Article

pubs.acs.org/ac

Longitudinal On-Column Thermal Modulation for Comprehensive

Two-Dimensional Liquid Chromatography
Mari E. Creese,† Mathew J. Creese,§ Joe P. Foley,†,‡ Hernan J. Cortes,⊥ Emily F. Hilder,†,×
Robert A. Shellie,†,∥ and Michael C. Breadmore*,†
†
Australian Centre for Research on Separation Science, School of Physical Sciences, University of Tasmania, Private Bag 75, Hobart,
Tasmania 7001, Australia
‡
Department of Chemistry, Drexel University, 3141 Chestnut Street, Philadelphia 19104, United States
§
Allison Laboratories Pty Ltd., Sandy Bay, Tasmania 7005, Australia
∥
Trajan Scientific and Medical, 7 Argent Place, Ringwood, Victoria 3134, Australia
⊥
HJ Cortes Consulting LLC, Midland, Michigan 48642, United States
×
Future Industries Institute, University of South Australia, GPO Box 2471, Adelaide, South Australia 5001, Australia

ABSTRACT: Longitudinal on-column thermal modulation for comprehen-

sive two-dimensional liquid chromatography is introduced. Modulation
optimization involved a systematic investigation of heat transfer, analyte
retention, and migration velocity at a range of temperatures. Longitudinal on-
column thermal modulation was realized using a set of alkylphenones and
compared to a conventional valve-modulator employing sample loops. The
thermal modulator showed a reduced modulation-induced pressure impact
than valve modulation, resulting in reduced baseline perturbation by a factor
of 6; yielding a 6−14-fold improvement in signal-to-noise. A red wine sample
was analyzed to demonstrate the potential of the longitudinal on-column
thermal modulator for separation of a complex sample. Discrete peaks in the
second dimension using the thermal modulator were 30−55% narrower than
with the valve modulator. The results shown herein demonstrate the benefits
of an active focusing modulator, such as reduced detection limits and increased total peak capacity.

M odulation is the core process in any comprehensive two-

dimensional chromatographic separation. The modulator
consecutively and systematically collects analytes as they are
eluted from the first dimension column and delivers them to the
second dimension column.1 There are many modulation
possibilities for comprehensive two-dimensional gas chromatog-
raphy (GC × GC): resistive heating of painted zones of
modulator capillary,2 rotational movement of a slotted heater,3,4
longitudinal movement of a cold trap,5−7 cryogen jets with or
without the combination of a hot jet,8 resistive heating with or
without a cryogen trap,9−11 and more.8 In contrast, compre-
hensive two-dimensional liquid chromatography (LC × LC) has
not been demonstrated in online-mode without use of switching
valves. LC × LC using switching valves may be performed with or
without trap columns.12−16 A trap column offers a focusing
mechanism; however, re-equilibration of the trap column is not
always reached and band compression efficiency is reduced
throughout the analysis unless the mobile phase strength of the
second dimension gradient greatly exceeds that of the first
dimension gradient or if an equilibration step is accounted for in
the modulation period.16 Further, switching valves have negative
effects on the 2D system17 such as pressure pulses (<10% of
operating pressure for 2DLC specific valve) induced by
switching, which also affects the second dimension column and
reduces its expected lifetime.
Within valve based LC × LC modulators, solvent dilution has
been demonstrated as an effective method for active focus-
ing.18,19 Dilution of the 1D effluent allows an improvement of the
on-column focusing at either the head of the 2D column or the
trap column. For example, the effect of introducing 10−20% less
organic solvent to the 2D allowed on-column focusing and
enhanced resolution.20−25 Dilution is achieved by a connection
of a weak solvent to the switching valve modulator and is easily
implemented. The limitation of any valve-based modulation lies
within the momentary blocking of flow as the valve is switched
from position a to b. This is particularly critical when operating at
or close to the pressure limit as the pressure increase, however
short in duration, can exceed the limitations of the system and
may result in termination of the analysis.
High temperatures are commonly employed in LC to achieve
faster separations, improve selectivity and resolution, and reduce
backpressure,4,26−30 although typically at isothermal conditions.
