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Anal. Chern. 1983, 55, 1752-1760
of many possible approaches to vaporization from a solid surface. As mentioned in the introduction, methods such as SIMS, FAB,etc. show real promise and may eventually allow the moving belt LC/MS interface to become practical for virtually any compound class. We have conducted this study for ultimate application of LC/MS to environmental problems. As an example, in the treatment of drinking water, unexpected substances are formed from the interaction of disinfection chemicals with unknown trace organic compounds present in the untreated water. In order to understand the disinfection chemistry and to assess the potentially adverse health effects, the unknown chemicals must be identified both before and after treatment. Many of these compounds are highly polar (22) and cannot be analyzed in a cost effective way by techniques such as gas chromatography/MS. LC/MS would appear to be of potential value in this analysis.
LITERATURE CITED (1) Eckers, C.; Games, D. E.; Lewis, E.; Nagaraja Rao, K. R.; Rossiter, M.; Weerasinghe, N. C. A. Adv. Mass Spectrom. 1980, 86,1396. (2) Henion, J. D. Adv. Mass Spectrom. 1980, 86, 1241. (3) Biakeiy, C. R.; Vestal, M. L. Anal. Chem. 1983, 55, 750-754. (4) Cairns, T.; Seigmund, E. G.; Doose, G. M. Anal. Chem. 1982, 5 4 , 953-957. (5) Henion, J. D.; Mayiin, G. A. 6iomed. Mass Spectrom. 1980, 7 , 115-12 1. (6) McFadden, W. H.; Bradford, D. C. J . Chromatogr. Scl. 1979, 17, 5 18-522. (7) Games, D. E.; Lewis, E. 6iomed. Mass Spectrom. 1980, 7 ,433-436. (8) Henion, J. D.; Thomson, B. A.; Dawson, P. H. Anal. Chem. 1982, 5 4 , 45 1-456. (9) Karger, B. L.; Kirby, D. P.; Vouros, P.; Foitz, R. L.; Hidy, B. Anal. Chem. 1979, 51,2324-2328.
(IO) Kirby, D. P.; Vouros, P.; Karger, B. L.; Hidy B.; Peterson, B. J. Chro-
matogr. 1981, 203, 139-152. (11) Smith, R. D.; Johnson, A. L. Anal. Chem. 1981, 53, 739-740. (12) Games, D. E.; Lant, M. S.;Westwood, S.A.; Cocksedge, M. J.; Evans, N.; Williamson, J.; Woodhaii, B. J. 6iomed. Mass Spectrom. 1982, 9 , 215-224. (13) Games, D. E.; Hewlins, M. J.; Westwood, S.A.; Morgan, D. J. J . Chromatogr. 1982, 250, 62-67. (14) Aicock, N. J.; et ai. J . Chromatogr. 1982, 251, 165-174. (15) Benninghoven, A.; Eicke, A.; Junack M.; Sichtermann, W. Org. Mass Spectrom. 1980, 15, 450-453. (18) Smith, R. D.; Burger, J. E.; Johnson, A. Anal. Chem. 1981, 53. 1603-1611. (17) Dobberstein, P.; Korte, G.; Meyerhoff, G.; Pesch, R. I n t . J. Mass Spectrom. Ion Phys. 1983, 46, 185-188. (18) van Dijk, J. H. J. Chromatogr. Scl. 1972, IO, 31-34. (19) Lankmayer, E. P.; Hayes, M. J.; Karger, 8. L.; Vouros, P.; McGuire, J. M. Int. J. Mass Spectrom. Ion Phys. 1983, 4 6 , 177-160. (20) Kutner, W.; Debowski, J.; Kemuia, W. J. Chromatogr. 1981, 218, 45-50. (21) Karger, B. L.; Martin, M.; Gulochon, G. Anal. Chem. 1974, 46, 1640-1647. (22) Christman, R . F.; Johnson, J. D.; Norwood, N. L.; Liao, W. T.; Hass, J. R.; Pfaender, F. K.; Webb, M. R.; Bobenrieth, M. J. PB 81-181952; NTIS: Springfield, VA.
RECEIVED for review February 7,1983. Accepted May 1,1983. The research described in this article has been funded by the U.S. Environmental Protection Agency under assistance agreement number CR807325-02 to Northeastern University. The contents of this article do not necessarily reflect the views and policies of the agency, and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsment or recommendation for use. This is Contribution No. 156 from the Institute of Chemical Analysis.
Gradient Elution Chromatography with Microbore Columns H. E. Schwartz and B. L. Karger* Institute of Chemical Analysis and Department of Chemistry, Northeastern University, Boston, Massachusetts 02115 P. Kucera Pharmaceutical Research Products Section, Quality Control Department, Hoffman-LaRoche, Inc., Nutley, N e w Jersey 07110
This paper deals wlth the development of practical approaches to mlcrobore column gradlent elution chromatography through the modlflcatlon of equlpment currently available for normal bore columns. Key to the deslgn Is the use of varlous hlgh-pressure, low volume mixers. The Influence of poor mixing on base llne nolse Is first examlned, and it Is shown that the column can amplify the slgnal due to unmlxed zones, partlcularly If one or more components of the moblle phase possess some UV absorbance. By use of the amplitude of the base llne nolse as a relative measure of mlxer performance, two static mlxers, a specially designed dynamlc mlxer and a combined dynamWstatlc mlxer, are examlned. I n additlon, response and delay volumes as well as retentlon and peak area preclslon are measured for each mixer. The mlcrovolume dynamlc mlxer Is shown to yield small delay (26 pL) and response (46 hL) volumes with low nolse characterlstlcs. By use of the dynamlc mlxer alone or in combination wlth a statlc mlxer, hlgh sensltlvlty mlcrobore separations of peptldes, proteins, and phenols are shown.
In the past several years there has been increasing interest in the use of microbore columns for HPLC (1-12). Packed
columns of 1to 2 mm i.d. are now commercially available, and there is potential for the possible use of open tubular columns, if appropriate instrument design can be achieved. Microbore columns as a consequence of low mobile phase flow rates have potential for coupling with specific detectors such as mass spectrometry (2, 13-15), flame photometry (16), Fourier transform IR (17), and electrochemical (18, 19). Solvent economy is another reason for the interest in microbore HPLC. It is worth noting that this economic factor can be significant when synthesized or expensive agents that permit high selectivity (e.g., chiral species) are used. In addition, the increasing low level detection of substances with highly sensitive detectors require improved purification of solvents and reagents. These added purification steps will inevitably increase the expense of such solvents. Another potential advantage of microbore columns is the improved mass detectability relative to normal bore columns (e.g., 4.6 mm i.d.), as a consequence of the low dilution in the narrow bore columns. In order to realize the full potential of microbore columns with respect to mass detectability, it will be necessary to use columns with small particle diameters, packed with high efficiency. Detectors which permit full realization of detection at low noise levels along with negligible contributions to band broadening will also be required.
