Anal. Chem. 2007, 79, 9302-9309
Microfluidic Gradient Formation for Nanoflow Chip LC Reid A. Brennen,* Hongfeng Yin, and Kevin P. Killeen
Agilent Laboratories, 5301 Stevens Creek Boulevard, Santa Clara, California 95051-7201
We report a method for forming a nanoflow liquid chromatography (nano-LC) gradient using a single fluid pump at flow rates below 1 µL/min by passively forming a gradient on a microfluidic device. This device works together with an Agilent HPLC-Chip to perform highthroughput nanoflow liquid chromatography/mass spectrometry (nano-LC/MS). The nanoflow gradient delay time is reduced from several minutes for a commercial LC nanoflow pump to only a few seconds with this microfluidic device, thus shortening the total analysis time and increasing the analysis throughput. With this microfluidic device, a nano-LC solvent delivery system can be greatly simplified and have increased robustness, reliability, reduced waste, and ease of use. Nanoflow liquid chromatography/mass spectrometry (nanoLC/MS) technology offers high detection sensitivity and is widely applicable in the field of proteomic research and biomarker discovery. Recently, microfluidic high-performance liquid chromatography (HPLC) chip technology has provided an alternative nano-LC separation platform to the traditional fused-silica capillary, with increased system reliability.1 However, one still must use a nanoflow pump to create a gradient and to drive the mobile phase through the separation channels on the HPLC chip. So, the gradient delay time of such system is still significant, especially at low flow rates because (1) the nanoflow pump itself has a delay volume and (2) the transfer capillary between the pump and the chip has a delay as well. With a flow rate of 200 nL/min, due to the pump and transfer capillary, the delay between the instruction to start the gradient and the gradient actually arriving at the LC column can be up to 2-5 min. This delay, while not significant for long separation analyses, becomes important for analyses with shorter separation times for high-throughput, low-flow LC. Conventional LC pumps perform well at certain flow rate ranges, generally between 10 µL/min and 1 mL/min. When a gradient is required, two pump heads must pump two mobile phases (A and B) independently with the A to B ratio changing during the LC run time. At the beginning of the gradient, mobile phase A is usually a few percent of the combined flow, which means that pump head A is pumping at a much lower flow rate than the combined flow rate, sometimes out of the optimum flow rate range of the pump. The situation is worse when microbore LC columns * Corresponding author. E-mail:
[email protected]. Fax: 408-5534464. (1) Yin, H.; Killeen, K.; Brennen, R.; Sobek, D.; Werlich, M.; van de Goor, T. Anal. Chem. 2005, 77, 527.
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are used and total flow below 10 µL/min is delivered. For example, when the pump is pumping at a flow rate of 1 µL/min and the gradient starts at 5% B, pump head B will be pumping as slow as 50 nL/min. It is very difficult to precisely pump at such a low flow rate due to temperature variations, degradation at the piston seal, and solvent compressibility. One solution to this problem is to measure the flow coming out of each individual pump head and use the measurement feedback to control the driving pressure to maintain the correct flow rate.2 The success of such a design relies on the precision and fast response of the flow sensor as well as the performance of the feedback control loop. The biggest challenge to such a design comes at the beginning and the end of a gradient cycle, at which point the sensor and feedback control loop is forced to work at extremely low flow rates. Such problems can be eliminated if the flow sensor is placed downstream from the solvent mixing point.3 With such a design, the pump heads still deliver mobile phase at a high flow rate between 200 and 800 µL/min, but only a small fraction of the pumped mobile phase is directed to the LC column, whereas the majority of the mobile phase is split off and diverted to a waste bottle. Over the years, many approaches for forming nanoflow gradients have been investigated. Ishii et al. designed an HPLC gradient pumping apparatus specifically for microflow. Their method of gradient formation involves continuously adding one solvent to a mixing vessel while the content of the mixing vessel is sucked into a 0.5 mm i.d. tubing. By doing so, they were able to store a preformed gradient in the tubing.4 This approach of storing