36433 Topic HA W9 R1Number of Pages 1 (Double Spaced)N.docx

36433 Topic: HA W9 R1
Number of Pages: 1 (Double Spaced)
Number of sources: 2
Writing Style: APA
Type of document: Essay
Academic Level:Master
Category: Nursing
Language Style: English (U.S.)
Order Instructions: Attached
Shawna Harris
Wednesday Mar 6 at 3:38pm
Manage Discussion Entry
The U.S. Preventative Services Task Force has prostate
screening recommendations. The U.S. Preventative Services
Task Force suggests discussing with the patient the benefits and
possible harm from obtaining a prostate specific antigen, PSA

test (U.S. Preventative Services Task Force). There is a small
percent of people for whom this test can correctly identify and
thus reduce the risk of mortality from prostate cancer (U.S.
Preventative Services Task Force). However, this test can often
have false positives, which could result in obtaining an
unnecessary biopsy (U.S. Preventative Services Task Force).
Invasive procedures, such as biopsies always have risk factors
of their own. Consequently, the U.S. Preventative Task Force
recommends a PSA screening test for men ages fifty-five to
sixty-nine, only if the patient is requesting this screening even
after discussing benefits and possible harms from testing and
biopsy. (U.S. Preventative Services Task Force).
The American Cancer Society, ACS, recommends that men age
fifty and over discuss the benefits and risks of screening in
order to make an informed decision with his provider (Wolf,
Wender, Etzioni,….& Smith, 2010). ACS also recommends if a
man is at a high risk, that this information is presented earlier
than fifty (Wolf et al…2010). Those at a higher risk include
African American men with a family history of prostate cancer
occurring in a family member who is not elderly (Wolf et
al…2010). ACS also does not recommend that men whose life
expectancy is less than ten years be screened for prostate cancer
(Wolf et al…2010). Providers need to provide men with the
benefits of early detection and treatment with the risk factors of
treatment for prostate cancer. The results of PSA testing are not
conclusive and therefore, the ACS reiterates the importance of
the patient having the knowledge and information to make an
informed decision. The ACS provides educational brochures and
handouts on PSA screening to help guide patients to a
discussion of this subject with his provider.
References
U.S. Preventative Services Task Force, (Accessed March 2019).
Screening Guidelines for Prostate. Retrieved from:

https://www.uspreventiveservicestaskforce.org/Page/Document/
RecommendationStatementFinal/prostate-cancer-screening1
(Links to an external site.)Links to an external site.
Wolf, A., Wender, R., Etzioni, R….& Smith, R. (2010).
American Cancer Society Guideline for the Early Detection of
Prostate Cancer: Update 2010. Retrieved from:
https://onlinelibrary.wiley.com/doi/full/10.3322/caac.20066
(Links to an external site.)
** Provide response writing with references. All references
must be in APA format and published within the last 5 years.
Determination of Suitable Column Geometries by Means of van
Deemter and Kinetic Plots for Isothermal and Isocratic Method
Development in High-Temperature Liquid Chromatography
Isotope
Ratio Mass Spectrometry
Paul Ermisch,† Steffen Wiese,† Harald Weber,‡ and Thorsten
Teutenberg*,†
†Institut fuer Energie- und Umwelttechnik e. V., Bliersheimer
Strasse 58-60, 47229 Duisburg, Germany
‡Niederrhein University of Applied Science, 47798 Krefeld,
Germany
*S Supporting Information
ABSTRACT: The method of high-temperature liquid chro-
matography isotope ratio mass spectrometry (HTLC-IRMS) is
used to determine the origin or authenticity of compounds.
Currently, the drawback of this hyphenation is the interface
which causes pronounced band broadening due to a large
extra-column volume. Therefore, the aim of this study is to

