Peaks" in Liquid Chromatography
Dr. Shulamit Levin
Analytical Department - Medtechnica
article refers to "legitimate" system peaks and not all other extra
peaks from unknown sources. A presentation about all other
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
and size is sensitive to the sample composition, but its origins are
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
at the column head. The equilibrium of each component of the mobile
can be disturbed and thereby manifested by one system peak for each
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
We obtain the elution chromatogram in the following figure (bottom
Then, the 2 components are added to the mobile phase (to the
and a sample containing just the mobile phase , acetonitrile:water, is
injected. A chromatogram of two negative peaks is obtained at exactly
same retention times as the 2 dichloroanthraquinones in the elution
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
the UV-Vizualization mode). The system peaks there originate mostly
the ion-pair reagents, which undergoe distribution between the two
stationary and mobile. The users of this chromatographic mode try to
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
moves down the column at a constant velocity Ui, dictated
by the adsorption properties of the corresponding additive i,
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
at the stationary and mobile phases respectively. These concentrations
are determined by the slope of the adsorption isotherm of additive
Formation of system peaks - Example:
When a new mobile phase is
introduced to a column, i.e., the
of the mobile phase is changed abruptly, a front is formed , (The
of such fronts is used for determination of an adsorption isotherm of a
substance in a chromatographic system). The front levels off into a
which indicates a new equilibrium in the system. The detector response
is zeroed on that plateu and then solutes are injected. When an
solvent is injected at this point, system peaks may be detected, if
permits. The following Figure shows a case where the composition of the
mobile phase was changed abruptly, the plateu leveled off, but the
was not zeroed on the new response. An additive-free sample was
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
the mobile phase additives are in the linear range of chromatography
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
* 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
those obtained in the injection of pure water.
System peaks are in fact
relaxation signals, whose study permits an investigation
of the underlying equilibria of the additives between the two phases.
to the system peak theory , when the mobile phase contains N
one of which is a weak solvent that is considered not-adsorbed, N-1
system peaks can be observed upon injection of a sample. The sample
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
chromatographic systems. In the first three systems there were
A, water:Component B and water:Component C in the mobile phase
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
the mobile phase all at low concentrations, well within linear
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
be assigned to one component of the system only if there is no
between them. Conversely, when there is competition for retention
the components of the system, the migration of a particular system peak
can no longer be related to a specific component. Furthermore, such
peaks are not pure, and each peak contains all the components involved
in the competition in variable amounts. The migration of each system
is related to the combination of velocities of all the components.
it is difficult to deconvolute or predict their chromatograms. The
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
of the nature of the sample injected. However, the size of the
perturbation caused by a given amount of sample depends on the nature
the injected sample.
Reversed Phase Liquid Chromatography Separation of
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
Column as Indicated by Their Adsorption Isotherms, S. Levin and S.
J. Chromatogr., 517, 285-295, 1990
Adsorption Isotherms of Phenylalanine in the Chromatographic
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
Chromatography Through Their System Peaks, S. Levin and S. Abu-Lafi,
6, 148-155, 1994
The Use of System Peaks for the Determination of the
Resorcinol, Catechol and Phenol in Liquid Chromatography, S. Levin, S.
Abu-Lafi, S. Golshan-Shirazi and G. Guiochon, J. Chromatogr., 679,
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
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.
61, 2428-2433, 1989
Please note: The text and images included herein or
for personal use only. NO reproduction is permitted for commercial use.
Photos and graphs may be used within archives if credit is included.
Dr. Shula Levin