Anal. Chem. 1988, 60,792-797
792
A characteristic ion at m l z 246 differentiates ortho,para’ and para,para’ isomers. The diphenylethanols give characteristic ions at mlz 250 and 338 for 4-chlorophenyl- and 4-bromophenyl-substituted compounds, respectively. Registry NO,1,72-55-9;2,3424-82-6;3, 1022-22-6;4,5432-01-9; 5 , 50-29-3; 6, 789-02-6; 7, 72-54-8; 8, 53-19-0; 9, 3563-45-9; 10, 72-43-5; 11,72-56-0; 12, 361-07-9; 13, 789-03-7; 14, 115-32-2; 15, 630-71-7; 16,510-15-6; 17, 18181-80-1; 18, 90-97-1; 19, 365-24-2; 20,90-98-2; 21,9262-4; 22, 530-48-3;23,119-61-9; 24, 2175-90-8; methane, 74-82-8.
(3) Safe, S.;Hutzinger, 0. Mass Spectrometry of Pesticides and Pollutants; CRC Press: Cleveland, OH, 1973; pp 113-121. (4) Dougherty, R. C. Anal. Chem. 1981, 5 3 , 625A-636A. (5) Dougherty. R. C.; Roberts, J. D.; Biros, F. J. Anal. Chem. 1975, 47, 54-59. (6) Stemmier, E. A.; Hites, R. A. Anal. Chem. 1985, 5 7 , 684-692. (7) Jensen, T. E.; Kaminsky, R.; McVeety, B. D.; Wozniak, T. J.; Hites, R. A. Anal. Chem. 1982, 5 4 , 2388-2390. (8) Lowry, T. H.; Richardson, K. S. Mechanism and Theory in Organic Chemistry; Harper and Row: New York, 1981. (9) Forrest, J.; Oliver, S.; Waters, W. A. J. Chem. SOC. 1946, 333-339. (10) Stray, H.; Mano, S.; Mikaisen, A.; Oehme, M. HRC CC, J . High Resoiut. Chromafogr. Chromatogr. Common. 1984, 7 , 74-82.
LITERATURE CITED
RECEIVED for review May 20, 1987. Accepted December 17,
(1) Jaffe, R.; Stemmier, E. A.; Eitzer. B. D.; Hites, R. A. J. Great Lakes Res. 1985, 1 7 , 156-166. (2) Rapaport, R. A.; Urban, N. R.; Capel, p. D.; Baker, J. E.; Looney, 8. B.; Elsenreich, S. J.; Gorham, E. Chemosphere 1985, 14. 1167-1173.
1987. This work was supported by the V.S. Department of Energy (Grant No. 80EV-10449) and the U.S. Environmental Protection Agency (Grant No. R808865).
Behavior of Liposomes in Flow Injection Systems Laurie Locascio-Brown,* Anne L. Plant, and Richard A. Durst Center for Analytical Chemistry, National Bureau of Standards, Gaithersburg, Maryland 20899
We have examlned the hydrodynamic behavlor and stablllty of llposomes In flow InJectIonanalysls, In order to evaluate thelr usefulness as analytlcal reagents In contlnuous flow systems. UpoaKHnes can be prepared wlth large numbers of water-soluble detectable or reactlve molecules, Le., fluorescent, electroactlve, or enrymatlc molecules, trapped In an aqueous center, and thus have the potential for providing tremendous M a l enhancement In many assay formats. We have found that liposomes contalnlng the fluorescent selfquenching dye, carboxyfluoresceln, are stable In the flow system and do not release thek trapped contents even at flow rates of 2 mL mln-‘. These structures are approxlmately 0.1 pm In diameter and showed flow profile behavior which Is quite dlfferent from solution (small molecule) behavlor in straight open tubing. Dmerences in concentration profiles for solution and liposome samples have been examined under condltlons of Induced radlai mbtlng In a knitted delay tube and a packed bead column. No aspects of ilposome behavlor have been observed that are not explalned by their small diffusJon coefflclent. Under condltlons of approprlate assay formats, Hpomnes wlll be hnpottant slgnai enhancement tools for flow Inlectlon analysls.
