Srbek, Coufal, Boskov, Tesařov
Jan Srbek1
Pavel Coufal1
Zuzana Boskov1
Eva Tesařov2
1
Department of Analytical
Chemistry, Faculty of Science,
Charles University, Prague 2,
Czech Republic
2
Department of Physical and
Macromolecular Chemistry,
Faculty of Science, Charles
University, Prague 2, Czech
Republic
1263
System peaks and their positive and negative
aspects in chromatographic techniques
Whenever a mobile phase contains more than one component, additional signals
commonly called system peaks can appear. The origin of these signals is explained
through loss of equilibrium in the separation column caused by injection of analyte
dissolved in a different solvent than the mobile phase. The system peaks are then
generated by a relaxation process started by the non-equilibrium state. An overview
of the theory and applications of the system peaks in separation methods, mainly in
liquid chromatography, is presented in this paper. Only a brief theoretical discussion
of the system peak origin is given as the theoretical aspects of system peak formation
have already been published in many papers. The main focus of this review is to summarize applications, in which system peaks were used to measure physical or physicochemical data. Signals of system peaks are often misinterpreted but they offer
valuable information about thermodynamics and kinetics of the separation process
that takes place in chromatographic column.
Key Words: System peaks; Applications;
Received: April 12, 2005; revised: June 2, 2005; accepted: June 3, 2005
1 Introduction
1.1 Origin of the system peaks
Sample injection into a multicomponent mobile phase frequently results in a chromatogram that includes more
peaks than the number of analytes in the sample. These
additional signals are often misinterpreted since they are
considered as analyte signals and experimentalists
search for the separation conditions which can eliminate
them from chromatograms. Many authors [1 – 21] termed
these signals system peaks, pseudo peaks, ghost peaks,
eigenpeaks, vacancy peaks, induced peaks, or dip peaks.
The term ghost peaks seems to be the most adequate at
first sight because these signals have an ability to appear
and disappear or to change their size and direction. However, the general term system peaks is probably the most
accurate one. These peaks originate from the chromatographic system itself and are caused by interactions of the
mobile and stationary phases. The main theoretical
description of the system peaks has been published by
Schill and co-workers [22 – 26], Knox and Kaliszan [27],
and Levin, Grushka, and Abu-Lafi [1, 3, 4, 28]. Applications of system peaks for determination of adsorption isotherms, calculation of column void volumes and retention
factors, use of indirect detection, and many other exploitations are described in this paper.
When the mobile phase flows through the chromatographic column, an equilibrium is established between the
stationary and the mobile phases in the chromatographic
system. If the mobile phase is suddenly changed, for
example by injection of a sample, the equilibrium is disturbed. The sample components move with various velocities different from the equilibrium velocity that was stabilized in the column before through the equilibrium distribution between the stationary and mobile phases. As a
result, a relaxation of the chromatographic system to
reach a new equilibrium state takes place. This process
can proceed in several different ways: (1) the sample is
further solvated with one or more mobile phase components, (2) the sample has a different concentration of one
or more mobile phase components, which leads to redistribution of these components on the stationary phase,
(3) interaction of the sample and the adsorbed mobile
phase components cause their release or ongoing
adsorption [1]. These processes generate a change in
velocities of the solutes and the mobile phase components, concentrations of which are different from the original ones. The relation between the velocity of the mobile
phase components and the mobile phase velocity is given
by the equation
Correspondence: Dr. Pavel Coufal, Department of Analytical
Chemistry, Faculty of Science, Charles University, Albertov
2030, 128 43 Prague, Czech Republic. Phone: +420 221951238.
Fax: +420 224913538. E-mail: pcoufal@natur.cuni.cz.
Abbreviations: TEA, triethylamine.
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mi = R i u
(1)
where mi is the velocity of the i-th mobile phase component, Ri is the equilibrium fraction of the i-th species in the
mobile phase, and u is the velocity of the mobile phase [1].
