American Journal of Anal yt ical Chemistry, 2011, 2, 934-937
doi:10.4236/ajac.2011.28108 Published Online December 2011 (
Copyright © 2011 SciRes. AJAC
Temperature and Pressure Behaviours of Methanol,
Acetonitrile/Water Mixtures on Chromatographic Systems
Talal Aburjai, Muhammed Alzweiri*, Yusuf M. Al-Hiari
Faculty of Pharmacy, The University of Jordan, Amman, Jordan
E-mail: *
Received September 9, 2011; revised October 17, 2011; accepted October 26, 2011
Temperature and pressure were shown to vary significantly with solvent mixing, showing maxima at differ-
ent solvent ratios. Acetonitrile/water mixing resulted in temperature reduction of solutions whereas metha-
nol/water mixing caused temperature increases. On the other hand, maximum recorded chromatographic
pressure of acetonitrile:water mixtures occurred at a solvent ratio of 1:6 compared with methanol:water,
which showed a maximum pressure at a solvent ratio of 1:1. These findings can be of use in stabilizing re-
tention time shifts during HPLC-based studies associated with compound identification based on retention
time such as analysis of complex mixtures.
Keywords: Retention Time, Pressure, Temperature, Methanol and Acetonitrile
1. Introduction
Methanol/water and acetonitrile/water mixtures are com-
monly used in chromatographic mobile phases. Many
physical characteristics used to understand solvent be-
havior within these mixtures, for example excess molar
volume and enthalpy change, have been reported inten-
sively in the literature [1-3]. Careful inspection in the
literature concerning solvent mixture interaction pro-
vokes a corner stone conclusion. Measurement of physi-
cal properties depicts nonlinear relation with the compo-
sition of mixtures. This might reflect the diversity of
possible interactions of solvent molecules
Understanding temperature and pressure behavior in-
fluencing retention time shift in gradient systems of
chromatography can be utilized to minimize the effect of
retention time shift on molecular identification.
Retention time shift in chromatographic separations
has undesirable effects on the results regardless of
whether a target molecule or a profiling analysis ap-
proach was used. Minimizing the shift becomes more
essential when the profiling analysis is being carried out
accompanied by chemometrics, in order to avoid the re-
sult of biasing the data [4-6]. In spite of the availability
of many software applications used to correct this shift,
the solutions are not always satisfactory [7-11]. Thus, it
is important to design HPLC mobile phases able to
minimize retention time shift by controlling, temperature
fluctuation and the excessive pressure of HPLC system.
Particularly, during the application of gradient systems
2. Experimental
2.1. Materials
Acetonitrile and water were HPLC grade (VWR Interna-
tional Ltd., Lutterworth, UK). A Minitherm HI 8751
thermosensor (Hanna instruments, Romania) was used to
measure the change of temperature of mixtures. Chro-
matographic pressure study was carried out by using
Spectra system P2000 HPLC pump (Thermo Separation).
HPLC columns used were ACE C8 50 × 3 mm 3 μm and
Kromasil 5ODS (C18) 250 × 3.2 mm 5 μm.
2.2. Procedures
HPLC backpressure was measured for two reversed
phases; C8 and C18, as the most common types of col-
umns used in HPLC systems, against several ratios of
methanol and acetonitrile in water with a constant flow
rate of 0.7 mL/min. The pressure readings were taken
directly from the LCD display of the HPLC pump.
The temperature change due to mobile phase solvent
mixing was studied by titrating water with either metha-
nol or acetonitrile and measuring the temperature at dif-
ferent ratios of solvents. The procedure was repeated but
by titrating the organic solvents with water instead in
order to take in consideration the anomaly of molecular
arrangement in mixtures at different solvent ratios.
3. Results and Discussion
One of the important chromatographic parameters that
contribute to retention time variability is the column
pressure, especially if a long analysis time is used. Thus
pressure changes of examples of reversed phase columns
were studied versus the organic modifier content in the
mobile phase. As the solvent gradient affects pressure it
was important to establish optimum conditions for anal-
ysis by assessing pressures under both gradient and iso-
cratic conditions. For this reason a gradient run from
zero to 100% was used in order to determine an ap-
proximate maximum value for column pressure. Follow-
ing this, elution was carried out isocratically in order to
accurately establish the solvent ratio that resulted in the
maximum pressure value for different solvent/column
conditions (Figures 1 and 2). While methanol induced
the highest pressure in the reversed phase columns at a
per centage o f m ethan ol in mobil e phase
backpressure (psi)
perc entage of m ethanol in m obile phase
backpressure ( psi)
Figure 1. HPLC backpressure of reversed phase columns
exposed to methanol-water mobile phases. (a) ACE C8
column; (b) Kromasil 5 ODS. () gradient of methanol and
water, while () isocratic (45:55) methanol:water.