Since the adaptation of low thermal mass (LTM) resistive
Received: August 21, 2016
Accepted: December 5, 2016
Published: December 5, 2016

© 2016 American Chemical Society 1123 DOI: 10.1021/acs.analchem.6b03279

Anal. Chem. 2017, 89, 1123−1130
Analytical Chemistry
heating from GC5−7,10 to LC,31 there have been a number of
studies on thermal gradient elution9−11,31−33 and temperature
pulsing for manipulating discrete sections of the separation.34,35
The LTM technology was also employed as a modulator for
offline LC × LC by Verstraeten et al.36 where a capillary trap
segment was temperature cycled to facilitate modulation and
fraction collection from the first dimension.
Temperature can also be used for peak focusing by inducing a
compression effect, exploiting the change in retention with
temperature and changing from low temperature for capture to
higher temperatures for release. Subambient temperatures were
used for large volume injections where a cold zone is created at
the head of the column and the remaining part of the column is
kept either at ambient or elevated temperatures.37,38 Remobiliza-
tion of the large volume injection zone is realized by moving the
column into the hot zone or by assisted heating. Similarly,
subambient temperature was used for peak compression prior to
detection39 where an alternating cold and hot zone is created by
flooding a reservoir surrounding the column. Gradual and
segmented temperature control for resolution manipulation has
also been achieved by a multiple Peltier element design where
each unit was individually controlled.40
The literature above suggests it is theoretically feasible to
create a thermal modulator for LC × LC. The physicochemical
differences between GC and LC, such as the incompressibility of
liquids compared to gases and slow heat transfer,41,42 are the
main challenges for nonvalve based modulators in LC × LC. The
approach undertaken in this work is inspired by the
longitudinally modulated cryogenic system (LMCS) developed
by Marriott and Kinghorn for GC × GC modulation5,6,43,44 and
the temperature cycled modulator for offline LC × LC by
Vertstraeten et al.36 Marriott and Kinghorn used a double walled
hollow cylinder fitted over the modulation segment of the
capillary column and purged the hollow with cryogenic liquid for
trapping. When the trap was moved away from the column, the
temperature of the GC column oven quickly heated the focused
band inside the cold zone of the capillary. During this process, a
zone further upstream (toward the first dimension) was cooled
before the column was moved back to the original position.
In this work, we explore the possibility to employ longitudinal
on-column thermal modulation (LOCTM) using LTM resistive
heating for LC × LC, using its performance toward a set of
alkylphenones as a proof of concept.
■ EXPERIMENTAL SECTION
Chemicals. Alkylphenones (Acetophenone (BDH); propio-
phenone, butyrophenone (Aldrich)); and valerophenone (Alfa
Aesar) were prepared to 500 μg/mL stock solutions in 100%
methanol. The standards were diluted to 50 μg/mL with 100%
water for analysis. The mobile phase was water (A) from an in-
house Millipore system (Millipore, North Ryde, NSW, Australia)
and methanol (B) from VWR (Tingalpa, QLD, Australia) both
with 0.1% formic acid from Sigma-Aldrich (Castle Hill, NSW,
Australia). Red wine was purchased from a local store.
Modulator. The modulator consisted of three parts: (i) a trap
column of 0.8 mm i.d. titanium tubing cut to 81 mm, slurry
packed with 5 μm porous graphitic carbon (PGC) particles
(Thermo Scientific, U.K.) in methanol/water using a packing
pump (Haskell, Burbank, CA). The column outlet was fitted with
a stainless steel frit (diameter 0.038 in. × 0.030 in. × 0.062 in., 0.5
μm pore size; Kinesis, Redland Bay, QLD, Australia). (ii) LTM
resistively heated sleeve modified to 2.1 mm × 20 mm with total
resistance of 97 Ω, and (iii) a modified longitudinally modulated
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cryogenic system (LMCS) was used to hold the LTM sleeve. A
brace was added to mechanically stabilize the trap in order to
keep the alignment and allow free movement of the sleeve. The
LTM was temperature controlled via an LTM module (Agilent
Technologies, Santa Clara, CA) and the LMCS was triggered by
remote start-out. Cold jets were employed to bring temperature
down to trapping conditions between mobilization steps in the
modulation process. Compressed air (6 bar) was passed through
an ice bath, split into two jets, and directed at two positions on
the modulator trap column. The temperature at the points of
cooling were recorded to ∼13 °C. The jets were purging cooled
air continuously throughout the analysis. A thermal camera
(FLIR E40), set to a temperature range of 0−650 °C ± 0.07 °C,
was employed to record thermal changes across the trap column
when modulating.
