GC Analysis Discussion Examples | Understanding Retention Time, Peak Area, and Quantitation

What Is GC Analysis?

GC analysis is an analytical method that uses gas chromatography to separate and detect volatile components in a mixture.
The sample is vaporized at the inlet and carried into the column by a carrier gas.
Because each component moves through the column with a different degree of ease, the time required to reach the detector differs, and the components appear as separate peaks on the chromatogram.

In GC analysis, the time at which a peak appears is treated as the retention time, while the size of the peak is treated as the peak area or peak height.
Retention time is often used to identify components, while peak area is used to quantify the amount of a component.
However, accurate identification and quantitation require comparison with standard substances, calibration curves, internal standards, and confirmation of the state of separation.

Main Items to Include in the Results

In GC analysis results, organize the sample name, standard substances, column conditions, temperature conditions, carrier gas, detector, retention time, peak area, peak height, separation state, calibration curve, quantitative values, and other information.
Because retention time and peak shape in GC change depending on measurement conditions, it is important to clearly state the instrument conditions.

Main Items to Include in the Results

• Sample name
• Analyte
• Standard substance
• Internal-standard substance
• Column used
• Type of stationary phase
• Carrier gas
• Flow rate
• Inlet temperature
• Column temperature
• Temperature-program conditions
• Type of detector
• Injection volume
• Split ratio
• Retention time
• Peak area
• Peak height
• State of peak separation
• Calibration-curve equation
• Correlation coefficient
• Quantitative value of the unknown sample
• Sources of error and points for improvement

Reference Experimental Values and Quantitative Calculation Examples for GC Analysis

Here, the process of identifying and quantifying components in a mixed sample using retention time, peak area, area percentage, and calibration curves obtained by gas chromatography (GC) is organized using reference experimental values.

In GC, volatile components are separated in a column and recorded as peaks by a detector.
Retention time provides a clue for component identification, while peak area provides an approximate indication of the amount of a component.
However, because detector sensitivity differs among components, accurate quantitation uses calibration curves prepared from standard solutions or the internal-standard method.

Reference Experimental Conditions

GC Measurement Results for a Standard Mixture

A reference example is shown for measurement of a standard mixture containing ethanol, acetone, ethyl acetate, and toluene.

If the retention time of a standard substance measured under the same conditions is close to the peak retention time of an unknown sample, that component may be present.
However, because retention time changes depending on temperature conditions and column condition, comparison with standard substances is important.

Example of Component Identification Using Retention Time

Similar retention times provide evidence for component identification, but another component may show a similar retention time.
In important analyses, confirmation is performed using standard addition, GC-MS, or similar methods.

Area Percentage From Peak Area

The relative amounts of components in a mixed sample can be determined from the peak areas of the GC chromatogram.
Here, the calculation is performed as a simple area percentage without correction for differences in detection sensitivity.

The area percentage is calculated using the following equation.

Area percentage (%) = Peak area of each component ÷ Total peak area × 100

If the peak area of ethyl acetate is 315000 and the total peak area is 1000000,

Area percentage = 315000 ÷ 1000000 × 100 = 31.5%

From this result, ethyl acetate can be said to have been detected as the largest component in terms of peak area.

When Correcting Detection Sensitivity

FID responds to many organic compounds, but the sensitivity is not necessarily exactly the same for every component.
Using correction factors may bring the calculated composition closer to the actual composition than simple area percentages.

The corrected percentage is calculated by dividing the corrected area by the total corrected area.
If detector response differs among components, performing this type of correction improves the reliability of quantitation.

Calibration Curve for Ethanol Standard Solutions

A reference example is shown in which ethanol standard solutions at multiple concentrations were measured and the relationship between concentration and peak area was examined.

In this reference example, the relationship between ethanol concentration and peak area is treated as the following calibration curve.

Peak area = 370000 × Concentration (%)

Therefore, the concentration in the unknown sample is determined using the following equation.

Concentration (%) = Peak area ÷ 370000

Example Calculation of Ethanol Concentration in an Unknown Sample

If the ethanol peak area of unknown sample B is 222000, the concentration is determined using the calibration curve.