a preformed gradient in a tubing has been further developed. Davis et al. described another low-flow gradient solventdelivering system that uses two syringe pump to preform and to store the gradient in a length of narrow bore tubing.5 Eschelbach and Jorgenson used a gradient pump to load a tube with a solvent gradient and then used a high-pressure pump to push the preformed gradient through a nanocolumn.6 Deguchi et al. used a 10-port valve to trace an LC gradient generated by a low-pressure gradient pump at high flow with another high-pressure, low-flow pump.7 With proper mixing, a smooth gradient profile can also (2) Neyer, D.; Hahnenberger, K.; Bailey, C. Am. Lab. (Shelton, Conn.) 2003, Dec, 11-15. (3) Agilent 1100 Series Nanoflow LC System; Publication No. 5989-0649EN. (4) Ishii, D.; Asai, K.; Hibi, K.; Jonokuchi, T.; Nagaya, M. J. Chromatogr. 1977, 144, 157-168. (5) Davis, M. T.; Stahl, D. C.; Lee, T. D. J. Am. Soc. Mass Spectrom. 1995, 6, 571-577. (6) Eschelbach, J. W.; Jorgenson, J. W. Anal. Chem. 2006, 78, 1697-1706. (7) Deguchi, K.; Ito, S.; Yoshioka, S.; Ogata, I.; Takeda, A. Anal. Chem. 2004, 76, 1524-1528. 10.1021/ac0712805 CCC: $37.00
© 2007 American Chemical Society Published on Web 11/13/2007
be formed from multiple mobile phase reservoirs filled with different concentration mobile phases.8,9 These alternatives add mechanical complexities that may work against system robustness. Microfluidic chips that can generate LC gradients based on electrokinetic pumping10 as well as electrochemical pumping11 have also been described. For high-throughput applications, delay times in an analysis run need to be minimized. A pressure damper and mixer are generally used in a conventional LC pump, but the added delay volume due to these features can be detrimental at nanoscale flow rates. For example, a 1 µL delay volume between the solvent mix point and the head of the LC column, which includes any fittings, transfer capillaries, valve grooves, and sample enrichment column, will translate to 5 min delay time at a 200 nL/min flow rate. In order to reduce this delay time and to simplify the gradient system, we have designed and tested a microfluidic device which uses a few microliters of both mobile phase A and B to passively form an LC mobile phase gradient on-chip. This gradient chip is sandwiched between an Agilent HPLC-Chip and the chip valve stator. The delay volume between the generation of the gradient and the LC separation chip is thereby reduced to a few nanoliters, which translates to a delay time in the range of a few seconds. Such designs significantly reduce gradient delay time as well as the total LC run time which translates to higher sample throughput. EXPERIMENTAL SECTION Chemicals and Materials. HPLC grade acetonitrile and formic acid as well as the two tracer compounds glucosamine and betaine (also known as trimethylglycine) were purchased from Sigma (St. Louis, MO). Tryptic digest of bovine serum albumin (BSA) was purchased from Michrom Bioresources (Auburn, CA). Deionized water was prepared using a Milli-Q system from Millipore (Bedford, MA). HPLC-Chips (G4240-62001) packed with Zorbax SB C18 HPLC 3.5 and 5 µm packing materials were obtained from Agilent Technologies (Delaware). The Agilent HPLC-Chip Cube (G4240A), microwell plate sampler (G1377A), NanoPump (G2226A), and CapPump (1376A) modules were used for all experiments as well as a KD Scientific 250 syringe pump. All mass spectrometry measurements were made using an Agilent LC/MSD time-of-flight (TOF) instrument (G1969A) except for the tracer measurements used to monitor the gradient profile. These measurements were made on an Agilent Ion Trap. Device Fabrication. The microfluidic gradient generation devices used in conjunction with the HPLC-Chip were fabricated using laser ablation of polyimide sheets which are subsequently laminated together using heat and pressure, resulting in an allpolyimide device.1 The laser ablation of the polyimide film was performed using a direct-write process with a nonlinear, upconverted, diodepumped, solid-state laser (Coherent Avia 355-1500) operating at 355 nm in combination with a fixed optics train and a high(8) Natsume, T.; Yamauchi, Y.; Nakayama, H.; Shinkawa, T.; Yanagida, M.; Takahashi, N.; Isobe, T. Anal. Chem. 2002, 74, 4725-4733. (9) Capiello, A.; Famiglini, G.; Fiorucci, C.; Mangani, F.; Palma, P.; Siviero, A. Anal. Chem. 2003, 75, 1173-1179. (10) Figeys, D.; Aebersold, R. Anal. Chem. 1998, 70, 3721-3727. (11) Xie, J.; Miao, Y.; Shih, J.; He, Q.; Liu, J.; Tai, Y.; Lee, T. D. Anal. Chem. 2004, 76, 3756-3763.