Instrumentation and Methods. The analyses were
performed using an Agilent 1290 Infinity 2D-LC solution system
(Wilmington, DE). The valve modulator was a four-port duo
valve (2DLC valve, Agilent Technologies) equipped with two
60-μL loops in a countercurrent flow configuration. The diode
array detector was set to 254, 260, 275, and 280 nm at a collection
rate of 20 Hz and 4 nm bandwidth. The injection volume was 1.5
μL of 50 μg/mL standards and 5 μL for red wine analysis. The
first dimension (1D) separation took place on a C18 column
(Zorbax Eclipse Plus RRHD, 2.1 mm × 150 mm, 1.8 μm; Agilent
Technologies, Mulgrave, Victoria, Australia) at a flow rate of
0.025 mL/min with a gradient from 55 to 85% B from 15.5 to 150
min (for alkylphenones) and 5−80% B from 15.5 to 215.5 min
(for separation of red wine). The second dimension (2D) column
was titanium tubing (0.3 mm × 34 mm) packed with 5 μm PGC
particles (Thermo Scientific) similar to the modulator trap
column. The 2D pump delivered 0.125 mL/min to a T-piece
adding to the flow from the first dimension, giving a total of 0.15
mL/min, which was then connected to the modulator trap
column. The second dimension mobile phase program was 73−
97% B (giving 70−95% after mixing with the 1D mobile phase)
for separation of alkylphenones. The 2D method used for the
valve modulation setup was set to 0.15 mL/min with a gradient of
70−95% methanol from 15.5 to 150 min. The method for
separating the compounds in the red wine sample was 5 μL
injected on the 1D column, which was operated at 0.025 mL/min
at a gradient from 5 to 85% methanol from 15.5 to 215.5 min.
The 2D pump delivered 0.125 mL/min giving effectively 0.15
mL/min through the modulator and the second dimension
column. The gradient was 15−73% methanol (effective 15−
70%). The second dimension when employing the valve
modulator was operated at 0.15 mL/min at a gradient from 15
to 70% methanol from 15.5 to 215.5 min. A modulation period of
60 s was employed for all methods.
■ RESULTS AND DISCUSSION
A homologous series of alkylphenones was used for development
and evaluation of the new modulator due to the well-defined
characteristics of these analytes allowing predictable retention in
both dimensions. Figure 1 shows a schematic of the LOCTM
system. Modulation is achieved by moving a resistively heated
sleeve (200 °C) from the inlet half to the outlet half of the PGC
trap column by aid of an LMCS controller system. Compressed
air is blown through two ports, one directly onto the trap column
to enhance cooling and the second onto a shield around the
heated sleeve. The air was cooled by first flowing through tubing
placed in an ice bath before the flow was split and directed to the
trap column. The modulation column (PGC) was always
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Analytical Chemistry
Figure 1. Setup of the LOCTM showing the resistive heating sleeve and
the longitudinal modulator controller: (a) trapping at inlet, remobilizing
at outlet, (b) trapping at outlet, remobilizing at inlet. (1) T-piece from
1
D column and 2D pump, (2) fitting (stainless steel at inlet, PEEK at
outlet), (3) PGC trap column (0.8 mm × 81 mm, 5 μm), (4)
longitudinal modulator, (5) resistively heated low thermal mass (LTM)
sleeve, (6) column brace, (7) to 2D column, (8) cooled compressed air.
shielded from the cooling jet that was directed to the alternating
halves of the column devoted to mobilization, so as to avoid
lowering the temperature and increasing the retention in the
mobilization region. The use of a second pump to mix the
effluent before the LMCS allowed independent control of the
flow rate in both dimensions as well as some manipulation of the
chemistry for trapping and separation on the 2D column.
