Instrumental Requirements for Nanoscale Liquid Chromatography

Using the down-scaling factor, which corresponds to the ratio of the column diameter in square, (dconv/dmicro)2, excellent agreement between the theor...
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Anal. Chem. 1996, 68, 1507-1512

Instrumental Requirements for Nanoscale Liquid Chromatography J. P. Chervet* and M. Ursem

LC Packings, Baarsjesweg 154, 1057 HM Amsterdam, The Netherlands J. P. Salzmann

LC Packings Inc., 80 Carolina Street, San Francisco, California 94103

Nanoscale liquid chromatography (nano-LC), with packed columns of typically 75 µm i.d. × 15 cm length, packed with C18, 5 µm of stationary phase, and optimal flow rates of 180 nL/min, can be considered as a miniaturized version of conventional HPLC. Using the down-scaling factor, which corresponds to the ratio of the column diameter in square, (dconv/dmicro)2, excellent agreement between the theoretically calculated values and the values obtained using the down-scaling factor (∼3800) has been observed. This factor was applied to all system components, including flow rate, injection and detection volumes, and connecting capillaries. Down-scaling of a conventional HPLC system to a nano-LC system is easy to realize in practice and involves using a microflow processor for nanoflow delivery (50-500 nL/min), a longitudinal nanoflow cell (e3 nL), a microinjection valve (e 20 nL), low-dispersion connecting tubing, and zero dead volume connections. Excellent retention time reproducibility was measured with RSD values of (0.1% for isocratic and (0.2% for gradient elution. Plates counts of more than 100 000/m indicate the excellent performance of the entire nano-LC system. With minimal detectable amounts of proteins in the low femtomole and subfemtomole ranges (e.g., 520 amol for bovine serum albumin), high mass sensitivity was found, making nanoLC attractive for the microcharacterization of valuable and/or minute proteinaceous samples. Coupling nanoLC with concomitant mass spectrometry using nanoscale ion spray or electrospray interfaces looks very promising and is obviously the next step for future work.

The growing interest in analyzing minute samples in various fields, such as environmental, clinical, forensic, and pharmaceutical chemistry and biotechnology, is one of the driving forces for the rapid development of microseparation techniques such as microLC and capillary LC in combination with mass spectrometry (MS). Today, micro-LC and capillary LC with column diameters of typically 300 µm i.d. and flow rates of 4 µL/min have found acceptance as routine techniques in various laboratories due to their superiority in situations with limited sample amounts and/ or sample concentration.1-12 0003-2700/96/0368-1507$12.00/0