0003-2700/83/0355-1752$01.50/00 1983 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 11, SEPTEMBER 1983
An important aspect of the successful utilization of microbore columns is the ability to employ gradient elution chromatography. For realistic sample mixtures, for example, of biochemical or environmental origin, substances with wide polarity differences will be found. In order to elute these substances in reason,zble time periodai and with reasonable signal intensities, one must change, continuously or discontinuously, the capacity factor. One of the most powerful and simplest approaches to achieving this is, of course, gradient elution (20). Two approaches to gradient elution have gained wide popularity in commeircial instrumentation: (1)low-pressure and (2) high-pressure mixing. In case '1 two or more solvents are mixed on the low pressure side prior to the pump, the composition being proportionally controlled by electronically actuated solenoid valves. Typically, i l single high-pressure pump is used to deliver the mixed solvent to the column. In case 2, the solvents are delivered by two high-pressure pumps to a low volume mixing chamber on the high-pressure side of the LC system. For both cases, adequate mixing of the solvents prior to reaching the column is a prerequisite. Generally, mixing is achieved by simple flow mixing devices (static) or by stirred mixing chambers (dynamic). Excellent reviews of gradient instrumentation for normal bore operations can be found in the literature (20-23). Flow rates using microbore columns of 1mm i.d. are roughly 20-fold lower than 4 6 mm i.d. columns for the same linear velocity. It is well-known that in orlder to maintain chromatographic fidelity of the eluted peaks the design of the detector flow cell must be carefully considered. In addition, the use of low flow rates requires either modification of pumps designed for normal bore columns or tlhe introduction of new pumping systems. For gradient elution, besides the pumps and detector, it is necessary to design carefully the scdvent mixing system. Previously, it has been shown that the 'Waters Associates dual reciprocating pumping system with high-pressure mixing can be modified for the low flow rates (Le., 20-100 pL/min) necessary for microbore gradient LC (24). The authors used a simple mixing T of very small volume for the analysis of standard test mixtures under relatively simple gradient elution conditions (i.e., 50-100% methanol). However, under wider gradient ranges with buffers and ion pairing reagents, we found that this mixing design is not sufficient. We were confronted with the occurrence of base line oscillations, especially in high water content gradients for reversed-phase chromatography and at relatively high sensitivity conditions. We felt it was necessary, therefore, to examine in more detail the design of the gradient mixing system with microbore LC. The purpose of this paper is to present and evaluate new approaches to microvolume mixing for gradient elution using 1mm i.d. columns. Our philosophy wa~ito modify, in as simple a fashion as possible, current commercial instrumentation for normal bore columns which has been optimized for flow rates of 0.5-2.0 mL/min. While specially designed gradient systems can be made for microbore HPLC @j), the flexibility of instrumentation available for both normal bore and microbore operation is appealing. In this regard, it is interesting to note that a commercial gradient system which can be used for both normal and microbore columns has recently been introduced (26). A split flow gradient system for capillary and microHPLC has also been reported (38). In this paper, we shall describe microvolume static and dynamic mixing approaches that can be easily developed for practical gradient microbore HPLC. In conjunction with this, we will discuss the causes of base line oscillations when using a UV detector with a modified reciprocating piston system On the basis of these studies, we will demonstrate high sensitivity analysis over wide
x
1753
i-/cDynomic or static mixing device
microbore column
Flgure 1. Block diagram of gradient microbore system. Components are specified in the Experimental Section.
Table I. Gradient Mixing Systems for Microbore LC dynamic total system static mixing mixing mixing no. volume tubing vol, PL vol, PL I 20 pL (400 X 0.25 mm) 20 400 X 0.25 mm
IIa
92~L{
92
t
92Xlmm 400 X 0.25 mm IIb 111 IV
172 pL{
+
194 X 1 mm 7 p L (300 x 0.178 mm) 300 x 0.178 mm 80pL{ t 92~1mm
172 31
38 111
solvent ranges for microbore gradient analysis.
EXPERIMENTAL SECTION Chromatographic Equipment. The experimental setup is shown in Figure 1. Two Waters Associates (Milford, MA) Model 6000A pumps were controlled by a Waters Model M660 gradient controller. The large volume pulse dampeners of the pumps were bypassed, as described previously (24). Two flow rate converters (Waters Associates) were installed between the gradient controller and the two pumps via appropriate gradient outlet cables. The converters stepped down the motor speed of the two pumps by either a factor of 10 or 100 and provided suitable micro LC flow rates in the 10-100 pL/min range. The pump outlets were connected to the mixing T via small bore (0.01 in. id.) stainless steel capillary tubing. A microvolume Micro-Vortex chamber (Kratos Analytical Instruments, Ramsey, NJ) was used to mix the two solvent streams. The mixing T described previously (24),with 1-2 pL volume, performed equally well. Small pieces (4 cm X 0.007 in i.d.) of stainless steel tubing were used to connect the microbore column to the injection valve and detector. The injection valves were either Model AC 1OU 10-port valves equipped with 1O-wL external loop (Valco Instruments, Houston, TX) or submicroliterValco valves, Model ACF 4U.5 or ACF 4U.2. Special microbore flow cells (0.5 pL volume, 1 mm path length) were installed in the Kratos Model 773 or 770 variable wavelength detectors and chromatograms and data were recorded with a Model 3385 automation system (Hewlett-Packard,Avondale, PA). Mixing Devices. Four mixing devices were used in this work systems I-IV, as detailed in Table I. In system I, the Kratos mixing T was connected directly t o the injection valve by means
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 11, SEPTEMBER 1983 2
6
13
0 valve
T' Figure 2. Diagram of dynamic micromixer: A, Valco 0.01 in. T; B, 4 cm of 0.007 in. i.d. stainless steel capillary tubing; c, ' / l e in. Swagelok nut and ferrule; D, X '/le in. drilled through Swagelok reducer; E, 8 X 1 mm Teflon coated stirring bars; F, In. Swaglok nut and in. zero dead volume Swagelok reducing union; ferrule; G, ' I 8to
H, 30 cm of 0.007 in. i.d. capillary tubing; I, stirring plate.