Ethanol concentration = 222000 ÷ 370000 = 0.600%

Therefore, the ethanol concentration in unknown sample B is determined to be 0.600%.

Example Calculation Including Dilution Factor

If the sample is diluted before measurement, the concentration in the measured solution determined from the calibration curve is multiplied by the dilution factor to obtain the concentration in the original sample.

If the dilution factor is forgotten in the calculation, the concentration in the original sample will be substantially underestimated.

Example of Quantitation Using the Internal-Standard Method

In GC, even small differences in injection volume affect peak area.
Therefore, there is a method in which an internal-standard substance is added and quantitation is performed using the peak-area ratio of the target component to the internal standard.

Here, a reference example using 1-propanol as the internal standard is shown.

In this reference example, the relationship between area ratio and concentration is treated as follows.

Area ratio = 1.85 × Ethanol concentration (%)

If the area ratio of the unknown sample is 1.30,

Ethanol concentration = 1.30 ÷ 1.85 = 0.703%

In the internal-standard method, even if the injection volume changes slightly, the peak-area ratio between the target component and internal standard is used, so the quantitative value tends to be more stable.

Example Calculation of Resolution

Resolution indicates how well adjacent peaks are separated.
If resolution is low, the peaks overlap and errors are more likely to occur in area calculation.

Resolution Rs = 2(tR2 − tR1) ÷ (w1 + w2)

For ethanol and acetone, if the retention times are 1.25 min and 1.80 min and the peak widths are 0.10 min and 0.11 min,

Rs = 2(1.80 − 1.25) ÷ (0.10 + 0.11)

Rs = 1.10 ÷ 0.21 = 5.24

This value is sufficiently large, so ethanol and acetone can be judged to be well separated.

Comparison of Resolution Between Peaks

Changes in Retention Time With Column Temperature

In GC, the higher the column temperature, the more quickly components elute and the shorter their retention times become.
However, if the temperature is too high, peaks may become closer together and separation may become poorer.

If shortening the analysis time alone is prioritized, separation may become poorer.
When the purpose is quantitation, temperature conditions that provide sufficient peak separation must be selected.

Changes in Peak Area With Injection Volume

When the injection volume changes, the peak area also changes.
If the injection volume is too large, the peak shape may deteriorate or the detector’s linear range may be exceeded.

If the injection volume is too large, the peak becomes asymmetric and errors occur in area calculation and reading of retention time.

Examples of Abnormal Peak Shapes

If the peak shape is poor, the quantitative value changes depending on the integration range used for the peak area, reducing the reliability of the result.

Example of Measurement Outside the Calibration-Curve Range

If the peak area of an unknown sample exceeds the range of the standard solutions, determining the concentration by extrapolation may result in a large error.
In this case, the sample is diluted and remeasured.

Example of Confirming Reproducibility

A reference example is shown in which the same sample was injected multiple times and variation in retention time and peak area was checked.

If variation in retention time and peak area is small, the injection operation and instrument condition are considered to have been relatively stable.

Example of How to Write the Results

When the standard mixture was measured by GC, peaks were observed at retention times of 1.25, 1.80, 2.65, and 4.40 min.
Comparison with the retention times of the standard substances suggested that these corresponded to ethanol, acetone, ethyl acetate, and toluene, respectively.
Because peaks were also observed at similar retention times in the unknown sample, the sample is considered highly likely to contain these components.

When area percentages were calculated from the peak areas, the values were 18.5% for ethanol, 26.0% for acetone, 31.5% for ethyl acetate, and 24.0% for toluene.
From this result, ethyl acetate was detected as the largest component in terms of peak area.
However, because detector sensitivity differs among components, area percentage cannot be regarded directly as mass percentage.

When a calibration curve was prepared from the ethanol standard solutions, the relationship between peak area and concentration was expressed as “Peak area = 370000 × Concentration.”
Because the ethanol peak area of unknown sample B was 222000, the ethanol concentration was determined to be 222000 ÷ 370000 = 0.600%.

Points for Connecting the Results to the Discussion

In a discussion of GC analysis, it is important to explain retention time, peak area, area percentage, calibration curves, resolution, and peak shape in relation to one another.