© 1996 American Chemical Society

Recent developments on new electrospray interfaces, such as “nano ion spray”, lead to even lower flow rates, in the range of 20 nL/min to 1 µL/min.13,14 Theoretically, such miniaturization increases the mass sensitivity and should allow for further enhancement of the limit of detection (LOD), in both mass and concentration sensitivity. The first attempts at using packed capillary columns with very small inner diameters, ranging from 20 to 70 µm, and flow rates on the order of 50-200 nl/min were reported several years ago by Novotny15,16 and Jorgenson.17,18 The direct coupling of packed microcapillary columns with ESI/MS was shown recently by Hunt et al.19,20 In this paper, instrumental requirements such as highly reproducible delivery of nanoliter flows under isocratic and gradient conditions, highly sensitive UV detection using Z- or U-shaped nanoliter UV flow cells, and the use of zero dead volume connections are presented, in order to realize the potential of (1) Henzel, W. J.; Bourell, J. H.; Stults, J. T. Anal. Biochem. 1990, 187, 228233. (2) Griffin, P. R.; Coffman, J. A.; Hood, L. E.; Yates, J. R. Int. J. Mass Spectrom. Ion Processes 1991, 11, 131-149. (3) Moritz, R. L.; Simpson, R. J. J. Chromatogr. 1992, 599, 119-130. (4) Kassel, D. B.; Luther, M. A.; Willard, D. H.; Fulton, S. P.; Salzmann, J.-P. In Techniques in Protein Chemistry IV; Angeletti, R. H., Ed.; Academic: San Diego, CA, 1993; pp 55-64. (5) Lane, S. J.; Brinded, K. D.; Taylor, N. L. Rapid Commun. Mass Spectrom. 1993, 7, 492-495. (6) Murata, H.; Takao, T.; Anahara, S.; Shimonishi, Y. Anal. Biochem. 1993, 210, 206-208. (7) Kassel, D. B.; Shushan, B.; Sakuma, T.; Salzmann, J. P. Anal. Chem. 1994, 66, 236-243. (8) Lewis, D. A.; Guzzetta, A. W.; Hancock, W. S.; Costello, M. Anal. Chem. 1994, 66, 585-595. (9) Roboz, J.; Yu, Q.; Meng, A.; van Soest, R. Rapid Commun. Mass Spectrom. 1994, 8, 621-626. (10) Battersby, J. E.; Mukku, V. R.; Clark, R. G.; Hancock, W. S. Anal. Chem. 1995, 67, 447-455. (11) Chowdhury, S. K.; Eshraghi, J.; Wolfe, H.; Forde, D.; Hlavac, A. G.; Johnston, D. Anal. Chem. 1995, 67, 390-398. (12) Simpson, R. C. J. Chromatogr. A 1995, 691, 163-170. (13) Mann, M.; Wilm, M. Intl. J. Mass Spectrom. Ion Phys. 1994, 136, 167ff. (14) Covey, T.; Shushan, B.; Kaudewitz, H. Proceedings of the 1st European Symposium of the Protein Society; Cambridge University Press: Cambridge, UK, 1995; Poster 456. (15) McGuffin, V. L.; Novotny, M. J. Chromatogr. 1983, 255, 381-393. (16) Karlsson, K. E.; Novotny, M. Anal. Chem. 1988, 60, 1662 ff. (17) Kennedy, R. T.; Jorgenson, J. W. Anal. Chem. 1989, 61, 1128-1135. (18) Kennedy, R. T.; Jorgenson, J. W. J. Microcolumn Sep. 1990, 2, 120-126. (19) Huczko, E. L.; Bodnar, W. M.; Benjamin, D.; Sakaguchi, K.; Zhu, N. Z.; Shabanowitz, J.; Henderson, R. A.; Apella, E.; Hunt, D. F.; Engelhard, V. H. J. Immunol. 1993, 151 (5), 2572-2587. (20) Henderson, R. A.; Cox, A. L.; Sakaguchi, K.; Apella, E.; Shabanowitz, J.; Hunt, D. F.; Engelhard, V. H. Proc. Natl. Acad. Sci. U.S.A. 1993, 90 (21), 10275-10279.

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Figure 1. Schematics of the nano-LC system. Conventional HPLC system with microflow processor, capillary column, and UV detecor with U-shaped nanoflow cell (modifications in black).

higher mass and concentration sensitivity of nanoscale LC (nanoLC). Further, it is demonstrated how conventional HPLC instrumentation can be modified for successful use in nano-LC. Preinjection flow splitting with high split ratios of 1:2000, the use of high-sensitivity nanoliter UV flow cells with volumes as small as 3 nL, and different injection techniques for optimal nondispersive injection are discussed in detail. EXPERIMENTAL SECTION (a) Reagents. A reversed-phase test mixture containing uracil (4.3 µg/mL), naphthalene (57.5 µg/mL), biphenyl (46.0 µg/mL), fluorene (8.21 µg/mL), anthracene (9.85 µg/mL), and fluoranthene (26.3 µg/mL) was used for efficiency (N/m) measurements. All compounds were purchased from Fluka AG (Buchs, Switzerland). Acetonitrile (ACN), methanol (MeOH), and water were all of HPLC grade (LabScan Ltd., Dublin, Ireland). For the separation of the protein standards, a test mixture was made containing 0.2 mg/mL each of ribonuclease, insulin, cytochrome c, lysozyme, and bovine serum albumin (Sigma Co., St. Louis, MO). Sample solvent was 0.1% trifluoracetic acid (TFA) in water/ACN (95:5 v/v). To assure high UV transparency at low wavelengths, supergradient ACN (Catalog No. C2527, LabScan) and TFA from ampules (Catalog No. T-6508, Sigma Co.) were used for the preparation of the mobile phases, which consisted of water/ ACN/TFA mixtures (see Chromatography, below). (b) Instrumentation. Figure 1 shows the schematics of the nano-LC instrumentation. The main system components were a conventional HPLC pump (either low- or high-pressure mixing gradient can be used), a conventional UV detector equipped with a U-shaped nanoflow cell, and a microinjection valve. A microflow processor (Acurate, Model AC-2000-NAN, LC Packings) placed between the pump and the injector was used to split the flow down to the required nanoflow. The microflow processor compensates for the viscosity changes during gradient elution and, hence, results in highly constant flow delivery. Further, it streamlines the baseline noise generated by the movement of the pump piston and/or stepper motor. More details about the function and working of this system have been published previously.21,22 Under isocratic conditions, the split flow was recycled back to the reservoir. Under gradient conditions, it was guided to waste. Typical pump settings were 300-400 µL/min to assure proper functioning of the conventional HPLC pump and accurate pro(21) Chervet, J. P.; Meyvogel, C. J.; Ursem, M.; Salzmann, J. P. LC-GC 1992, 10 (2), 140-148. (22) Chervet, J. P. (LC Packings). Eur. Pat. Appl. 0597552A1, 1993.

