurities.

Standard solution RRF PrOH RRF EtAc RRF MeBuOH

1 0.944 0.548 1.0121

2 0.946 0.549 1.0127

3 0.953 0.546 1.0365

Average of RRF 0.947 0.5476 1.0202

Calculation for concentration of PrOH by RRF value:

Con, of PrOH = Peak area PrOH/ Peak area of BuOH x 1 / RRF x conc, of

BuOH
= 101594 / 96556 x 1/0.947 x 1000 ppm = 1110.87 ppm.
Table 9. Concentration of three compounds with average RRF values:
Compound PrOH Concentration EtAc
Concentration MeBuOH
Concentration RRF PrOH RRF EtAc RRF MeBuOH
1 1110.87 ppm 982.84 ppm 992.79 ppm 0.947 0.547 1.020
2 1112.98 ppm 982.90 ppm 1018.76 ppm 0.947 0.547 1.020
3 1110.80 ppm 965.87 ppm 1011.26 ppm 0.947 0.547 1.020

Discussion:
The observed retention times of the impurities were the compound with 1.469 retention time was eluted first is 1- propanol followed by 1.780 EtAc, and 1- butanol is at 2.371 and finally with 3.775 is MeBuOH for almost every chromatogram. So here the column used is Supelco SPB -5 and was coated with phase 5% phenyl 95% dimethylpolysiloxane which is hydrophobic so this can be explained that 40% ABV solution of ethanol in water can be used in the column.so almost more polar compounds will elute first from the column so ethanol in water is more polar than compared to remaining impurities in the whiskey simulated sample so it was observed as 1.025, 1.195, 1.255 as retention time for ethanol in water. But ethylacetate is more polar than propanol but it will elute later due to its hydrophobic nature. The retention time can be explained from the polar nature as well as the hydrophobic property of the impurities present in the standard solutions. In FID the components will experience a combustion that cracks the ions by the current flow that will generate the peaks for each analytes in the solution thus, the RF is dependent on the FID. The average RF values for the impurities was calculated as BuOH was 93.57, PrOH as 89 ethyl acetate as 51 and finally MeBuOH AS 96 So the concentration of unknown 1- propanol was calculated from the peak areas of PrOH was found to be 1141.5 ppm for the first run followed by 1115.06 ppm for second run and 1110.35 ppm for the third run of the sample 1- propanol, this is repeated for all two whiskey samples all the three runs by using peak areas of EtAc, MeBuOH and their relative RF values and their concentration was calculated as for ethyl acetate it is 1107.5 ppm, 1080.05 ppm, 1058.86 ppm respectively. For the MeBuOH the concentration for all the three runs was found to be 1103.94 ppm for the first run, 1081.88 ppm, 1071.41 ppm for the third run. From these concentrations of the three impurities the average concentration was calibrated using the three concentration values for each of the compounds PrOH, EtAc, MeBuOH as 1122.30, 1082.13, 1085.46 resp., with the standard deviation for each of the impurities as PrOH with 16.78, EtAc as 24.38 and MeBuOH with 16.60 respectively. The average RF for ethyl acetate is lowest compared to other impurities and found as 51, and the highest RF is 96 for MeBuOH followed by butanol as 93.57.
The RRF for all the three impurities were calculated for the three runs and their average was noted, for propanol it was 0.947, ethyl acetate as 0.547 and MeBuOH as 1.0202. Finally from RRF values the average concentration for all the three impurities was calculated and found to be for propanol as 1111.57 ppm, ethyl acetate as 977.20 and MeBuOH with 1007.60 ppm respectively. So it was clear that use of RF values for all the components in the mixed standard solutions was between its peak area and concentration of the internal standard with all the components in the mixed solutions and the use of internal standard (IS) is to provide the same signal as to the analyte in the solutions, so in this experiment the IS used was 1- butanol (BuOH) which showed a good result as similar signal for all the analytes.