1368
Anal. Chem. 1986, 58,1368-1372
Preformed Gradient Technique for Microbore High-Performance Liquid Chromatography Steve Schachterle* a n d Tom Alfredson
Varian Instrument Group, Walnut Creek Instrument Division, 2700 Mitchell Driue, Walnut Creek, California 94598
A technique Is described that allows gradients to be run at microbore flow rates uslng conventional lnstrumentatlon without the long delay times normally encountered. By preforming the gradlent at a higher flow rate and storing H In the hydraullcs, the technique allows any Iiquld chromatograph that Is capable of proportioned lsocratlc microbore operation to run mlcrobore gradlents. The chromatographic performance was tested by uslng 1-mm-1.d. columns at 50 pL/mln. Retentlon tlme relatlve standard devlatlons of up to 1% and peak area relatlve standard devlatlons of 1-3 % were obtalned. The analyses of peptldes and PTH amlno acids, two problems requiring a gradlent for separation and a microbore column for sensltivlty, were demonstrated at low concentrations.
There has been significant interest in the miniaturization of HPLC columns as a means of achieving higher sensitivity, greater detection selectivity, and reduced solvent consumption. The increased mass detectivity obtainable from microbore HPLC makes it very attractive for application to samplelimited analysis, while reduced flow rates provide the capability for interface of the HPLC with a specific detector such as a mass spectrometer or a GC detector. Yang (1) has recently reviewed developments in column technology and instrumentation for microbore HPLC. For a given linear velocity, the use of small-diameter packed columns (0.3-1.0 mm i.d.1 results in greatly reduced peak volumes and volumetric flow rates. The total volume required of gradients is also greatly reduced. Schwartz and Berry (2) recently reviewed currently available instruments for gradient elution microbore HPLC. Pumping systems that were optimized at flow rates suitable for 4.6-mm columns generally have large delay volumes which lead to prohibitively long delay times at microbore flow rates. While one solution to this problem is to reduce the delay time by removing downstream volume from the pump, this can lead to unacceptable performance (3). If the delay time can be reduced without altering the hydraulic volume (and without degrading the performance of the pump), then commercially available solvent delivery systems can be adapted for use with microbore columns to solve routine analysis problems that require small-diameter columns for higher sensitivity and gradienta for samples with a wide range of polarities. One way of separating the delay time from the delay volume is to preform the gradient a t a higher flow rate. The gradient may be formed and displaced into a holding vessel at low pressure, moving the start of the gradient to the injector. The gradient may then be delivered to the analytical column a t the operating flow rate and pressure. Preformed gradient techniques for HPLC were initially applied to normal-phase separations using conventional columns by Snyder and Saunders (4).More recently Katz and Scott (5) have applied the concept of preformed gradients to reduce the gradient delay time in high-speed HPLC separations. A 4 mm X 25 cm holding tube packed with 40-gm glass beads was employed to receive a solvent composition gradient 0003-2700/86/0358-1368$01.50/0
that was blended at 1 mL/min. The gradient was subsequently delivered to the column at high speeds (4-8 mL/min). The purpose of this report is to evaluate the application of a preformed gradient concept to gradient elution microbore HPLC using a single-piston reciprocating pump and standard hydraulics without major modification. The performance of such a system for gradient elution microbore HPLC was investigated and applications demonstrated for the analysis at low concentration of complex samples such as phenylthiohydantoin (PTH) amino acids and peptide mixtures. EXPERIMENTAL PROCEDURE Instrumentation and Columns. All chromatography was performed on a Varian Model 5500 HPLC, which employs a microstepped, single-piston reciprocating pump. Solvent blending is accomplished by high-speed proportioning valves. Figure 1 displays the system configuration used for gradient elution microbore HPLC. To the system hydraulics were added an autopurge valve (SSI, State College, PA) and a check valve (Varian Associates, Walnut Creek, CA) for use in the preformed gradient technique. No other system or hydraulic modifications were undertaken. A Rheodyne Model 7410 injection valve with 1-pL internal loop volume was used throughout this work. A Microbore-1 