determine suitable column geometries and particle sizes at
different temperature and to study the effect of extra-column
band broadening. The tools to assess the efficiency of columns
are van Deemter and kinetic plots. By comparison of different
column geometries and particle sizes, it could be shown that 3.0
mm ID columns achieve a higher performance than 2.1 mm ID
columns and a particle size of 1.7 μm is advantageous over 3.5
and 5.0 μm particles when the injection volume is adjusted to 2
μL
and the temperature is higher than 60 °C. Because water was the
mobile phase, the retention factor could not be kept constant at
different column temperatures. The lower retention factor at
elevated temperatures leads to a decrease of the plate number,
because of the relatively larger contribution to extra-column
band broadening at lower retention factors. This is the reason
why
3.0 mm ID columns should be preferred for the HTLC-IRMS
hyphenation when the separation is carried out under isothermal
and isocratic conditions.
The use of elevated temperatures in liquid chromatographyhas
some advantages compared to liquid chromatography
at ambient conditions.1−6 By increasing the temperature, the
viscosity of the mobile phase and hence the pressure drop over
the column decreases.7 Therefore, a longer column can be used
at the same flow rate or shorter analysis times can be achieved
using higher flow rates without sacrificing efficiency.
Furthermore, the diffusion coefficient DM, described by the
Wilke-Chang equation, increases with increasing temperature.8
= *
Ψ
η
−D

M
V
T7.4 10M
8 2 2
1
0.6
(1)
Here, Ψ2 is the association factor of the solvent (1 for nonpolar
solvents and 2.6 for water), M2 [g mol
−1] is the molecular
weight of the solvent, T [K] is the absolute temperature, V1
[mL] is the molar volume of the solute, and η [cP] is the
viscosity of the solvent which strongly depends on temperature.
Another effect is that the static permittivity and hence polarity
of the mobile phase will be reduced when the temperature is
increased.9 At high temperatures, water has a similar polarity
like an organic solvent at ambient temperature. Consequently, a
solvent gradient can be replaced by a temperature gra-
dient.10−12 This allows the use of special hyphenation
techniques, where only water can be used as mobile
phase.13−18
One of these techniques is isotope ratio mass spectrometry
(IRMS), which is used to determine the origin or authenticity
of samples.14,15,19,20 The variation in stable isotope ratio of
different samples is due to fractionation effects during chemical
or biological reactions. Before determining the isotope ratio of
components, complex samples must be separated chromato-
graphically. For this reason, the isotope ratio mass spectrometer

is coupled to an HPLC system. In the interface, all carbon
which is in the mobile phase is oxidized to CO2. Afterward, the
CO2 is separated from the mobile phase for the isotope
analysis. To prevent a high blank value, only water can be used
as eluent in the HPLC unit.
The drawback of this hyphenation is the interface which
causes pronounced band broadening due to a long residence
time of the analytes in the oxidation reactor. The flow rate of
the mobile phase is adapted to the oxidation time and cannot
be increased further. Currently, a maximal flow rate of 0.7 mL
min−1 can be adjusted on commercially available instrumenta-
Received: October 25, 2011
Accepted: January 4, 2012
Published: January 20, 2012
Article
pubs.acs.org/ac
© 2012 American Chemical Society 1565
dx.doi.org/10.1021/ac202819v | Anal. Chem. 2012, 84,
1565−1571
pubs.acs.org/ac
tion. However, it would be advantageous to increase the flow
rate in order to prevent work in the B-term dominated region
of the van Deemter curve at elevated temperatures. A loss in
separation efficiency would also negatively effect the resolution
between adjacent peaks. One of the basic requirements for
IRMS analysis is that a baseline separation of all compounds in
the mixture has to be achieved. Therefore, the best possible
separation in the HPLC unit is necessary to prevent a coelution

of the CO2 fractions at increased flow rates. The aim of this
study was to determine suitable column geometries and particle
sizes for a temperature range of 60 to 180 °C using water as the
mobile phase and to study the effect of extra-column band
broadening on the overall efficiency.
■ THEORY
When assessing the efficiency of columns, van Deemter plots
can be used. They show the lowest achievable plate height Hmin
and the corresponding optimal linear velocity uopt. Van
Deemter plots are based on the van Deemter equation. Here,
H [μm] is the plate height, A [μm] is the Eddy diffusion, B
[μm2 s−1] is the longitudinal diffusion, C [μm s−1] is the mass
transfer, and u [mm s−1] the linear mobile phase velocity.
= + +H u A
B
u
Cu( )
(2)
Another tool to compare the efficiency of columns is the kinetic
plot method. Kinetic plots are derived from experimental van
Deemter plots and show the highest plate number a column
can achieve in the shortest time when working at the maximum
pressure of the system.21 A transformation of experimentally
generated van Deemter plots to kinetic plots can be performed
with some simple equations. For the basic plate number (N)
versus dead time (t0) kinetic plot, the following equations are
applied
=
Δ
η