• Can retention time be compared with that of a standard substance to provide a basis for component identification?
• Can it be explained that confirmation by standard addition or GC-MS is necessary when appropriate rather than making a definitive judgment from retention time alone?
• Can the area percentage be calculated from peak area?
• Can it be explained that area percentage does not necessarily represent mass percentage?
• Can the concentration in an unknown sample be determined using a calibration curve?
• If the sample was diluted before measurement, has the dilution factor been correctly taken into account?
• Can it be explained that the internal-standard method can correct for variation in injection volume?
• Can the effects of column temperature and flow rate on retention time and separation be discussed?
• Can it be explained that peak overlap, tailing, and fronting lead to quantitative errors?

Example Discussion

In this experiment, volatile components in a mixed sample were analyzed using gas chromatography.
In the standard mixture, ethanol, acetone, ethyl acetate, and toluene eluted at 1.25, 1.80, 2.65, and 4.40 min, respectively.
Because peaks with retention times close to these were also observed in the unknown sample, the same components were considered highly likely to be present.

When the area percentages were calculated from the peak areas, ethyl acetate was the largest at 31.5%, followed by acetone, toluene, and ethanol.
However, peak area is affected not only by the amount of a component but also by detector sensitivity and the ease of ionization or combustion.
Therefore, area percentage should be treated as an approximate indication of the mixing ratio, and calibration curves or correction factors must be used for accurate quantitation.

For quantitation of ethanol, a calibration curve prepared from the standard solutions was used.
Substituting the peak area of 222000 for unknown sample B into the calibration curve gave an ethanol concentration of 0.600%.
If the sample is diluted before measurement, the concentration in the measured solution must be multiplied by the dilution factor to obtain the concentration in the original sample.
If the dilution factor is not taken into account, the concentration will be substantially underestimated.

In the internal-standard method, quantitation was performed using the peak-area ratio between the target component and the internal-standard substance.
Even if the injection volume changes slightly, the areas of both the target component and the internal standard change in the same direction, so using the area ratio makes it possible to correct for variation in the injection procedure.
This method is particularly effective for manual injection and analysis of volatile samples.

Possible sources of error include variation in injection volume, fluctuations in column temperature, peak overlap, column deterioration, and abnormal peak shape.
Increasing the column temperature shortens the retention time, but the spacing between peaks may become narrower and separation may become poorer.
In addition, if the injection volume is too large, peaks may show fronting and the readings of retention time and area may become unstable.
Therefore, in GC analysis, conditions must be set while checking not only the analysis time but also peak separation and peak shape.

Summary

In GC analysis, components are estimated by comparing retention times with those of standard substances, and component amounts are evaluated from peak areas.
Area percentage can be calculated easily, but because it does not take differences in sensitivity among components into account, calibration curves or the internal-standard method are required for rigorous quantitation.

This reference example used a mixed sample containing ethanol, acetone, ethyl acetate, and toluene to examine retention time, peak area, area percentage, calibration curves, dilution factor, the internal-standard method, resolution, and the effects of peak shape.
In a report, it is useful to discuss identification by retention time, quantitation by peak area, changes in retention time caused by measurement conditions, and sources of error in relation to one another.

How to Read a Chromatogram

A chromatogram obtained by GC is a graph with time on the horizontal axis and detector signal intensity on the vertical axis.
Each component appears as a peak when it reaches the detector.
Components that produce early peaks are considered to be weakly retained in the column, while components that produce late peaks are considered to be strongly retained in the column.

When examining a chromatogram, attention is paid to the number of peaks, retention times, peak areas, peak shapes, and separation.
If peaks overlap, it becomes difficult to accurately identify and quantify the components.
If the peak shape is asymmetric, there may be a problem with the column condition or injection conditions.

What Is Retention Time?

Retention time is the time from injection of the sample until each component reaches the detector and is observed as a peak.
Retention time changes depending on the volatility of the component, interaction with the stationary phase, column temperature, carrier-gas flow rate, type of column, and other factors.
If measured under the same conditions, the same component shows a peak at approximately the same retention time.

Retention time is used for component identification, but it is risky to conclude that components are completely identical simply because their retention times agree.
Because another component with similar properties may show a similar retention time, confirmation is performed when necessary by standard addition, measurement under different conditions, GC-MS, or similar methods.

