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8
A Rugged Capillary HPLC System for High Throughput,
Sample-Limited Chromatographic Analysis
System maximizes detection, supports high sample throughput.
by Rainer Schuster and Cornelia Vad
I
In order to accelerate the discovery of new drug leads, techniques
such as combinatorial synthesis and high throughput screening are
increasingly being used to synthesize and evaluate limited amounts
of large numbers of analytes (combinatorial libraries). The need to
isolate and characterize these materials prior to screening is creating a demand for liquid chromatography systems appropriately
scaled for sample-limited analyses. This scaling requirement is also
evident in many other life science and biopharmaceutical applications, among them peptide and protein purification.
A significant reduction in sample size can be problematic for
analyses that require chromatographic separation using conventionally sized packed HPLC columns. Under these conditions, the volume of mobile phase needed to maintain satisfactory flow rates can
dilute the already low level of sample analytes to a concentration
below the limit of detection. A logical way to overcome this limitation is to perform the chromatography using much smaller volumes
of mobile phase. To do this, the chromatographic column must be
downsized to capillary dimensions (50 µm <1.0 mm ID) in the context of a high performance chromatographic system configured to
operate under the constraints and conditions encountered at this
operational scale. (Experiments have demonstrated that detection
sensitivity actually increases as column ID decreases.) (Figure 2.)
In addition to proper instrument scaling, an appropriate capillary
chromatographic system must maximize detection sensitivity, provide precise, reproducible performance, and be sufficiently
rugged to support requirements for high sample throughput with minimal downtime. The Agilent 1100 Series
Capillary HPLC system meets these standards
(Figure 1). The modular configuration and other
user-friendly design features incorporated into this
instrumentation make for easy set up, validation,
routine operation, and maintenance.
Figure 1. The Agilent
1100 Series Capillary
HPLC system with solvent reservoir, capillary
pump, micro vacuum
degasser, thermostatted
column compartment,
microwell plate sampler,
diode array detector,
and 500 nl flow cell. A
handheld controller simplifies setup and provides full control of all
system modules and online display of operations. The graphic user
interface with colorcoded system display
shows the progress of
an analysis. Agilent’s
ChemStation chromatographic software performs all data management
from acquisition to report generation.
Screening
libraries requires
appropriately scaled
Active flow rate control
Until now, capillary HPLC systems have attempted to
control flow rate by means of a passive splitter set to a
desired split ratio. This approach has proven to be unreliable in maintaining a constant flow rate independent of
changes in back pressure—changes which can be caused by variations
in pump performance, change in a solvent gradient, or the buildup of
obstructions anywhere in the system during an analysis. Since precise
control of flow rate is essential for quality chromatographic separations and for properly characterizing and collecting analytes based on
predictable chromatographic retention times, a method based on
active flow control rather than a passive splitter is called for.
The Agilent 1100 Series Capillary HPLC system employs a highpressure capillary pump with electronic flow control (EFC) to
actively maintain constant flow. A special sensor monitors flow
resistance in the column flow path and signals any changes to an
electromagnetic proportioning valve (EMPV) which compensates
by appropriate alterations in the split ratio (Figure 3). EFC has
proven to be a highly robust method of flow control as demonstrated by retention time precision across replicate runs (Figure 4).
The pump design also provides flexibility in solvent selection and
mixing with independent sets of valves for each operation in each
LC systems.
Figure 2. The Relationship Between Decreasing Column I.D. and
Sensitivity. The results demonstrate that detection sensitivity increases
as the I.D. of the column decreases. Since column permeability also
decreases with decreasing column I.D., less pressure is required to
pump solvent at a specific flow rate. This means that in comparison
with normal-bore columns, capillary columns allow a fuller pressure
range of the pump to be utilized for solvent delivery. Stationary phase:
ZORBAX® SB-C18; length: 150 mm; solvent: water/acetonitrile, 40/60;
flow rate: see diagram; sample: isocratic checkout sample; injection
volume: 0.1 µl; third peak: biphenyl, 200 ng; temperature: 25 C; detection wavelength: 230 nm
chamber, enabling precise gradient delivery from 0-100 %. An inline micro vacuum degasser employs new gas exchange membrane
technology that provides a level of degassing efficiency comparable
to larger-volume solvent degassers. The degasser’s low internal volume (1 ml) facilitates a 10-fold increase in rate of system purging
compared to a conventional degasser and fast solvent changeover
while minimizing solvent usage.