⎜ ⎟
⎛
⎝
⎞
⎠N
p K
uH
max v
exp (3)
=
Δ
η
⎛
⎝⎜
⎞
⎠⎟t
p K
u
0
max v
2
exp (4)
Here, for every experimentally determined (u, H)-couple, the
column is prolonged to the length which yields a pressure drop
equal to the system pressure limit Δpmax at the given linear

velocity u. Hence, every data point of a kinetic plot curve is
achieved with another column length at different dead times.
The system pressure limit can be freely chosen. For a
theoretical consideration, the pressure limit can be set to
values beyond the real system limit. Thus, one obtains
information about the performance a column can achieve on
a UHPLC system even though the van Deemter data are
generated on a conventional HPLC system. However,
extrapolating HPLC data to UHPLC conditions can over-
estimate the performance of the system at ultrahigh pressures
because of effects like viscous heating.
■ EXPERIMENTAL SECTION
Chemicals and Columns. The model compounds
sulfadiazine, sulfamerazine, and sulfamethazine, with a purity
grade of >99%, were purchased from Sigma-Aldrich (Seelze,
Germany) and dissolved in water/acetonitrile (75/25, v/v) at a
concentration of 100 μg mL−1. High-purity deionized water was
produced by an Elix 10-Milli-Q Plus water purification system
(Millipore, Eschborn, Germany) and acidified with 0.1% formic
acid, obtained from Fluka (Seelze, Germany). Acetonitrile
(Optigrade) was purchased from LGC Standards (Wesel,
Germany). The columns, which were used in this study and
filled with ethylene-bridged hybrid (BEH) C18 particles, were
provided by Waters (Eschborn, Germany). Columns containing
different particle sizes from 1.7, 3.5, and 5.0 μm and with
internal diameters of 2.1 and 3.0 mm ID were used in this
study. The column length was always 5 cm in order to obtain a
better signal-to-noise ratio on the 2.1 mm i.d. column, because
HPLC-IRMS is not a trace analytical technique. For monitoring
the degradation of the stationary phases, a mixture of uracil,
naphthalene, and acenaphthene, purchased from Fluka, was
measured with a water/acetonitrile (50/50, v/v) mobile phase
after finishing measurement series at 60, 120, and 180 °C,

respectively.
Instrumentation. The experiments were always performed
in triplicate on two HPLC systems at temperatures of 60, 120,
and 180 °C. Columns with 3.5 and 5.0 μm particles were used
on an Agilent 1100 Series HPLC system (Agilent Technologies,
Waldbronn, Germany) in combination with a commercially
available SIM HT-HPLC 200 high temperature column oven
(SIM, Scientific Instruments Manufacturer, Oberhausen,
Germany). For the column packed with 1.7 μm particles, an
UltiMate 3000 UHPLC System (Dionex GmbH Deutschland,
Idstein, Germany) in combination with a Metalox Model 200-C
high-temperature column oven (Systec, New Grighton, USA)
was used. The SIM HT-HPLC oven included an eluent
preheater and an eluent cooler, which decreased the mobile
phase temperature to 50 °C before it was introduced into the
detector. The investigated flow rate range was between 0.05
and 1.5 mL min−1. The injection volume was always 2 μL
which
was regarded to be the lower injection limit. A lower injection
volume would not make sense for HPLC-IRMS hyphenation
due to the relatively high amount of carbon which has to be
injected on column. Detection was carried out at a wavelength
of 270 nm.
To simulate the above-described HPLC-IRMS-Interface, a
PEEK capillary with an internal diameter of 0.17 mm and a
length of 1 m was connected between the column and the
detector and heated at 80 °C. The volume of this additional
capillary was about 23 μL. All measurements were carried out
first without and subsequently with additional capillary.
Methodology. Data fitting to the van Deemter curves was
performed by minimizing the sum of error squares. From the
experimentally determined van Deemter data, kinetic plots
were transformed using the Kinetic Plot Analyzer software