Understanding the thermal behavior of the heated sleeve and
trap column is critical for constructing a suitable modulator for
comprehensive LC. For the modulator to work most effectively,
there needs to be a large and rapid thermal change between the
heating and cooling stages of the modulator. To enhance cooling,
jets are positioned to blow cold air over the trap column when it
is not being heated. To examine the influence of cooling, the
heating sleeve was kept at the inlet (Figure 2, left) and outlet
(Figure 2, right) position for 600 s before it was moved to the
alternate position. Temperatures were measured as the heating
Figure 2. External cooling rates at (a) inlet and (b) outlet of the modulator trap column. Impact of active cooling is seen by the black, red, and blue trace
and passive cooling by the yellow, green, and pink trace. Positions 1−6 are illustrated on the modulator trap inset to the right. The infrared camera (FLIR
E40) was recording from −10 to 120 s after the heating sleeve, kept at 200 °C, was moved away from the position of measurement. The heating sleeve
had been held at the measuring position for 600 s before cooling rates were recorded. For active cooling the cold jets, ∼13 °C, were directed at positions
1 (for cooling at inlet) and 4 (for cooling at outlet).
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sleeve moved away from the initial position, at 1, 2, 3, 4, and 5 s
and then at 5 s increments until 120 s. A thermal camera was
chosen to record this data as moving the heating sleeve could
have damaged a thermocouple if placed in the small gap (∼0.3
mm) between the heating sleeve and the trap column. As seen in
Figure 2, immediately after the trap is moved, the temperature of
the trap decreases until it reaches thermal equilibrium, which
occurs within 10 s with cooling and about 30 s without cooling.
Also, the equilibrium temperature is about 20−40 °C cooler
when active cooling is employed. The figure also shows that the
equilibrium temperature reached with active cooling for the
outlet section is slightly higher than the inlet section. This is due
to the warmer incoming mobile phase, which has passed through
the heated inlet half of the trap, as opposed to the 25 °C mobile
phase that enters the inlet of the trap column. This higher outlet
temperature allows more efficient release of the captured targets
into the second dimension column. As a result, with active
cooling, the minimum modulation period is approximately 40 s
based on a two-stage modulation movement (inlet−outlet). On
the basis of Figure 2, it is beneficial to implement a cooling
process to rapidly lower the temperature back to improve
trapping of the analytes. In this proof-of-concept study, cooled
compressed air was employed. Further investigation on efficient
cooling mechanisms would be of benefit to minimize the
modulation period and improve the focusing effect.
It is important that analytes are trapped and eluted from the
trap within a single modulation cycle in order for the LOCTM to
function properly. As the trap column capacity and retention may
be significant, modulation periods must be optimized not only
based on heat transfer rates, but also based on the Murphy-
Schure-Foley rule,45 and according to the analyte velocity at the
modulation temperature. The retention and migration velocity of
alkylphenones as a function of temperature (up to 150 °C) are
given in Table 1. Retention factor values were obtained by
heating the whole trap column using a 50 mm long resistively
heated sleeve. The retention data and column void were obtained
by elution at isotherms ranging from 25 to 150 °C and were
corrected for the effect of extra column times (measured at the
same temperature) by subtraction of the latter from the former.
Triplicate analyses were performed. As seen in Table 1, the
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Analytical Chemistry
Table 1. Temperature Dependence of Alkylphenone
Retention Factor and Analyte Velocity on PGC Trap
Columna
alkyl- temperature retention factor analyte velocity
phenone (°C) (k) (mm/s)
aceto- 25 0.51 ± 0.04 3.7
150 0.01 ± 0.01 8.8
propio- 25 1.95 ± 0.06 1.9
150 0.05 ± 0.02 7.5
butyro- 25 3.96 ± 0.07 1.1
150 0.07 ± 0.03 6.5
valero- 25 10.26 ± 0.11 0.5
150 0.17 ± 0.03 4.7
a
Values showing 95% confidence interval (n = 3). Flow rate 0.15 mL/
min.
retention factor is reduced to approximately 0 for the all of the
test alkylphenones and analyte velocities increased by a factor of
2−9. The large change in retention factors from 25 to 150 °C
should be sufficient for modulation as both trapping and thermal
remobilization are essential for the performance of the
modulator, although slight retention remains at 150 °C. The
consequence of excessive retention at the remobilization
condition will cause the fraction to elute in a later cycle (wrap-
around) and potentially interfere with the early eluting
components from the next fractions. Ultimately, this can lead
to a reduction of the 2D column’s peak capacity and loss of overall
resolution.