Causes of Changes in Retention Time

Retention time is a value readily affected by measurement conditions.
As column temperature increases, components move more readily into the gas phase and retention time generally becomes shorter.
If the carrier-gas flow rate is high, the time required for components to pass through the column also becomes shorter, and the retention time may decrease.

Column deterioration, condition of the stationary phase, condition of the inlet, sample amount, solvent, and contamination inside the column also affect retention time.
Measuring standard substances and unknown samples on the same day under the same conditions improves the accuracy of retention-time comparison.

What Is Peak Area?

Peak area is the total area of a peak on the chromatogram.
In many cases, peak area is proportional to the amount of the detected component.
Therefore, GC quantitation generally uses peak area rather than peak height.
This is because even if a peak broadens slightly, the area tends to reflect the amount of the component.

However, appropriate baseline setting and peak separation are important for determining peak area correctly.
If peaks overlap or the baseline is unstable, errors occur in peak-area calculation.
In addition, because detector response differs among components, the area cannot always be treated directly as a concentration ratio.

Difference Between Peak Height and Peak Area

Peak height represents the maximum signal value of a peak.
Peak area, on the other hand, represents the total amount of signal from the entire peak.
If the peak is sharp and symmetrical, peak height can also serve as an approximate indication of the amount of a component, but if the peak broadens or tails, height alone becomes less able to accurately reflect the amount of the component.

In GC quantitation, using peak area is generally more reliable.
However, area calculation is also affected by the baseline and peak separation, so the peak shape and overlap must be checked.

How to Think About Retention Time and Component Identification

The basic method for identifying a component by GC is to compare the peak retention time of the unknown sample with the retention time of a standard substance.
If the standard substance and unknown sample are measured under the same GC conditions and the retention times agree, the peak may be the same component as the standard substance.

However, retention time alone does not provide complete identification.
Multiple components may appear near the same retention time.
For more reliable identification, it is effective to confirm an increase in the peak after addition of the standard substance, perform measurements under different column conditions, or confirm the mass spectrum by GC-MS.

How to Think About Peak Area and Quantitation

GC quantitation uses the relationship in which peak area is proportional to the amount of a component.
Standard solutions of known concentration are measured to prepare a calibration curve showing the relationship between concentration and peak area, and the concentration is determined by substituting the peak area of the unknown sample into the calibration curve.
This method is sometimes called the external-standard method.

However, variation in injection volume, volatilization of the sample, differences in detector response, and peak overlap produce errors in the relationship between peak area and concentration.
For accurate quantitation, it is important to measure the standard solutions and unknown sample under the same conditions and confirm the linearity of the calibration curve.

Discussion of the Calibration Curve

A calibration curve is a graph showing the relationship between the concentration of standard solutions and peak area.
In GC analysis, standard solutions at multiple concentrations are measured to confirm whether peak area increases linearly with concentration.
If the calibration curve shows good linearity, concentration can be determined using peak area within that range.

A high correlation coefficient of the calibration curve is important, but it is not sufficient by itself.
The accuracy of standard-solution preparation, reproducibility of injection volume, outliers, size of the intercept, and linear range of the detector must also be checked.
If the peak area of the unknown sample is outside the calibration-curve range, it is desirable to dilute or concentrate the sample and remeasure it.

Discussion of the External-Standard Method

The external-standard method is a method in which standard solutions and unknown samples are measured separately and the concentration of the unknown sample is determined from a calibration curve prepared using the standard solutions.
It has the advantage of being relatively simple to perform and making calibration-curve preparation easy.
On the other hand, it has the disadvantage of being readily affected by variations in injection volume and instrument condition.

In GC, even small differences in injection volume may affect peak area.
Therefore, in the external-standard method, it is important to improve the reproducibility of the injection procedure.
Reproducibility becomes higher when an autosampler is used, while variation may become larger with manual injection.

Discussion of the Internal-Standard Method

The internal-standard method is a method in which a fixed amount of an internal-standard substance is added to the sample and standard solutions and quantitation is performed using the ratio of the peak area of the target component to that of the internal standard.
Using an internal standard makes it easier to correct for the effects of variation in injection volume and losses during sample preparation.