Figure 1. Schematic of a six-stage gradient generation concept. Each stage is made up of the four sections. Section 1 is the inlet manifold. Section 2 is the restrictor area. The section 3 timing channels define each stage’s dwell time. Section 4 is the outlet manifold.
Figure 2. Conceptual gradient profiles for a simple six-stage gradient generator. For a flat-front velocity profile in the gradient generator channels, the output would be the stair step shown above, but for pressure-driven flow as in HPLC, the velocity profile across the channels is parabolic and therefore results in a softened gradient profile as shown.
precision x-y table controlled from a personal computer. The laser beam was focused onto the polyimide film, and the irradiated area was ablated. The width and depth of the ablated features are determined by the laser intensity and the velocity of the stage holding the film. Ablated microfluidic features can range in size from 5 µm by 5 µm cross sections to cross sections of 250 µm wide by 75 µm deep and larger. Three layers of filmstop, middle, and bottomswere used to fabricate each chip. Holes 200 µm in diameter were ablated through the top and bottom layers of the device to provide access to the channels and reservoirs inside the finished device. Each layer had a thickness of between 50 and 125 µm. After laser ablation, the film layers were cleaned using mechanical abrasion in the presence of a solvent solution and then laminated under pressure between heated, flat metal plates in a vacuum. Laser ablation was then used to trim the structure to its final shape and, for the LC/MS chip, to cut the electrospray tips using a circular path around the desired tip channel. The final tip shape was conical with a circular end with dimensions from 35 to 100 µm o.d. and up to 2 mm in length. Gradient Generator Concept and Design. The core feature of gradient generation on the microfluidic chip is the gradient generator which consists of n stages (also called channels) in parallel. Inlet and outlet manifolds are used to create a single fluidic inlet and a single outlet for these stages, as shown schematically in Figure 1. Each stage has “timing channels” of different length from the others, but all have the same crosssectional area. The stages are initially filled with liquid A and then liquid B at a high pressure, and a controlled low flow rate is introduced into the single entrance point. Since liquid B takes different amounts of time to travel through each stage (the dwell Analytical Chemistry, Vol. 79, No. 24, December 15, 2007
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Figure 3. Photograph and close-up of a 20-stage gradient generation (GG) chip. (Only the center, feature-defining layer is shown in this photograph.)
Figure 4. Fluid loading of the gradient generator. (a) Prerun preparation: liquids introduced for the prerun preparation of the LC chip and the gradient generation (GG) chip. (b) Run configuration: flow path for the LC run. Note that the high-pressure, controlled flow of mobile phase A used to flush the LC chip in the prerun preparation is also used to drive the separation during the LC run. Note: LP is low pressure, HP is high pressure.