of 400 X 0.25 mm i.d. tubing (total volume 20 pL). In system IIA, static mixing, the mixing T was connected via 400 X 0.25 mm i.d. tubing and a Valco l/ls in. union to an open tube 92 X 1 mm i.d. The open tube dimensions of the second static mixer (system IIB) were 194 X 1 mm i.d. Details of the dynamic mixer of system I11 are given in Figure 2. The mixer was placed on top of a thermolyne Type 7200 stirring plate (VWR Scientific, Boston, MA). The mixer contained two microstirring bars (1/2 in. x l / * in. o.d., VWR Scientific). These bars required miniaturization with a razor blade in order to fit into the l/s in. Swagelok reducer and to provide adequate stirring motion. The approximate dimensions of the smaller stirring bars were 8 mm X 1mm 0.d. In system IV (system IIA and I11 combined), the dynamic mixer (including 30 cm X 0.007 in. connecting tubing) was connected via 92 X 1mm i.d. tubing to the injection valve. It was determined that mixing was significantly improved when the stirring bars were rotating relative to when the bars were motionless. Microbore Columns. All microbore columns were homemade and packed into 1 mm i.d. stainless steel tubing with a Model DSXHT6Q air-drivenfluid pump (Haskell Engineering, Burbank, CA) at 25000 psi pressure. Special Valco Model CEF-1 (0.01 in. hole) end fittings in which small 2-pm frits were inserted (Mott Metallurgical, Farmington, CN) terminated the microbore columns. The columns (25-50 cm in length) were packed with 7-10 pm bonded phase material. Under isocratic test conditions, reduced plate heights of 2.3-3 at the optimum flow rate of 10-20 gL/min were typically achieved. Three commercial bonded phase packings were used: Zorbax BP-ODs, 7-8 pm (Du Pont, Wilmington, DE); Vydac 201 TPB C18, 10 gm (The Separations Group, Hesperia, CA); and LiChrospher RP-8, 10 gm (MCB, Gibbstown, NJ). Chemicals. All solvents (Baker Analyzed Reagents, VWR Scientific) were HPLC grade. Five buffer systems, commonly used in peptide analysis (27-29), were selected in order to assess their compatibility with the microbore gradient analysis under high sensitivity conditions. The following reagents were added in equal amounts to the "A" and "B" solvent of a water/acetonitrile or a water/propanol gradient system (see figure captions): (1) 0.1% (v/v) trifluoroacetic acid (Pierce Chemical, Rockford, IL); (2) 0.14 M triethylamine "sequanal" grade (Pierce Chemical)/ phosphoric acid (Baker) adjusted to pH 3.5 with triethylamine; (3) 10 mM NH,HC03, adjusted to pH 6.5; (4) 0.1% phosphoric
20
10 -L
30
40
time, min
Flgure 3. Gradient elution microbore separation of test mixture of 16 polynuclear aromatic hydrocarbons: column, 35 X 0.1 cm, Vydac 201 TPB C18, 10 pm; solvent A, water; solvent B, acetonitrile; gradient, 50-+100% B in 20 mln; flow rate, 50 pL/min; injection volume, 0.5 pL; detection Wavelength, 254 nm; peak identification, (1) naphthalene, (2) acenaphthylene, (3)acenaphthene, (4) fluorene, (5) phenanthrene, (6)anthracene, (7) fluoranthene, (8)pyrene, (9) benz[a ]anthracene, (10) chrysene, (1 1) benzo[b]fluoranthene, (12) benzo[k]fluoranthene, (13) benzo [a ] pyrene, (14) dlbenz [a ,h ]anthracene, (15) benzo[ghi] perylene, (16) indeno[lP2,3-cd]pyrene. ~
acid; ( 5 ) 0.1% phosphoric acid and 10 mM KH2P04. A test mixture of 16 polynuclear aromatic hydrocarbon priority pollulanta and the 10 phenol standards were purchased from Supelco, Inc. (State College Park, PA). Bovine serum albumin, cytochrome c, a-chymotrypsinogen A, and ovalbumin were obtained from Sigma Chemical (St. Louis, MO). The insulin hydrolysate sample was obtained from S. Cohen, Institute of Chemical Analysis.
RESULTS AND DISCUSSION Preliminary Studies. Figure 1 shows a block diagram of the basic components of a microbore gradient elution system. As noted in the Experimental Section, the Waters pumps were appropriately modified for low flow rate operation. After the pumps a micromixing T was used in all designs. In the simplest design, system I, which is comparable to that of ref 24, no dynamic or static mixing device was included. For systems I1 through IV, microvolume static or dynamic mixing was added. Our first studies involved an examination of system I. With simple gradients, i.e., gradients run under normal phase conditions or in the reversed-phase mode with relatively high organic modifier compositions and no buffers or ion pairing reagents, good gradient elution separations were achieved. Figure 3 shows an example in the separation of 16 priority pollutant polynuclear aromatic hydrocarbons on a reversedphase C18 column. As a basis of comparison with later designed systems, we next examined the precision in retention, peak height, and peak area for the gradient run in Figure 3 with a flow of 50 gL/min. Table I1 summarizes results of this study based on nine consecutive gradients. The repeatability in retention times, t,, is quite good: 50.46% relative standard deviation (RSD). The more retained the solute the smaller the %RSD, a trend also found in the literature (30). The %RSDs for the peak height and peak area measurements are 1 3 % for most solutes, which for most applications is acceptable. The values on repeatability correspond well with previously published data (24). The chromatogram in Figure 3 was run under relatively low sensitivity conditions with a simple gradient. Problems can arise in applying system I to other types of gradient analyses where a larger gradient range, lower UV wavelength detection
ANALYTICAL CHEMISTRY, VOL. 55, NO. 11, SEPTEMBER 1983
rn
~~
Table 11. Precision in Retention ( t,), Peak Area ( A), and Peak Height ( h )under Gradient Elution Conditions with Microbore Columns and System I (See Figure l ) a 0 10
%RSD t,, min
solute
uracil acetophenone toluene pyrene triphenylene benzo[u]pyrene
5.13 10.76
15.64 21.64 22.26 24.45
t,
A
h
0.46 0.38 0.15 0.12
1.98 2.48 1.55
2.45 2.98
3.20
0.11 0.07
1.34 1.92
2.73 1.86 1.70
I
I
I1
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I
i
1.91 AU
50 x 0.1. cm, LiChrospher RP-8, 1 0 urn; 50a Column: 100% acetonitrile in 20 min; F = 50 pL/min, inject 0.2
pL. Based on nine consecutive runs.