Initial optimization of the modulation period was performed
by injecting individual analytes into the system without the 1D
and 2D columns and with well-defined movement of the heated
sleeve. This allows direct observation of the dual stage trapping
and remobilization capability of the modulator. The analyte was
initially trapped on the first half of the modulator trap with the
heated sleeve at the outlet position. Then the analyte was
mobilized onto the second half of the trap by moving the heated
sleeve to the inlet position. Finally, the heated sleeve was moved
back to the outlet position and the peak was eluted. The trap
column was kept at isocratic conditions with 70% methanol at
0.15 mL/min, and the modulation period was explored in the
range of 45−150 s. The 60 s modulation period is shown in
Figure 3 where the analytes are eluted within one modulation
period except valerophenone. It can be seen from the figure that
the analytes are injected and trapped during the first 30 s when
the heating sleeve is in the outlet position, transferred to the
second half of the trap from 30 to 60 s when the heating sleeve is
in the inlet position, and eluted from 60−90 s when the heating
sleeve was back at the outlet position. Moving the heating sleeve
up and down the trap column causes pressure fluctuation, seen as
the black trace, due to thermal changes in the system. At face
value, it would be anticipated that the pressure should not
change; however, the system is asymmetric with the temperature
of the liquid entering the cold half of the trap different depending
on whether the heating sleeve is at the inlet or outlet. The pump
pressure transducer recognizes this as a change in thermal
expansion due to local hot and cold spots. There is a void
between the column and detector due to a resistor coil needed for
the prevention of solvent evaporation, as such the elution times
in Figure 3 are 15 s later than the actual elution from the trap
column. The actual elution times are acetophenone = 60 s,
propiophenone = 69 s, butyrophenone = 81 s, and
valerophenone = 96 s. These elution times were translated
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Figure 3. Modulation optimization showing peak elution at 60 s
modulation period for acetophenone (green), propiophenone (orange),
butyrophenone (blue), and valerophenone (pink). The vertical dashed
lines show when the heating sleeve (200 °C) was moved along the trap
column and the associated pressure fluctuation (black) due to
temperature change. The analytes were individually injected onto the
modulator with the heating sleeve at the outlet position from 0 to 30 s, at
the inlet position from 30 to 60 s, and back to the outlet position from 60
to 90 s (when the peak is required to be eluted). This was repeated until
5 min (5 modulation cycles). Note the void time of 12 s between trap
and detector.
back to the temperature dependent retention study to estimate
the effective modulation temperature (heating sleeve pro-
grammed to 200 °C). Fitting the converted retention factors
(0.03, 0.15, 0.35, and 0.59) to the trend lines of a thermal
retention study of aceto-, propio-, butyro-, and valerophenone
gives an estimated internal trap column temperature of
approximately 105 °C.
Comparison of modulation at different conditions was
performed, and a score table (Table 2) for each of the
Table 2. Score Table of Modulation Period Effect on
Alkylphenone Elutiona
system constants effect on alkylphenone
modulation period (s) aceto propio butyro valero
150 − −−− + +
120 − + + +
90 −−− + + +
60 + + + −
45 + + − −−−
a
Peak not eluted within one modulation cycle (−), peak splitting (−
−), and peak not eluting within one modulation cycle and splitting (−
− −).
alkylphenones at the different modulation times was constructed
with negatives of (i) peak eluted outside the modulation cycle
(−), (ii) peak splitting (− −), and (iii) the combination of (i)
and (ii) (− − −). The successful elution within one modulation
cycle was annotated with a positive (+).