time), a gradient profile is created after recombination of the liquids flowing through all the stages. On the basis of the total flow rate and the volume of each stage, a reproducible gradient profile with respect to time can be created from a given gradient chip structure. Further, the gradient-generating chip is connected directly to the HPLC-Chip containing the LC column such that the LC mobile phase gradient is introduced by the chip instead of an LC pump. The delay volume is therefore reduced to a few nanoliters, resulting in a delay time of a few seconds even at the low flow rates which are best for high-sensitivity electrospray. Although the gradient profile can be designed simply by changing the lengths of the timing channel within each stage with respect to the other timing channels, it is easier to design and control the flow rate in each stage using flow restrictors within each stage that provide almost all the restrictive pressure drop 9304
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for that stage. Thus, if the flow restrictors’ dimensions are the same for all the stages, the result is a flow rate that is similar in all stages. The schematic design in Figure 1 shows a six-stage gradient generator. The different length, larger timing channels in section 3 define the gradient, whereas the restriction channels in section 2, which are much smaller in cross section, define the flow rate through all six stages. The inlet and outlet manifolds in sections 1 and 4 are made up of channels that are large compared to the restriction channels but small in comparison to the timing channels. If the timing channel cross sections are all the same and their lengths have a linear progression, as is schematically shown in Figure 1, then the output gradient profile will also have a generally linear profile, as shown conceptually in Figure 2. In order to produce a smoother profile, a 20-stage gradient generation design was created, as shown in the photograph in Figure 3. The
Figure 5. Schematic layout of prerun and run concentric valve rotor configurations for LC using the gradient generation (GG) chip with the HPLC-Chip. (a) The green lines represent the channels within the gradient generator chip, the dotted yellow lines represent the outer rotor channels, and the black dotted lines represent the inner rotor channels. The brown lines represent the enrichment column (center) and the LC column (right) that are within the HPLC-Chip. (b) The different flow paths through the valve and chip are shown in different colors for the prerun and run modes. Note that because the gradient generator chip is made up of five different layers, channels may overlap one another, and some of the channels that appear, from a top view, to be connected at their ends may, in fact, not be connectedsone channel may connect to the top of the GG chip while the other channel exits out through the bottom of the HPLC-Chip directly below the top port.
20 timing channels in this design have a rectangular cross section of 75 µm by 75 µm, range in length from 2 mm for the shortest to 40 mm for the longest, and are curved into nested arcs to keep the device compact. The total combined volume of the timing channels is 2.4 µL. The restriction channels for this design have a triangular cross section 9 µm deep by 11 µm wide. Gradient Generator Chip and HPLC-Chip Interface. The HPLC-Chip interface inside the HPLC-Chip Cube is based on a concentric, two-rotor switching valve in which an inner rotor has three rotor grooves connecting 6 ports while an outer rotor has five rotor grooves that connects 10 ports. The basic chip-valve interface concept is described by Yin et al.1 For LC runs using the gradient chip, all 16 ports are used. Essentially, the outer rotor controls the reservoir and the gradient generator features, visible in Figure 3, whereas the inner rotor controls the sample, the sample enrichment column, and the LC column connections of the HPLC-Chip. The gradient chip is placed directly in contact with the HPLCChip such that the ports on each chip are directly aligned to one another, as shown in Figure 4. The chip interface consists of, in ascending order, the valve inner and outer rotors, the HPLC-Chip, the gradient generator chip, and the valve clamping stator. To allow direct connection between the rotor and the gradient generator chip, 10 small ports, visible in Figure 4, corresponding
to the outer rotor channel positions, were laser-ablated completely through the HPLC-Chip. This configuration allowed the HPLCChips to be used with no other modification to the system. The HPLC-Chip has a 40 nL enrichment column filled with Zorbax SB C18 HPLC 5 µm particles and an LC column 43 mm long with a cross section of 50 µm by 75 µm. Use of the Gradient Generator. A single high-pressure, controlled flow rate pump was used for run preparation and LC analysis. As shown in Figure 4a, a 6 µL reservoir on the gradient generation (GG) chip is first filled with mobile phase B during the prerun process. During the subsequent switch to the LC running mode, the high-pressure, controlled flow of mobile phase A is switched from feeding into the LC column to feeding into the reservoir, as shown in Figure 4b. The mobile phase B liquid in the reservoir then pushes into the GG section, thereby creating the gradient. During the prerun preparation, this system configuration requires only two low-pressure pumps or syringes to fill the on-chip reservoir and GG stages, one pump to load sample, and one high-pressure, controlled flow rate pump to push mobile phase A through the LC column. No gradient pump is needed. The full interconnection switching and valving process is presented in Figure 5. Prior to the HPLC run start, the gradient structure and the reservoir were filled with mobile phases A and B, respectively, Analytical Chemistry, Vol. 79, No. 24, December 15, 2007
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Figure 6. Plots of theoretical results for 6- and 20-stage gradient generators. The blue lines show the theoretical B fluid concentration at the exit of each individual restrictor/timing channel stage over time. The black line shows the theoretical B fluid concentration after recombination of the six stages at a 200 nL/min flow rate. For the six-stage design, the restrictor channels are 2 mm long with a 7 µm equivalent diameter corresponding to the 9 µm by 11 µm triangular cross section of the experimental restrictors; the timing channels are 130 µm in diameter and range in length from 2.0 to 40 mm long. The 20-stage design has the same dimensions except the timing channels are 75 µm in diameter so as to attain nearly the same gradient. The calculations are for circular cross section channels assuming a parabolic velocity profile, identical viscosities, and no mixing between the A and B fluids.