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2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Review
DOI 10.1002/jssc.200500168
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Srbek, Coufal, Boskov, Tesařov
The resulting chromatogram then contains peaks of analytes as well as peaks of some mobile phase components.
The maximum number of system peaks is n – 1 in a system with n components, solvent that is considered not to
be adsorbed will never give rise to a system peak. An
important feature of system peaks is that, when the sample size is small, their retention time is independent of the
nature of the sample injected. The position of the system
peaks is constant but their areas or directions may change
depending on the nature and amount of the injected sample. System peaks are characterized by constant retention in the given chromatographic system. They can be
positive or negative with respect to the baseline and their
areas depend on the nature of the injected solute. System
peaks can also affect the shape of the analyte peak when
co-eluting with the analyte. In most cases, the analyte
peaks will be deformed, but under certain conditions
extremely narrow and sharp peaks are obtained. Deformations, which are certainly not desired, will occur more
frequently than compressions. The risk of deformation
increases when the system peaks are large. The influence
of co-eluting system peaks on the shape of analyte signals
was studied by Westerlund and co-workers [29]. Some
useful information about the processes in the chromatographic column can be obtained by careful choice of the
injected sample. For readers needing a deeper understanding of system peak theory, papers by Schill and coworkers [22 – 26], Knox and Kaliszan [27], and Levin,
Grushka, and Abu-Lafi [1, 3, 4, 28] can be recommended.
2 Applications of the system peaks
2.1 Calculation of retention factors of the mobile
phase components
Levin and Grushka [3, 4] studied system peaks in a
reversed phase chromatographic system consisting of a
typical column and an aqueous mobile phase containing
acetate buffer, copper acetate, and alkylsulfonates as the
mobile phase additives. Four system peaks appeared in
this chromatographic system when pure water was
injected as shown in Fig. 1 [1]. The peak related to cupric
cations was very sensitive to the presence of alkylsulfonate and acetate anions in the sample. Whenever a compound that could complex cupric ions was injected, the
area of this peak decreased. At a certain concentration of
the acetate or heptanesulfonate ions in the sample the
peak representing cupric cations suddenly disappeared
from the chromatogram. The peak area decrease or its
disappearance at a certain additive concentration could
be explained by the following mechanism. The anionic
solute was injected as the sodium salt and therefore, the
sample zone did not contain any cupric ions compared
with the mobile phase. Injection of the sample without cupric ions caused their desorption from the stationary phase.
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Figure 1. Chromatograms showing four system peaks, identified by the letters A, B, C, and D. The top chromatogram is
the result of the injection of pure water. The bottom four chromatograms are the results of the injections of tyrosine,
methionine, valine, and threonine, respectively. In all cases,
the mobile phase included acetate buffer (0.1 M, pH 5.6),
0.5 mM copper acetate, and 5.0 mM heptanesulfonate. Thermostated Lichrosorb RP18 column with laboratory made
Lichroprep Si-60 and Lichroprep C18 precolumn. Reproduced with permission from Ref. [1]. Copyright 1986 American Chemical Society.
Anionic solutes could then interact with these desorbed
cupric ions and formed ion pairs or complexes. The
amount of free cupric ions in the sample zone decreased
in this way and at the certain, above mentioned, concentration of anionic solute in the sample the cupric ion concentration was zero. This caused the disappearance of
the copper related peak from the chromatogram. Comparison of the experimentally obtained and calculated retention factors then served to explain the physical-chemical
background of the observed phenomena. If the interactions between the mobile phase components are not too
strong, the retention factor can be calculated from the system peaks through the thermodynamic definition as
k¼
qs
qm
ð2Þ
where qs and qm are the mole amounts of the given compound in the stationary and mobile phases, respectively.
