EXPERIMENT 10

GAS CHROMATOGRAPHY
Group 4: Lopez, Palmario, Sibug

What is gas chromatography?

Analytical technique for separating compounds based on their volatilities

For

compounds that can be vaporised without decomposition

Compounds move through a GC column as gases, partition between a stationary phase and a mobile phase
The

differential partitioning into the stationary phase allows the compounds to be separated in time and space

Uses of gas chromatography..

To test the purity of a particular substance To separate different components of a mixture and determine relative amounts of such components Can be used to prepare pure compounds from a mixture in preparative chromatography

Components of a gas chromatograph

1.

2.
3. 4. 5.

Gas supply Injector / column inlet Column Detector Data recorder

Gas Supply

Carrier gas must be inert Commonly used gases: N, He, Ar, CO2 Gas depends on detector type

Sample Injection Port = Injector

provides the means to introduce a sample into a continuous flow of carrier gas Types include:
Split/Splitless

type- most common On-column inlet PTV injector Gas source inlet Purge-and-Trap system

Column

Houses the stationary phase composed of the high-boiling liquid General types:
Packed-

inert solid material that is coated w/ liquid stationary phase Capillary- more efficient
Wall-coated

open tubular (WCOT)- wall ang coated ng

SP Support-coated open tubular (SCOT)- more efficient

Thin layer of support material then diatomaceous earth where SP adsorbed

Detector

Different detectors give different types of selectivity

non-selective

detector responds to all compounds except the carrier gas selective detector responds to a range of compounds with a common physical or chemical property specific detector responds to a single chemical compound

concentration dependant detectors vs mass flow dependant detectors

Data Recorder

Plots the signal from the detector over time the chromatogram Retention time - qualitatively indicative of the type of compound

Gas Chromatography

Theoretical Plate Model of Chromatography

Theoretical plates- analytes move down the column by transfer of equilibrated mobile phase
Separate

layers in column Separate equilibrations of the sample between the stationary and mobile phase occur in these "plates

Plates serve as a way of measuring column efficiency by stating the number of theoretical plates in a column

Column Efficiency

Related to the number of compounds that can be separated by the column Measured by determining number of theoretical plates (N) and resolution (Rs)

Capacity Factor

Rate of migration of solutes in the column ratio of the mass of the compound in the stationary phase relative to the mass of the compound in the mobile phase

Factors Affecting GC Separation

Volatility of compound- more volatile mas una Polarity of compounds and column Column temperature- increase T -> faster movement -> lesser separation Flow rate of the gas through the columnhigh flow rate -> higher speed Length of the column- greater separation using longer column

Quantitative Analysis in GC

Quantitative Analysis Using GC

Peak area is directly proportional to the amount of respective component in sample BUT: response of a detector varies from one component to another
Peak A and B have about the same peak areas BUT: actual concentration of A > B due to response factor Peak area alone does not indicate concentration of sample
B

Component B

Component A

The report of peak area % not indicative of concentration ratios, but more useful for

Calibration

Calibration involves correlation between known concentration of a component and resultant detector response
A = kC
A = peak area, k=response factor, C = concentration

Single-level vs. Multi-level calibration

Standard Methods of Calibration

External Standard Calibration

Mixtures

of the sample components at varying concentrations are analyzed and peak areas recorded

Plot

Area vs. Concentration to determine response factor k Requires very precise sample injection Prone to error due to varying injected sample

Standard Methods of Calibration

Internal Standard Calibration

Errors

arising from varying injected sample volume (or any other variations) eliminated by use of internal standard Procedure:
Internal

standard added to the standard mixtures prior to analysis Determine response factor (k) for each component by plotting area ratios vs. concentration ratios (or Cx only if Cint is constant for all standard mixtures):

Standard Methods of Calibration

The ideal internal standard:

Should

give completely resolved peak but that which is close to components of the sampleseparate! Peak should be similar in magnitude to those of the components Should be chemically similar but not present in the original sample

Sample could then be analyzed by adding internal standard to the unknown

Determination of Peak Areas

Cut and Weigh Triangulation Integration

Peak Area Determination: Cut and Weigh Method

Cut-and-Weigh Method
Area

under the peaks is cut and weigh together with a reference paper with known weight Economical Disadvantages
incorrect

drawing of the base line, incorrect cutting of the peaks lack of homogeneity of the paper tedious

Peak Area Determination: Triangulation Method

A geometric method that approximates the peak area Assumes that the peaks are perfect Gaussian-curve shaped Errors can arise if peak shape is irregular

height (h)

width at h (w1/2)

Area = hw1/2

Peak Area Determination: Integration Method

Automated

method less to human error measurements of relative peak areas are given by the electronic integrators or detectors Limitations
distinguish

peaks from noise, correctly identifying the underlying baseline maintaining correct peak and baseline detection throughout a sequence of chromatograms correctly handling rider peaks and other unresolved peaks

Objectives
-To successfully perform GC -To separate and quantify components of a mixture -To test different calibration method

Methodology

General Procedure for Quantitative GC Analysis

1.

2.

3.