using two 250 µL syringes that were mounted on the syringe pump. The syringe pump was set to run at a flow rate of 2 µL/ min. The sample was loaded using the autosampler backed by the microflow CapPump pumping mobile phase A, usually running at 4 µL/min. The nanoflow pump was connected to a single port on the HPLC-Chip Cube and was pumping mobile phase A only during the entire LC run. Both the inner rotor and the outer rotor of the valve were controlled by the instrument control computer. After the sample was loaded on the trapping column, the inner valve switched to the run position and triggered the start signal. The outer rotor switched at the same time the inner rotor switched in order to minimize the gradient delay time. When both inner and outer valves were at the run position, the nanoflow pump delivered mobile phase A which pushed the mobile phase B from 9306
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the reservoir into the gradient structure generating the gradient just before it reached the enrichment column and subsequently the LC column. After the analysis was done, the outer rotor switched back to the prerun position so that both gradient structure and reservoir can be filled with mobile phase B and A again. The inner rotor was switched back to the prerun position as well. When both rotors were at prerun positions, the nanoflow pump was connected directly to the LC column and the LC column was again flushed with mobile phase A. RESULTS AND DISCUSSION Modeling of the Gradient Generation. As shown schematically in Figure 2, a plug flow model will result in a step gradient with the same number of steps as stages in the gradient generator.
Figure 7. Tracer profiles using a 20-stage gradient generator during an LC run are represented by extracted ion chromatograms (EIC) of 118 (betaine, red trace, showing liquid A) and 180 (glucosamine, blue trace, showing liquid B). (a) The nanoflow pump delivers 200 nL/min during the entire LC run. (b) The pump flow rate time table is set to 200 nL/min between 0 and 5 min and 600 nL/min between 5 and 9 min.
