What Is The Gas Chromatography Technique Biology Essay

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Gas chromatography is a technique used to separate and measure volatile components of mixtures. A small sample of the mixture is injected into the instrument and it is vaporized before entering the column. The sample is moved by helium gas through the column until it reaches a detector. In this experiment, solutions with various known concentrations of caffeine were analyzed using gas chromatography and a calibration curve was created from the data acquired. A sample of caffeine of unknown concentration was analyzed using gas chromatography and its caffeine concentration was found to be 0.14 % by using the created calibration curve for caffeine concentration.

Introduction:

Gas chromatography (GC) is a technique used to separate mixtures of highly volatile liquids. As in all chromatographic methods, the mobile phase is a gas (Hydrogen or Helium) that carries the components of the mixture over a stationary phase. In GC, the stationary phase is a column packed with silica or coated with a high boiling point liquid. The components of the mixture leave the column in order of volatility, the most volatile first. About a micro-liter of the sample is injected to the GC instrument and is transported into the column which is kept in a temperature controlled oven. Columns are often wind up into spirals to save space. The properties of the column and its packing materials are chosen based on the particular separation that is to be carried out. The components of the mixture are carried through the column by a stream of inert of helium (or hydrogen) gas. The rate at which the mixture is separated through the column depends on the volatility of the components and their interaction with the stationary phase. At the other end of the column is a detector that detects each components of the mixture as it comes out of the column and measures its amounts. The GC instrument used for this experiment has a flame ionization detector (FID) which consists of hydrogen flame burning on air. As a substance leaves the column, it burns into a flame producing ions which can be detected by measuring the electrical conductivity of the flame. Before beginning a separation the FID must be lit, the flow rate of the carrier gas and temperature of the oven must be determined to ensure optimum results. The temperature of the inlet port is set at a level that the sample is fully vaporized before it enters the column.

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The objective of this experiment was to understand the theoretical aspects of gas chromatography by analyzing caffeine solutions of different concentrations and creating a calibration curve for caffeine concentrations. The calibration curve was used to determine the concentration of a caffeine sample of unknown concentration.

Experimental:

50 mL of a 0.25 mg/mL biphenyl stock solution were prepared by dissolving 12.50 milligrams of biphenyl in 50 mL dichloromethane. The biphenyl solution was treated as the internal standard for the experiment. The solutions with caffeine concentrations of 1, .8 and .6 mg/mL were prepared by dissolving 10, 8 and 6 milligrams of caffeine respectively in 10 mL of an internal standard solution. In order to create less concentrated caffeine solutions, 1:2 serial dilutions were performed to the .8 mg/mL solution to create solutions with caffeine concentrations of .4, .2 and .1 mg/mL.

A 50 mL sample of caffeinated water was transferred to a 125 mL separatory funnel. Extractions were performed with 10 mL portions of dichloromethane and were collected into a beaker. High pressure air flowing through a needle was used to evaporate the dichloromethane and reduce the volume to about 2 mL.

All solutions were transferred to properly labeled GC vials and the parameters in Table # 1 were used in the GC instrument.

Table # 1: GC instrument parameters

Settings

Helium carrier gas flow rate

1.0 mL/min

Initial oven temperature

140 °C

Ramp rate

10 °C/min

Final oven temperature

240 °C

Total run time

10 min

Inlet temperature

200 °C

Detector temperature

250 °C

Split ratio

1:2

Injection volume

1-2 µL

Retrieved from:

Results:

Analysis of the caffeine solutions and unknown sample was conducted a total of three times inside the gas chromatograph in order to minimize error and display reproducibility. (Tables 2-4 Appendix).

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Data obtained from the chromatograph shows three peaks appearing at different retention times.

Table # 2: Data from the gas chromatograph for caffeine solutions and unknown - Trial 1

Caffeine solutions with ISTD biphenyl

(mg/mL)

Peak 1

Peak 2

Peak 3

Time

(min)

Peak Area

Time

(min)

Peak Area

Time

(min)

Peak Area

1.00

1.28

1.14 x

2.997

1062.24

6.695

955.34

0.80

1.28

1.12 x

3.003

1047.76

6.711

1168.01

0.60

1.28

1.12 x

2.995

1043.38

6.692

859.41

0.40

1.28

1.11

3.001

1085.49

6.667

508.24

0.20

1.28

1.12

2.999

1155.87

6.637

213.64

0.10

1.28

1.17

3.002

1159.68

6.623

62.67

Unknown

1.28

1.25 x

-

-

569.69

Caffeine solutions with ISTD biphenyl

(mg/mL)

Peak 1

Peak 2

Peak 3

Time

(min)

Peak Area

Time

(min)

Peak Area

Time

(min)

Peak Area

1.00

1.28

1.13 x

2.999

1067.20

6.697

954.97

0.80

1.28

1.12 x

2.999

1051.05

6.710

1173.36

0.60

1.28

1.15 x

3.000

1070.20

6.694

879.87

0.40

1.28

1.11

2.995

1084.09

6.667

512.08

0.20

1.28

1.11

3.000

1137.18

6.636

209.07

0.10

1.28

1.13

3.003

1125.24

6.623

62.43

Unknown

1.28

-

-

Caffeine solutions with ISTD biphenyl

(mg/mL)

Peak 1

Peak 2

Peak 3

Time

(min)

Peak Area

Time

(min)

Peak Area

Time

(min)

Peak Area

1.00

1.28

1.13 x

2.999

1061.39

6.696

957.36

0.80

1.28

1.12 x

2.998

1052.44

6.711

1177.01

0.60

1.28

1.12 x

2.999

1047.43

6.692

865.05

0.40

1.28

1.11 x

2.998

1086.39

6.667

512.25

0.20

1.28

1.11 x

3.001

1142.85

6.636

210.14

0.10

1.28

1.15 x

3.004

1145.47

6.623

61.91

Unknown

1.28

1.25 x

-

-