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portioning of the gradient. The flow was split by a ratio of ∼1: 2000 to achieve the required nanoflow of 150-200 nL/min. Low-pressure in-line filters (e.g., 0.2 µm disposable filters from Whatman Inc., Clifton, NJ, placed between the reservoir and the pump) are recommended to avoid clogging of the nano-LC system. Further, the microflow processor contains built-in, replaceable, high-pressure in-line filters to trap particles originating from normal pump seal wear and/or the mobile phase. A commercial microinjection valve equipped with a 20 nL internal loop (Model C4-1004.02, Valco Europe, Schenkon, Switzerland) was used for nanoscale injections. To allow system evaluation (plate count measurements), injections of 2-3 nL are required. Hence, an additional 1:10 split of the sample was applied by connecting the column to the injector via an additional split T. For 20 nL large volume injections, the above-mentioned valve can be used without any sample split, simply by installing the appropriate rotor loop (Model C4-10R4.02, Valco). However, sample focusing (peak compression) conditions must be assured. On the injection side, the column is directly connected to the injection valve with a 1/16 in. o.d. PEEK sleeve connector, thus guaranteeing minimal dispersion. On the detection side, the column is directly connected to the nanoflow cell with a Teflon connector (TF-250, LC Packings) that allows for a dead volume free butt connection. Detection was performed with a conventional UV detector (model 1050 VWD, Hewlett Packard, Waldbronn, Germany) equipped with a U-shaped nanoflow cell of 8 mm path length and 3 nL cell volume (Model UZ-HP-NAN, LC Packings). The flow cell had connecting capillaries (inlet and outlet) of e20 µm i.d. and short length (e15 cm) to assure minimal extracolumn dispersion. For further details, see refs 23-25. (c) Chromatography. For isocratic system evaluation a 75 µm i.d. × 30 cm column, packed with C18, 5 µm particles was used (Fusica, Catalog No. NAN75-30-05-C18, LC Packings). The mobile phase consisted of acetonitrile/water (80:20 v/v) at a flow rate of 180 nL/min. Injection volume was ∼2.0 nL using the 20 nL injection valve and a 1:10 sample split (made by a T-piece, Valco, and a fused silica restrictor capillary). Detection was UV at λ ) 254 nm. A step gradient was measured without a column by directly connecting the microflow processor to the UV detector, equipped (23) Chervet, J. P.; Ursem, M.; Salzmann, J. P.; Vannoort, R. W. J. High Resolut. Chromatogr. 1989, 12 (5), 278-281. (24) Chervet, J. P.; Van Soest, R. E. J.; Ursem, M. J. Chromatogr. 1991, 543, 439-449. (25) Chervet, J. P. (LC Packings). Eur. Pat. Appl. 0495255A1, 1991.