Automated sample introduction and handling
Samples can be introduced into the system by means of a micro
autosampler or a micro well-plate sampler, both of which enable
sampling over a 3-order of magnitude range from 30 nl to 40 µl.
Each sampler can be equipped with Peltier temperature control (4 to
Bioscience TECHNOLOGY 7 • 2002
10
[Tools & Techniques] www.biosciencetechnology.com
Figure 3. Schematic Representation of the Agilent 1100 Series
Capillary Pump with Electronic Flow Control (1). (Source: Page 3,
First figure: Agilent 1100 Series Capillary LC System: A one-vendor
solution for highest sensitivity and robustness - Brochure, Agilent
publication number: 5988-1729EN.
Solvent from each reservoir passes through the micro vacuum
degasser and into one of the two pump chambers and from there
into a gradient mixing chamber. One set of valves controls the solvent selection and a second set of valves determines the gradient
ratio. Solvent downstream from the mixing chamber is split between
the solvent waste path and the column flow path, the latter of which
contains a flow sensor. (In practice, two separate sensors are used
for two different capillary flow rate ranges-one for 1-20 µl/min and
the other for 10-100 µl/min.) Active flow control is maintained by
the Electromagnetic Proportioning Valve (EMPV) which compensates for changes in back pressure signaled by the flow sensor by
altering the split ratio, thereby restoring the original flow rate. The
relatively high solvent flow rate upstream of the EMPV (0.2-1.0
ml/min) relative to the downstream capillary flow rate (1-100 µl/min)
coupled with the very small dead volume between sensor and capillary column, minimize the delay in the response to gradient
induced flow rate changes, providing highly reproducible flow rates.
(The Agilent 1100 Series Capillary HPLC system is designed to
bypass EFC sensor and operate with wider-bore columns at flow
rates up to 2.5 ml/min with appropriate hardware changes.)
40 C ) for protection of thermally-labile
samples. Up to 100 2 ml sample vials can
be accommodated in this way. The micro
well-plate sampler has the ability to handle
different types of sample containers e.g.,
thermostatted 96 and 384 well plates as
well as 2 ml vials, and a large sampling
capacity (runs up to 768 samples unattend-
ed using 384 well plates), gives users considerable flexibility in sample containment
and delivery while accelerating throughput.
A micro Rheodyne® valve increases the
speed of sample introduction while improving take-up and delivery precision tenfold
compared with a standard autosampler.
Together with an optimized design of nee-
dle seat, loop and seat capillaries, the new
metering device also minimizes dispersion. Further increases in throughput are
gained by the sampler’s ability to overlap
sampling and chromatographic separation
cycles. A significant reduction in dead volume further limits dispersion and the resulting band broadening that can decrease
Table 1. Analysis of Depressants– Equipment and Conditions.
Conditions
Equipment
Agilent 1100 capillary LC system
components:
Sample mobile phase
standard mixture (A=125 pg/µl, B=10 ng/µl)
A = 25 mM NaH 2 P0 4 , pH 2.5 (H2 S04)
• Capillary pump
• Micro vacuum degasser
B = acetonitrile
15 µl/min
Flow rate gradient
• Thermostatted column compart
0 min 22 % B, 5 min 35 % B, 7 min 35 %
• Micro autosampler
B,8 min 22 % B, 11 min 2 % B
• Diode array detector
Injection volume
100 nl
• Agilent ChemStation
Column compartment temp
40 C
• Column: ZORBAX SB C-18,
150 ⫻ 0.5 mm, 3.5 µm
Diode array detection
signal 225/10 nm, reference
• 500 nl flow cell
450/80 nm, 500-nl volume
Table 2. Analysis of Sulfonamides—Equipment and Conditions
Equipment
Agilent 1100 Capillary LC system
components:
Conditions
Sample mobile phase
A = 0.1 % formic acid in water
• Capillary pump
• Micro vacuum degasser
• Thermostatted column compart.
