Chromatography is an analytical technique used to separate and analyze the individual components within a complex mixture. The process works by passing a sample through a system with two phases: a stationary phase and a mobile phase. Different substances travel at different speeds based on their physical and chemical properties, allowing them to be separated. The visual output of this separation process is called a chromatogram, a graph that serves as a fingerprint of the mixture’s composition. The chromatogram provides the data necessary to determine both what substances are present and their concentration in the original sample.
Deciphering the Chromatogram Structure
The chromatogram is a two-dimensional plot that records the detector’s response as the sample components exit the separation system. The horizontal axis (X-axis) represents time, specifically the retention time (\(R_t\)). \(R_t\) is the period from the moment the sample is injected until a specific component reaches the detector. This measurement indicates how long a substance was retained by the stationary phase.
The vertical axis (Y-axis) shows the signal intensity or detector response. This signal is generated by a detector that measures a property of the substance as it passes, such as light absorbance or electrical conductivity. The height of the graph indicates the strength of the signal, which is proportional to the amount of substance passing through the detector. A distinct, bell-shaped curve rising above the baseline is called a peak, and each peak represents a separated component of the original mixture.
The flat line at the bottom of the graph, existing before and between the peaks, is the baseline. The baseline reflects the background signal when only the mobile phase is flowing and no separated components are present. Small, random fluctuations in this baseline are referred to as noise. A consistently low, straight baseline indicates that the instrument is operating effectively.
Qualitative Analysis: Identifying Components
Qualitative analysis focuses on determining the identity of the substances present within the mixture. This determination relies on a substance’s unique retention time (\(R_t\)). Under fixed experimental conditions, such as temperature and flow rate, a particular chemical compound will always reach the detector at the same \(R_t\). This time measurement acts like a chemical signature for that compound.
To identify an unknown peak, its \(R_t\) must be compared to the \(R_t\) of a known standard (a pure, reference material) run under identical conditions. If the \(R_t\) of the unknown peak matches the standard’s \(R_t\), it suggests the identities are the same. For complex samples, analysts may inject the unknown sample mixed with a known standard; if the target peak increases in size without changing its \(R_t\), the identity is confirmed.
\(R_t\) is not an absolute proof of identity because multiple compounds could potentially share the same \(R_t\). Analysts must maintain consistent conditions, as minor changes in factors like mobile phase composition or column temperature can alter a compound’s \(R_t\). Comparing the peak’s \(R_t\) to reference databases or utilizing advanced detectors like mass spectrometers can further validate the identification.
Quantitative Analysis: Measuring Concentration
Quantitative analysis determines the concentration of an identified substance in the sample. This measurement relies on the principle that the size of a peak is proportional to the compound’s concentration. Peak size is calculated by determining the peak area, which is the space enclosed by the peak curve and the baseline. This calculation process is called peak integration, typically performed by the instrument’s software.
Peak area is preferred over peak height for quantification because peak shape can be influenced by slight variations in instrument conditions. These variations can alter the height without changing the total area. The area provides a more robust and accurate measure of the total detector response generated by the compound. A greater peak area indicates a higher concentration of that component in the original mixture.
To convert a measured peak area into a meaningful concentration value, an analyst must use a calibration curve. This curve is created by running a series of standard solutions, each containing a known, precise concentration of the target compound. The peak area measured for each standard is plotted against its known concentration, establishing a linear relationship. The unknown sample’s peak area is then applied to this established curve, allowing its concentration to be accurately extrapolated. This calibration process ensures the instrument’s response is reliably translated into a numerical concentration.