_ _ _ _ _ I -
I _
4.
0.01
-
X(nrn)
Figure 5. Amplitude oscillations at 15% B vs. Wavelength, system I: solvent A, water; solvent B, 50% acetonitrile, 50% water; column, 35 X 0.1 cm, Zorbax-BP-ODS, 7.5 pm; flow rate, 80 pLlmin; (0)with column and above solvents, ( 0 )with column and 0.1 YO TFA added to solvents A and B, (A)capillary and 0.1 % TFA added to solvents A and B, back pressure provided by connecting column to outlet of flow cell, (El) UV absorbance spectrum 0.1 % TFA in water (reference, water).
be no flow from one of the pumps, since this flow can only be resumed when the piston has compressed the solvent up to the back pressure (22). Consequently, depending on the extent of high-pressure mixing, unmixed small slugs of more or less pure A or B solvent can periodically move through the LC system. At higher back pressures, the pistons have to compress the solvents to a greater extent before delivery can occur, leading to larger solvent slugs and larger oscillations I I on the base line. 0 5 10 timn min It is interesting to note that the oscillations also occur when the absorbance of solvents A and B is the same. This result Flgure 4. Base line noise at 15% B for three buffer systems using means that the noise can arise from effects other than simple system I: column, 35 X 0.1 cm, Zorbax-BP-ODS, 7.5 pm; solvent A, differences in UV background of components of the two 5 % acetonitrile, 95 % water; solvent B, 50% acetonitrile, 50 % water; [A] = 0.1 % TFA added1 to both solvents; [ B] = 10 mM NH,HCO, solvent mixtures. While refractive index differences between added to both solvents; [C] = 0.15 M triethylamine phosphate added solvents A and B could cause some of the noise, we found to both solvents; flow rate, 200 pL/min; pressure drop, 3300 psi; another factor to be important, namely, the UV absorbance detection wavelength, 2‘14 nm. of the buffer employed. and/or higher sensitivity is required. Due to incomplete Figure 4 shows the base line noise obtained at 3300 psi when mixing of the two liquid streams from pumps A and B, large three commonly used buffer systems (trifluoroacetic acid oscillations can be observed in the base line blank run. Under (TFA), ammonium bicarbonate (NH,CO,), triethylamine these circumstances, iamall sample peaks would be difficult phosphate (TEAP)) are compared in terms of base line noise to discern from the background noise. Thus, it was necessary at 214 nm, with a constant composition of 12% acetonitrile. to improve on the system I design by providing more efficient In this case we have mixed solvent A which contained 5/95 mixing for more demanding gradient elution analyses. As (v/v) acetonitrile/water with solvent B containing 50/50 shown later (see Figures 11-13), with proper design of a acetonitrile/water. In all three cases the absorbance of solvent microvolume mixer, high sensitivity analysis is readily A and B was “matched” by adding roughly equal amounts of achievable. buffer to the two solvents and measuring the absorbance. It Base Line Oscillations. While the noisy base line above can be seen in Figure 4 that the amplitude of the base line can be generally attributed to poor mixing of solvents A and noise is a function of which buffer system is used. Indeed, B (20),we decided to explore the causeEi of this noise in more the largest amplitude is observed for TFA which at the same detail. First, it was found that the amplitude of the oscillations time has the largest extinction coefficient at 214 nm. The in the base line increased as the flow rate or back pressure noise was found to be lowest when no buffer was used. increased. This result suggested that the oscillations are Figure 5 shows a plot of the amplitude of the base line related to the compressiion of the liquid in each piston chamber oscillations in absorbance units as a function of wavelength of the dual reciprocating piston pumps employed. In parof the UV detector using 0.1% TFA buffer. (The amplitude ticular, it is possible that for a short period of time there will was measured as the maximum absorbance range of the noise
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 11, SEPTEMBER 1983 I
0.04
I
I
1
I
1
-
Yo B
Flgure 6. Amplitude oscillations vs. % B for an acetonltrile/water/TFA system: system I; solvent A, water, 0.1 % TFA added; solvent B, 50% acetonltrile, 50% water, 0.1 % TFA added: column, 35 X 0.1 cm, Zorbax-BP-ODs, 7.5 pm; flow rate, 80 pL/min; (0)wlth column, (0) capillary, back pressure provided by connecting column to outlet of flow cell; detectlon wavelength, 214 nm.
over an interval of several minutes. This range included on an absolute basis both positive and negative deflections of the base line.) In this case, solvent A, pure water or buffered water, was mixed with 50/50 (v/v) acetonitrile/water (pure solvent or buffer added) such that 15% B was obtained. The absorbance spectrum of 0.1% TFA in water is shown as well in this figure. It is first noted that with TFA added to both the A and B solvents, the amplitude of the noise as a function of wavelength follows the spectral absorbance variation observed for TFA itself in water. This result indicates that the noise amplitude is a consequence of TFA, in spite of the fact that the A and B solvents have the same overall absorbance. Next, we placed a capillary tube between the injector and detector. The microbore column was then connected to the outlet of the flow cell, in order to maintain the same pressure at the pumps. As shown in Figure 5, the absorbance for the noise is significantly less a t all wavelengths below 250 nm, relative to the column operation, with 15-fold difference a t 214 nm. Finally, we include the amplitude of the noise when using solvents A and B without buffer but with a column present, and, as expected, the amplitude of the noise is quite low, a t least to 200 nm. The results in Figure 5 show that the noise arising from poorly mixed slugs of solvent is amplified by the column since the back pressure was maintained constant for the capillary and column. This amplifcation is most likely a consequence of equilibrium disturbances a t the head of the column from the unmixed solvent slugs. It is well-known that such nonequilibrium can lead to bands which can propogate through the column (22,31,32).The amplitude of these disturbances will be a function of the absorptivity of the components of the mobile phase (as well as the distribution of these components between the mobile and stationary phase). Hence, the base line oscillations will be a function not only of the buffer but also of the wavelength selected. The equilibrium disturbances due to unmixed solvent slugs are also dependent on the composition and percent B of the mixture. Figure 5 shows a plot of the amplitude of the noise at 214 nm for the 0.1% TFA system as a function of the percent B solvent. In this experiment, each point was obtained in an isocratic mode by mixing solvent A and B in the correct proportion. I t can be seen that a maximum in amplitude is observed at roughly 15% B. The explanation for the amplitude variation in Figure 6 is complex. Most likely, it is a consequence of several factors acting together including, among others, the relative flow rate of pumps A and B, the distribution of the buffer between the two phases (Le.,