(version 6.7), provided by the Vrije Universiteit Brussel.22 The
permeability K [mm2 s−1 bar−1], specific permeability Kv [mm
2],
and porosity ε needed to create kinetic plots were calculated as
specified in the literature.23 The mobile phase dynamic
viscosity
η [mPa s] was calculated according to ref 24. Lc [mm] is the
column length, Δpcol [bar] is the pressure drop across the
column, t0 [s] is the dead time, F [mL min
−1] is the flow rate,
and dc [mm] is the internal column diameter. The dead times
were calculated with the simulation software DryLab 2000 Plus,
Ver. 3.8.
= ηε−K K10v
8
(5)
Analytical Chemistry Article
1565−15711566
=
*
Δ
=
Δ *
K

u L
p
L
p t
c
col
c
2
col 0 (6)
ε =
*
*
F t
d L
21.2 0
c
2
c (7)
The column pressure drop (Δpcol) was determined by
subtracting the system pressure drop without column (Δpcap)
from the entire system pressure drop including the column
(Δpsys) at every flow rate:
Δ = Δ − Δp p pcol sys cap (8)

To investigate the influence of the temperature on the
separation efficiency, the retention factor should be kept
constant. This condition can be fulfilled by adapting the
composition of the mobile phase to the temperature.25,26 In
this
work, however, only water was used as mobile phase and hence
the retention factor could not be kept constant.
■ RESULTS AND DISCUSSION
All experiments were performed on silica ethylene-bridged
hybrid (BEH) stationary phases because of their excellent
stability at high temperatures.27 Figure 1 shows the influence
of
an additional extra-column volume on the separation power of
two columns with an ID of 3.0 and 2.1 mm at 60 and 120 °C.
When the 3.0 mm ID column is used at 60 °C which is
depicted in Figure 1(a), an additional extra-column volume has
almost no influence on the plate height minimum. This can also
be observed when the temperature is increased to 120 °C as is
shown in Figure 1(b). There is just a slight increase of the plate
height at higher flow rates. In contrast, the 2.1 mm ID column
is subjected to a decreased efficiency at temperatures of 120 °C
and higher when an extra-column volume is added (Figure
1(d)). Here, the minimal plate height is increased from 28 to
31 μm. At 60 °C, no loss in efficiency is observed (Figure 1(c)).
At a temperature of 180 °C, however, a loss of the separation
efficiency for the 3.0 mm ID column is also observed. The
minimal plate height of sulfamerazine increases from 43 to 47
μm when an additional extra-column volume is added (data not
shown). For the 2.1 mm ID column, the data could not be
Figure 1. van Deemter plots of sulfamerazine on a 3.0 mm ID
(a, b) and 2.1 mm ID (c, d) column with 5 μm particles at 60 °C
(a, c) and 120 °C (b,
d). The black solid curves ( × ) were obtained without and the

red dashed curves (○) were obtained with additional extra-
column volume.
Table 1. Minimum Plate Height [μm] for Sulfamerazine at
Optimal Linear Velocity uopt and at Flow Rates of 0.7 and 1.5
mL min−1 Including the Influence of an Additional Extra-
Column Volume (aecv)
2.1 mm ID 3.0 mm ID
T, °C uopt 0.7 mL min
−1 1.5 mL min−1 uopt 0.7 mL min
−1 1.5 mL min−1
60 without aecv 15 20 33 13 13 17
with aecv 15 19 33 14 14 18
120 without aecv 28 51 96 18 20 31
with aecv 31 56 103 18 20 33
180 without aecv 43 83 162
with aecv 47 81 148
1565−15711567
fitted to a valid van Deemter curve when the temperature was
increased to 180 °C, because the data points showed no
minimum and a large scatter.
In Table 1, the plate heights for the above-discussed curves at