Flow injection analysis (FIA) has many advantages in analytical chemistry ( I ) . Besides providing on-line, automated reaction systems, FIA is a versatile technique that is being exploited for many diverse applications including biomedical applications such as soluble enzyme activity measurements (2), macromolecular affinity constant determinations (3),and immunoreactions (4). A review of current applications of FIA in clinical chemistry is provided by Linares et al. (5). One practical difficulty in clinical diagnostics employing FIA systems has been associated with the nonideal hydrodynamic behavior of red blood cells (6). The present work addresses the behavior of macromolecular assemblies and heterogeneous samples in FIA and hopefully will enhance the usefulness of FIA in applications involving biological samples. We are studying the use of phospholipid vesicles, or liposomes, in FIA. Liposomes are spherical structures that form
spontaneously when phospholipid molecules are dispersed in an aqueous solution. The bilayer membranes of liposomes are similar to cellular membranes and surround an entrapped aqueous volume. Liposomes are similar to red blood cells in this way, but they are at least an order of magnitude smaller. Liposomes can be prepared with as many as 1 X lo5watersoluble molecules trapped inside of their enclosed volume. These structures have great potential for application in drug delivery (7) and are also attractive for use in diagnostic and other assay systems where they provide signal enhancement in the form of the many molecules that can be released from them for detection. Other possible uses of liposomes in FIA systems include measurement of activity of membrane-associated proteins or quantification of phospholipid esterase activity. Memon and Worsfold (8)have used microemulsions of micelles (lipid structures that do not contain an aqueous center) for signal enhancement in fluorescence FIA. In addition, micelles have been used to quantify Tb(II1) in FIA (9), and bilayer vesicles have been used in a FIA method for cyanide analysis (IO). Our objective is to use liposomes containing a marker compound as analytical tools for signal enhancement in an immunochemical-based FIA system. The interaction between an antibody and antigen is converted to an optically detectable signal through an encapsulated marker which is released from the liposome cavity by lysis with detergent. A single binding event may, therefore, be amplified by a factor of lo5. Our goal is to study the feasibility of performing a competitive assay, although both competitive and noncompetitive assay formats can be used. Successful application of liposomes in competitive reactions requires that liposomes and solution species have identical and reproducible flow behavior so that liposomes and soluble analytes are uniformly mixed within the reacting volume. The criteria for development of a noncompetitive reaction scheme are less rigorous; however, the competitive reaction will not involve a prereactor incubation step thus resulting in a faster sample throughput time. In this work, we examine and compare the behavior of liposomes in various FIA system configurations so that appropriate liposome assay formats and system designs can be developed. We have examined the mixing behavior of liposomes and solution in the absence of chemical interactions on an unmodified,
This article not subject to U.S. Copyright. Published 1988 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 8, APRIL 15, 1988
packed bead column, in open straight tubing and in knitted delay tubing.
EXPERIMENTAL SECTION Apparatus. The flow system was designed from the valve scheme described by Harrow et al. (11). A microprocessor was used to control the opening and closing of solenoid pinch valves (Automatic Switch Co., Florham Park, NJ). These valves and the tubing used are rated to pressures of up to 2.1 bar, suitable for use with FIA. The sequence and timing of events were microprocewor-controlled,thereby ensuring precise and reproducible injections. The rate of flow was varied with a four-channel peristaltic pump that was placed at the outlet of the system. In this configuration, the system could accommodate several additional reagent lines without the need for a larger pump. All solutions were degassed for 15 min under low vacuum while stirring. Reagent bottles were pressurized with nitrogen to ensure that no air was aspirated into the system at the connections. Samples were aspirated into the sample loop by using a vacuum with a negative pressure of 0.03 bar. Tubing was 0.5 mm i.d. Teflon with interconnections through the valves of 1.56 mm (1/16 in.) i.d. sterile silicon tubing. The knitted delay tube used in some experiments was prepared from a 152.4-cm piece of Teflon tubing (12). Tees and crosses were made of Kevlar with Altex fittings. The reactor used in some of the experiments was a glass column 2 mm i.d. and 9.9 cm in length and packed with nonporous soda lime glass beads (Cataphote, Jackson, MS). The beads used were approximately 210-250 pm in diameter (60-70 mesh) and were slurry packed into the column with no prewashing. The void volume of this column was calculated to be 124 pL. For most experiments, the volume flow rate was 0.5 mL m i d , providing a Reynolds number of 0.28. AU experiments were performed by using fluorescence detection. Peak profiles of buffered solutions (0.02 M Tris, 0.15 M NaC1, 0.01% NaN3, pH 8.0) containing 5- (and 6)-carboxyfluorescein (CF, 1 or 2 pM) (Molecular Probes, Junction City, OR) or liposomes containing CF were determined by monitoring CF fluorescence over time. Samples of CF solution and CF liposomes were injected separately. Samples of buffer-containingliposomes were diluted with CF solution. The fluorescence flow cell (p/n 8830, NSG Precision Cells, Inc., Farmingdale, NY) had