equilibrium disturbances) and the mixability of the solvents. The dotted line a t the bottom of Figure 6 represents the noise levels obtained when a capillary tube was placed between the injector and detector and the column connected to the outlet of the flow cell. A comparison of this curve to that with the column dramatically shows the amplification of the column toward unmixed zones. The finite level of noise for the capillary tube case is probably related to refractive index differences between the plugs from the A and B solvents and the specific mobile phase composition. The results of Figures 4 and 6 point to the need to have good mixing for sensitive operation at low wavelength detection. In the next section we discuss various mixing designs for microbore HPLC. Mixing Devices. Fundamentally, two different mixing devices have been used in gradient LC, one based on flow with an open or flattened tube (static) and a second based on convection with active mixing (dynamic). These devices have been discussed in the literature (20,26,34,35).Beyond their capability to produce uniform mixing of solvents from the two pumps, there are two other aspects of the mixing device that need to be assessed. First, the delay volume from the start of the gradient until its appearance a t the beginning of the column must be considered. If the volume of the mixer is large relative to the chromatographic column, then large delay volumes will occur. While these can be subtracted out from the chromatograms, they will slow down the chromatographic analysis by adding additional time to both the gradient elution run and the recycling time to starting conditions. Thus, the delay volume should be as small as possible. The second characteristic of importance of a mixer is the response volume, here defined as the time from the first appearance at the end of the column of a step change in a gradient until 63% of the final value is reached. High response volumes will result in gradients which are inaccurate, particularly a t regions near the beginning and end of the gradient. Since 1 mm bore columns have much lower dead volume than corresponding 4.6 mm i.d. columns, it is necessary to reduce the volume of the mixer in order to reduce both delay and response volumes. On the basis of tube diameters, similar mixing devices should be 21-fold lower for the microbore than the conventional system. At the same time that we try to obtain microvolume mixing, we must keep in mind the efficiency of mixing in terms of smoothing out base line noises. In this work we have examined both static and dynamic mixers with the microbore system using the modified Waters Associates pumps. In order to provide a sensitive probe of the mixing capability of the various devices, we have selected the acetonitrile/water system with 0.1% TFA at the detection wavelength of 214 nm. Since we have already shown that other buffers and/or other wavelengths can reduce the noise, it is clear that the studies here do not represent the lowest noise level attainable. Nevertheless, they do point to the relative performance of the various mixing devices. Our first studies dealt with static mixing devices in which stainless steel tubing was placed between the mixing T and the injector. As previously noted, a related design is used commercially (26). It is first interesting to note that the tube diameter can play a role on the extent of mixing for a given tube mixing volume. In Figure 7 the amplitude of the base line oscillations (peak to peak amplitude) is plotted vs. tube diameter while maintaining the mixing volume of the static mixer constant a t either 35 or 75 pL. I t can be noted that for any given tube diameter the noise level is lower and thus the mixing better for the 75-wL volume than for the 35-pL volume. It is well-known that larger mixing volumes will improve the extent of mixing, as shown in Figure 7. For both static mixing volumes, the noise level decreases with increasing tube diameter (and shorter column length).
ANALYTICAL CHEMISTRY, VOL. 55, NO. 11, SEPTEMBER 1983
rIs
-7
L-d 001 002
'0
-ID
I
141
0004AU
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System
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003 004
TUBING (inches)
Flgure 7. Amplitude oscillations vs. inslde diameter tubing of statlc mixing device: solvent A, water, 0.1 % TFA added; solvent B, 50 % acetonitrile, 50% water, 0.1 % TFA added; columm, 35 X 0.1 cm, volume mixer 65 Zorbax-BP-ODs, 7.5 pm; flow rate, 80 pL/mln; (0) pL, (0)volume mixer 75 ALL;detection wavelength, 214 nm.
The fact that wider tubes yield better mixing for the same tube volume can be ratiionalized as follows. A wider-shorter tube will result in poorer radial mixing than a narrower-longer tube. Poorer radial miring will cause more sample dilution by the mobile phase in elution chromatography. Correspondingly, this poorer mixing will result in better solvent blending in a static mixing device. Barred on Figure 7, we decided to use 1mm i.d. tubing (0.04 in.) for the static mixer. As shown in Table I, two static mixers were constructed, one consisting of a tube of 72 pL volume and the other of 152 pL volume. With the 20 p L mixing T the total static mixing volumes were 92 and 172 pL, respectively. Figure 8 compares the base line noises obtained for these two static mixers and the dynamic mixer (to be discussed shortly) to that of system I which consisted of the mixing T alone The mobile phase conditions consisted of 0.1% TFA in 7.5% AcN, made by blending solvent A (8!5%) and solvent B (15%) with the gradient device. Note that the attenuation is 4-fold greater in the case of system I relative to the other systems. It can be seen that systems IIa and IIb yield sigrdficantly lower noise levels than system I and that system IIb, which has the larger mixing volume, also results in lower noiise than system IIa, as expected. Again, note that lower base line noises would be obtained if other buffers such as ammonium bicarbonate or phosphoric acid weIe used. The frequency of the major pulses in trystem IIb in Figure 8 (8-9 min intervals) can be used to show that the piston cross over points of the pump are the causes of the noise signal. The flow rate in pump B is 12 pL/min in a total flow rate of 80 pL/min (Le., 15% B). Furthermore, since each piston volume of a Waters pump is 100 pL, the time necessary to empty one piston chamber is 8.3 min. Thus, the major spikes appearing in system IIb and comparable time intervals in system IIa and system I are a consequience of slugs of relatively unmixed A solvent moving through the chromatographic system. Pump B does not flow for a specific period of time as the solvent in the piston chamber must first be compressed before it empties from the chamber. Note also that the shapes of these pulses are not fully regular as a consequence of the random superposition of other oscillations in the base line noise. Pump A, on the other hand, will operate a t a flow rate of 68 pL/min which means that each piston chamber will empty in 1.5 min. Thus, the higher frequency spikes observed in
Ub
System
0
LY
I
i
IO
20
-time,
min.