The internal-standard substance is selected so that it has chemical properties somewhat similar to those of the target component, is not originally present in the sample, and is sufficiently separated from the target component.
If the internal-standard peak overlaps with the target-component peak, the area ratio cannot be determined correctly.

Discussion of the Area-Percentage Method

The area-percentage method determines the component ratio from the proportion of the target peak area relative to the total area of all peaks in the chromatogram.
It is convenient for determining the approximate composition of a mixture, but because detector response is not necessarily the same for every component, it does not necessarily represent an accurate concentration.

Particularly with FID and similar detectors, the response may differ depending on the number of carbon atoms and structure of the component.
Therefore, caution is required when treating area percentage directly as weight percent or mole percent.
Accurate quantitation may require correction of response factors using standard substances.

Discussion of Peak Separation

In GC analysis, it is important that the peaks of the target component and other components be sufficiently separated.
If peaks overlap, identification by retention time becomes difficult and the peak area cannot be determined accurately.
If separation is insufficient, the amount of the target component may be overestimated or underestimated.

To improve separation, the column temperature, temperature program, type of column, carrier-gas flow rate, and injection volume are reviewed.
In general, lowering the temperature may improve separation, but the analysis time becomes longer.
Adjustment of the temperature-program conditions is particularly important in multicomponent analysis.

Discussion of Resolution

Resolution is an indicator of how well two peaks are separated.
The higher the resolution, the less the peaks overlap and the higher the reliability of identification and quantitation.
If the resolution is low, division of peak areas becomes inaccurate and quantitative errors increase.

Resolution is related to the difference in retention time and peak width.
Even if the retention times are sufficiently separated, broad peaks readily overlap.
Conversely, even if the retention times are close, sharp peaks are easier to separate.

Discussion When Peaks Overlap

If peaks overlap in GC, multiple components may be eluting at similar retention times.
If the peaks overlap completely, they may appear as a single peak, causing the number of components to be underestimated.
Even when they overlap only partially, errors occur when dividing the peak areas.

Countermeasures for peak overlap include changing the temperature conditions, changing the temperature-program rate, using a column with different polarity, and adjusting the sample concentration or injection volume.
Adding a standard substance and observing how the peak changes can also provide a clue for identification.

Discussion When Peaks Are Broad

Causes of broad peaks include excessive injection volume, column deterioration, excessively low column temperature, inappropriate carrier-gas flow rate, excessive retention of the sample in the column, and excessive diffusion within the instrument.
Broad peak width reduces resolution and also reduces the accuracy of reading peak area.

Peaks may also broaden if the sample solvent is incompatible with the column conditions or if the sample is not properly vaporized at the inlet.
Peak width is important information for judging the quality of separation.

Discussion When Peaks Tail

The phenomenon in which a peak trails toward the back is called tailing.
Tailing may occur when a component interacts strongly with the column, inlet, liner, or active sites.
Tailing may occur readily with polar compounds, acidic or basic compounds, and components that readily adsorb.

When tailing occurs, calculation of peak area and reading of retention time become inaccurate.
Possible countermeasures include replacing the column, replacing the liner, cleaning the inlet, derivatization, and changing the column conditions.

Discussion When Peaks Broaden Toward the Front

The phenomenon in which the front side of a peak broadens is called fronting.
Fronting may occur when an amount of sample exceeding the column capacity is injected or when the retention capacity of the column is insufficient.
If the injection volume is too large, the peak shape deteriorates and accurate quantitation becomes difficult.

Methods for preventing fronting include diluting the sample, reducing the injection volume, increasing the split ratio, and reviewing the column conditions.
If the peak shape is poor, it is important to check not only the area value but also the injection conditions.

Effect of Column Temperature

Column temperature is a very important condition in GC analysis.
The higher the temperature, the more readily components move into the gas phase and the shorter their retention times become.
On the other hand, at lower temperatures, retention times become longer and separation may improve, but the analysis time may increase and peaks may broaden.

For multicomponent mixtures, a temperature program may be used to analyze low-boiling and high-boiling components simultaneously.
If the temperature ramp is too fast, peaks are more likely to overlap, while if it is too slow, the analysis time becomes long.