However, since this is a pressure-driven flow, the velocity profiles within the channels are parabolic, not a constant velocity. Figure 6 shows theoretical results for the 6- and 20-stage systems, each stage consisting of a restrictor and a timing channel, using a simple model which assumes a parabolic velocity profile in each of the circular cross section restrictors and timing channels while also assuming that the two liquids, A and B, have the same viscosity. The model does not include diffusive mixing between liquids A and B. The parabolic velocity profile in each stage obviously slows the emptying of each timing channel, but this also serves to smooth the gradient profile. The modeling showed that increasing the number of stages above about six increases the linearity of the gradient over much of the time span but also that smaller timing channel diameters reduce the time at which the gradient starts, i.e., the time for fluid B to go through the first stage. A 20-stage design was chosen for initial experiments. Gradient Generation Data. In order to study the gradient profile, tracer compounds were added to water to fill both the gradient formation structure and the reservoir. Betaine and glucosamine were selected to be the tracer compounds because of their electrospray properties and minimum retention on the reversed-phase media. However, some signal fluctuations were observed at the ion trap mass spectrometer since the ion trap was not optimized for such a low mass range. Such fluctuations can be seen in Figure 7. The gradient structure was filled with water plus 20 ng/µL betaine (m/z ) 118) plus 0.1% formic acid while the reservoir was filled with water plus 20 ng/µL glucosamine (m/z ) 180) plus 0.1% formic acid. Figure 7a plots both
tracers. The design of the tested gradient formation chip gives a delay time of less than 90 s at 200 nL/min. Note that the signal response time includes both gradient delay time and t0, the time it takes for the mobile phase to travel through the LC column. In Figure 7, t0 is time at which the blue trace starts to rise. Gradient delay time can be measured between the blue trace and red trace start. The differences between the theoretical and experimental results in Figures 6 and 7 are due to two factors. First, the greater delay time before the gradient starts in the experimental data is due to t0, which is not included in the model. Second, during the gradient generation, the model does not include radial diffusive mixing between the two liquids which serves to reduce the total time to reach 100% B. In order to further speed up the analysis, the flow rate was set to 600 nL/min between 5 and 9 min. The gradient profile changed due to the increase in flow rate (Figure 7b). LC/MS Analysis. The Agilent nanoflow pump achieves a stable and accurate flow throughout the gradient using a flow splitter design which has a primary flow upstream of the splitter usually running between 0.2 and 0.5 mL/min which results in a flow delivered to the chip’s separation column of 200-400 nL/ min. For peptide separations, mobile phase A was water plus 0.1% formic acid and mobile phase B was 95% acetonitrile/5% water plus 0.1% formic acid. The capillary pump pumped 4 µL/min through the autosampler to load the sample onto the on-chip sample enrichment column. For peptide samples, the maximum scan range of the TOF MS was set to 1700. At 5000 transients per scan, the data rate was 2.67 scan cycles/s. A 6 L/min nitrogen Analytical Chemistry, Vol. 79, No. 24, December 15, 2007
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Figure 8. Experimental data comparing the separations and run times produced using (a) a standard nanoflow-pump-generated gradient and (b) a gradient generator chip gradient, both run at a 200 nL/min flow with the same HPLC-Chip. The sample is a 20 fmol/µL tryptic digest of BSA (1 µL injected). The plots are based on 12 extracted ions to increase the clarity of the plot, but the full data sets were similar as well.
drying gas was used to heat the MS inlet capillary but was not allowed to enter the spray chamber. The spray voltage was set to 1950 V during the entire LC gradient. With the use of the same HPLC-Chip on the TOF MS for both cases, a direct comparison was made between the traditional pump-generated gradient and the gradient created by the GG chip. Separation performance and retention time reproducibility are the key parameters used in order to characterize a chromatographic system such as the gradient that is formed with a gradient chip. A tryptic digest of BSA was used as a test mixture because it is extensively used by many research groups to characterize and troubleshoot their nano-LC systems. All nanoflow separations were performed on an Agilent HPLC-Chip (G4240-62001). The gradient chip was placed on top of the HPLC-Chip as described earlier. Figure 8 compares chromatographic separation of a 20 fmol BSA digest using the same HPLC-Chip with the gradient generated by a nanoflow pump (Figure 8a) and by a