-
Flgure 8. Comparison of noise levels at 15% B using systems I IV: A, system I ; B, systems 11, 111, and IV; column, 35 X 0.1 cm, Zorbax-BP-ODs, 7.5 pm; solvent A, water, 0.1 % TFA added; solvent
B, 50% acetonitrlle, 50% water; flow rate, 80 pLlmln; detection wavelength, 214 nm.
systems I and IIa and IIb can be attributed to plugs of relatively unmixed solvent B traveling through the system when the compression of the solvent in a piston chamber occurs in pump A. The amplitudes of the oscillations are observed to be lower for the case when solvent in pump A is compressed than when the solvent in pump B is compressed. This result may arise in part from the fact that a larger volume of relatively unmixed solvent A will flow through the system than solvent B, since pump B moves at a lower flow rate. Of course, the specific equilibrium disturbance at the head of the column may also influence the amplitude of the noise. We next examined the use of a dynamic mixer shown in Figure 2, which was designed to keep the internal volume as small as possible in order to yield acceptable delay and response volumes. This mixer contained two stirring bars that rotated on a vertical axis by virtue of their enclosure within the small volume of the fittings. It is worth noting that the mixer in Figure 2 can be easily assembled from readily available parts. Whereas the static mixers reduced the amplitude of the noise from system I by 5- (system IIa) and 12-fold (system IIb), system 111, as seen in Figure 8, reduced the noise by approximately 11-fold. Thus, the dynamic mixer appeared nearly as efficient as a static mixer of almost 4 times the volume (IIb). If desired, the dynamic mixer can be combined with the static mixer of system IIa to form system IV which
1758
ANALYTICAL CHEMISTRY, VOL. 55, NO. 11, SEPTEMBER 1983
I !y, ,,,/,
i
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ref 23 static (H)a dynamic ( D ) a dynamic (B)a dynamic ( K ) a a
delay volume,
2 5, t
,/'
t
/'/
75
-
Table 111. Delay and Response Volumes for Three Microbore Gradient Systems; Comparison with Normal Bore Commercial Instrumentation system
B
400
Figure 9. Response to step gradient for systems I1 IV: (---) system Ira, (--) system IIb, system 111, (-) system IV; A solvent, methanol; B solvent, methanol -I-20 pL/L benzene: gradient, 0 100% B in 0 min; flow rate, 80 pL/min; back pressure provided by connecting column to outlet of flow cell; detection wavelength, 254 nm. For illustration purposes, the measurements of the delay and response volumes of system I I A are shown. (-e-)
Yo
PL
response volume, pL
71 136 26 78
37 75 46 87
1300 ( : 2 1 = 6 2 ) 600 (:21 ='29) 8200 ( : 2 1 = 390) 6000 ( : 2 1 = 285) 3600 ( : 2 1 = 171) 5100 ( : 2 1 = 243) 2500 ( : 2 1 = 119) 1700 ( : 2 1 = 81)
From ref 23, Table 11, systems H, D, B, and K.
yielded noise levels of 28-fold less. Thus, where appropriate and necessary, the dynamic mixer can be modified by the addition of a short piece of 1 mm tubing for further reduction of noise level. However, as we have already noted, the mobile phase may often be appropriately selected to minimize the need for the additional static mixer. We next studied the delay and rebponse volumes for systems IIa, IIb, 111, and IV. In this experiment, solvent A was methanol and solvent B was methanol plus a trace amount of benzene. The wavelength for detection was 254 nm, and the flow rate was 80 pL/min. Solvent A was stepped to solvent B and the response recorded on a chart recorder. Figure 9 shows the results of this experiment. Similar response curves have been published by others (23,36). The delay volume is measured a t the start of the appearance of benzene and is indicated for the case of system IIa in Figure 9. Table I11 summarizes the data for the four systems of Figure 10. It is observed that the smallest delay volume is obtained for the dynamic mixer, as expected, since the total volume of that mixer is the smallest of the four systems of Figure 9. The static mixer with the largest open tube yields the largest delay volume (system IIb). On the other hand the response volume is lowest for the small static mixer (system IIa); however, the response volume of the dynamic mixer (system 111)is only slightly larger. It is known that dynamic mixers in general yield larger response volumes by virtue of their mixing characteristics relative to static mixers of the same volume (23). In the bottom part of Table I11 we have assembled the characteristics of several commercial gradient systems used
,
I
0
IO
20
0
time, min
10
1
20
- -
Figure 10. Linear gradient profiles for systems I IV: (A) system I, (B) system Ira, (C) system 111, (D) system IV; gradient, 0 100% B in 20 min; other conditions, as in Figure 9. with normal bore (4.6 mm) diameter columns, as measured in ref 23. The number outside the parentheses represents the actual delay or response volume of that gradient system. Within the parentheses we have divided the number by a fador of 21 to correct for the narrower tube diameter of 1mm, rather than 4.6 mm i.d. It can be seen that the static mixer (system IIa) yields roughly comparable characteristics to static system H from ref 23. Most interesting is the fact that the microdynamic mixer shown in Figure 2 has proportionately much lower delay and response volumes than the lowest commercial dynamic mixer, system K. Returning to Figure 9, the relationship of the response volume of the mixer to the accuracy of the gradient is seen. Thus, system IIa yields a response closest to the step gradient, whereas system IV, having the largest response volume, yields the least accurate results. Note further that the greatest inaccuracy occurs near the ends of the gradient. Figure 10 shows gradient accuracy in terms of linear profiles for systems I, IIa, 111,and IV. In this experiment, the solvents A and B are similar to those of Figure 9, and a linear gradient is run from A to B in 20 min. In system I (a simple T) the slope of the imposed gradient is accurately followed; however, a noisy base line is observed. The static mixer of system IIa improves the base line noise, as seen in Figure 10B. In Figure 10C,D the profiles obtained in systems I11 and IV are shown, and it can be observed that good linearity is obtained, particularly in the 10-90% region. As expected, the gradient accuracy decreases at the gradient ends. Nevertheless, the smooth base line is to be noted. We next addressed the issue of precision for the mixing systems. A test mixture was chosen which would be indicative of the repeatability in retention and peak area over a fairly large gradient range (e.g., 1040% acetonitrile), while initiating the gradient a t high water content. Eight peptides, eluting for the most part in the early portion of the chromatogram, were chosen as test solutes; see Figure 11A for sample chromatogram. Table IV shows the results of nine consecutive runs for systems IIa and 111. It can be seen that the relative standard deviation in retention is as low as 0.2% and the relative standard deviation in peak area is in the range of 2-3%. No differences in precision are observed for systems IIa and 111. Comparison with Table I1 indicates that the precisions in t, and area are roughly similar. The slightly better relative standard deviation in t, in Table I1 may be a consequence of the use of polynuclear aromatic solutes in Table I1 and peptides in Table IV. Precision in t, and peak area comparable to the simple gra-
ANALYTICAL CHEMISTRY, VOL. 55, NO. 11, SEPTEMBER 1983
1759
I
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i 0.004AlJ
T 3
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5
T
0 024 AU
ik
0
20
+
40
time, min
Flgure 12. Microbore gradient elution separation of a protein mixture using system 111. Also shown Is the base line of a blank run: column, 35 X 0.1 cm, Vydac 201 TPB C18, 10 ym; solvent A, water, 0.1% phosphoric acid added; solvent B, 45% propanol-1, 55% water, 0.1 YO phosphoric acid added; gradient, 5 80% B in 40 min; flow rate, 40 yL/min; temperature, 23 'C; injection volume, 5 pL; detection wavelength, 214 nm; peak identification, (1) cytochrome c (250 ng), (2) bovine serum albumin (250 ng), (3) a-chymotrypsinogen A (175 ng), (4) ovalbumin (325 ng).