The molar amount of the given component in the mobile
phase qm can be expressed by its concentration in the
mobile phase, cm, and the injection volume, Vi.
qm = cm Vi
(3)
The molar amount of the same component in the stationary phase can be calculated similarly
qs = cs Vi
i
(4)
2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
System peaks in chromatographic techniques
The value cs can be taken as the component concentration that causes peak disappearance. This type of calculation was repeated in different systems as well as at different copper concentrations in the mobile phase with similar
results [1]. Retention factors calculated in the acetate buffer were in good agreement with the experimental retention factors. The main advantage of this calculation
approach is that both stationary and mobile phase concentrations were normalized to the injection volume. Any
injection volume should give similar results. The use of
the column void volume for calculation of the retention factor was avoided, since dimensionless quantities were
applied. In turn, values of the retention factors calculated
in this way were used for the calculation of the column
void volume. On the other hand, retention factors obtained
from the heptanesulfonate and other alkylsulfonate systems were smaller than the experimental ones. This difference could be caused by a non-linear isotherm of heptanesulfonate adsorption on the stationary phase. There is
a close relation between the retention factors of system
peaks and the adsorption isotherms of corresponding
components. The retention factor of a particular mobile
phase component depends on the slope of the adsorption
isotherm, and hence the retention factor can serve for
characterization of the adsorption isotherm.
2.2 Calculation of column void volume from the
system peaks
Determination of the column void volume is often a problem in chromatographic techniques [30 – 34]. In most
cases, the solute that does not interact with the stationary
phase is chosen as the void time marker. This way of
determination of the void time depends on the choice of
the marker and sometimes several different values of the
column void volume are obtained [31]. If the calculated
retention factors based on the system peaks are in a good
agreement with the experimental ones (see Section 2.1),
the column void volumes can be calculated from the retention factors of the system peaks as
V0 ¼
VR
k þ1
ð5Þ
where k is retention factor of the solute, VR is retention
volume of the solute and V0 is the column void volume [3].
In other words, if the adsorption isotherm is linear, the system peaks are to be used not only for determination of the
solute retention factors but also for calculation of the column void volume.
2.3 Relation of the system peaks to adsorption
isotherms
It is obvious from the facts mentioned above that there is a
close relation between the retention factors of the system
peaks and the adsorption isotherms of the corresponding
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1265
compounds. The retention factor of a particular mobile
phase component is proportional to the slope of the component’s adsorption isotherm. The retention factors thus
unambiguously characterize the adsorption isotherm. The
thermodynamics of separation is based on adsorption isotherms of the sample components. The concentration of
each solute i in the stationary phase cs,i depends clearly on
the solute concentration in the mobile phase cm.i and the
function of this dependence is described by the adsorption
isotherm. The adsorption isotherms can be used in prediction of the peak shape and the solute retention [35 – 45].
There are several methods for determination of adsorption isotherms [46]. Frontal analysis is the most frequent
one; the system peaks can be applied as well. With a
mobile phase, in which one or more components are
adsorbed on the stationary phase, injection of a sample
differing in composition from the mobile phase causes a
system peak or peaks. As mentioned above, system
peaks can be employed for calculation of the retention factors and the column void volume [1, 4]. It has also been
shown that the adsorption isotherm of any mobile phase
component can be measured through the system
peaks [1, 3, 4, 28]. Levin and Abu-Lafi [28] described such
a method, in which the chromatographic system consisted
of a reversed phase column and an aqueous acetate buffer with phenylalanine as the mobile phase. Injection of
pure water in such a system resulted in a chromatogram
containing three system peaks. The third, negative peak
corresponded to phenylalanine. Using the third system
peak of this chromatographic system, the adsorption isotherm of phenylalanine was determined. The concentration of phenylalanine in the mobile phase cm,p was changed stepwise and the retention factor of phenylalanine kp
was measured at each step. The concentration of phenylalanine in the stationary phase cs,p was calculated from an
integral equation at each step [28]. The appearance of the
system peak allows the calculation of the retention factor
of phenylalanine without any need for determination of the
column void volume. This approach to the measurement
of the adsorption isotherms has the advantage of simplicity and speed. The adsorption isotherms obtained from
the system peaks were identical to those measured by the
frontal analysis. A detailed explanation of the use of the
system peaks for determination of adsorption isotherms
was described in another work published by Levin and
Abu-Lafi [47].
