Experiment C: Gas Chromatography Safety notes
! In addition to the normal obligations to safety in laboratories, the Gas Chromatography Laboratory has some specific hazards.
At all times follow the direction of your demonstrators and always ask if you’re unsure of a procedure to operate an apparatus.
PPE You must wear safety glasses, a laboratory coat and fully enclosed shoes covering your forefoot, toe and heel at all times whilst in the laboratory during this experiment.
Goals
Perform correct injections to ensure optimum separation and use standard addition to calibrate a spectrometer. Using a series of standards perform a calibration to determine the quantity of eucalyptol in eucalyptus oil.
Observe how intermolecular forces can be exploited to separate a mixture. Relate absorption to concentration through Beer’s law to obtain a calibration curve.
Summarising and comparing results. You will compare results between groups to ascertain accuracy and precision of a series of analyses and summarise them, either during the session or in a lab report if this experiment is allocated to you.
Assessment
If this experiment is allocated to you for your laboratory report the assessment details can be found on Blackboard and this will constitute 10% of your final grade in this unit. For all other students feedback will be provided by your demonstrator in the laboratory. The concept of chromatography will be found in the final examination.
Aim
Understand the operation of common gas chromatographs and become familiar with correct injection technique. Utilise the technique to identify a compound in a mixture and quantitatively determine its concentration.
Introduction
Gas chromatography achieves separation of analytes by passing a mixture through a column that contains a stationary phase, usually an immobilised liquid or silica compound. A small volume (microlitres) of a solution of analytes is injected into the system and the solution is immediately volatilised by the injector oven. The analytes must be able to be volatilised easily and not be degraded by heat. The analyte gas is then passed into the carrier gas, which must be chemically inert, and the varying interactions between the analyte and stationary phase achieve separation of the analytes. The presence of an analyte is determined using a detector, which in this experiment is a flame ionisation detector or FID.
With gas chromatography it is necessary that users become familiar with the day-to-day procedure for setting up an instrument and gaining practice in overcoming any problems that may be encountered. The simple task of igniting the flame in a FID can become a nightmare if
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gas flow rates are incorrectly set. Poor operating parameters and/or poor injection techniques can hinder the quality of results during routine analysis.
Early GC technology used packed columns that allowed high carrier gas flows into the detector, i.e. 30 mL/min of nitrogen. The detector also required hydrogen gas as the fuel at a flow rate of 30 mL/min and air at a rate of 300 mL/min, giving a total gas flow of 360 mL/min. If this total gas flow was not achieved, the detector may have been difficult to light and in the event of successful ignition, detector responses may be noisy and insensitive.
More recent GC technology utilises capillary columns, where the carrier gas flow rate is between 0.7 and 3.5 mL/min. The hydrogen, flame gas, and airflow rates are still maintained at 30 and 300 mL/min. With these flow rates, there is a short fall in the total gas flow to the detector. It is necessary to use a “make-up” gas i.e. nitrogen at 30mL/min to make up the difference.
STOP Follow the instructions provided with each GC and the directions of your demonstrator to conduct the following four exercises.
Exercise 1: Injection technique
Introduction
In gas chromatography, several factors contribute to errors in quantitative analysis. One of these is operator skill in the introduction of samples. To be able to obtain the best precision for quantitative analysis, that is reproducible quantitative data, one must have a proper injection technique. The following steps should yield good results.
1. Rinse the syringe with solvent, completely filling and expelling syringe several times.
2. Wipe excess solvent from the syringe needle.
3. Draw in 1 μL of air. Ensure that the end of the plunger is aligned with the 1 μL mark.
4. Draw in 0.5 μL of sample. This is achieved by drawing the plunger and aligning it with the 1.5 μL mark. This ensures the required injection volume is achieved, i.e. 0.5 μL.
5. Wipe excess sample from the needle.
6. Draw in air until the sample/solvent is entirely within the syringe barrel. The sample is ready for injection as shown below.
7. With the syringe in a vertical position, push the needle into the injector and inject the sample in a continuous, rapid manner. Do not use heavy force, as this will damage the syringe.
8. Remember to clean the syringe with solvent after use to prevent ceasing of the plunger and cross contamination.
This method results in the syringe filled as shown in the diagram below.
Figure C.1: Properly filled syringe for split or splitless injection.
Solvent following the sample (about 0.7 μL) helps to wash components from the syringe and needle bore.