Table 1. Names and Definitions for HPLC Techniques column i.d.

flow rate

name

3.2-4.6 mm 1.5-3.2 mm 0.5-1.5 mm 150-500 µm 10-150 µm

0.5-2.0 mL/min 100-500 µL/min 10-100 µL/min 1-10 µL/min 10-1000 nL/min

conventional HPLC mirobore HPLC micro-LC capillary LC nano-LC

with an ∼100 pL on-column flow cell (20 µm i.d. fused silica) to assure linear response over the entire range and minimal dispersion. Gradient separations were performed at ambient temperature on a 75 µm i.d. × 15 cm column, packed with Vydac, C18, 5 µm diameter, 300 Å pore stationary phase (Fusica, Catalog No. NAN75-15-05-C18P3, LC Packings). Gradient was from 25% B to 55% B in 20 min, whereby the mobile phase consisted of (A) 0.1% TFA in water/ACN (90:10 v/v) and (B) 0.08 TFA in water/ACN (10:90 v/v). To compensate for the gradient delay (dwell volume), the 20 nL sample was injected 2 min after the gradient start at a flow rate of 180 nL/min. Detection was UV at λ ) 220 nm. The mobile phase was renewed every second day and used under continuous helium sparging. RESULTS AND DISCUSSION Names and Definitions. Table 1 lists the most common names for the different HPLC techniques. So far, most of the techniques have been named on the basis of the type or the inner diameter of the column tubing. With the use of new tubing materials (e.g., fused silica capillaries, polymeric tubing, stainless steel tubing, micromachined channels on silicon wafers, etc.), this nomenclature is not sufficiently precise. Therefore, we suggest defining the chromatographic technique according to the flow rate range used rather than by the inner diameter of the tubing or its material. Hence, for packed microcolumns with 10-150 µm i.d. and flow rates of 10-1000 nL/min, the technique would be called nano-LC. Fundamental Factors in Nano-LC. (a) Down-Scale Factor. In nano-LC, as well as in all other microcolumn LC techniques, all volumes must be down-scaled by a factor (f), 2 f ) d2conv/dmicro

(1)

where dconv and dmicro are the diameters of the conventional and microscale HPLC columns, respectively. This down-scaling is inevitable in order to maintain the performance of the microseparation system and to work under optimal conditions. Down-scaling from a 4.6 mm i.d. conventional HPLC column to a 75 µm i.d. packed nano-LC column equals a factor of ∼3800 (3762). This factor applies to all components or parameters of the nano-LC system, such as flow rates, injection and detection volumes, and connecting capillaries. (b) Required Flow Rates. The volumetric flow rate (F) in a chromatographic system is defined as26,27 (26) Ishii, D., Ed. Introduction to Microscale HPLC; VCH Publishers, Inc.: New York, 1988. (27) Kucera, P., Ed. Microcolumn HPLC; Journal of Chromatography Library 28; Elsevier Publishers: Amsterdam, 1984.

F ) uπdc2/4

(2)

where u is the linear velocity of the mobile phase, dc the column diameter, and  the column porosity. With a typical linear velocity of u ) 1 mm/s and a column porosity of  ) 0.7, the volumetric flow rate for a 75 µm i.d. column can be calculated as 186 nL/min. This value compares favorably with the value obtained by using the down-scaling factor and a flow rate of 0.8 mL/min for the conventional column. (c) Maximum Injection Volume. As in conventional HPLC, the maximum injection volume (Vmax) that can be injected onto the microcolumn can be expressed by the following equation,

Vmax ) θKπdc2L(k + 1)/xN

(3)

where θ is the fractional loss of the column plate number caused by the injection, K is a constant describing the injection profile, L the column length, k the retention factor,28 and N the theoretical plate number. Allowing a typical value of 5% in volume dispersion (θ ) 0.05) and column porosity  ) 0.7, assuming that the injection profile is almost an ideal rectangular plug (K ) 4) and that the columns have good efficiency with reduced plate height h ) 2, and substituting N by

N ) L/hdp

(4)

where dp is the particle size of the stationary phase, eq 3 then becomes

Vmax ≈ 0.622dc2(k + 1)xLdp

(5)