2.4 Indirect detection
The sensitivity of detection of an analyte in liquid chromatography and other separation techniques depends
strongly on the analyte molecule structure. For detection,
the molecule of analyte must contain a chromophore or
luminophore or the molecule must be electrochemically
active. Lack of chromophore and luminophore in the ana-
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Srbek, Coufal, Boskov, Tesařov
lyte molecule can be solved by derivatization of the analyte but this often causes serious technical complications.
One of the solutions to the low-sensitivity detection is use
of an indirect detection method. This technique has long
been used for ionic analytes [48 – 51]. In the indirect
detection method, a detectable ion is added to the chromatographic system and at the same time an oppositely
charged analyte is introduced into the mobile phase as an
ion pair agent. The ion pair formed between the detectable
ion and the analyte ion is then visible at the detector. In
reversed phase liquid chromatography, the indirect detection method can be applied to the charged and even to the
uncharged analytes. Any compound, not necessarily an
ion, with detectable properties and an affinity for the stationary phase could be added to the mobile phase.
Injected analytes then give a response, the magnitude of
which depends on the distribution of the added system
compound between the stationary and the mobile phases.
Two kinds of peaks appear in the chromatogram. One that
represents the analytes and an additional peak which is
characteristic for the system as it is the system peak. The
analyte peaks are negative when eluted before or positive
when eluted after the system peak. This orientation of
peaks is obtained if the analyte and the added system
compound have opposite charges. In the case that the
analyte is neutral or has the same charge as the added
system compound, the analyte peaks are positive before
and negative after the system peak [52]. The retention
time of the system peak depends only on the properties of
the chromatographic system and is independent of the
nature of the analyte, whereas its direction changes with
the injected sample composition. A mobile phase component gives a system peak, which can be recognized by
injection of the pure mobile phase solvent [22]. It is evident that the orientation of the analyte peak depends on
the analyte charge and its retention relative to the added
system compound. Moreover, the peak area is proportional to the analyte amount injected into the chromatographic system. Chromatographic systems based on
other kinds of common interactions, such as complexation
or protolysis reaction, also show the indirect detection
effect. Detection sensitivity of the indirect detection system is controlled by three factors: absorptivity of the
added system compound, affinity of the system compound to the stationary phase, and relative retention of
the analyte [53]. A high absorptivity of the system compound is advantageous but elution of the analyte peak
close to the system compound may have a higher impact
on the detection sensitivity. As the system should be as
stable and simple as possible it is recommended that the
mobile phase contain just one retained component, i.e.
the system compound. Several retained system compounds affect the analyte response significantly [54 – 56].
When the mobile phase contains unknown impurities, problems of indirect detection may occur [57]. Moreover, parJ. Sep. Sci. 2005, 28, 1263 – 1270
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ticularly large system peaks appear if the mobile phase
gives a detector response itself and the injected sample is
dissolved in a different solvent [22, 56, 58]. Indirect detection is a sensitive method for visualization of all kinds of
compounds and can be combined with any sensitive and
stable detector. However, indirect detection is not specific
and is especially suitable for samples with a limited number of analytes. An understanding of the indirect detection
principle is important not only for optimization of the detection sensitivity but also for elimination of disturbances and
unexpected effects related to the system peaks.