For split sampling, with high gas velocity through the inlet, injection must be made in a continuous, rapid manner. Any lack of smooth motion may cause “multiple” injections and hence extra peaks.
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Note: Retention times can depend upon amount injected; so total sample volume should be kept constant.
Procedure
1. Use the instructions with the GC and select “Injection Technique” as the method file.
2. Record the detector, injector and column temperature from the instrument.
3. Inject the first sample press [START] on the GC. Wait for the analyte to elute before injecting the next replicate. Continue until you have three analyte peaks of equal height. Following is an example of the expected resultant chromatogram.
4. Click on the [STOP] button located in the top right of the computer screen. From the eDAQ menu bar, select Data then Analyse Runs.
5. Select Windows on the menu bar; then Run Info. Enter your name(s) and sample details in the run info comments section.
6. Select File from the menu bar, then Print. Use the data in the eDAQ report to complete the laboratory report sheet for this exercise.
Exercise 2: Identification of eucalyptol in eucalyptus oil using temperature programming
Introduction to temperature programming
One of the key elements in the efficient separation of components on a GC column is the degree of partitioning of these components between the liquid stationery phase and the gaseous mobile phase. The partition coefficient of a compound in a mixture is strongly influenced by its vapour pressure, which in turn is directly related to column temperature. For example, the increase in vapour pressure from a 30 ºC rise in column temperature will approximately halve the partition coefficient, and thus double the rate of migration of a component through the column. Clearly, the most immediate effect of an increase in column temperature is a reduction in analysis time, often a very important factor to be considered when the sample contains components having a wide range of boiling points.
A further consideration is the influence of column temperature on resolution, the ability of the system to separate components, which have similar retention times, on a given column. A temperature increase will reduce differences between the partition coefficients of components and decrease the separation efficiency of the column. Conversely, a reduction in column temperature will improve the resolution of components, which may well co-elute at higher temperatures.
When analysing a complex sample using an isothermal run, it is often found that no single column temperature can produce an entirely satisfactory chromatogram.
A low temperature isothermal run, whilst probably adequately separating the components has some limitations.
1. It may require a very long analysis time to elute all components.
2. Those with long retention times will usually elute as poorly shaped peaks, being both flat and broad or irregular in profile.
Time / min 1 2 3 4 5 6 7 8 9
Injection 1 Injection 2 Injection 3
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3. The operator is often unsure whether all the high boiling components have been eluted from the column.
A high temperature isothermal run does not suffer from the above problems, but will most often produce a poorly resolved chromatogram having the low boiling components co-eluting at a very short retention time.
A temperature-programmed run is therefore the best option:
1. It can start at a sufficiently low temperature to ensure that all low boiling components are efficiently separated. A short isothermal interval at this temperature can also be used for enhanced separation of these components.
2. The rate of increase in column temperature can be adjusted to produce satisfactory resolution, sharp peaks and reduced analysis time as required.
3. The column temperature can be held at the maximum selected until all high boiling components are eluted.
The main limitation to the use of temperature programming is the choice of a suitable liquid stationary phase. Many phases produce a background signal at higher temperatures due to the stationary phase bleeding from the column, and this may become excessive at high temperatures, with increasing signal to noise also apparent.
Introduction to standard additions and spiking
The eucalyptus tree is indigenous to Australia. The genus contains about 300 species with differing quantities of essential oils. People are often fooled into thinking that a plant oil extract has only one constituent. That is not the case. The oils may be roughly divided into three classes of commercial importance.
1. The medicinal oils.
2. The industrial oils, containing terpenes, which are used for flotation purposes in mining operations.
3. The aromatic oils, which are characterised by their aroma.
The essential oil of eucalyptus is obtained by the steam distillation of fresh leaves. It is a colourless or straw-coloured fluid with a characteristic odour and taste. Eucalyptol may be the predominate constituent, depending on species. Other components present include large amounts of terpene, cymene and limonene.
To determine the presence and quantity of eucalyptol, the technique of standard addition or “spiking” is employed. The sample is first analysed by GC. The sample is then enriched with a known quantity of analyte, in this case eucalyptol. The additional signal produced by the addition of standard increases the area of the original signal.