Thus, the maximum injection volume is proportional to the square of the column diameter (dc2), the retention factor (k), and the square root of the column length (L) and the particle size (dp). For a typical 75 µm i.d. × 15 cm microcolumn, packed with C18, 5 µm particles, the injection volume for an slightly retained peak (k ) 1) should be not larger than 6.1 nL. Similar values are obtained by dividing the typical injection volume of a conventional column, e.g. 20 µL, by the down-scaling factor. In practice, however, there are no commercial injection valves available that allow for the injection of volumes less than 20 nL. Therefore, a 1:10 sample split was used for performance measurements of the nanocolumns (h/u curve). For large volume injections, however, the sample should be dissolved in a weaker solvent than the mobile phase, thus allowing the enrichment (adsorption) of the analytes on top of the column and avoiding peak-broadening. (d) Detection Volume. To maintain the high efficiency of the microcolumn and to avoid peak dispersion during detection, the volume of the sensing region must be adapted. Using UV detection, the sensitivity is proportional to the path length of the flow cell (Lambert-Beer), and due to shot noise limitations, the flow cell aperture should be relatively large. Consequently, a (28) Ettre, L. S. Pure Appl. Chem. 1993, 65 (4) 819-872. (29) Chervet, J. P.; van Soest, R. E. J.; Salzmann, J. P. LC/GC Int. 1992, 5, 3338. (30) Chervet, J. P.; Ursem, M. GIT Spez. Chromatogr. 1992, 12, 38-44.

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Table 2. Flow Rate and Volume Requirements for Nano-LC (50 and 75 µm i.d. Packed Columns) packed column

(nL/min)a

flow rate injection volume (nL)b flow cell volume (nL) connecting capillaries (µm)c

50 µm i.d.

75 µm i.d.

80 e1.5 e1.5 e10

180 e3.0 e3.0 e20

a For maximum plate counts according to the h/u curve. b For nonretained peak, k ) 0. c Maximum length, 15 cm.

compromise between maximum sensitivity and minimum peak dispersion must be found. Using Z- or U-shaped capillary flow cells, Chervet et al.21 have demonstrated excellent sensitivities with minimal dispersion even under CE conditions. Ideally, the maximum detection volume should be not more than one-tenth of the volume of the peak that is leaving the separation column. For a nonretained peak the dilution over the column is ∼4-fold. From eq 1 or 3, the detection volume for an ideal nanoflow cell can be calculated as 1 nL. For peaks with k > 1, it has been shown in conventional HPLC that flow cell volumes are usually similar to the injection volumes. Hence, in our experiments, we used flow cells of 3 nL volume and 8 mm path length. (e) Maximum Sample Mass (Loadability). Similar to the maximum injection volume as defined in eq 3, the maximum sample mass (Mmax) that can be injected onto a column can be expressed as26,27

Mmax ) Cmπdc2Lk/2xN

(6)

where Cm is the maximum sample concentration of a peak eluted from the column. Thus, for two columns of the same length and with identical packing and performance characteristics, differing only in the column diameter, the ratio of the maximum injectable mass that can be placed on each column to give the same sample concentration (Cm) is equal to the ratio of the column diameters in square,

d2conv

(Mmax)conv (Mmax)micro

)

2 dmicro

)f

(7)

and equals the down-scaling factor (f) of eq 1. In practice, this means that, for a 75 µm i.d. × 15 cm packed column, the maximum injected sample mass (loading capacity) for a nonretained compound (k ) 0) should not exceed 50 ng. In all experiments, the injected sample mass was significant lower to avoid the risk of any mass overload and to assure optimal separation conditions. Flow rate and volume requirement for nano-LC are summarized in Table 2. System Evaluation. To 1evaluate the performance of the entire nano-LC system, chromatographic reproducibility (isocratic and gradient) as well as efficiency and sensitivity measurements have been conducted. (a) Isocratic Elution. For reproducibility and efficiency measurements, the reversed-phase test mixture was injected under isocratic conditions, using ACN/water (80:20 v/v) for elution. To 1510 Analytical Chemistry, Vol. 68, No. 9, May 1, 1996

Figure 2. Isocratic separation of test mixture for system evaluation: uracil (1), naphthalene (2), biphenyl (3), fluorene (4), anthracene (5), and fluoranthene (6); flow rate, 180 nL/min (other conditions, see Experimental Section).