2.5 Methods for studying of drug-protein
interactions
Studying drug-protein interactions is important for determination of the pharmaceutical activity of drugs. Various
chromatographic and electrophoretic methods have been
developed to study these interactions. Methods used for
this determination are zonal elution [59 – 63], frontal analysis [59, 64], and vacancy peak measurements. Zonal
elution is actually limited in terms of applications for drugprotein studies because it requires that there is little or no
dissociation of the drug-protein complex during the separation. This situation is rarely present in drug-protein systems. The main drawback of frontal analysis is its need for
large-volume samples of the drug and protein. If protein
and drug show fast interaction kinetics and, at the same
time, the column gives different retention times for the
drug-protein complex and the free drug, then vacancy
techniques are the most appropriate methods for examining drug-protein interactions. The vacancy peak measurement methods are based on application of a mobile phase
containing the protein with drug, or just the drug of interest. The mobile phase without one or more of its components is then injected as the sample. The equilibrium
between the drug and protein in the chromatographic column is perturbed by the sample injection, which results in
formation of one or more system peaks in the chromatogram. These system signals can be used for determination of concentrations of the free drug and the proteindrug complex, which are present in the chromatographic
system under equilibrium conditions.
2.5.1 Hummel-Dreyer vacancy peak method
Hummel and Dreyer first described this vacancy peak
technique in 1962 [65]. A mobile phase containing a
known concentration of the drug of interest is applied in
the method and injection of a small amount of the protein
into the mobile phase is performed. If the protein and the
drug show fast interaction kinetics and at the same time
the column gives different retention times for the drug-protein complex and the free drug, the chromatogram will
contain positive and negative peaks as demonstrated in
Fig. 2 [66]. The first positive peak represents the eluted
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System peaks in chromatographic techniques
Figure 2. Typical chromatogram for the binding of warfarin
to HSA in the Hummel-Dreyer method. The results were
generated using a 12.5 lL injection of 2 g/L HSA into a
mobile phase containing 0.5 lM warfarin in pH 7.4 phosphate buffer flowing at 0.5 mL/min through a 15 cm64.2 mm
ID. Glycophase G column held at 378C. Reprinted with permission from Ref. [66]. Copyright 1978 Elsevier.
protein and its associated bound-drug fraction. The second peak, i.e. the negative drug vacancy peak, is eluted at
the free drug retention time. The second peak is produced
by binding of the protein sample with drug molecules in
the mobile phase and its area may be used for calculation
of the amount of drug in the chromatographic system. The
amount of protein bound to the drug molecules can be
estimated from the drug vacancy peak using an internal
calibration method. The same protein sample applied in
the presence of several different drug concentrations is
used in this calibration method [65, 66]. In the external
calibration method, the absolute area of the vacancy peak
is compared with the drug peak area measured when the
same drug is injected to the pure mobile phase buffer without any drug in the same chromatographic system [67].
Both calibration methods give similar results for certain
model systems [68]; however, the detector linearity has to
be examined first to ensure that reliable results can be
obtained. To obtain satisfactory results, there must be fast
interaction kinetics between the drug and the protein and
a good resolution between the two peaks as well.
2.5.2. Equilibrium saturation method
The equilibrium saturation method was firstly published by
Sebille et al. in 1979 [69]. This method is performed on a
size-exclusion chromatographic column, which can identify both the drug and the drug-protein complex. Both the
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1267
drug and the protein are used as mobile phase additives
and injection of a sample containing only the mobile phase
buffer is carried out. The resulting chromatogram contains
two vacancy peaks that correspond to the retention times
of the drug-protein complex and the free drug. The area of
these peaks can be used with internal or external calibration methods, as in the Hummel-Dreyer vacancy peak
method, to determine fractions of the free and the bound
drug at equilibrium. This method was applied to study
interactions of human serum albumin with diazepam [70]
and effects of fatty acids [69] and sodium dodecyl sulfate
on the binding of human serum albumin to warfarin [71].