As can be seen from the longest modulation time of 150 s, this
is too long for acetophenone and propiophenone, which were
both eluted before the modulation cycle was complete, which is
expected due to their lower retention than butyro- and
valerophenone. The shortest modulation time of 45 s is also
inadequate, being too short for butyro- and valerophenone,
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Analytical Chemistry
Figure 4. Raw chromatogram of butyrophenone showing the number of
cuts of the 1D peak with 60 s modulation period.
which are eluted in subsequent modulations. This provides
opportunity for components from different cuts in the first
dimension to be coeluted in the second, against the rules defined
by Giddings.1 With the modulation periods from 60 to 120 s,
there is better coverage, with shorter periods not ideal for
valerophenone and longer periods not ideal for acetophenone
Having demonstrated the ability to trap and elute the
alkylphenones using the LOCTM, it was tested for the capability
to modulate online with the first dimension at gradient
conditions. The1D column, second dimension pump and the
modulator trap column were connected to a T-piece. The mobile
phase flow delivered from the 2D pump was matched to the 1D
mobile phase to deliver 0.15 mL/min to the modulator trap. The
percentage methanol from the 2D pump was adjusted to give an
effective gradient from 70% to 95% methanol assuming good
mixing in the T-piece. The repeatability of the modulator’s
performance is good where 95% confidence intervals (n = 3)
were found to be less than ±1 s for gradient 2D elution. The raw
chromatogram in Figure 4 shows that this modulator clearly has
the capability to perform according to the 3−4 cuts per 1D peak
rule.45
A suitable gradient in the 2D is important not only for the
separation on the 2D column but also for the modulator
performance. Considering the columns in this setup are serially
Figure 5. Separation of alkylphenones using (a) longitudinal on-column thermal modulator and (b) valve modulator.
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connected, high elutropic strength causes breakthrough and
incomplete sampling of the first dimension peaks whereas a
mobile phase that is too weak will induce wrap-around and
excessive retention. Wrap-around is an effect of excessive elution
times in the second dimension resulting in broad peaks or in the
worst-case scenario “streaking”.46 This is of particular concern
with the trap column setup used herein.
The physical dimensions of the trap column were chosen on
the basis of direct connection with standard size HPLC fittings
and the smallest practical size of the heating sleeve (20 mm). The
diameter of the trap column was justified based on the thin
column wall for better heat transfer and for the retention
capacity. The 1D column had to offer broad peaks to allow
sampling; a long column operated at a low flow rate could offer
this. The 2D column had to be operated fast, have low
backpressure due to the accumulation of pressure through the
serially coupled setup, and express moderate retention at the
operating temperature (75 °C) to avoid extensive wrap-around,
thus a short capillary titanium column was packed with PGC.
Online comprehensive two-dimensional separations were
achieved by connecting the 2D column (PGC, 0.3 mm × 34
mm, 5 μm) to the modulator trap column via a stainless-steel
transfer capillary (0.12 mm × 20 mm) and operated at the same
elution conditions as the modulator, gradient 70−95% methanol
at 0.15 mL/min. The 2D contour plots of alkylphenones by
longitudinal on-column thermal modulation and an equivalent
separation by valve modulation are shown in Figure 5a,b,
respectively. Although 40 s modulation is possible with LOCTM
(Figure 2), a 60 s modulation was found to be more suitable for
the separation of the alkylphenones on the columns used to
demonstrate the first thermal modulator for LC × LC.
The four alkylphenones fall on a straight line indicating
correlated retention in the 1D and 2D columns and demonstrates
that the modulator is working correctly, as each homologue in
the same series is expected to behave similarly within the two
reversed phase separation mechanisms, C 18 and PGC,
respectively. The 2D peak width is one metric with which to
quantitatively compare the modulator performance. For the
alkylphenones, the longitudinal thermal modulator shows wider
2
D peaks than the valve-based modulator. This is caused by the
relatively low retention on the trap and 2D column, which limits
compression on the modulator column. For acetophenone, this
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Figure 6. Separation of red wine on (a) thermal modulator and (b) valve modulator. Numbered peaks are considered for peak width comparison in the
second dimension (Table 3).
Figure 7. Contour plot of discrete peak (1) from red wine analysis by (a) thermal modulation and (b) valve modulation.
results in breakthrough, as the modulation period is too long.