Effect of Carrier-Gas Flow Rate

The carrier gas is the gas that transports vaporized sample components through the column.
If the flow rate is high, components move more quickly and retention times become shorter.
However, if the flow rate is too high, components may reach the detector before they have been sufficiently separated, resulting in poorer separation.

Conversely, if the flow rate is too low, the analysis time becomes long and peaks may broaden.
There is an optimum range for the carrier-gas flow rate, and the balance between separation efficiency and analysis time must be considered.

Interaction Between the Stationary Phase and Components

The stationary phase inside a GC column greatly affects component retention time and separation.
The more strongly a component interacts with the stationary phase, the longer it remains in the column and the longer its retention time becomes.
Polar compounds tend to be retained by polar stationary phases, while in nonpolar stationary phases, boiling point and dispersion forces may have a greater effect.

The elution order can be explained by considering not only the boiling points of components but also polarity, molecular weight, functional groups, and interactions with the stationary phase.
Even components with similar boiling points may have different retention times if their interactions with the stationary phase differ.

Relationship Between Boiling Point and Retention Time

In general, on a nonpolar column, components with lower boiling points tend to elute earlier, while components with higher boiling points tend to elute later.
This is because lower-boiling components exist more readily in the gas phase and move through the column more quickly.
On the other hand, higher-boiling components tend to remain in the stationary phase and have longer retention times.

However, for polar compounds or components having particular functional groups, interactions with the stationary phase may be strong and the elution order cannot always be explained by boiling point alone.
GC retention time is determined by both volatility and interaction with the stationary phase.

Effect of Injection Volume

In GC, injection volume greatly affects peak area and peak shape.
The larger the injection volume, the larger the peak area becomes, but if it is too large, the column becomes overloaded and peaks may broaden or show fronting.
If the injection volume is too small, peaks become small and more susceptible to noise.

In quantitative analysis, the injection volumes of standard solutions and unknown samples must be kept constant.
With manual injection, variation in injection volume and injection speed may occur readily and affect the reproducibility of peak area.

Effect of Split Ratio

In split injection, only part of the injected sample is introduced into the column and the remainder is discharged.
The larger the split ratio, the smaller the amount of sample entering the column.
For concentrated samples, increasing the split ratio can prevent column overload and improve peak shape.

On the other hand, if the split ratio is too large for a low-concentration sample, the amount of sample entering the column becomes small, causing the peak to become too small and making detection difficult.
It is important to select an appropriate split ratio according to sample concentration.

Discussion of Detector Response

In GC, the components that can be detected and the sensitivity differ depending on the type of detector.
FID is commonly used for detecting organic compounds and shows high sensitivity to many compounds containing carbon.
TCD is relatively versatile, but its sensitivity may be lower than that of FID.
ECD is highly sensitive to halogenated compounds and similar substances.

Because detector response differs among components, caution is required when simply comparing peak areas to judge relative component amounts.
For accurate quantitation, it is desirable to prepare a calibration curve using a standard substance for each analyte.

Discussion of GC-FID

GC-FID is GC analysis using a flame-ionization detector.
Organic compounds are combusted and ionized in a hydrogen flame, and the resulting ion current is detected.
FID is characterized by high sensitivity to many organic compounds and relatively good quantitative performance.

However, the FID response may vary depending on the number of carbon atoms and structure of the component.
It may respond poorly to water, carbon dioxide, inorganic gases, and similar substances.
Therefore, although peak area obtained by FID reflects the amount of organic components, response factors for individual components must be taken into account.

Difference From GC-MS

GC-MS is a method in which components separated by GC are detected with a mass spectrometer.
With GC alone, components are estimated from retention time, whereas GC-MS provides molecular-weight and fragment information from mass spectra, increasing the reliability of identification.

On the other hand, ordinary GC-FID has excellent quantitative performance, but identification based on retention time alone may be insufficient.
GC-MS is effective for identifying unknown components, while GC-FID may be easier to use for quantitation of known components.

Causes of Quantitative Values Being Too High

Causes of GC quantitative values being higher than the actual value include peak overlap, errors in baseline setting, excessive injection volume, mistakes in preparing standard-solution concentrations, mistakes in calculating dilution factors, and components other than the target component eluting at the same retention time.
When peaks overlap, the area of another component is included in the peak area of the target component, potentially causing the concentration to be overestimated.