As an example of this, one of the peaks shown on the chromatogram of eucalyptus oil in Figure C.2 is believed to be eucalyptol. By adding (enriching or “spiking”) a small volume of pure eucalyptol to the eucalyptus oil, an increase in peak size (height and area) can be observed when inspecting the resultant chromatogram (Figure C.3) and comparing it to the original unspiked sample, Figure C.2. This allows the identification of eucalyptol as a component of eucalyptus oil. This is however not conclusive when GC-FID is used alone. Techniques such as gas chromatography-mass spectrometry can confirm the result.
The original concentration can be calculated either mathematically or graphically. Increasing the number of additions produces a straight-line calibration. The x-axis intercept value (made positive) represents the original concentration. A typical calibration graph is shown in Figure C. 4. It is necessary to normalise the areas or height before carrying out any calculation. This is achieved by selecting a reference compound appearing in the chromatogram and adjusting the area of this peak such that it is the same for each chromatogram. The areas (or height) ratio should be calculated using the reference compound.
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Figure C.2: Illustration of the effect of standard addition to the sample
Time / min 1 2 3 4 5 6 7 8 9
Sample + eucalyptol
Sample
Spike
Figure C.3: The area of the largest peak (say, just before 7 mins, not the solvent) in Figure C.2 is 8000. The area of the eucalyptol peak is 800. The ratio is therefore 0.1. Therefore the ratio can be used to plot the calibration curve for the standard addition.
From the plot, eucalyptol content = 9%.
Calibration for eucalyptol
Procedure
The following solutions are provided:
(a) Eucalyptus oil diluted with hexane sample
(b) Eucalyptol diluted with hexane standard
(c) Eucalyptus oil spiked with 10% eucalyptol
(d) Eucalyptus oil spiked with 20% eucalyptol
Your demonstrator will instruct you on the operation of the GC using an autosampler and the explain the different temperature profiles used.
If this experiment is allocated to you for your laboratory report then the questions will guide you to the type of information needed in this report. If this experiment is not assessed your demonstrator will provide you with additional activities to complete for feedback.
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Name Student IDStudent IDStudent IDStudent IDStudent IDStudent IDStudent IDStudent ID
Pre-laboratory questions: GC 1. Draw the structure of eucalyptol, find its reported boiling point, and cite the source where
you obtained this information from.
2. Based on the structure and boiling points above, what aspect/s of this molecule might allow it to be separated from the solvent hexane using chromatography?
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3. After rinsing the syringe with solvent and wiping excess from the needle, you draw up
………………. μL of ………………., followed by ………………. μL of sample.
4. Suggest three reasons why you may not achieve repeatable injections.
1 :
2:
3:
5. Circle which temperature option is best for separating a complex mixture such as in eucalyptus oil?
(a) Low temperature isothermal run.
(b) High temperature isothermal run.
(c) Temperature program run.
6. What advantages are there in using the best temperature option as selected in Q5?
Submit this sheet to your demonstrator when you enter the laboratory at the start of the session.
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Results: Exercise 1
1. Obtain the peak heights from the eDAQ integration report and copy them into the table below then calculate:
(a) average height
(b) relative error
Peak Time – tR(min) Height – HNorm Area – ANorm
Average
% RSD
If the relative error exceeds 3%, and if time permits, repeat the exercise.
2. Repeat the calculation using peak area instead of heights and compare the two results.
3. How do the RSD values compare?
Results: Exercise 2
1. Record the retention time for eucalyptol from the programmed run for the pure sample and use this to identify and label the eucalyptol peak on the eucalyptus oil and spiked eucalyptus oil chromatograms.
2. Complete the table below showing the retention time – tR(min) and peak areas – ANorm for eucalyptol and a nearby reference peak in each chromatogram.
Sample Eucalyptol Peak Time
Eucalyptol Peak Area
Reference Peak Time
Reference Peak Area
Ratio Eucalyptol/ Reference
Pure eucalyptol
Eucalyptus oil
Eucalyptus oil spiked with 10% Eucalyptol Eucalyptus oil spiked with 20% Eucalyptol Experiment C: Gas Chromatography 114
3. Construct a calibration curve as described in the introduction and determine the original eucalyptol concentration in the sample (the unspiked sample).
% Eucalyptoleucalyptol in Eucalyptus Oil =………………………………..
4. Discuss the advantages of temperature programming when analysing a complex mixture.
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5. Does the spiking technique prove conclusively the identity of an unknown peak? Explain your answer. How might this be achieved?
STOP If this experiment forms part of your laboratory report, you should scan and insert all chromatograms into your report before submission.
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