C18-5 column and Microbore-1 C18 IP-4 column, each 1 mm i.d. X 30 cm length, were usd for analysis of phenyl alkanone standards. A Microbore-1 C18 Protein column (1 mm i.d. x 30 cm length) was employed for analysis of peptide standards. A Microbore-1 PTHAA column (1mm i.d. X 15 cm length) was employed for analysis of PTH amino acid mixtures. All of these columns were manufactured by Vapian Associates (Walnut Creek, CA). A Varian Model UV-200 automated variable-wavelengthUV-vis detector was employed throughout this study. A 0.5-pL-volume, 2-mm-path-lengthflow cell was used for all microbore applications. Connection tubing employed in the Model 5500 HPLC was 0.005 in. i.d. stainless steel (SSI,State College, PA) for injector to column and column to detector connections. Peptides were detected at 215 nm. PTH amino acid standards were detected at 270 nm and the phenyl aklanone standards at 254 nm. Data acquisition, reduction, and automatic sampling were accomplished with a Varian Vista CDS 402 data system and 8085 Autosampler. Solvents and Chemicals. Phenyl alkanone standards (Pierce Chemical Co.) were filtered through a 0.45-pm membrane filter prior to injection. All peptide standards and the individual PTH amino acid standards were obtained from Sigma Chemical Co. (St. Louis, MO). A premixed PTH standard was obtained from Pierce Chemical Co. PTH standards were prepared in methanol, stored at -20 "C, and diluted daily to appropriate concentrations with the initial mobile phase. Ternary gradient mobile phases for HPLC of PTH amino acids included methanol and acetonitrile (Burdick and Jackson, Muskegon, MI). Buffers were prepared by using HPLC grade sodium acetate and potassium dihydrogen phosphate (Fisher, Pittsburgh, PA). HPLC grade water was generated with a Hydro Service water purification system (Varian, Sunnyvale, CA). RESULTS AND DISCUSSION Preformed Gradient Methodology. Preformed gradients differ from conventional gradients in that the gradient formation process is separate from the delivery step. The system hydraulics, shown in Figure 1, consist of the flow controller, 0 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
1369
Waste
t H draulics
0.05 ml/min
Valve
3%/min
I
Injection Valve
Event
4.3%/min
Microbore Column ( l m m ID)
uv-200 Detector
Flgure 1. Schematic of LC system configured for microbore preformed gradient operation.
Figure 3. Gradient profile as a function of flow rate: requested gradient, from 0 to 100% B In 20 mln at all flow rates; solvent A, water; solvent B, 1% acetone in water. Curves have been offset along time axis for clarity. (00%
OPEN
1OO%B
T
/
SOLVENT BLENDING
lml/rnin
2T
j
--
LLL?!?L
O%B I
0
I
I
I
I
I
1
I
I
I
I
I
I
I
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I
l
l
I
I
1
Flush HydrauliCI volume and Prm.Form O-lOO%B Gladlent
2 TIME(MIN)
3
l p ”
0% B
Flgure 4. Gradient profile at constant volume: requested gradient, from 0 to 100% B In 1.0 mL; at 1 mL/min, from 0 to 100% B in 1 mln; at 0.05 mL/mln, from 0 to 100% B In 20 min; solvent A, water; solvent E, 1% acetone In water.
TIME PROGRAM OF PARAMETERS T (MINI 0 0 1.80 1.81 2.00 2.01
lml/min) 1 .o 1.0 1.0 1.0 0.05
E
EVENTI
0 100
OPEN
100 100 100
CLOSE
-
-
DESCRIPTION
Deliver Gradient 8n 36 mm at 50111 nun
‘OPEN = FLOW TO WASTE
Figure 2. Timing diagram for microbore preformed gradient technique: delivered gradient, from 0 to 100% B In 36 mln. mixer, and damper. The approximately 2 mL of volume contained in these components is used as the volume into which the gradient is preformed and stored. During the gradient-forming step the autopurge valve (Figure 1)is open to waste to allow the gradient to fill the hydraulics at 1 mL/min and to displace the solvent previously contained. Following gradient formation, the purge valve is closed and the system pressurized by continuing to pump at 1mL/min for a short time. The pump is then slowed to the delivery flow rate prior to injection. The gradient, contained in a small volume, is displaced through the column by solvent of the final composition until all peaks have eluted. At this point the column may be reequilibrated to the initial solvent composition for the next run by reopening the autopurge valve and pumping the initial solvent composition at a high flow rate. The purge valve is closed and the system pressurized as before. The solvent is maintained at the initial composition until the column has been equilibrated. The timing diagram in Figure 2 shows the relationship between flow, solvent composition, and relay programming. By use of air-actuated purge and injector valves this entire procedure was automated via the program-controlled relays.