Figure 3. h/u curve. Column: 75 µm i.d. × 30 cm, packed with C18, 5 µm.

avoid peak dispersion, injection volumes were set at ∼2 nL. In Figure 2, a typical chromatogram is shown for the separation of the test mixture. In Figure 3, the h/u curve for fluorene (peak 4) and fluoranthene (peak 6) is shown. The data for the h/u curve are listed in Table 3. At a linear velocity (u) of 1.16 mm/s, which corresponds to a flow rate of 175 nL/min, maximum column plate counts of more than 100 000/m (reduced plate heights h e 2.0) were measured. These high plate counts illustrate the high performance of the nano-LC system. Furthermore, the experimentally determined flow rate corresponds well with the calculated value of 186 nL/ min. With RSD values of (0.1% on retention times (n ) 20), excellent reproducibility under isocratic conditions was observed. (b) Gradient Elution. Proper gradient elution at nanoliter flow rates is possible only when accurate proportioning, homogeneous mixing with minimal delay, and reproducible delivery of the mobile phase are realized. At the present time, there are no micropumping systems available that allow for nanoflow delivery

Table 3. Data for the h/u Curve N

h

flow (nL/min)

t0

E0 (porosity)

u (mm/s)

fluorene

fluoranthene

fluorene

fluoranthene

25 50 100 150 175 200 225 250 275 300 350 400 450 500 550 600

31.48 13.45 7.45 4.97 4.30 3.74 3.30 2.90 2.67 2.54 2.24 1.91 1.57 1.48 1.34 1.26

0.594 0.508 0.562 0.563 0.568 0.565 0.561 0.547 0.554 0.575 0.592 0.577 0.533 0.559 0.556 0.571

0.16 0.37 0.67 1.01 1.16 1.34 1.52 1.72 1.87 1.97 2.23 2.62 3.18 3.38 3.73 3.97

13 150 24 574 28 907 29 799 30 127 28 188 26 331 25 483 23 087 22 233 21 712 19 281 17 767 16 657 16 070 15 184

12 536 23 549 27 561 28 161 27 872 25 982 23 457 23 711 21 842 21 388 20 687 18 332 17 274 16 190 15 261 14 893

4.56 2.44 2.08 2.01 1.99 2.13 2.28 2.35 2.60 2.70 2.76 3.11 3.38 3.60 3.73 3.95

4.79 2.55 2.18 2.13 2.15 2.31 2.56 2.53 2.75 2.81 2.90 3.27 3.47 3.71 3.93 4.03

Figure 4. Step gradient at 180 nL/min using conventional HPLC pump with microflow processor. Gradient from 0 to 100% B; step, 10% every 5 min. (A) MeOH, (B) MeOH + 10% acetone; UV detection at λ ) 254 nm.

under these conditions. Therefore, the concept of microflow processing, using conventional HPLC pumps combined with flow spitting and simultaneous viscosity change compensation, was applied.21,22 In comparison to microflow processing in micro-LC and capillary LC, the main difference for nano-LC was the use of higher splitting ratios, typically 1:2000. Thus, almost any conventional HPLC pumping system can be successfully used for nano-LC (isocratic and gradient). The flow rate of the conventional HPLC pumping system is usually set at 400 µL/min, which results in a nanoflow of 200 nL/min using a 1:2000 splitting rate. If syringe pumps are used, lower spitting ratios (∼1:500) and smaller input flows (100 µL/min) can be applied (data not shown). To measure the proportioning accuracy, a step gradient was programmed. Figure 4 shows the step gradient ranging from 0% B to 100% B with steps of 10% B every 5 min at a total flow rate of 180 nL/min (for experimental conditions, see Chromatography, above). Over the entire gradient range, only minimal deviation from the theoretical step profile was observed, thus illustrating homogeneous mixing and accurate proportioning of the mobile phases. Further, the system provides a minimal gradient delay of only 3 min (without column installed). That corresponds to a dwell volume of less than 600 nL. Baseline noise over the entire gradient is similar to that of a conventional HPLC step gradient, indicating good mixing and streamlining capabilities of microflow processors. The use of proteinaceous samples rather than small organic molecules is recommended for sensitive monitoring of gradient

Figure 5. Separation of protein standards: ribonuclease (1), insulin (2), cytochrome c (3), lysozyme (4), and bovine serum albumin (5); flow rate, 180 nL/min (other conditions, see Experimental Section).

reproducibility.31 Owing to their strong dependency on the retention factor k from the mobile phase composition, they are ideally suited as test compounds. Minor changes in the gradient composition result in notable retention time variations. Therefore, gradient reproducibility was measured by consecutive injections of a test sample containing five standard proteins, as shown in Figure 5. To circumvent the need for sample splitting during injection and to avoid the loss of valuable sample, a 20 nL sample was injected directly onto the packed 75 µm i.d. nanocolumn, using sample focusing (peak compression). In the case of our protein standards, the proteins were dissolved in acidified water with only a small amount of organic modifier (0.1% TFA in water/ACN, 95: 5, see Experimental Section). This allows for effective sample focusing without any measurable peak dispersion and the direct use of microinjection valves without any sample loss due to the absence of the sample split. Optimal handling of minute samples using large and extra-large volume injection in nano-LC will be (31) Dolan, J. W. LC-GC 1989, 7 (1), 18-24.

