CHROMATOGRAM: WHAT IT IS FOR AND TYPES - CHEMISTRY - 2025

The chromatogram is a two-dimensional graphic record obtained on an absorbent medium, showing the separation of substances by chromatography. A visible pattern, peaks or spots, forms on the chromatogram, reflecting the physical separation of the components of a mixture.

The lower figure is a chromatogram with three peaks, A, B and C, of ​​three components of the sample separated by chromatography. It is observed that each of the three peaks has a different height and location on the time axis of the chromatogram.

Typical chromatogram. Source: Heliagon, from Wikimedia Commons

The ordinate or Y axis records signal intensity information (in millivolts mV in this case). Represents the record, depending on the detector, of some physical property of the substance or separate component of the mixture.

The height of the peak is proportional to the concentration of the component separated from the sample in an optimal system. Thus, for example, it is easy to visualize that component B is in a higher proportion than A and C.

On the abscissa or X axis, the retention time of the components of the sample or mixture is represented. It is the time that elapses from the injection of the sample until it stops, being it different for each pure substance.

What is a chromatogram for?

It is the final record of the entire chromatography process. Parameters that are of analytical interest are obtained from it. This can be obtained as an electronic file, a printed histogram or on the process medium; on paper, for example.

The Y axis is generated by signal or intensity response detectors, such as spectrophotometers. An optimal analysis of the time, of the characteristics of the peaks or spots obtained is essential; the size, location, color, among other aspects.

The analyzes of the chromatograms generally require the use of controls or standards, substances of known identity and concentration. The analysis of these controls allows to establish by comparison with the characteristic samples of the components of the investigated sample.

In the chromatogram you can observe and analyze how the separation of the components of a mixture was carried out. Its optimal study allows to identify a substance, demonstrate its purity, quantify the amount of substances present in a mixture, among other aspects.

The information extracted can be of a qualitative nature; for example, when substances are identified and their purity determined. Quantitative information relates to the determination of the number of components in the mixture and the concentration of the separated analyte.

Identification of substances

By analyzing the results of the chromatogram, various substances can be identified by comparing the retention times with that of known substances. It can be observed if the substances under study travel the same distance if they have the same time as the known substances.

For example, the chromatogram can detect and identify metabolites of drugs such as stimulants and steroids in the urine of athletes. It is an important support in the study and research of some metabolites produced by genetic disorders in the newborn.

The chromatogram facilitates the detection of halogenated hydrocarbons present in drinking water, among other substances. It is essential in quality control laboratory analysis, since it allows detecting and identifying the contaminants present in the various products.

Classification of purity of substances

In a chromatogram you can distinguish between pure and impure substances. A pure substance would produce a single peak on the chromatogram; whereas an impure substance would produce two or more peaks.

By properly adjusting the conditions under which chromatography is performed, two substances can be prevented from forming a single peak.

Quantification of substances

By analyzing the peak area of ​​the chromatogram, the concentration of the sample components can be calculated.

Therefore, the area of ​​the peak is proportional to the amount of the substance present in the sample. These quantitative data are obtained in highly sensitive systems, such as those generated by gas or liquid chromatography, for example.

Types

One of the classifications of chromatograms is closely related to the different types of chromatography, which generate the corresponding chromatogram.

Depending on the running conditions, the detectors, among other aspects, the chromatogram will vary in its content and quality.

Chromatograms on paper or thin layer

The chromatogram can be generated directly on paper or thin layer, directly showing the distribution or distribution of the components of the sample.

It is very useful for the separation and study of colored substances that have natural pigments, such as chlorophyll. It can be subjected to development processes in case the substances do not have natural color, and it is useful for qualitative studies.

Chromatograms generated by detectors

The chromatogram can also be obtained using a detector that records the response, output, or final signal of the chromatography. As mentioned before, this detector is usually a spectrophotometer, a mass spectrometer, automatic sequencers, electrochemicals, among others.

Chromatograms generated in columns, whether of gases or liquids, as well as those of high resolution in thin layers, use detectors.

Depending on the type of detector, the chromatogram can be classified as differential or integral, depending on the response form of the detector.

Differential chromatogram

A differential detector continuously measures the response signal from the chromatogram, while integral detectors cumulatively measure the corresponding signal.

A differential chromatogram is a chromatogram obtained by a differential detector. These detectors include, for example, spectrophotometers and detectors for changes in electrical conductivity.

This type of chromatogram has shown the result of the separation of anions from a sample, detected by indirect photometry. The same results have also been obtained for the study of ions, for example, with final detection by conductimetry.

Differential chromatogram. Source: Pixabay

The upper graph shows the example of a differential chromatogram, obtained by automatic DNA (deoxyribonucleic acid) sequencers. The graph clearly shows peaks of four colors, one color for each of the nitrogenous bases in DNA.

Through the support of a computerized program, the interpretation of the sequence of the bases of the analyzed DNA is facilitated, as well as for more complex analytes.

Integral chromatogram

The integral chromatogram corresponds to that obtained by an integral detector. This chromatogram shows the output of a single component under study. Multiple peaks are not obtained as in the differential.

In the integral chromatogram, a record is obtained with a shape described as a step. This shape is the portion of the chromatogram that corresponds to the amount of a single substance that comes out of the column.

References

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