a volume of approximately 20 pL with dimensions of 1 mm X 1mm X 21 mm. The incident light beam contacted approximately 25% of the total area of the cuvette,thereby exciting 5 pL of sample. This is important in flow injection schemes where the sample size is typically between 50 and 500 pL. In these experiments, the sample volume was 187 pL. A monochromator was used to select an excitation wavelength of 450 nm, and a 515-nm long-pass filter was used to isolate the emission. Data were collected from the fluorometer at a rate of 1 s-'. Data Manipulation. Peak heights and peak asymmetries were calculated by interpolation of the data using a cubic spline program which was written in BASIC. Peak asymmetry (PA) was calculated according to Snyder and Kirkland (13) using the following expression: PA = BO.l/AO.l where Ao,l and Bo,lare, respectively, the number of seconds before and after the peak maximum at which the intensity is 10% of the maximum peak height. Dispersion was calculated as follows:
D = c,/cm, where C, is the original sample fluorescence intensity and, ,C is the maximum intensity of the peak height. Peak profie overlaps were calculated from fluorescence intensity data by comparing integrated intensities for areas common to peaks with integrated intensities for common areas plus areas not common to both peaks. Data collected on at least two different days were compiled for computation of means and standard deviations. Preparation and Characterization of Liposomes. Liposomes were prepared by the injection method (14) from a stock mixture of dimyristoylphosphatidylcholine (Avanti Polar Lipids, Birmingham, AL), cholesterol, and dicetyl phosphate (Sigma Chemicals, St. Louis, MO) in a molar ratio of 5:4:1. Two micromoles of the stock lipid mixture in chloroform was dried under a stream of nitrogen in a small test tube, and residual solvent was
793
FLUORESCENCE DETECTOR ERlSTALTlC PUMP
BUFFER
SAMPLE VACUUM
WATER
-1
WASTE
Flgure 1. Schematic of FIA system with all system components connected. Data for Figure 2A were collected with the system of this configuration. Other data were collected separately for the individual components.
removed by vacuum desiccation for at least 24 h. Dried lipid was solubilized in 50 pL of 2-propanoland the entire volume was slowly injected with a 100-pLsyringe into 1mL of rapidly stirred Tris buffer (pH 8) containing CF at concentrations of either 3 or 25 mmol/L. Liposomes containing entrapped dye were separated from free dye by gel filtration on Sephadex G50 (15). Liposome size was calculated from diffusion coefficients which were measured by autocorrelation of scattered laser light with equipment described by Lodge et al. (16). Data were analyzed by the cumulants method (17). Liposomes used in these experiments were determined to have an average diffusion coefficient of 5 X cm2 s-l, an average diameter of 0.1 pm, and a polydispersity of 0.1, indicating a size distribution of about f0.5% (18). Negative staining electron microscopy showed that there were between 1 and 5 lamellae per liposome. These liposomes remain suspended in solution and cannot be centrifuged easily to form a sediment. Their net negative charge due to the dicetyl phosphate prevents aggregation. Light scattering data show no evidence of change in liposome size after storage at room temperature for several months.
RESULTS AND DISCUSSION Since we are evaluating the feasibility of using liposomes in a competitive reaction mode, we are particularly concerned with the similarity of flow profiles of small molecules and liposomes in solution. The concentration profiles have been analyzed by three criteria: peak asymmetry, peak dispersion, and residence time. We have examined these characteristics in the various components of our FIA system: a 187-pL (95 cm of 0.5 mm i.d. tubing) sample loop, a packed bead column, a knitted delay tube, and straight open bore tubing, as well as in the combination of these components. A schematic of our FIA system is shown in Figure 1. Figure 2A shows representative peak profiles of separate injections of CF solution and liposomes after flow through the column, knitted tubing, and 40.5 cm of straight connecting tubing arranged in series, a t a flow rate of 0.5 mL min-l. Fluorescence intensity is monitored as a function of time. In this system configuration, the coefficient of variation of peak heights measured on the same day is 1% for CF solution and less than 3% for liposome samples. The error is larger if the samples are not degassed prior to injection. This error is caused by the release of bubbles in the detectcr flow cell caused either by flow into the larger diameter tube which induces a significant pressure drop or by heat introduced by the light source in the chamber where the flow cell is housed. Data from experiments performed on the entire system as shown in Figure 1 were compiled to calculate parameter means. In this configuration, the CF solution showed a residence time of 59.3 & 4.0 s, a dispersion of 1.69 f 0.03, and a peak symmetry of 2.12 f 0.14. Liposomes, on the other hand, have a shorter residence time (52.8 f 3.1 s), greater dispersion (1.85 f 0.10), and poorer symmetry (2.95 0.37), indicating that these FIA components do not cause sufficient mixing of the liposomes or CF solution to produce identical profiles. From theory (12,19,20),it is expected that the component that makes the greatest contribution to profile differences between solution and liposomes is straight open tubing. The large difference in the diffusion coefficients of CF molecules
*
794
ANALYTICAL CHEMISTRY, VOL. 60, NO. 8, APRIL 15, 1988
---
LIPOSOMES
--8 4 4 6-
1
t m z
W
t-
z UI
C) 1u
u m w oc
0
0 ' 2'0
60 80 ' 100' 120 1 4 0 T 1 % 0 TUBING LENGTH (CM) Flgure 3. Peak asymmetries for liposomes, H, and solution, , were calculated as descrlbed in the text.