the optimal linear velocity as well as flow rates of 0.7 and 1.5
mL min−1 are summarized. These flow rates have been chosen
because 0.7 mL min−1 is the highest flow rate which can be
adjusted on commercially available instrumentation for HPLC-
IRMS hyphenation. In order to increase the speed of analysis,
an increase of the flow rate by a factor of 2 is desirable. When
intending to increase the flow rate from 0.7 to 1.5 mL min−1,
the loss of efficiency has to be as low as possible. From Table
1,
it can be derived that a two times higher flow rate leads to an
increase in the plate height. However, when a 3.0 mm ID
column is used, the loss of efficiency is lower than with a 2.1
mm ID column. Especially when increasing the temperature
from 60 to 180 °C, the plate height increases from 13 to 43 μm
for the 3.0 mm ID column. Interestingly, the plate height
minimum without the extra column capillary is slightly higher
than with additional extra column volume. We assume that this
is because of the extremely low retention factors (k < 1) at
these temperatures so that the error in calculating H is rather
high.
As already mentioned above, an increase of the temperature
in theory leads to a shift of the van Deemter minimum to
higher linear velocities, but the plate height minimum remains
constant.25 This is the result of a higher B-term- and lower C-
term-contribution at elevated temperatures. However, this
behavior can only be observed when the retention factor is
kept constant at all temperatures by adjusting the mobile phase
composition. Figure 2(a) shows the influence of temperature
when the retention factor is not constant due to the use of
water as sole mobile phase. By increasing the temperature, the
curve minimum increases from 15 μm at 60 °C to 82 μm at 180
°C. Furthermore, the slope of the C-term increases, and the
curve fit is worse than at lower temperatures. This reduced
separation power is caused by a lower retention factor at higher

temperatures, because of the relatively larger contribution to
extra-column band broadening at lower retention factors. The
model compound in Figure 2(a), sulfadiazine, has a retention
factor of 10 at 60 °C, 2.3 at 120 °C, and 0.7 at 180 °C. Neue
already showed that the plate number will decrease when the
retention factor decreases.28 Here, the loss of efficiency is
dependent on the internal diameter of the column. The lower
the internal column diameter, the higher is the efficiency loss at
a given retention factor.
This is also underlined by the comparison of the separation
power of the two column IDs (2.1 and 3.0 mm) at different
temperatures (60 and 120 °C) in Figure 2(c,d). At 60 °C, both
columns generate a minimal plate height of 15 μm (Figure
2(c)). When increasing the temperature to 120 °C (Figure
2(d)), the 3.0 mm ID column still achieves the same efficiency
at the optimal linear velocity in the plate height minimum. In
contrast, the separation power of the 2.1 mm ID column
decreases, and the plate height minimum has a value of 22 μm.
When the temperature is increased further to 180 °C, the
performance of both columns is significantly reduced. The 3.0
mm ID column has a doubled minimal plate height (15 to 28
μm). Because of an invalid van Deemter curve at 180 °C, no
minimal plate height for sulfadiazine on the 2.1 mm ID column
could be calculated. The curve could not be fitted because it
showed no minimum and a large scatter of data points. These
Figure 2. (a) van Deemter plots of sulfadiazine at 60 °C (black
solid curve, × ), 120 °C (red dashed curve, ○), and 180 °C (blue
dotted curve, □),
obtained with a 3.0 mm ID column with 3.5 μm particles. (b)
van Deemter plots of sulfamethazine on columns with an ID of
2.1 mm and 1.7 μm
(black solid curve, × ), 3.5 μm (red dashed curve, ○), and 5.0
μm (blue dotted curve, □) at a temperature of 120 °C. (c and d)
van Deemter plots of

