What is chromatography?
Chromatography is an analytical technique used to separate the various components of a mixture, operating on the principle of phase separation. First, the sample containing the mixture of substances to be separated is prepared; the amount of sample depends on the type of chromatography and on the dimensions of the column or plate used.
The stationary phase is a fixed material that remains immobile during the procedure, and may be a solid (as in thin-layer or column chromatography) or a gel (as in size exclusion chromatography). The chemical nature of the stationary phase is chosen according to the properties of the substances to be separated, such as polarity and molecular size.
The mobile phase, in turn, is a liquid or gas that moves through or over the stationary phase. The choice of mobile phase is also related to the properties of the substances present in the sample. In liquid chromatography, the mobile phase may be an aqueous or organic solution, whereas in gas chromatography an inert gas is used.
During the process, the sample is applied to the stationary phase and the mobile phase is then passed through it. As the mobile phase advances, the sample components are carried along with it. The interaction of the different sample components with the stationary and mobile phases varies: some move quickly, while others, owing to stronger interaction with the stationary phase, move more slowly. This difference in migration rates leads to the separation of the components along the stationary phase.
After the components are separated, they are detected and quantified. Detection can be carried out in several ways, depending on the type of chromatography employed. For example, a UV detector can be used to identify the compounds as they pass a specific point of the stationary phase.
The result of a chromatographic analysis is usually presented as a chromatogram, a plot showing the different sample components separated along the horizontal axis (time or distance) and the amount of each component along the vertical axis (detector signal intensity).
Applications of Chromatography
Chromatography has a wide range of applications in many fields. The company Openscience has several applications in different research areas
Application of High-Resolution Mass Spectrometry and Chemometrics to the Evaluation of Coffee Sensory Profiles
This study presents a rapid method for estimating sensory parameters of commercial roasted coffee capsules using flow injection analysis coupled to high-resolution mass spectrometry (FIA-HRMS), as an alternative to traditional sensory analysis, which is laborious and subjective. More than 25 types of coffee capsules were studied. The samples were divided into aqueous and organic extracts, analyzed by FIA-HRMS in positive and negative ionization modes. Data fusion of these mass spectra was performed to explore complementary information from sample preparation and ionization conditions. Orthogonalized partial least squares discriminant analysis (OPLS-DA) models were built and trained to determine the capsule type and estimate important coffee parameters (such as acidity, bitterness, body, intensity and roast level), achieving accuracy values above 91.1%. In addition, variable importance in projection (VIP) scores made it possible to assign elemental compositions and, in some cases, the putative identification of compounds in coffee (such as caffeine, caffearine and quinides) that played an important role in class discrimination.
Analysis of chemosensory markers in cigarette smoke from different tobacco varieties by GC×GC-TOFMS and chemometrics
This study proposes a new approach to identify chemosensory markers in cigarette smoke from different tobacco varieties, using two-dimensional gas chromatography (GC×GC) coupled to time-of-flight mass spectrometry (TOFMS), in combination with chemometric techniques. Four main types of tobacco were analyzed, considering different curing stages and geographic regions. The aim was to link the sensory markers to the tobacco varieties and to their cultivation and curing practices. Partial least squares discriminant analysis (PLS-DA) was used to classify the samples and identify the key chemosensory markers. The study was able to establish a clear link between the compounds identified in cigarette smoke and the specific characteristics of the tobacco. In total, 1,098 markers were highlighted, of which 173 were tentatively identified, providing significant insights into the chemical composition of cigarette smoke and its sensory implications.
Increasing analytical response by optimizing the splitless injection system in gas chromatography using the ideal gas law
This article discusses the development of a simple, fast and effective procedure to optimize the splitless injection system in gas chromatography, using the ideal gas law to improve the analytical response. The methodology was applied to the analysis of a group of pesticides to demonstrate its effectiveness. The procedure made it possible to establish experimental parameters based on theoretical considerations, enabling a better instrumental response, a reduction in the required sample volumes, savings in time and cost, and simplified sample preparation. The study focused on optimizing chromatographic parameters, particularly the injection system, to increase analytical sensitivity, allowing more efficient analysis of volatile and thermally stable compounds, such as pesticides, in environmental and health contexts where low quantification limits are required.
Applications of high-performance liquid chromatography to the study of emerging organic pollutants
This work developed and validated a method for the multiresidue determination of 20 pesticides in drinking water, employing solid-phase extraction followed by gas chromatography coupled to mass spectrometry in selected ion monitoring (SIM) mode. The method aimed to meet the limits set by Brazilian legislation for the presence of pesticides in drinking water, which requires high analytical sensitivity because of the low concentrations of interest. The method's limits of quantification ranged from 0.003 to 0.093 µg L-1, and the mean recoveries of the analyzed compounds were between 51 and 116%. The study highlighted the importance of adequate sample preparation, leading to a highly selective and sensitive chromatographic analysis. Method validation showed excellent precision and suitability for routine analysis, satisfying the quality control and compliance requirements for drinking water.
Types of Chromatography
Chromatography is a separation technique used to divide the components of a mixture based on their physical and chemical interactions. There are several types of chromatography, depending on the nature of the sample and the compounds to be separated!
Get to know some chromatography techniques:
Gas Chromatography (GC): Uses a gas as the mobile phase 🌬️ and a column coated with a solid or liquid material as the stationary phase. It is especially useful for separating and analyzing compounds that can be vaporized without decomposition.
High Performance Liquid Chromatography (HPLC): Uses a liquid as the mobile phase and a column packed with a solid material as the stationary phase. It is ideal for analyzing a wide variety of compounds, including those that are non-volatile or thermally unstable.
Thin Layer Chromatography (TLC): A simple and fast technique in which the sample is applied to a glass, plastic or metal plate coated with an adsorbent material (usually silica gel). The mobile phase (a solvent or mixture of solvents) migrates up the plate, carrying the sample components with it.
Ion Exchange Chromatography: Based on the attraction between charged ions in the sample and oppositely charged ions bound to the stationary phase. It is commonly used to purify proteins and other biomolecules.
Size Exclusion Chromatography (SEC) or Gel Permeation Chromatography: Separates molecules based on size, allowing small molecules to penetrate the pores of the stationary phase while larger molecules are excluded and elute first.
Affinity Chromatography: Exploits the specific interaction between a ligand and a molecule of interest to separate and purify that molecule from a mixture. It is often used in biochemistry to purify proteins and nucleic acids.
Reverse Phase Chromatography (RPC): A variant of HPLC in which the stationary phase is hydrophobic (usually silica-derived) and the mobile phase is more polar. It is widely used to separate compounds based on their hydrophobicity.
References:
Cardoso, V. G. K., Sabin, G. P., & Hantao, L. W. (2021). Fast High–Resolution Mass Spectrometry and Chemometrics for Evaluation of Sensory Parameters of Commercial Coffee Blends. Brazilian Journal of Analytical Chemistry, 8(32), 91-106.
Schwanz, T. G., Bokowski, L. V., Marcelo, M. C., Jandrey, A. C., Dias, J. C., Maximiano, D. H., ... & Kaiser, S. (2019). Analysis of chemosensory markers in cigarette smoke from different tobacco varieties by GC× GC-TOFMS and chemometrics. Talanta, 202, 74-89.
Sabin, G. P., Prestes, O. D., Adaime, M. B., & Zanella, R. (2009). Multiresidue determination of pesticides in drinking water by gas chromatography-mass spectrometry after solid-phase extraction. Journal of the Brazilian Chemical Society, 20, 918-925.