-
I 10
-*
I 20
I 30
time, min
Figure 11. (A) Microbore giradient elution separatlon of a 5 yL sample of a peptide mixture using system 111: column, 35 X 0.1 cm, Zorbax-BP-ODS, 7.5 ym; solvent A, 5% acetonitrile, 95% water, 0.1 % phosphoric acid, 10 mM KIi2P0, added; solvent B, 50% acetonitrile, 50% water, 0.1 % phosphoric acid, 10 mM Kti2P04added: gradlent, 100% B in 36 min; flow rate, 80 pL/min; detection wavelength, 10 214 nm; peak identification, see Table IV. (B)Microbore gradient elution separation of a 5 pL sample of an insulin hydrolysate. Conditions are given in Figure 11A. -+
Table IV. Precision in Retention Time and Peak Area in Microbore Gradient Elution with Static (System IIa) and Dynamic (System 111)Mixinga %RSD system Ila tg, - system I11 solute niin t, A t, A 0.38 2.50 '2.35 0.20 2.68 1 GlySer 2 AlaVal 4.08 0.51 2.90 0.59 1.92 '7.42 0.33 2.40 0.49 1.79 3 PheGly 8.92 0.42 2.31 0.48 2.16 4 TyrTyr 5 AlaPhe 110.39 0.31 3.02 0.37 1.69 6 ValAlaAlaPhe 14.98 0.22 2.27 0.30 1.17 7 GlyPhePhe 22.32 0.21 2.54 0.25 3.06 8 TrpTrp 25.37 0.26 2.10 0.22 2.34 Based on nine consecutive runs. Column: 35 X 0.1 cm, Zorbax-BP-ODs, 7.8pm. Solvent A : 5% acetonitrile/ 95% (v/v) water; 0.1% phosphoric acid, :LO mM KH,PO, added. Solvent B: 50% acetonitrile/50% (v/v) water; 0.1% phosphoric acid, :LO mM KH,PO, added. Gradient: 10 to 100% B in 36 min. Flow rate: 80 pL/min. Injection amounts: 0.2,ug except for GlyPhePhe (0.02p g ) and TrpTrp (0.03og). Injection volume: 5 pL. dient conditions studied previously (24) is thus achieved. Applications. Empliasis has been placed in the literature on the need to inject submicroliter volumes of sample into microbore columns ( 2 , 3 ) .Today, commercial switching valves are available for injecting these small Bample volumes on
microbore columns. Also, frontal analysis has been suggested as a technique to overcome the limitation of very small volumes (37). In gradient elution, however, it is possible, under proper conditions, to use conventional injection valves with loops of 5-10 pL. In this case, one takes advantage of the preconcentration step of the sample at the head of the column upon injecting into a weak solvent. This technique, wellknown in normal bore chromatography, has also been used in microbore operation (9,24). We have found the use of 5-10 p L of sample to be convenient for gradient elution microbore analysis as well. The applications we show below all use a regular injection valve and 5 yL sample volumes. Figure 11A shows the separation of the standard peptide mixture of Table IV with the dynamic mixer (system 111). At moderately sensitive attenuation, low base line noise is observed. It is noted that for this gradient which covers the range of 10-50% acetonitrile, system I would have produced large base line oscillations. Comparable peak widths to those of Figure 11A (5 yL injection) are found when the 0.5 pL injection valve was employed. Figure 11B shows a chromatogram of a bovine insulin hydrolysate with the same instrument conditions as in Figure 11A. I t is well-known that the mixing of 1-propanol and water is difficult to accomplish. Hence, as in the case of 0.1% TFA shown earlier, it can be anticipated that a gradient of the above two solvents would require efficient mixing. Figure 12 shows the room temperature separation of four proteins using the gradient conditions described in the figure caption, again with system 111. (A higher column temperature would narrow peak widths of the proteins.) Base lines with low noise levels are observed, even with a 2-fold increase in sensitivity relative to Figure 11. Finally, Figure 13 shows the separation of a series of phenols over a wide range of acetonitrile composition. Here, we have increased the sensitivity by a factor of 8 over that in Figure 11. In this case we have used system IV (dynamic mixer plus static mixer) in order to reduce the noise level to a greater extent than possible with system 111 alone. (Of course, the
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 11, SEPTEMBER 1983 2,0005AU 7
the trace enrichment step upon injecting under weak solvent conditions. In general, as noted, we would advocate the use of the micromixer alone as a reasonable trade-off between low noise level and low response and delay volumes. If a specific solvent system required more efficient mixing, it would be a simple matter to add 1 mm i.d. tubing of a short length after the dynamic mixer or a second dynamic mixer in series. As in d l aspects of chromatography, a compromise must be struck, in this case between efficiency of mixing and accuracy of the gradient.