The requirements for this method are similar to those for
the Hummel-Dreyer method, such as fast drug-protein
interaction kinetics and good resolution between the
vacancy peaks. Compared to the Hummel-Dreyer method
the equilibrium saturation method needs more protein but
it has some other advantages. There are no problems
with dilution because the protein and drug are applied at
fixed concentrations and the presence of protein in the
mobile phase helps to keep the drug, which can have a
low solubility in aqueous buffers, in solution [59]. Not only
size-exclusion but also affinity chromatography columns
can be used in the vacancy techniques and this was
demonstrated in the work of Soltes et al. [72]. Both methods could applied to the study of protein binding to chiral
solutes [73]. The Hummel-Dreyer and the equilibrium
saturation methods can also be used in capillary electrophoresis, as was demonstrated by Kraak et al. [74], who
employed these methods in the determination of warfarin
and bovine serum albumin interactions.
2.6 Vacancy gel chromatography
Gel permeation chromatography is often applied for characterization of polymer molecular weight distribution [75].
When a polymer solution is injected into a chromatographic column with a solvent as the mobile phase, a regular, positive elution curve is observed. If the polymer
solution is used as the mobile phase and the pure solvent
is injected as the sample, the elution curve will be mirror
image of the regular one. Otocka and Hellman [76]
reported the vacancy method with a conventional gel permeation chromatography column. As a result, the calibration curves obtained for the regular and the vacancy gel
permeation chromatography were not identical. There
was a difference in elution volumes between the regular
and the vacancy modes as the molecular weight and the
flow rate increased. It was suggested that this difference
may be minimized or even eliminated using high-performance vacancy gel permeation chromatography, which
was studied by Ye et al. [77]. The calibration curves
obtained were also not identical and the differences again
increased with molecular weight. However, when the concentration of analyte was decreased, the calibration
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2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Srbek, Coufal, Boskov, Tesařov
graphs became identical. This indicates that the differences in elution volumes between the regular and
vacancy mode should be ascribed to the concentration
dependence rather than the column performance. Gel
chromatography has also been employed to study the
solubilization of drugs since Draper et al. [78] used elution
gel chromatography for investigation of drug solubilization
in micellar solutions. This method differs from conventional gel permeation chromatography in the use of a surfactant solution as the eluent, and injection of a small
volume of this solution at different surfactant concentrations is carried out to test for an associate equilibrium in
the eluent. The elution curves, which were observed in
this gel permeation chromatography experiment, showed
system peaks demonstrating the solubilization of analytes
in the used surfactant system.
2.7 Negative aspects of system peaks
2.7.1 System peaks observed in liquid
chromatography with mobile phase
recycling
Mobile phase recycling has become a frequently used
technique in liquid chromatography but its effects on sample quantification are not yet well documented. Abreu and
Lawrence [10] spiked mobile phase with three different
concentrations of two analytes (tartaric acid and sodium
nitrate) to simulate mobile phase recycling. These mobile
phases were used to analyze eight different concentrations of the two analytes mentioned above in standard
solutions. When the mobile phase is recycled, the analyte
exiting the detector is fed into a reservoir and diluted with
mobile phase. The mobile phase in the reservoir is considerably larger in volume than the injected sample and, as a
result, the concentration of the analyte in the reservoir is
diluted by a large factor. As the volume of the mobile
phase is large relative to the sample, there is almost no
change in its composition for a single injection. In analysis
with mobile phase recycling, most samples injected into
the chromatographic system show analyte concentrations
greater than those in the mobile phase, which results in
positive peaks. However, there could be negative peaks
or no peaks at all in the case that the concentration of the
analyte in the sample is lower than or the same as its concentration in the recycled mobile phase. The negative
peaks are the system peaks and their number corresponds to the number of impurities in the recycled mobile
phase. It was proven possible to determine the concentration of analyte in recycled mobile phase by performing linear regression analysis of the peak areas for a set of standards. The intercept of the linear regression gives the
absolute value for the peak area that would be observed
for the concentration of analyte in the pure mobile phase.
When the analyte concentration in the mobile phase
approximates to the lowest concentration of analyte in
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samples, new mobile phase without impurities has to be
prepared.