• Micro autosampler
• Diode array detector
• 500 nl flow cell
• Agilent ChemStation
sulfonamide mixture
B = 0.1 % formic acid in acetonitrile
Flow rate
13 ml/min
Gradient
0 min 15 % B, 10 min 95 % B, 11 min 15 % B
Injection volume
100 nl
Column compartment temp
25 C
Diode array detection
278/10 nm (reference 450/80 nm)
• Agilent 1100 Series LC/MSD SL
• Electrospray source
MS conditions:
• Capillary LC nebulizer
Source
ESI
• Column: ZORBAX SB C-18, 150
3 0.5 mm, 3.5 mm
Ionization mode
positive
Vcap
4000V
Nebulizer
10 psig
Drying gas flow
7 l/min
Drying gas temp
150 C
peak resolution, sensitivity, and reproducibility. To enhance retention time stability,
the system incorporates a column oven
equipped with fast Peltier cooling and
heating capability (10 degrees below
ambient to 80 C). A micro column switching option is also available.
High performance capillaries
Connections are an important component of
an HPLC system and can contribute significantly to band spreading. For this reason,
system designers counter the dispersive
effects in connections by minimizing dead
volume. Normally, in a modular system
such as the Agilent 1100 Series Capillary
HPLC, the relatively long lengths of connection tubing required would be viewed as
having a detrimental effect on band broadening. Fortunately, the effect on overall band
spreading is negligible at capillary diameters (1) and the i.d. of the connection capillaries need be reduced less than that of the
column diameter.
HPLC capillaries are designed to be
both rugged and to facilitate uniform
flow. This is accomplished with PEEK
(polyetherether ketone) coated fused silica
capillaries with the required internal
diameter. The polymer sheath protects the
capillary against handling and breakage,
while the fused silica inner tube provides
the precision bore and clog-resistant
smooth inner wall needed for consistently
reproducible flow. Capillaries are secured
with standard swagelok fittings.
Optimum sensitivity
The need to minimize extra column band
broadening is an important constraint in
the design of a detection flow cell. The
Agilent 1100 Series Capillary HPLC system employs a low-volume 500 nL flow
with geometry optimized to minimize peak
dispersion while maximizing path length
for high sensitivity (Figure 5). The configuration of the flow cell and the material
from which it is constructed are selected
for low RI sensitivity and reduced light
scattering to minimize background noise,
ensuring low baselines with long-term stability and good peak separation (Figure 6).
Application examples
Analysis of nanoliter volumes of antidepressants by capillary LC.
To demonstrate the ability of the Agilent
1100 Series Capillary LC system to handle small sample volumes accurately and
reproducibly, mixtures of tricyclic antidepressants were used as exemplary analytes. Figure 7 shows the analysis of the
analytes near the method detection limit.
Excellent data on precision, linearity and
repeatability are given for 100 nl injections on a 500 µm ID capillary column.
This pseudo non-destructive analysis
makes the system ideal in situations
where sample volumes are limited and
multiple analyses are necessary on a single sample. Table 2 lists equipment and
experimental conditions.
Analysis of sulfonamides in the low pg
range by capillary LC using diode array
and mass spectrometric detection.
continued on page 16
Bioscience TECHNOLOGY 7 • 2002
[Tools & Techniques] www.biosciencetechnology.com
10 ng/µl
Repeatability - 100 nl injections
0.18
1.20
0.27
1.08
mAU
20
0.20
1.17
15
0.17
1.34
0.18
1.23
10
5
0
3.5
% RSD RT
% RSD RT
3.75
4
4.25
4.5
4.75
5
5.25
5.5
0.23
1.49
mAU
2 ng/µl
16
0.30
1.49
4
0.24
1.28
3
5.75
min
0.21
1.76
0.24
1.26
2
1
0
3.5
4
4.5
5
5.5
min
Figure 4. Flow Rate Reproducibility as Indicated by Retention Time Precision. In the illustration
shown, retention time precision is 0.21-0.30% and 0.17-0.27% at 2 ng/ml and 10 ng/ml sample
loadings at a flow rate of 4 ml/min.