Causes of Quantitative Values Being Too Low

Causes of GC quantitative values being lower than the actual value include volatile loss of the sample, insufficient injection volume, decomposition at the inlet, adsorption, underestimation of peak area, errors in standard-solution concentration, and losses during sample preparation.
For highly volatile components, part of the component may evaporate during sample preparation or storage, causing the measured concentration to be lower than the actual concentration.

In addition, polar components and components that readily adsorb may adsorb at the inlet or inside the column, reducing the amount that reaches the detector.
In such cases, the peak area becomes smaller and the concentration may be underestimated.

Discussion of the Baseline

The baseline is the reference signal line in portions where no peak is present.
If the baseline is stable, peak area can be determined accurately.
However, if the baseline is sloped, drifting, or contains large noise, errors occur in peak-area calculation.

Baseline disturbances may be caused by column bleed, the temperature program, detector stability, sample contamination, and effects of solvent peaks.
Particularly when quantifying small peaks, baseline setting greatly affects the result.

Discussion of Noise

Noise is unwanted fluctuation contained in the detector signal.
If noise is large, detection of small peaks and calculation of their areas become difficult.
In low-concentration samples, the peak signal is small, so the effect of noise becomes larger and quantitative accuracy decreases.

Causes of noise include detector instability, fluctuations in gas flow rate, electrical noise, contamination, column deterioration, and unstable temperature control.
If noise is large, stabilization of the instrument, blank measurements, and checking the condition of the column and detector are necessary.

Importance of Standard Substances

In GC analysis, standard substances can be used to confirm retention time and prepare calibration curves.
Without a standard substance, it becomes difficult to identify a component reliably from retention time alone.
Measuring a standard substance under the same conditions makes it easier to compare peaks in the unknown sample.

In quantitative analysis, it is important to prepare the concentration of the standard substance accurately.
If there is an error in the concentration of the standard solution, the entire calibration curve shifts and the quantitative value of the unknown sample is also affected.

Errors Caused by Sample Preparation

In GC analysis, sample preparation greatly affects the results.
Errors in dilution factor, mistakes in preparing standard solutions, volatile loss of the sample, inappropriate solvent selection, sample decomposition, and adsorption to containers are sources of error.
When handling volatile components, it is important not to leave the sample open for a long period.

In addition, if the sample contains large amounts of water or nonvolatile components, the inlet or column may become contaminated.
Pretreatment such as extraction, dilution, filtration, or drying is performed when necessary.

Discussion of Decomposition and Adsorption at the Inlet

In GC, the sample is vaporized at a high-temperature inlet.
Thermally unstable components may decompose at the inlet.
In addition, highly polar or highly reactive components may adsorb to the inlet liner or column entrance.
If these occur, peaks may become smaller, show tailing, or separate peaks from decomposition products may appear.

Possible countermeasures include setting an appropriate inlet temperature, replacing the liner, derivatization, and using a low-activity column.
For components that readily undergo thermal decomposition, it is necessary to consider whether the GC conditions themselves are appropriate.

When GC Analysis Can Be Judged to Have Given Good Results

GC analysis can be judged to have given good results when the peak of the target component appears clearly at the same retention time as the standard substance, is sufficiently separated from adjacent peaks, has a symmetrical peak shape, and the calibration curve shows good linearity.
It is also important that the peak area of the unknown sample be within the calibration-curve range and that similar values be obtained upon remeasurement.

Agreement of retention time is important for identification, while the validity of peak area and the calibration curve is important for quantitation.
The less peak overlap, tailing, and baseline disturbance there is, the easier it is to judge the result as reliable.

Example Discussion When the Experiment Did Not Go Well

When GC analysis does not go well, possible causes are considered from results such as no visible peak, shifted retention time, overlapping peaks, broad peaks, tailing, variation in peak area, a nonlinear calibration curve, or a quantitative value that deviates greatly.
Organizing the causes according to sample preparation, injection procedure, column conditions, temperature conditions, detector, and baseline makes the discussion easier.