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Table 4. Limits of Detection for Proteins Using Nano-LC (UV Detection, λ ) 220 nm) compound

S/N

LODa (pg)

LODa (fmol)

ribonuclease insulin cytochrome c lysozyme bovine serum albumin

330 710 290 560 340

37 17 42 22 35

2.7 2.9 3.2 1.4 0.5

a

S/N ) 3.

Figure 6. Reproducibility nanoscale gradient using protein standards (3D plot). Flow rate, 180 nL/min.

discussed in an upcoming paper.32 With the injection of 20 nL of the protein standard solution (0.2 mg/mL of each protein), a total of 4 ng of each protein was injected. These amounts correspond to 292 fmol of ribonuclease, 696 fmol of insulin, 308 fmol of cytochrome c, 264 fmol of lysozyme, and 60 fmol of bovine serum albumin. With signal (peak height)-to-noise (S/N) levels ranging from 286 for cytochrome c to 710 for insulin, the LODs shown in Table 4 can be calculated. To confirm the calculated LOD values, the protein standard solution was diluted 1:100 (using 0.1% TFA in water/ACN, 95:5) and reinjected under the same conditions as described above. With 2 times lower LOD, similar values were observed, as reported in Table 4 for the five proteins. These data demonstrate unambiguously the high mass sensitivity of nano-LC systems. In Figure 6, the reproducibility of seven consecutive injections is shown as a 3D plot. With RSD values of (0.2% (n ) 7) for retention times, excellent reproducibility was found, indicating the high performance of the entire nano-LC system. CONCLUSIONS The use of nano-LC with packed columns of 75 µm i.d. and typical flow rates of 180-200 nL/min looks very promising for the analysis of minute and/or valuable samples. (32) Chervet, J. P.; et al. Manuscript in preparation.

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Conventional HPLC instrumentation can be easily upgraded to nano-LC. Modifications thereof include the use of microflow processors for flow splitting, longitudinal capillary flow cells (Uor Z shape) for sensitive UV detection, microinjection valves for nanoliter injections, and connecting capillaries of small inner diameter (e20 µm) to avoid peak dispersion. Installation is very easy, extremely economical due to the use of existing hardware (pumps and detector), and done within a few minutes. Further, the use of conventional HPLC instrumentation in nano-LC allows the entire range of HPLC techniques (conventional HPLC, microLC, capillary LC, and nano-LC) to be covered with one instrument versus the need for dedicated hardware. Over the last 2 years, we have been working with such nano-LC systems. We have found great ease of use and almost no downtime. Both isocratic and gradient elutions are feasible at nanoflows, with excellent retention time reproducibility, similar to that of conventional HPLC. The reduced plate height of 2 reflects the overall high performance of the nano-LC system. Further, large volume injections of several nanoliters (g20 nL) can be applied directly onto the packed capillary column with sample focusing to allow trace analysis and to enhance the minimal detectable concentration. With minimal detectable amounts on the order of 35 pg or 523 amol for bovine serum albumin, extremely low limits of detection (LODs) were realized using simple UV detection. Such LODs are not attainable by either conventional HPLC or microLC. Also noteworthy is the high resolution of the protein separation. The increased sample-to-phase ratio in nano-LC might be an explanation. Using nano-LC in conjunction with new MS techniques and new interfaces, such as the “nano ion spray” or ion trap ESI/MS, one might expect further enhancements of the LOD, required for the microcharacterization of proteins and peptides. In an upcoming paper,32 results will be presented for the use of large and extra-large volume injections to further enhance the concentration sensitivity in nano-LC.

Received for review September 1, 1995. January 20, 1996.X AC9508964

X

Abstract published in Advance ACS Abstracts, March 1, 1996.

Accepted