LITERATURE CITED L
0
15
-time,
I
I
30
45
(1) Ishii, D.; Asai, K.; Hlbi, K.; Jonokuchi, T.; Nagaya, M. J . Chromatogr. 1977, 144, 157. (2) Ishii, D.; Tsuda, T.; Hibi, K.; Takeuchi T.; Nakanishi, T. J. HRC CC, High Resoiut Chromatogr Chromatogr , Commun . 1979, 2 , 2 1. (3) Scott, R.P.W.; Kucera, P. J . Chromatogr. 1979, 169, 51. (4) Scott, R.P.W. J. Chromatogr. Sci. 1980, 18, 49. (5) Kucera, P. J . Chromatogr. 1980, 198, 93. (6) Knox, J. H. J . Chromafogr. Sci. 1980, 18, 453. (7) Guiochon, G. Anal. Chem. 1981, 5 3 , 1318. (8) Novotny, M. Anal. Chem. 1981, 5 3 , 1294A. (9) Kok, W. Th.; Brinkman, U. A. Th.; Fret, R. W.; Hanekamp, H. 6.; Nooitgedacht, F.; Poppe H. J . Chromatogr. 1982, 237, 357. (10) Takeuchi, T.; Ishil, D. J . Chromatogr. 1981, 213, 25. (11) Yang, F. J. J . Chromatogr. 1982, 238, 265. (12) Scott, R . P. W.: Kucera, P.; Munroe M. J . Chromatogr. 1979, 186, 475. (13) Henlon, J. D. J . Chromatogr. Sci. 1981, 19, 57. (14) Krlen, P.; Devant, G.; Hardy M. J . Chromatogr. 1982, 257, 129. (15) Games, D. E. et ai. Biomed. Mass Spectrom. 1982, 9 , 215. (16) McGuffin, V. L.; Novotny, M. J . Chromatogr. 1982, 218, 179. (17) Taylor, L. T.; Brown R. S.; Johnson, C. C. "Book of Abstracts", 184th National Meeting of the American Chemical Society, Sept 12-17, 1982; American Chemical Society: Washington, DC, 1982 (18) Hirata, Y.; Lin, P. T.; Novotny M.; Wightman, R. M. J . Chromatogr. 1980, 181, 287. (19) Goto, M.; Koyanag, Y.; Ishli, D. J . Chromatogr. 1981, 208, 261. (20) Snyder, L. R. "High-Performance Liquld Chromatography, Advances and Perspectlves"; Horvath, Cs., Ed.; Academic Press: New York, 1980; Vol. 1. (21) Snyder, L. R.; Kirkland, J. J. "Introduction to Modern Liquid Chromatography"; Interscience: New York, 1979. (22) Martin, M.; Guiochon, G. "Instrumentation for HPLC"; Huber, J. F. K., Ed.; Eisevier: New York, 1978. (23) Biiliet, H. A. H.; Keehnen, P. D. M.; DeGaian, L. J . Chromatogr. 1979, 185, 515. (24) Scott, R. P. W.; Kucera, P. J . Chromatogr. 1979, 185, 27. (25) Sjcdahi, J.; Lundln, H.; Eriksson, R.; Ericson, J. Chromatographia 1982, 16, 325. (26) Waters Associates, Milford, MA, 1982. (27) Rivier, J. E. J . Liq. Chromatogr. 1978, 1 (3), 343. (28) Hearn, M. T. W. J . Lip. Chromatogr. 1980, 3 , 1255. (29) Waterfield, M. D.; Scrace, G. T. "Biological/Biomedicai Application of Llquid Chromatography"; Hawk, G. L., Ed; Marcel Dekker: New York, 1981; Vol. 3. (30) Bakalyar, S. R.; Henry, R. A. J . Chromatogr. 1979, 126, 327. (31) McCormick, R. M.; Karger, B. L. J . Chromatogr. 1980, 199, 259. (32) Slaats, E. H.; Markowski, W.; Fekete, J.; Poppe, H. J . Chromatogr. 1981, 207, 229. (33) Ha, N. L.; Ungvari, J.; Kovats, E. Anal. Chem. 1982, 5 4 , 2410. (34) Korpi, J.; Bidlingmeyer, B. A. Am. Lab. (FairfieiU, Conn.) 1981, 13, 110. (35) Jandera, P.; Churacek, J. Adv. Chromatogr. 1981, 19, Chapter 5. (36) Gonion, R. D.; Ettie, L. S.; Schmid, C. E.; Schwartz, A. Am. Lab. (Fairfield, Conn.) 1982, 14, 104. (37) Kucera, P.; Moros, S. A.; Mlodozeniec, A. R. J . Chromatogr. 1981, 210, 373. (38) Van der Wal, Si.; Yang, F. J. HRC CC, High Resolut. Chromatogr. Chromatogr. Common. 1983, 6 , 216.
.
rntn
Flgure 13. High sensitivity microbore gradient elutlon separation of a mixture of phenol standards using system IV: column, 35 X 0.1 cm, Zorbax-BP-ODS, 7.5 pm; solvent A, 5% acetonitrlle, 95% water, 0.1 % phosphoric acid added; solvent 8, 95% acetonitrile, 5% water, 0.1 % phosphoric acid added; gradient, 20 100% B In 80 min; flow rate, 40 pLlmln; injection volume, 5 pL; detection wavelength, 214 nm; peak identification, (1) phenol (4 ng), (2) 4-nitrophenol (19 ng), (3) 2-chlorophenol (4 ng), (4) 2,44initrophenol (1 1 ng), (5) 2-nltrophenol (4 ng), (6) 2,4dlmethylphenol (4 ng), (7) 4-chloro-3-methyiphenol (19 ng), (8) 2,4dichlorophenol(4 ng), (9) 4,6dinitr~2-methylphenol(19ng), (IO)2,4,6-trichlorophenoi (11 ng).
-
response and delay volumes for system IV are larger than those for system 111.) On the basis of the results of this paper, the dynamic mixer of Figure 2 would appear to yield a good compromise in terms of efficiency of mixing (base line noise), delay volume, and response volume. In most cases, it is believed that this mixer should be sufficient. Such a mixer with low flow rate reciprocating pumps provides a practical approach using modified current instrumentation for high-performance gradient elution chromatography with 1 mm microbore columns. If more efficient mixing is necessary, it is a simple matter to add a short piece of 1 mm tubing as a static mixer in addition to the dynamic mixer (system IV), as in Figure 13.
CONCLUSIONS This paper has shown that it is possible to modify conventional liquid chromatographic gradient systems for use in microbore gradient analysis. By means of a dynamic microvolume mixer, we have obtained accurate and reproducible high-performance gradient elution with short delay and response volumes. In addition, the efficient mixing permitted low level detection as base line noise was minimized. The results in this paper further show that by using the Waters dual reciprocating piston pumps the extent of base line noise in gradient elution for a given mixer will be a function of the wavelength and the constituents of the two solvents. Selection of mobile phase components with base line noise in mind can often prove useful. In addition, it is also shown that conventional sample valves can be used with microbore columns in gradient elution, taking advantage of
.
RECEIVED for review March 2,1983. Accepted June 9, 1983. B. L. Karger gratefully acknowledges NSF for support of this research. Contribution number 148 from the Institute of Chemical Analysis.