Determine the retention times for each component in the sample mixture Establish calibration curve using standards of each component Analyzed sample in GC and correlate peak areas obtained to concentration using the calibration curves for each component

Preparation of Standard Solutions

Procedures Rationale

Gas chromatograms of pure benzene, toluene, xylene and ethylbenzene in diethylether were obtained Standard solutions were prepared and GC were obtained

To know the Rt of each solvents To have standard calibration curves for the unknown analysis

Preparation of Standard Solutions

Procedures Areas under the peaks were determined by: -Triangulation -Cut and Weigh (not done) -Integration Plot Area vs. Concentration Rationale - for standard calibration curve - to test the different methods

Unknown Mixture Analysis

Procedures GC of unknown mixture of the used hydrocarbons Identify and quantify each component of the mixture Determine the capacity factor, resolution and average number of theoretical plates of the column

Results and Discussion

Determination of Retention Times

Determination of Retention Times

BUT WAIT!
Whats this peak?? tR = 1.21 (same as benzene) Benzene contamination?! Possible implications to be discussed later

Order of Elution
CH3 CH3 CH3 CH3

<
bp
tR
80.1 C
o

<
110.6 C
o

<
136.0 C 1.59 min
o

144.4 C

1.21 min

1.37 min

1.72 min

Order of elution is based on the boiling point and affinity to stationary phase
similar

structure same affinity Increasing boiling point longer tR

Standard 1 Chromatogram

Standard 2 Chromatogram

Standard 3 Chromatogram

Benzene as Internal Standard

Benzene fits the requirements for a good internal standard of a mixture containing toluene, ethylbenzene, and o-xylene:
Completely

resolved peak close to components Peak similar in magnitude Chemically similar but not originally present

Triangulation | External Standard

Triangulation | External Standard

Triangulation | Internal Standard

Triangulation | Internal Standard

Gives improved correlation, in particular to ethylbenzene

Integration | External Standard

Toluene Ethyl Benzene Standard % Peak % Peak Number Comp Area Comp Area 1 0.10 45858 0.20 54305 2 0.20 399893 0.30 387321 3 0.30 601619 0.10 328382 o-xylene % Peak Comp Area 0.30 55322 0.10 299751 0.20 290815

Integration | External Standard

Toluene Ethyl Benzene Standard % Peak % Peak Number Comp Area Comp Area 1 0.10 45858 0.20 54305 2 0.20 399893 0.30 387321 3 0.30 601619 0.10 328382 o-xylene % Peak Comp Area 0.30 55322 0.10 299751 0.20 290815

Note the peak areas obtained for standard 1, which appear to be much lower in magnitude than the other peak areas. This might suggest a significant change in sample volume injected

Integration | External Standard

700000 Peak Area (uVs) 500000 400000 300000 200000

Toluene

Peak Area (uVs)

600000

100000
0 0 0.1 0.2 % Composition 0.3 0.4

450000 400000 350000 300000 250000 200000 150000 100000 50000 0 0

Ethyl benzene

0.1

0.2 % Composition

0.3

0.4

400000 350000 300000 250000 200000 150000 100000 50000 0 0 0.1 0.2 % Composition

Peak Area (uVs)

O-xylene

Toluene

Ethyl benzene 294695 197731 0.0275

0-xylene -12221 459725 0.7774

Slop e
0.3 0.4

2778809 -206638 0.9756

y-int R2

Integration | External Standard

700000 Peak Area (uVs) 500000 400000 300000 200000

Toluene

Peak Area (uVs)

600000

100000
0 0 0.1 0.2 % Composition 0.3 0.4

450000 400000 350000 300000 250000 200000 150000 100000 50000 0 0

Ethyl benzene

0.1

0.2 % Composition

0.3

0.4

400000 350000 300000 250000 200000 150000 100000 50000 0 0 0.1 0.2 % Composition

Peak Area (uVs)

O-xylene

Toluene

Ethyl benzene 294695 197731 0.0275

0-xylene -12221 459725 0.7774

Slop e
0.3 0.4

2778809 -206638 0.9756

y-int R2

- points from standard 1 show the most deviation

Integration | Internal Standard

Integration | Internal Standard

- Sample injection error most evident with benzene internal standard, which should have approximately same peak area since same concentration is in each standard mixture - Error should be corrected with plotting area ratios instead - Relatively high deviations in benzene peak area between standard 2 and 3 can be related to the mysterious peak found in the toluene standard earlier

Integration | Internal Standard

1 0.95 0.9 0.85 0.8 0.75 0.7 0.65 0.6 0 y = 1.5167x + 0.4825 R = 0.9977 AA/IA 0.85 0.8 0.75 0.7 0.65 0.6 0.55 0.5 0 0.1 0.2 % Composition 0.3 0.4 y = 1.1946x + 0.4354 R = 0.7404

AA/IA

0.1

0.2 % Composition

0.3

0.4

0.8 0.75 0.7 AA/IA 0.65 0.6 y = 0.9373x + 0.4152 R = 0.3501

Ethyl Toluen Benzen e e

Oxylene 0.9373 0.4152

0.55
0.5 0.45 0.4 0 0.1 0.2 0.3 0.4 % Composition

Slop e
Y-int

1.5167 0.4825

1.1945 0.4354

Still2gives hideous0.7404 but the linearity, 0.3501 R 0.9977 correlations are relatively improved!

What method gave the best correlation?

Comparing correlation coefficients, triangulation/internal standard method appears to give better linear fits

Theoretically, Integration/Internal standard should have given the best correlation

Triangulation

method prone to a number of errors; not very precise The more automated (integration), the better

That Mysterious Peak

The toluene standard used throughout the experiment could have been contaminated with benzene (internal standard)! This leads to:
Less

toluene in each standard mixture (but still somewhat proportional) More benzene (internal standard)

Ideally, this error could be masked for ethylbenzene and xylene in external standard method IF consistent sample injection wasnt a problem

The Hypothetical Contamination

Say the toluene used was actually 40% toluene and 60% benzene:

Since Cint no longer constant, now must plot concentration ratio instead when using internal standard (integration) method

The Hypothetical Contamination

Analysis of Unknown

Analysis of Unknown

5.1333 7.778 2.380 5.1333

0.04+ 0.05

Conclusions

GC is a good method to separate mixtures of components with similar structures Experimentally, it was determined that triangulation with internal standardization provided the best correlation
BUT:

a number of errors arose due to possible standard contamination