Gas Chromatography (GC) is an analytical technique used to separate and analyze the individual chemical components within a complex mixture. The method involves vaporizing a sample and carrying its components through a specialized column using an inert gas, known as the mobile phase. As substances travel through the column, they separate based on their unique physical and chemical properties. The output of this separation is a graphical representation called a chromatogram, which is used for interpreting the sample’s composition.
Visualizing Separation in a Chromatogram
The chromatogram provides a visual map of the separation achieved inside the gas chromatograph’s column. This graph is structured around two primary axes that convey specific information about the sample’s components. The horizontal axis, or X-axis, represents the Retention Time, which is the total time elapsed from the moment the sample is injected until a component reaches the detector.
The vertical axis, or Y-axis, measures the Detector Response, which is the electrical signal generated when a separated component passes through the instrument’s detector. This response is directly related to the amount of substance present at that specific moment. As each compound exits the column and hits the detector, the signal rises sharply from the flat baseline and then returns, creating a distinct, bell-shaped feature known as a peak.
Each peak on the chromatogram signifies a different, separated chemical compound. The position of the peak along the Retention Time axis helps to identify the substance, while the size of the peak provides an indication of its quantity. A larger peak suggests a greater amount of that specific compound was present in the sample.
The size of the peak is not measured by its height alone, because variations in the chromatography system can broaden the peak without changing the total amount of substance. Therefore, the total area enclosed by the peak and the baseline offers a more reliable measure of the compound’s quantity.
Calculating Peak Area Through Integration
The peak area is defined as the total space enclosed beneath the chromatographic curve, measured from the point where the signal first deviates from the baseline to where it returns to the baseline. This area represents the cumulative detector response over the time the entire component passed through the detector. Measuring this area is a process called integration.
Modern gas chromatographs use specialized data processing software to perform this integration automatically and precisely. The software effectively divides the area under the curve into thousands of tiny, vertical slices. It sums the area of all these minute slices to yield a single numerical value for the peak area.
For this integration to be accurate, the software must correctly establish the baseline, which is the signal level when no substance is eluting from the column. The software must also correctly identify the start and end points of the peak, a process that relies on detecting subtle changes in the signal’s slope.
The resulting numerical value for the peak area is typically reported in arbitrary units, such as volt-seconds, representing the signal intensity integrated over time. This value does not inherently represent a mass or concentration until it is correlated with a known standard. The peak area serves as the primary metric for quantitative analysis.
Using Peak Area for Quantification
The detector response is linearly proportional to the amount of substance passing through it, making peak area the direct link for determining mass or concentration. For example, doubling the compound’s quantity will result in a near-doubling of the peak area, assuming instrument parameters remain constant. This linear relationship is the foundation of quantitative analysis in gas chromatography.
To translate the arbitrary area units into a tangible unit like parts per million (ppm) or milligrams per liter (mg/L), analysts employ standardization techniques. External standardization involves running several samples of the compound at known concentrations and plotting their resulting peak areas to create a calibration curve. This curve establishes the mathematical relationship between area and quantity, which is then used to calculate the concentration of the compound in an unknown sample.
Internal standardization involves adding a fixed, known amount of a reference compound, called the internal standard, to every sample and standard. Instead of using the absolute peak area, the analyst calculates the ratio of the target compound’s peak area to the internal standard’s peak area. This area ratio is then plotted against the concentration ratio to create the calibration curve.
The benefit of internal standardization is that it accounts for slight variations in the volume of sample injected into the instrument. Since both the target compound and the internal standard are affected equally by injection variability, the resulting area ratio remains consistent.