The time lag in the first dimension experienced with the LOCTM
arises from the difference in the operating pressure of the two
columns. In the valve interface, the pressure is essentially that
from the analytical column alone, while in the LOCTM, the
pressure is higher due to the contribution of the trap and the 2D
column. This change in pressure has an effect on the first
dimension interaction due to the change in solute partial molar
volume.47−50 The cumulative backpressure with thermal
modulation experienced by the 1D column is 7 times larger
compared to the backpressure when using the valve setup. A
difference of 26% for acetophenone, 22% for propiophenone,
18% for butyrophenone, and 15% for valerophenone is not
unreasonable given that McGuffin and Evans reported an
increase of 9 to 24% for a homologous series of fatty acid
derivatives under isocratic conditions.47 The pressure-dependent
retention observed here is presumably due to the decreasing
backpressure as the viscosity of the mobile phase decrease during
gradient elution, i.e., the smaller the homologue, the earlier it
elutes during the gradient when the backpressure is greater and
thus the usual pressure-dependent effect on homologue
retention is inverted. Overall, the larger total pressure by
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LOCTM to valve modulation induces a time shift in the separation
space.
One advantage of the thermal modulation are the minimized
pressure fluctuations. The valve modulator used here produced a
10% increase in pressure upon switching, compared to a 2%
decrease in pressure due to the thermal modulator. The resulting
baseline noise, as an effect of change in refractive index of the
mobile phase observed by the detector,51 translates to improved
signal-to-noise by a factor of up to 14 for the thermal modulator.
The longitudinal movement of the heated sleeve and the
online implementation are the fundamental differences between
LOCTM and the approach by Verstaeten et al.36The offline
approach relied on the rapid temperature transfer to and from the
trap column, which was packed with PGC as in this study, by
temperature cycling the heating sleeve. LOCTM is a continuous
system where temperature is kept isothermal, circumventing the
time lag for heat transfer by temperature programming,
particularly in this case where a 0.8 mm i.d. trap column is
employed and as cryogenic cooling was not available. LOCTM
may also be gentler on the trap column stationary phase as it is
operated at a lower temperature than the maximum according to
stationary phase specifications. The improvement in signal-to-
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noise ratio corresponds to the improvement of signal response
reported by Verstraeten et al.36
In a complex sample, the range of molecules with different
retention on both the first and trap/second dimension stationary
phase will give a variety of relative retentions (kPGC/kC18), as such
breakthrough and wrap-around will be observed. There is also
potential for focusing through the thermal modulator, which is
not possible with the simple valve approach. Red wine was
analyzed as an example of a complex sample using both
modulation techniques with the chromatograms shown in Figure
6. The separation is not intended as a quantitative study but to
illustrate the performance of the modulators and to provide some
evaluation of the potential of the LOCTM as a modulator for
comprehensive two-dimensional LC.
Both modulation techniques show a comparable peak pattern
in the 2D space and both show streaks. The effect of baseline
noise in Figure 6b is seen as a blue smear along the bottom length
of the plot. The streaks are attributed to the 1D effluent fraction
migrating through the modulator and second dimension column
without being modulated. Wrap-around is also seen for both
modulation techniques for the wider second dimension bands; in
cases of excessive wrap-around, this results in streaking. The
vertical lines in the contour plot using the valve modulator
(Figure 6 b) are due to wrap-around and/or sample
concentration overload. To provide some quantitative compar-
ison of performance, an initially well-resolved peak was selected
for analysis, Figure 7. The peak width using LOCTM gave 50%
narrower bands in the second dimension compared to the valve
modulator. On the basis of this result, three more peaks were
carefully selected based on the difference in retention, the 2D
peak widths of the longitudinal thermal modulator and the valve
modulator shown in Table 3.
Table 3. Peak Widths, With 95% Confidence Interval (n = 3),
in the Second Dimension for Longitudinal Thermal and Valve
Modulation of Red Wine, Peak Identification As in Figure 6
peak LOCTM tw (s) valve tw (s)
1 11 ± 2 23 ± 2
2 19 ± 3 41a ± 3
3 18 ± 1 25 ± 2
4 13 ± 1 26a ± 7
a
Baseline noise/peak overlap interferes with peak width determi-
nation. An estimated peak boundary is used to give an approximate
peak width value.