gradient chip (Figure 8b). The reduction in run time is clearly visible. For visual comparison purposes, the extracted ion chromatogram (EIC) of 12 ions were selected and merged to represent the entire elution window. These selected ions are listed in Table 1. The total run time on the TOF MS at a 200 nL/min flow rate when using the pump gradient was 19.5 min with a 20% duty cycle while the total run time for the gradient generator chip was 11.5 min with a 40% duty cycle. (Total run time is the time interval between adjacent injections in a sequence of automated LC/MS runs. The duty cycle is the sample elution window divided by total run time, i.e., the percentage of time in which the MS measures sample components as opposed to baseline noise.) The outer rotor was not actuated when using the nanoflow pump gradient. The nanoflow pump was running a steep gradient in order to achieve a fast duty cycle so as to compare it directly with the gradient chip results. The %B increases from 0% to 40% in 5.5 min before it reached 90% for another 2 min to flush the LC column. Although there are retention time shifts between the two separations, due to differences in gradient profile, the separation performance is comparable. 9308 Analytical Chemistry, Vol. 79, No. 24, December 15, 2007
Table 1. Run Values for 12 Selected Ions for the Nanoflow Pump and Gradient Generator Runsa GG chip gradient RT (min) mass value 1344.592 915.3436 1499.414 911.3658 822.3511 828.4571 909.376 1071.479 1622.814 2102.819 1927.874 996.5034 average
monitored charge m/z state value 449.2 458.7 750.7 456.7 412.2 415.2 455.7 536.7 541.9 701.9 483.0 997.5
3 2 2 2 2 2 2 2 3 3 4 1
2.56 2.66 3.03 3.21 3.40 3.50 3.64 4.18 4.51 4.80 5.52 6.14
SD 0.006 0.008 0.014 0.011 0.009 0.010 0.011 0.013 0.008 0.012 0.015 0.017
nanoPump gradient RT (min)
PW (min) value 0.05 0.05 0.07 0.07 0.04 0.04 0.05 0.08 0.05 0.10 0.06 0.08
0.011 0.061
5.06 5.18 5.56 5.70 5.92 6.07 6.27 6.77 7.13 7.43 8.06 8.54
SD
PW (min)
0.012 0.012 0.011 0.012 0.011 0.011 0.011 0.016 0.010 0.013 0.007 0.006
0.06 0.05 0.05 0.08 0.06 0.05 0.06 0.07 0.06 0.08 0.06 0.07
0.011 0.061
a The RT value is the average retention time in minutes. RT values for the gradient from the gradient chip are an average of seven replicated runs, and for the nanoPump gradient, they are an average of five replicate runs. The peak width values (PW) are from single representative runs.
The retention time reproducibility and peak width for the separations are listed in Table 1. Twelve ions were selected and values were calculated for comparison between the nanoflow pump and gradient chip. Mass value was calculated based on m/z and the charge state of each ion by the Agilent Mass Hunter analysis software. Monitored m/z was used for EIC in Figure 8. The average retention time and its standard deviation of seven replicates using the GG chip and five replicates using nanoflow pump gradient is listed. The peak width was calculated based on one representative run in each case. Peak width value is the product of peak area divided by peak height. Overall, the comparison shows that the peak widths and the standard deviations for the retention times are similar for the two methods for generating the gradient. This is shown graphically in Figure 9. Replicate runs are overlaid and plotted in different colored dots. Each of the 70 dot groups in Figure 9 is a single “feature” extracted
Figure 9. Retention time reproducibility of the 70 most abundant molecular features for the BSA digest when measured using (a) the gradient generator system and (b) a standard nanoflow pump.
by the Agilent Mass Hunter software. The chromatographic peaks were automatically detected and integrated over the entire retention time range and mass range. From this plot, one can conclude that the retention time reproducibility from a gradient chip is comparable to that of a nanoflow pump gradient.
Extensions to the GG concept can enable defined-gradient profiles which are tailored to specific LC analyses. Further improvements in performance and the simplicity of the fluid delivery system are anticipated from refinements of the concepts described in this paper.
CONCLUSION The gradient generation on a chip concept was implemented and tested both for gradient characterization and with actual sample analyses. Gradient profile tests were measured for a 20stage linear gradient design. The separation of a test mixture of BSA digest was demonstrated and showed good reproducibility, chromatographic performance, and stable MS electrospray. The direct connection of the GG chip to the HPLC-Chip resulted in a 2-fold increase in effective LC duty cycle from 20% to 40% due to the reduced gradient delay time.
ACKNOWLEDGMENT The authors extend thanks to Debbie Ritchie of Agilent Technologies for her support on chip fabrication and to Uwe Effelsberg, also of Agilent, for hardware and software support.
Received for review June 18, 2007. Accepted October 5, 2007. AC0712805
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