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"System Peaks" in Liquid Chromatography

Dr. Shulamit Levin
Analytical Department - Medtechnica

This article refers to "legitimate" system peaks and not all other extra peaks from  unknown sources.  A presentation about all other peaks:  "HPLC_detective"

A chromatographic system peak is a peak that originates from the chromatographic system itself: mobile phase and column and not from the sample. It's mere appearance and size is sensitive to the sample composition, but its origins are the mobile phase components.
When a mobile phase is introduced to a column, its components undergo distribution until an equilibrium is attained. Then, injection of a sample different in any way from the mobile phase causes a small equilibrium perturbation at the column head. The equilibrium of each component of the mobile phase can be disturbed and thereby manifested by one system peak for each mobile phase additive, using the appropriate detection conditions.
Example of system peaks: Lets say that the mobile phase is acetonitrile:water and we inject a mixture of 1:1 1,5-dichloroanthraquinone and 1,8-dichloroanthraquinone. We obtain the elution chromatogram in the following figure (bottom chromatogram). Then, the 2 components are added to the mobile phase (to the acetonitrile:water) and a sample containing just the mobile phase , acetonitrile:water, is injected. A chromatogram of two negative peaks is obtained at exactly the same retention times as the 2 dichloroanthraquinones in the elution chromatogram. The 2 chromatograms and the UV spectra of the 2 peaks are miror images of one another.

In practice, the chromatographic mode in which system peaks are mostly abundant is Ion-Pair Reversed-Phase liquid chromatography (especially in the UV-Vizualization mode). The system peaks there originate mostly from the ion-pair reagents, which undergoe distribution between the two phases, stationary and mobile. The users of this chromatographic mode try to eliminate these additional peaks from the chromatogram.
When a perturbation in the distribution of a mobile phase additives is created, i number of peaks are created, and each system peak moves down the column at a constant velocity Ui, dictated by the adsorption properties of the corresponding additive i, according to the expression:

Ui = Uo/(1+dCs,i/dCm,i)

Where Uo is the mobile phase velocity, dCs,i and dCm,i are the infinitesimal disturbances in the concentrations at the stationary and mobile phases respectively. These concentrations are determined by the slope of the adsorption isotherm of additive i.

Formation of system peaks - Example:

When a new mobile phase is introduced to a column, i.e., the composition of the mobile phase is changed abruptly, a front is formed , (The velocity of such fronts is used for determination of an adsorption isotherm of a substance in a chromatographic system). The front levels off into a plateu, which indicates a new equilibrium in the system. The detector response is zeroed on that plateu and then solutes are injected. When an additive-free solvent is injected at this point, system peaks may be detected, if detection permits. The following Figure shows a case where the composition of the mobile phase was changed abruptly, the plateu leveled off, but the detector was not zeroed on the new response. An additive-free sample was injected on that plateu andnegative system peaks were obtained (vacancy peaks):

Each system peak can be related to a specific mobile phase component, and each component' peak behaves independently, as long as concentrations of the mobile phase additives are in the linear range of chromatography (Cs/Cm=constant). The following Figure shows what happens in the following cases:
The mobile phase contains two components in water: 0.001M:0.001M each component.
* Injection of pure water (C=0.0) yields two negative system peaks - vacancy peaks.
* Injection of 0.001M of each component yields straight line baseline.
* Injection of concentration higher than 0.001M of each component yields a positive peak.
* Injection of concentration lower than 0.001M of each component yields also negative peaks, but the absolute value of their area is smaller than those obtained in the injection of pure water.

LINEAR CONDITIONS:

System peaks are in fact relaxation signals, whose study permits an investigation of the underlying equilibria of the additives between the two phases. According to the system peak theory , when the mobile phase contains N components, one of which is a weak solvent that is considered not-adsorbed, N-1 additive system peaks can be observed upon injection of a sample. The sample creates a perturbation when its composition is different in any sense than that of the mobile phase. These N- 1 peaks propagate at different velocities characteristic to the mobile phase additives.
The following Figure brings such an example: there are 5 chromatograms that were resulted from the injection of just pure water into 5 different chromatographic systems. In the first three systems there were water:Component A, water:Component B and water:Component C in the mobile phase respectively. In the 4th chromatogram there was water:Component A and B in the mobile phase, and in the 5th chromatogram there was water:Components A and C in the mobile phase all at low concentrations, well within linear conditions.

NON-LINEAR CONDITIONS

System peaks can either be positive or negative, depending on the nature of the sample and on the detection mode. Each one of these N-1 peaks can be assigned to one component of the system only if there is no competition between them. Conversely, when there is competition for retention between the components of the system, the migration of a particular system peak can no longer be related to a specific component. Furthermore, such system peaks are not pure, and each peak contains all the components involved in the competition in variable amounts. The migration of each system peak is related to the combination of velocities of all the components. Therefore, it is difficult to deconvolute or predict their chromatograms. The following Figure shows an example 5 injections of pure water to the same 5 system as above, in the previous Figure, but this time the concentrations are much higher, well above the linear range.
An important feature of system peaks is that, when the sample size and thus the perturbation is small, their retention time is independent of the nature of the sample injected. However, the size of the perturbation caused by a given amount of sample depends on the nature of the injected sample.


References
Reversed Phase Liquid Chromatography Separation of Amino Acids with Aqueous Mobile Phase Containing Copper Ions and Alkyl Sulfonates., E. Grushka and S. Levin, Anal. Chem., 57, 1830-1835, 1985

Competition Between Phenylalanine and Acetic Acid in the Chromatographic Column as Indicated by Their Adsorption Isotherms, S. Levin and S. Abu-Lafi, J. Chromatogr., 517, 285-295, 1990

Adsorption Isotherms of Phenylalanine in the Chromatographic Column Measured Simultaneously by System Peaks Analysis and Frontal Analysis, S. Levin and S. Abu-Lafi, J. Chromatogr., 556, 277-285, 1991

Experimental Studies of Competition Between Enantiomers in Chiral Liquid Chromatography Through Their System Peaks, S. Levin and S. Abu-Lafi, Chirality, 6, 148-155, 1994

The Use of System Peaks for the Determination of the Distribution of Resorcinol, Catechol and Phenol in Liquid Chromatography, S. Levin, S. Abu-Lafi, S. Golshan-Shirazi and G. Guiochon, J. Chromatogr., 679, 213-229, 1994

System Peaks in Liquid Chromatography, Their Origin, Formation and Importance, S. Levin and E. Grushka, Anal. Chem., 58, 1602-1607, 1986

Systems Peaks in Liquid Chromatography: Their Relation to the Adsorption Isotherm, S. Levin and E. Grushka, Anal. Chem., 59, 1157-1164, 1987

Calculation of Capacity Ratios of the Mobile Phase Components and Column Void Volumes Through System Peaks, S. Levin and E. Grushka, Anal. Chem., 61, 2428-2433, 1989



Please note: The text and images included herein or attached are for personal use only. NO reproduction is permitted for commercial use. Photos and graphs may be used within archives if credit is included. Thanks, Dr. Shula Levin



















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