sulfamethazine on columns containing 5 μm particles with an ID
of 2.1 mm (black solid curves, ×) and 3.0 mm (red dashed
curves, ○) at a
temperature of 60 and 120 °C.
1565−15711568
results are related to the above-mentioned fact of a much lower
retention factor at higher temperature when the solvent
strength of the mobile phase cannot be adjusted. A small
internal column diameter and a low retention factor leads to
very short interaction times of the analytes with the stationary
phase. In this case, the influence of the column volume on
efficiency is extremely high.
To determine the influence of the particle size on the column
efficiency, particles with dimensions of 1.7, 3.5, and 5.0 μm
were investigated. The comparison at 120 °C in Figure 2(b)
shows that the 1.7 μm particles achieve the lowest plate height.
In addition, the slope of the C-term at higher linear velocities
decreases with smaller particles. In consequence, the minimum
is shifted to higher linear velocities. These results are in
complete agreement with theoretical considerations and could
be shown in other studies.29,30
The following results refer to kinetic plots, transformed from
experimentally obtained van Deemter plots. Figure 3(a,b)
shows kinetic plots of plate number N versus dead time t0 to
compare the three different particle sizes. The plots differ in the
chosen working pressure of 400 (Figure 3(a)) and 1200 bar
(Figure 3(b)), respectively. At low dead times, both plots show

that small particles generate a higher plate number than larger
particles. At high dead times, larger particles are superior over
small particles. The intersection is dependent on the operating
pressure. Working at 400 bar, 1.7 μm particles are advantageous
up to a dead time of 24 s. In the dead time range of 24 to 107 s,
3.5 and 5.0 μm particles generate almost the same plate number
but more than 1.7 μm particles. For dead times higher than 107
s, 5.0 μm particles are suited best. This means that for fast
analysis times lower than 10 min small particles and for
analysis
times higher than 10 min large particles should be used. When
the working pressure is increased to 1200 bar (Figure 3(b)),
the intersection points shift to three times higher dead time
values and the curves reach higher values. The advantage of
small particles over larger particles increases proportional to
the
increase in the working pressure. Furthermore, for maximizing
the absolute plate number, only large particles should be used.
However, a very high number of plates is only achievable at
excessively long retention times. For example, the marked data
point (*) in Figure 3(a) refers to a plate number of
approximately 130.000 at a dead time of 170 min. With a
retention factor for sulfamerazine of 25 at 60 °C, a retention
time of almost 74 h results! For usual chromatography a plate
number of 10.000 is sufficient and can be generated much faster
with 1.7 μm particles than with larger particles.
In Figure 3(c), a kinetic plot of column length L versus plate
number N is shown. From this plot, it can be derived which
column length is needed to reach a certain plate number. Short
columns are filled ideally with small, long columns with large
particles. Here, the performance of the three particle sizes to
each other also changes. The intersection points are obtained at
a column length of 24 and 69 cm, respectively. Because of the
fact that conventional columns have lengths of 5, 10, and 15
cm, only the small particle packed columns should be used. Of

course, longer columns can be obtained by coupling columns
together, but this approach leads to excessively long retention
times31 and possible analyte degradation when working at
elevated temperatures.
Figure 4 also shows N versus t0-kinetic plots as a function of
the maximal column pressure. It illustrates the separation
efficiency of columns with internal diameters of 2.1 and 3.0
mm. As can be seen in Figure 4(a), at a temperature of 60 °C,
the efficiency of the 3.0 mm ID column is slightly higher than
for the 2.1 mm ID column. This holds true for the whole dead
time/retention time range. When increasing the temperature to
120 °C as is shown in Figure 4(b), the difference in efficiency
becomes more pronounced. The 3.0 mm ID column achieves a
much higher plate number compared to the 2.1 mm ID
column. The vertical, dotted lines mark the retention times of
10, 30, and 60 min, respectively. For a given retention time,
both columns generate a higher number of plates when the
temperature is increased from 60 to 120 °C.
Figure 3. Kinetic plots of sulfamerazine showing a comparison
of
different particle sizes (1.7 (black ×), 3.5 (red ○), and 5.0 μm
(blue
□)) at 60 °C. (a and b) N versus t0-plot at 400 and 1200 bar,
respectively. (c) Column length L versus N-plot at a pressure of
400
bar.
1565−15711569