2.7.2 System peaks observed with eluents
containing triethylamine
When basic analytes, such as acridines or pyridoquinolines [79, 80], are analyzed in reversed phase liquid chromatography triethylamine (TEA) is often added to the
mobile phase. TEA interacts strongly with the free acidic
silanol groups of the stationary phase [81]. In this way
TEA inhibits interactions of basic analytes with the stationary phase, significantly reduces analyte retention times,
and improves peak symmetry. As TEA is distributed
between the stationary and the mobile phases and at the
same time strongly absorbs ultraviolet radiation at lower
wavelengths, it may cause system peaks. Two system
peaks were observed in the mobile phase containing
methanol-triethylamine 99:1 (v/v) [82]. The first system
peak originated from the local decrease of TEA in the
mobile phase and the second peak was caused by the
local increase or decrease, respectively, of TEA on the
stationary phase surface. The latter peak represents the
retention time of TEA in the investigated chromatographic
system. A possible misinterpretation of a system peak in
analysis of an acridine derivative is shown in Fig. 3 [83].
The chromatograms A and B represent the analysis of
1 mM acridine derivative dissolved in water and the chromatograms C and D correspond to the analysis of a
0.1 mM solution of the derivative in the same sample
matrix. In these chromatograms, the first positive peak
represents the analyte and the second positive one is the
system peak caused by TEA as the mobile phase additive.
It is evident that in some cases the identification and interpretation of analyte peaks in chromatograms containing
also system peaks might be rather complicated since the
system peaks can give higher signals than the analyte
peaks. The combination of direct and indirect detection
not only complicates identification of the analyte but also
considerably reduces the response of the analyte. There
could be no peak at all when both responses are the
same. The careful selection of the detection wavelength is
essential when dealing with the systems affected by the
presence of the system peaks.
3 Concluding remarks
System peaks originate from disturbance of the equilibrium between mobile and stationary phases in a chromatographic column, which takes place during sample injection. System peaks offer very useful information about the
chromatographic system itself and allow calculation of
many of its thermodynamic and kinetic characteristics.
Based on these facts, in some cases the system peaks
may be even more important than the analyte signals
themselves. On the other hand, system peaks may com-
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System peaks in chromatographic techniques
1269
Figure 3. Chromatograms of (A,B) 1 mM and (C,D) 0.1 mM acridine derivative dissolved in water. Stainless steel capillary column (300 lm ID6250 mm) packed with 5 lm LiChrosorb RP-select B. Methanol-triethylamine 99.9:0.1 (v/v) mobile phase; flow
rate 1 lL/min; detection wavelengths (A,C) 214 and (B,D) 230 nm. The peak areas are expressed as percentages. Reproduced
with permission from Ref. [83].
plicate the interpretation and evaluation of analyte peaks
especially when high concentrations of additives are used
in the mobile phase for analyses of samples of low concentration. Carefully chosen detection conditions could
visualize or suppress system peaks, respectively, in
cases where they are helpful or where they complicate the
chromatographic signal.
[6] D. Berek, T. Bleha, Z. Pevna, J. Chromatogr. Sci. 1976, 14,
560 – 563.
[7] D.J. Solms, T.W. Smuts, V. Pretorius, J. Chromatogr. Sci.
1971, 9, 600 – 603.
[8] W.R. Melander, J.F. Erard, C. Horvath, J. Chromatogr.
1983, 282, 229 – 248.
[9] D.S. Hage, S.A. Tweed, J. Chromatogr. B 1997, 699, 499 –
525.
Acknowledgments
Financial support by Grant no. 203/03/0161 of the Grant
Agency of the Czech Republic and by international project
COST Action B16 is gratefully acknowledged.
[10] O. Abreu, G.D. Lawrence, Anal. Chem. 2000, 72, 1749 –
1753.
[11] J.J. Stranahan, S.N. Deming, Anal. Chem. 1982, 54, 1540 –
1546.
[12] Z. Iskandarani, D.J. Pietrzyk, Anal. Chem. 1982, 54, 1065 –
1071.
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