percentage of Acetonitr ile in m obil e phase
backpressur e (psi)
percentage of Acet onit r ile in m obil e ph as e
backpressure (psi)
Figure 2. HPLC backpressure of reversed phase columns
exposed to Acetonitrile-water mobile phases. (a) ACE C8
column; (b) Kromasil 5 ODS. () gradient of acetonitrile
and water, while () isocratic (15:85) acetonitrile:water.
methanol:water ratio of 45:55, acetonitrile produced its
maximum back-pressure at an acetonitrile:water ratio of
15:85. However, this not only represents methanol-water
or acetonitrile-water interactions but may also reflect the
interaction of the mobile phase with the stationary phase
of reversed phase columns. Methanol has the character of
accepting and donating hydrogen bonds, while acetoni-
trile only accepts hydrogen bonds. Thus, stronger inter-
actions between water and methanol molecules are ex-
pected as well as other interactions between methanol
molecules and silica silanol groups are speculated. These
interactions of methanol resist fluid flow through the
HPLC column and consequently higher back pressures of
methanol/water systems were obtained in comparison
with acetonitrile/water systems. Comparison of Figure
1(a) with Figure1(b) depicts higher back pressure ob-
tained from C18 column compared with C8 column. This
might be due to more availability of silanol groups on the
surface of C18 columns resulted from limited surface
coverage during derivatization by large C18 molecules.
Furthermore, Figure 2(b) shows higher back pressure
Copyright © 2011 SciRes. AJAC
comparing with Figure 2(a) due to the same reason men-
tioned previously. However, the difference of backpres-
sure is with less extent in case of acetonitrile. This might
be due to the limited interactions of acetonitrile with si-
lanol groups in comparison with methanol. Moreover,
distribution of data points of backpressure produced from
methanol/water systems is closer to normal distribution,
while acetonitrile/water has a significant deviation from
the norm. This is due to inconsistency of water/acetone-
trile interactions resulted from acetonitrile ability in
forming clathrates with water as proved by our team [14].
Another factor affecting retention time is the tempera-
ture change due to mobile phase solvent mixing. Thus
temperature change was studied by simple titration of
methanol with water and vice versa, the same procedure
being repeated for acetonitrile and water.
In the case of methanol/water mixing a considerable
rise in temperature was detected up to approximately
40% contribution of either solvent to the mixtures (Fig-
ure 3(b)). This might indicate a relation between the
formation of new hydrogen bonds among water and
methanol molecules within the liquid with consequent
decrease in entropy. Methanol/water mixtures did not
show hysteresis in the thermal behavior during mixing
procedure. Whether the addition started with water or
with methanol, the system produced identical thermal
behaviors. In contrast the mixing of acetonitrile/water
caused a fall in temperature when a constant volume of
acetonitrile titrated drop-wise with water (Figure 3(a)).
This might indicate the increased disorder within the
liquid and the overall breaking of hydrogen bonds.
However, it is clear that there is a different behavior
when water titrated with acetonitrile indicating several
possibilities of arrangement between water and acetoni-
trile molecules depending on their relative molar ratios.
These findings match NMR and IR studies of acetoni-
trile/water systems conducted by Alzweiri et al. [14]. It
was also observed that drop in temperature occurred
when water titrated with acetonitrile until acetonitrile
reached 20% of the mixture then rapid increase in tem-
perature was occurred. This might be due to possible
clathrate formation between water and acetonitrile at
certain molar ratios. Anomaly of thermal behavior re-
sulted from Water titrated with acetonitrile compared
with acetonitrile titrated with water matches Satoh and
Nakanishi suggestion in existing different arrangements
of water and acetonitrile in mixtures [15].
Variations in temperature and backpressure show that
there are critical solvent ratios at which these physico-
chemical properties change dramatically. The knowledge
of these critical ratios might be exploitable for control-
ling retention time shifts in different chromatographic
Figure 3. Temperature change of water titrated drop wise
with acetonitrile ((a)-A), acetonitrile titrated with water
((a)-W), water titrated with methanol ((b)-M) and methanol
titrated with water ((b)-W).
4. Conclusions
Retention time shift of chromatographic peaks is a par-
ticular source of errors in molecular identification. In this
study an attempt has been made to examine the pressure
and temperature factors that influence retention time shift
to allow future development of LC methods that would
reduce intrinsic shift errors to a minimum. Temperature
and pressure variation revealed that critical maxima oc-
cur during solvent mixing which can be taken in consid-
eration during the setup of the chromatographic devel-
opment. Despite the close physicochemical properties of
acetonitrile and methanol, differences were observed in
their behaviour and response as follows: 1) while ace-
tonitrile/water mixing resulted in temperature reduction
of the solution, methanol/water mixing caused tempera-
ture increase; 2) the maximum recorded column back-
pressure for an acetonitrile:water mixture occurred when
the solvent ratio was 1:6, whereas a methanol:water
Copyright © 2011 SciRes. AJAC
Copyright © 2011 SciRes. AJAC
mixture resulted in a maximum backpressure at a solvent
ratio of 1:1.
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