The dashed curves represent the efficiency which can be
obtained when the columns are operated at a working pressure
of 1200 bar. It is seen that the columns can generate a higher
number of plates compared to a maximal pressure of 400 bar
(solid lines). For a retention time of 60 min and a working
pressure of 1200 bar, a 3.0 mm ID column is able to generate
100 000 plates. However, for this performance a column length
of about 2.5 m is necessary!
The stationary phase degradation was monitored with a
mixture of uracil, naphthalene, and acenaphthene. As expected,
the columns showed a loss of retention time up to 12% after
the experiments at elevated temperatures. The loss of retention
is independent of the column dimension or particle size and
occurs for all columns at the same degree (Figure S.1, Table
S.1, Supporting Information).
Finally, the effect of frictional heating has to be addressed.
The basis data for the calculated kinetic plots were almost
exclusively determined at pressures below 400 bar. When
calculating kinetic plots for any higher pressures, e.g., 1200
bar,
frictional heating was not taken into account. When frictional
heating occurs, the peak may be subjected to a band broadening
due to axial and radial temperature gradients.6 In this case, the
real column efficiency will be much lower than the calculated
efficiency. Therefore, it is useful to extrapolate kinetic plots
just
for operation pressures where frictional heating can be
excluded. By this means, a realistic assessment of the possible
column efficiency can be made.
In order to derive the conclusions, the obtained results now
have to be discussed in the context of the hyphenation of high-
temperature HPLC with isotope ratio mass spectrometry. As

was already explained in the introduction, this hyphenation
technique completely relies on the requirement that only a
water mobile phase can be used. This means that any addition
of an organic solvent in order to facilitate the elution of
compounds is prohibited. The conventional solvent gradient
elution cannot be applied. Instead, temperature is the most
important parameter to influence retention, selectivity, and
efficiency. When the temperature is increased, the retention
factor will decrease for most compounds in reversed phase
HPLC. This in turn leads to a much higher influence of the
extra-column volume to band broadening and hence efficiency.
Operating the column under isothermal and isocratic
conditions means that the analytes cannot be focused at the
column head, especially when the temperature is increased. In
order to obtain high efficiencies at isothermal conditions, a low
temperature would always be preferred because of a much
higher retention. In this respect, the influence of the column
diameter will only exert a negligible influence on the column
efficiency. Working at low temperature has the inherent
disadvantage, however, that extremely long retention times
will be observed.
Applying higher temperatures will increase the sample
throughput because of a much lower retention of the target
analytes. However, the extra-column volume exerts a strong
influence on the efficiency. Therefore, a compromise has to be
found between the column inner diameter and the temperature.
In our experiments, the injection volume was kept constant.
The reason is that IRMS is not an analytical technique for trace
analysis. The injection volume or the injected sample amount
has to be high enough to get a signal. Therefore, an injection
volume below 2 μL will not be considered for HPLC-IRMS
hyphenation. Applying higher injection volumes up to 20 μL
would lead to an even higher discrepancy between the
performance of the 2.1 and 3.0 mm ID column. Increasing

the column length to 10 cm for the 2.1 mm ID column would
not lead to a better peak focusing at elevated temperatures
when the injection volume is kept constant. Furthermore, a
higher signal-to-noise ratio can also not be expected because of
a longer elution of the analyte bands through the column.
From the discussion above, it becomes clear that temperature
programming instead of the isothermal operation mode would
offer the benefit of a sample focusing on the head of the
column as well as a higher retention of all compounds. In this
respect, low and highly retained solutes could be eluted in a
much narrower time window, which we have shown else-
where.10 Currently, the maximal flow rate which can be
adjusted in high-temperature liquid chromatography (HTLC)-
IRMS is around 0.7 mL min−1, although it would be desirable
to
increase the flow rate in order to compensate the loss in
efficiency when working in the B-term dominated region of the
van Deemter curve. However, our findings also demonstrated
that increasing the flow rate to 1.5 mL min−1 will cause a
slightly negative influence on band broadening but a doubled
sample throughput when working at 60 °C and using a column
diameter of 3.0 mm.
■ CONCLUSION
Our results clearly show that columns with an inner diameter of
3.0 mm should be preferred over columns with an inner
diameter of 2.1 mm for HPLC-IRMS hyphenation at
Figure 4. N versus t0-kinetic plots of sulfamerazine showing a
comparison of different column internal diameters (2.1 mm
(black ●, green ○) and
3.0 mm (red ■, blue □)) at 60 °C (a) and 120 °C (b). Solid lines
at 400 bar (full symbols) and dashed lines at 1200 bar (open
symbols),
respectively.