How to Write Points for Improvement

In a discussion of GC analysis, including not only sources of error but also points for improvement makes the report easier to organize.
Improvements can be organized by dividing them into sample preparation, injection procedure, separation conditions, quantitative methods, and instrument management.

Improvements to Sample Preparation

• Prepare standard solutions accurately
• Manage dilution factors accurately
• Prevent loss of volatile components
• Store the sample in a sealed container
• Perform filtration or extraction when necessary
• Reduce water and nonvolatile components in the sample

Improvements to the Injection Procedure

• Keep the injection volume constant
• Standardize the injection speed
• Remove bubbles from the syringe
• Use an autosampler
• Adjust the split ratio according to sample concentration
• Set an appropriate inlet temperature

Improvements to Separation Conditions

• Adjust the column temperature
• Review the temperature ramp rate
• Optimize the carrier-gas flow rate
• Consider a column with different polarity
• Reduce the injection volume to improve peak shape
• Find conditions that avoid peak overlap

Improvements to Quantitation and Analysis

• Confirm the linear range of the calibration curve
• Confirm that the unknown sample is within the calibration-curve range
• Use the internal-standard method
• Check baseline settings
• Confirm reproducibility of peak area
• Perform multiple measurements and show the average value and variation

Difference Between a Superficial Discussion and a Good Discussion

In a discussion of GC analysis, simply writing that “a peak appeared,” “the retention times were the same,” or “the area was large” results in a superficial discussion.
A good discussion relates retention time, peak area, separation state, calibration curve, injection conditions, and sources of error.

Examples of Expressions That Can Be Used in Reports

The following expressions can be used when writing the results and discussion of GC analysis.
Adjust the necessary parts according to your own experimental results.

• Retention time indicates the time required for a component to move through the column and reach the detector.
• Because the peak retention time of the unknown sample agreed with that of the standard substance, the presence of the target component was suggested.
• Peak area corresponds to the amount of detected component and can be used for quantitative analysis.
• Because the calibration curve showed linearity, quantitation using peak area was possible within this concentration range.
• If peaks overlap, it becomes difficult to determine peak area accurately and errors occur in quantitative values.
• A shift in retention time may be caused by changes in column temperature, carrier-gas flow rate, or column condition.
• Variation in injection volume directly affects peak area and therefore causes variation in quantitative values.
• Tailing may occur when a component adsorbs to active sites in the column or inlet.
• In the area-percentage method, differences in detector response among components must be considered.
• Using the internal-standard method makes it easier to correct for the effects of variation in injection volume.

Points to Check When Discussing GC Analysis

Checking the following points before writing the report makes the discussion easier to write.

• Are the GC conditions clearly stated?
• Has retention time been compared with that of a standard substance?
• Has the basis for using peak area for quantitation been stated?
• Are peak height and peak area kept separate?
• Are the calibration-curve equation and correlation coefficient shown?
• Has it been confirmed that the unknown sample is within the calibration-curve range?
• Has it been confirmed that peak separation is sufficient?
• Have the effects of peak overlap and tailing been considered?
• Have the effects of injection volume and split ratio been considered?
• Have the effects of column temperature and carrier-gas flow rate been considered?
• Are the differences between the internal-standard and external-standard methods understood?
• Do the points for improvement correspond to the sources of error?

Summary

In GC analysis, volatile components in a mixture are separated by a column and identified and quantified based on retention time and peak area.
Retention time is the time required for a component to reach the detector and can be used to estimate components by comparison with standard substances.
Because peak area corresponds to the amount of detected component, it can be used for quantitation with a calibration curve.

However, retention time changes depending on column temperature, carrier-gas flow rate, and column condition.
Peak area is also affected by injection volume, baseline, peak separation, detector response, and sample preparation.
Therefore, in a discussion of GC analysis, identification should not be concluded solely from agreement of retention time, and peak separation and comparison with standard substances must also be confirmed.

In a report, rather than simply writing that “a peak appeared” or “the area was large,” discuss retention time, peak area, calibration curves, resolution, injection volume, column conditions, and sources of error in relation to one another.
For accurate quantitation, it is important to measure within the linear range of the calibration curve, use the internal-standard method when necessary, and analyze under conditions in which the peaks are sufficiently separated.