The data show that the peaks widths are reduced by 53% for
peak 1, 55% for peak 2, 28% for peak 3, and 48% for peak 4 by the
LOCTM compared to the valve. This is a good indication of the
benefit of active focusing modulation. Peaks 2 and 4 are partly
incorporated in the baseline noise when employing valve
modulation, making it difficult to determine the exact second
dimension peak width.
Like solvent-dilution active modulation,18,19 improved second
dimension peak widths are obtained with LOCTM. However,
solvent-dilution modulation outperforms LOCTM in terms of
reduced modulation periods and shorter total analysis times. The
20 s modulation period and half the analysis time were obtained
with active solvent-dilution modulation compared to passive
valve-modulation,19 allowing the first dimension to operate at
closer to optimum conditions provided the 2D separation is fast
enough. Cooling rates and the physical dimensions of the trap
column currently restricts the speed of the LOCTM and thus the
1129
Article
overall performance of the modulation technique. This shows
that much work remains in further developments and
optimization of LOCTM before it can be implemented as a
routine approach to LC × LC to meet the fast modulation
periods required for narrow first dimension peaks. While these
results show the first experimental implementation of a thermal
modulator for LC × LC, there are several aspects that are not
ideal. The first is the discriminating nature of the trap column,
where the analyte interaction on the trap phase may lead to
breakthrough or wrap-around and in the worst-case scenario
induce significant retention due to polar retention interaction on
graphite.52−55 An analyte specific trap column phase may be
required to obtain a desirable peak focusing effect, keeping in
mind the operating temperature using LOCTM. In the current
system, the 2D time is limited by both the speed of the 2D
separation and the modulator. New materials, particularly those
that are thermally responsive, may be required to obtain a
desirable peak focusing effect, keeping in mind the operating
temperature using LOCTM. The second limitation is the speed of
the modulation cycle. A third shortcoming is the accumulation of
pressure, which restricts column selection and flow rates. The
entire system can be improved through addressing improve-
ments in the 1D and 2D column and mobile phase chemistry, as
well as a more rapid thermo-cycling interface. Despite the
drawbacks, this work has demonstrated equal repeatability to
valve modulation and reduced baseline distortion. The
longitudinal on-column thermal modulator shows promise for
improvement of comprehensive two-dimensional liquid chro-
matography as a valveless, online, dual stage focusing modulator.
■
CONCLUSION
An important condition for successful compression by
modulation is the retention on the modulator trap relative to
the first dimension stationary phase. Also, the rapid and large
changes of analyte retention and migration velocity on the
modulator trap at trapping (low) and remobilizing (high)
temperatures are critical parameters. The longitudinal on-
column thermal modulator has paved a new path for
comprehensive two-dimensional liquid chromatography. The
recognition of a dual stage active modulator is of utmost
importance for the performance of LC × LC. Peak capacity is
critical and reducing 2D peak widths is one of the possibilities to
achieve this. The LC × LC separation of red wine using the
longitudinal on-column thermal modulator achieved up to 60%
narrower bands of discrete peaks in the second dimension
compared to the valve-based modulator at otherwise identical
conditions. Furthermore, the signal-to-noise ratios were up to an
order of magnitude larger by the longitudinal thermal modulator.
■ AUTHOR INFORMATION
Corresponding Author
*Phone: + 61 3 6226 2154. Fax: + 61 3 6226 2858. E-mail:
Michael.Breadmore@utas.edu.au.
ORCID
Michael C. Breadmore: 0000-0001-5591-4326
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
M.E.C. would like to acknowledge the University of Tasmania for
receiving a graduate research scholarship. J.P.F. gratefully
acknowledges the 2015−2016 sabbatical leave provided by
DOI: 10.1021/acs.analchem.6b03279
Anal. Chem. 2017, 89, 1123−1130
Analytical Chemistry
Drexel University and the Visiting Fellowship provided by the
University of Tasmania and the Australian Centre for Research
on Separation Science (ACROSS). M.C.B. is the recipient of an
ARC Future Fellowship (Grant FT130100101). The authors
would also like to thank Dr. Jim Luong (Dow Canada, Analytical
Technology Center, Fort Saskatchewan, AB, Canada) for
valuable discussions and technical guidance and Thermo
Scientific for the porous graphitic carbon.
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