Figure 6. Analysis of
nanoliter volumes of antidepressants by capillary
LC. An absolute injection
of 12.5 pg gave signal-tonoise values of 3.3 - 8.7.
In figure 1B an overlay of
10 runs at the 10 ng/µl
level is depicted. Relative
standard deviation (RSD)
was <0.5 % for retention
time and <2 % for area,
respectively. Analyzing a 2
ng/µl sample resulted in
RSD for retention time of
<0.5 % and RSD for area
of <3 %. The assay was
linear in a range from 125
pg/µl to 4 ng/l (r 2 >0.999).
Column: ZORBAX SBC18, 3.5 µm. Equipment:
Agilent 1100 Capillary LC
system with capillary
pump, micro vacuum
degasser, thermostatted
column compartment,
micro autosampler, diode
array detector, 500 nl flow
cell, Agilent ChemStation
software.
Figure 5. Agilent 1100 Series Capillary HPLC Flow Cell. The 0.7 ⫻ 0.7 ⫻ 10 mm dimensions
combine low-volume for minimal dispersion with a long detection path length for maximum sensitivity. Spectral integrity for diode array spectrophotometric detection is maintained by the use of
flat detection cell windows. The configuration and materials used to construct the cell were selected for low RI sensitivity in order to achieve flat baselines at low flow gradients. A combination of
deuterium and tungsten lamps provides an extended detection range from 190 to 950 nm.
When dealing with extremely small
sample volumes, adding mass spectrometric capability to a capillary LC system can
often enhance the sensitivity and specificity of the analysis. Figure 8 shows the
analysis of seven sulfonamides with 100 pg
and 10 pg on-column with diode-array
detection. With a signal-to-noise ratio of 3,
the 10 pg injection is at the detection limit.
Figure 9A shows the MS signal (total ion
chromatogram) for the same analysis. The
injected amounts for MS are 10 pg and 1 pg
(10 times less than with DAD). Several of
the sulfonamides elute closely and extraction of the ion of interest enables better
integration and calibration, especially at
low levels. In addition, using extracted ion
chromatograms (EICs) can enhance the
visibility of smaller components in a mixture. Figure 9B illustrates the use of EICs
for three of the components of the sulfonamide mixture at 1 pg on-column. Relative
standard deviation for the sulfonamides was
<0.1 % for retention time and <2.8 % for
area respectively, based on ten runs with
100 pg and 100 nl injection volumes. The
assay was linear in a range from 1 pg/ml to
1 ng/ml (r2 >0.9999) for the MSD and 10
Figure 7. Analysis of a mixture of 7 sulfonamides with diode array at 10 pg and100
pg absolute with 100 nl injection volume.
pg/ml - 100 ng/ml for DAD. Table 2 lists
equipment and experimental conditions.
About the authors
Rainer Shuster is an application chemist and
Cornelia Vad is a product manager at Agilent
Technologies, Waldbronn, Germany.
More information is available from:
Agilent Technologies, Palo Alto, CA.
800-227-9770; agilent.com.
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References
1. Gerard Rozing, Maria Serwe, HansGeorg Weissgerber, and Bernd Glatz, A
system and columns for capillary HPLC,
American Laboratory, Vol. 33, page 26 38, May 2001.
2. Maria Serwe, Analysis of nanoliter volumes of antidepressants by capillary LC,
Agilent Technologies Application Note,
Pub. Number 5980-2172E, July, 2000.
3. Rainer Schuster and Christine Miller,
Analysis of sulfonamides in the low pg
range by capillary LC using diode array
and mass spectrometric detection, Agilent
Technologies Application Note, Pub.
Number 5980-2499EN, September, 2000.
5988-8255EN (KAISER, ASTRID, mer. 06 nov. 2002)
Figure 8. Analysis of the standard mix with 10 pg and 1 pg with MSD and SIM - total
ion chromatogram from SIM (A) and extracted ion chromatogram from SIM(B).
Bioscience TECHNOLOGY 7 • 2002