1565−15711570
temperatures above 60 °C if the following restrictions
concerning method development exist: (a) The mobile phase
consists of water, and the retention factor cannot be adjusted at
higher temperatures by changing the mobile phase composi-
tion. (b) The injection volume cannot be decreased because of
the detection limit given by the detector. In this study, the
injection volume was always 2 μL which can be regarded as the
lower limit of injection for HPLC-IRMS hyphenation. (c) The
sample is eluted under isothermal conditions.
These requirements are specific for the hyphenation of high-
temperature HPLC with isotope ratio mass spectrometry, but
they can also be transferred to the LC-FID hyphenation which
is well described in the literature. Although a higher inner
column diameter will always be better, the particle size of the
stationary phase should be as low as possible if the column is
shorter than 24 cm. Columns containing 5 or 3.5 μm particles
will always show a lower efficiency. A small extra-column
volume of about 23 μL does not exert a strong influence on
column efficiency for a 3.0 mm ID column if the temperature is
below 120 °C. In order to overcome these inherent drawbacks,
temperature programming should be used instead of isothermal
elution, which will be addressed in a forthcoming publication.
■ ASSOCIATED CONTENT
*S Supporting Information
Additional information as noted in text. This material is
available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION
Corresponding Author
*Phone: +49 2065 418 179. Fax: +49 2065 418 211. E-mail:
[email protected]
■ ACKNOWLEDGMENTS
The authors are thankful for the financial aid supported by the
German Federal Ministry of Economics and Technology on the
basis of a decision by the German Bundestag (Project number
KF 2025405MK9). We would also like to thank Dionex for the
loan of the UltiMate 3000 UHPLC system.
■ REFERENCES
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1565−15711571
http://pubs.acs.org
mailto:[email protected]
Chromatography Article:
1. Using the information provided below derive an expression
using Equation 1 given in the
article to describe the change in the diffusion coefficient of an
analyte as a function of
temperature. The information below shows the change in
viscosity as a function of temperature
at pressures similar to those used in this paper. You will need
to fit these data to a polynomial
function in Excel and then use that function in Equation 1.
Show all work.
Viscosity Temperature (°C)
(Pascal seconds (Pas))
9 x 10-4 25
3 x 10-4 100
2 x 10-4 150
1.7 x 10-4 175

1.5 x 10-4 200
1.35 x 10-4 225
1.25 x 10-4 250
1.2 x 10-4 275
b) Graph the resulting expression from room temperature to 180
C (highest temperature used in
this paper).
c) Use the expression derived in part a) to find the change in H
(equation 2 in the paper) as a
function of temperature. You will need to use the equations
given in your book for A, B, and C.
Assume changes to diffusion in the stationary phase are not as
important as changes in the
diffusion in the mobile phase. You will have to make some
assumptions for the retention factor
k. State all assumptions
d) Graph the change in each of the components of Equation 2
(A, B, C) and H as a function of
temperature (do these on separate graphs).

e) Does the data in Figures 1 and 2 of the paper correlate to
what you would expect based upon
the behavior of equation 2 plotted in part d of this question?
Specifically address the differences
seen in internal column diameter and particle size. If the
behavior does not match the expected
results propose some explanations of why it does not match the
expected results.
2. For the 2.1 mm I.D. column no minimum plat height can be
calculated at 180 °C. From a
thermodynamic point of view why is this the case?
3. Figure 3 shows that the efficiency of smaller particles in the
separation decreases for longer
dead volume times. Why is this the case and is that result
predicted from the equations given in
the paper?

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