Both binary and ternary gradients developed for larger bore columns can be readily transposed to run on microbore columns. To achieve the same linear velocity, theory predicts that the flow rate must be reduced by the squared ratio of the column inner diameters, all other factors being held constant. To change from a 4.6-mm4.d. column to a 1-mm4.d. column requires a reduction in the flow rate of approximately 21. For similar analysis times, similar gradient slopes can be used, and the total gradient volume will be reduced by this same factor of 21. The time to preform each segment is then the desired gradient volume divided by the preforming flow rate. For example, a 20-min gradient at 50 pL/min has a total volume of 1mL and requires a 1-min gradient preformed at 1 mL/min. Preformed Gradient Technique Performance. Figure 3 shows the performance of the system at three flow rates. For all three profiles a gradient from 0 to 100% in 20 min was requested. Keeping the gradient slope constant while changing the flow rate resulted in a different total volume for each gradient. At 1 mL/min the requested slope of 5%/min is achieved. At the lower flow rates, however, the gradient slope is lower. These lower slopes are caused by the increased relative effect that the volume of the hydraulics has on the gradient. The 2.2-mL delay volume of the hydraulics has very little effect on the 20-mL gradient (1 mL/min) but has an observable effect on the 2-mL gradient (0.1 mL/min) and an even more substantial effect on the 1-mL gradient (0.05 mL/min). Quarry et al. have defined parameters that allow the effect of the hydraulics on the gradient shape to be quantitated (7).They express the two volumes as a ratio. At 1mL/min the gradient volume is 20 mL and the ratio of the hydraulics dispersion variance, V,, to the gradient volume, V,, is 1.5 mL/20 mL or 0.08. A t 0.05 mL/min the ratio
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
Table I. Reproducibility of Peak Retention Times and Areas for an Automated Preformed Gradient peak no.
compd acetophenone 1-phenyl-1-propanone 1-phenyl-1-butanone 1-phenyl-1-pentanone 1-phenyl-1-hexanone 1-phenyl-1-octanone 1-phenyl-1-decanone 1-phenyl-1-dodecanone 1-phenyl-1-tetradecanone
" Relative standard deviation, N
=
tR
(mean), min
% RSD"
area (mean), pV s
% RSD"
0.57 0.48 0.59 0.86 1.15
651 577 524 079 576 819 503 449
1.38 1.30
371 803
1.55
1.13 0.82
351 774
2.49
0.53
314 867 269 573
0.38
258 853
2.09 2.06 1.93
7.008 9.086
11.715 15.362 19.902 29.774 42.685 56.206 72.054
1.53 1.36
18.
becomes 1.5. The curves in Figure 3 show that the larger this ratio is, the more pronounced is the smoothing effect of the hydraulics on the gradient. Figure 4 shows that this is indeed a volume effect and independent of flow rate. Both gradients, when plotted vs. volume, exhibit the same shape after passing through the same hydraulics volume in spite of the 20-fold difference in flow rates. The effect of the hydraulics volume on the gradient is due nearly entirely to the volume of the gradient and the amount of associated mixing. The speed at which the gradient is pumped through the hydraulics has a very small effect due to the low diffusion rates found in liquids. Because the residence time of the gradient in the system hydraulics had no measurable effect on gradient shape, no holding column was used in this work. The suitability of a given HPLC system for running preformed gradients in the manner described here can be predicted by calculating the ratio of VM/V, and comparing this value to the figures in ref 7. If the volume of the hydraulics is too large in comparison to the microbore gradient volumes that will be used, then the dispersion of the gradient as it moves through the hydraulics will probably render the slope of the gradient too low to be useful. It should be noted that an HPLC system does not need to have any minimum volume in order to be useful for running preformed gradients, since it is not a requirement that the entire gradient be stored in the hydraulic volume of the pump. As most HPLC pumping systems are capable of producing solvent gradients as a function of time, a gradient could easily be programmed in two segments: the first segment would be preformed as described above to fill the hydraulics volume, and the second segment would be formed a t the delivery flow rate as it followed the first segment through the hydraulics. The major purpose of gradient preforming is to move the start of the gradient rapidly through the hydraulics and thus reduce the delay time. If properly calculated, the two gradient segments would, together, produce the desired gradient shape, because they would both pass through the same hydraulics volume (Figure 4). As an alternative to the two-stage gradient program, a holding column of sufficient volume could be added to the pump hydraulics to simplify programming of the gradient. Automated preformed gradients give reproducible peak retention times and areas. A mixture of nine phenyl alkanone standards was injected onto a Microbore-1 C18 IP-4 column using the system shown in Figure 1. A preformed linear gradient from 55 to 100% acetonitrile in 32 rnin a t 50 pL/min was.used for analysis. Results for 18 consecutive runs are summarized in Table I. The
