What is Chromatography?
Chromatography is a analytics technique used to separate the various components of a mixture, operating on the principle of phase separation. Initially, the sample containing the mixture of substances to be separated is prepared, with the amount of sample depending on the type of chromatography and the dimensions of the column or plate used.
The stationary phase consists of a fixed material that remains immobile during the procedure, which can be a solid (as in thin-layer or column chromatography) or a gel (as in size exclusion chromatography). The choice of the chemical nature of the stationary phase is determined by the properties of the substances to be separated, such as polarity and molecular size.
On the other hand, the mobile phase refers to a liquid or gas that moves through or over the stationary phase. The selection of the mobile phase also relates to the properties of the substances present in the sample. In liquid chromatography, the mobile phase can be an aqueous or organic solution, while in gas chromatography, an inert gas is used.
During the process, the sample is applied to the stationary phase, and then the mobile phase is driven through it. As the mobile phase moves, the sample components are carried along with it. The interaction of different sample components with the stationary and mobile phases varies: some move quickly, while others, due to greater interaction with the stationary phase, move more slowly. This difference in movement rates leads to the separation of the components along the stationary phase.
After the components are separated, they are detected and quantified. This detection can occur in various ways, depending on the type of chromatography employed. For example, a UV detector can be used to identify the compounds as they pass through a specific point of the stationary phase.
The result of chromatography is usually presented in the form of a chromatogram, which is a graph showing the different components of the sample separated along the horizontal axis (time or distance), and the quantity of each component along the vertical axis (intensity of the detector signal).
Applications of Chromatography
Chromatography has a wide range of applications in various fields. The company Openscience has several applications in different research areas.
Application of High Resolution Mass Spectrometry and Chemometrics in 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 with high resolution mass spectrometry (FIA-HRMS), as an alternative to traditional sensory analysis, which is a laborious and subjective method. More than 25 types of coffee capsules were studied. The samples were divided into aqueous and organic extracts, analyzed by FIA-HRMS in both positive and negative ionization modes. Data fusion from 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 constructed and trained to determine the type of capsule and estimate important coffee parameters (such as acidity, bitterness, body, intensity, and roast level), achieving accuracy values higher than 91.1%. Moreover, variable importance in projection (VIP) scores allowed for the assignment of elemental composition and, in some cases, the putative identification of compounds in coffee (such as caffeine, caffearine, and quinides) that played a significant role in class discrimination.
Analysis of Chemiosensory Markers in Cigarette Smoke from Different Tobacco Varieties by GC×GC-TOFMS and Chemometrics
This study proposes a new approach to identify chemiosensory markers in cigarette smoke from different tobacco varieties, using comprehensive two-dimensional gas chromatography (GC×GC) coupled with time-of-flight mass spectrometry (TOFMS), in combination with chemometric techniques. Four main types of tobacco were analyzed, considering different stages of curing and geographic regions. The goal was to link sensory markers to tobacco varieties and their cultivation and curing practices. Partial least squares discriminant analysis (PLS-DA) was used to classify the samples and identify key chemiosensory markers. The study successfully established a clear connection between the compounds identified in cigarette smoke and specific tobacco characteristics. In total, 1,098 markers were highlighted, of which 173 were provisionally identified, providing significant insights into the chemical composition of cigarette smoke and its sensory implications.
Enhancing Analytical Response through Optimization of 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 analytical response. The methodology was applied to the analysis of a group of pesticides to demonstrate its effectiveness. The procedure allowed for the establishment of experimental parameters based on theoretical aspects, enabling better instrumental response, reduction in sample volumes needed, minimization of time and costs, and simplification of 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.
High-Efficiency Liquid Chromatography Applications for the Study of Emerging Organic Pollutants
This work developed and validated a method for the multi-residue determination of 20 pesticides in drinking water, employing solid-phase extraction followed by gas chromatography coupled with mass spectrometry in the selected ion monitoring (SIM) mode. The method aimed to meet the limits regulated by Brazilian legislation for the presence of pesticides in drinking water, requiring high analytical sensitivity due to the low concentrations of interest. The quantification limits of the method ranged between 0.003 and 0.093 µg L-1, and the average recoveries of the analyzed compounds were between 51 and 116%. The study highlighted the importance of proper sample preparation, leading to highly selective and sensitive chromatographic analysis. The validation of the method showed excellent precision and suitability for routine analysis, meeting 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 various types of chromatography, depending on the nature of the sample and the compounds to be separated.
Here are some chromatography techniques:
Gas Chromatography (GC): Uses a gas as the mobile phase 🌬️ and a column coated with 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 solid material as the stationary phase. It is ideal for analyzing a wide variety of compounds, including those that are not volatile or thermally unstable.
Thin Layer Chromatography (TLC): A simple and fast technique where the sample is applied on a plate made of glass, plastic, or metal 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.
Ion Exchange Chromatography: Based on the attraction between charged ions present in the sample and oppositely charged ions fixed 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 smaller molecules to enter the pores of the stationary phase, while larger molecules are excluded and eluted 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 where 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.
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