3
U. _1
W
P k-
5W
40
U
11l.lC
(sccotrus)
Flguro 2. Flow profiles were determined by monitoring fluorescence intensity with time in the flow-through cuvette. CF was elther solubked In aqueous buffered solution (-) or was trapped inside liposomes (- - -). (A) The system was configured as depicted in Figure 1 using a straight sampie loop. (B) Samples were injected from a knitted injection loop dlrectly to the detector.
(approximately5 X lo4 cm2s-l based on an estimation of the cm2 s-l) is size of the dye molecule) and liposomes (5 x expected to have radical effects on both radial and axial dispersion. The magnitude of these effects was examined with a series of experiments in which the packed column and mixing coil were removed and only straight tubing of various lengths was used. Under these conditions, characteristics of the system could be accurately calculated. Tubing lengths ranged from 5.8 to 175 cm. For each length, the dimensionless time unit, 7,was calculated from the expression r
= tD,/R2
where t is time and is calculated from tube length (in centimeters) divided by linear velocity (in centimetersper second), D, is the diffusion coefficient of the species, and R is tubing radius. This term was introduced for the numerical solution of the convection-diffusion equation by Anathrikrishnan et al. (21),where it has been shown that 7 must be at least 0.8 before radial diffusion is sufficient to produce a homogeneous mixing within each element of the sample zone solution. for liposomes Values for 7 ranged from 1.1 X lo4 to 3.3 X and from 0.01 to 0.33 for CF in solution; therefore, neither preparation is expected to show purely Gaussian profiles. As will be shown, CF solution profiles are nearly Gaussian at the longer tubing lengths, while liposome profiles are less Gaussian as predicted. Tube Length Effects. For lengths of straight tubing, the following expression applies as derived by Ruzicka and Hansen (22): D = kL'I2 where k is a constant which is inversely dependent on the molecular diffusion coefficient and L is tube length in centimeters. Plots of D 2vs tubing length are linear for both samples with correlation coefficients of 0.99 and 0.96 and slopes of 0.0065 and 0.0038 for CF solution and liposomes, respectively. Dispersion of both solution and liposomes increases with increasing tubing length, but the slope of the liposome data is smaller indicating a smaller effect of tubing length on the liposome profile. A t short tubing lengths, dispersion of liposomes is slightly greater than dispersion of
solution, but at longer lengths dispersion is less for liposomes than for solution. Residence times are shorter for liposomes than for solution except in very short tubing, and the difference in residence times becomes more significant with increasing tubing length. Plots of residence time versus tubing length have correlation coefficients of 0.99 for both CF solution and CF liposomes and slopes of 0.15 and 0.12 for solution and liposomes, respectively. Figure 3 shows the effect of tubing length on peak asymmetry of solution and liposome profiles. Initially, peak asymmetry increases for both samples up to about 61 cm. *ith longer lengths, solution profiles become more symmetric, but peak asymmetry of the liposome profiles continues to be large. These data can be explained by the difference in diffusion coefficients of liposomes and CF molecules. Theory predicts that a small molecular diffusion coefficient inhibits both axial and radial diffusion. This is described in the following expression for the determination of theoretical plate height, H, which is defined as the length of one well-stirred mixing stage in straight open tubing according to Tijssen (23):
H
= (2DJu)
+ (2KR2u/D,)
where u is linear velocity, K is a velocity profile factor and is equal to 1/48 in straight tubing (23), and D, is the radial diffusion coefficient and is equal to D, when considering straight tubing. The first term on the right side of the equation represents axial molecular diffusion; because of the difference in molecular diffusion coefficients, axial diffusion is 2 orders of magnitude larger for CF solution molecules than for liposomes. The relative magnitude of this term is small, however, and mixing is adequately described by the second term. The second term is the combined effect of the stratified velocities of carrier in the tubing (which causes dispersion) and the counteractive force of radial diffusion in the carrier phase (24,25). Because of the inverse relationship with the molecular diffusion coefficient, this term is 2 orders of magnitude smaller for the dye molecules than for liposomes and predicts significantly more mixing of the dye solutions with carrier. Plate height in straight tubing calculated by this method is 22 cm for solution and 2209 cm for liposomes. Thus, theory predicts that in long lengths of tubing, flow profiles of solution, but not of liposomes, should reflect a high degree of mixing. This prediction is confirmed experimentally. Dispersion of solution as a function of tubing length is much larger than that of liposomes and increases as a function of residence time, indicating more mixing of solution with carrier. Also, the shorter residence time for liposomes in straight tubing reflects a much slower rate of radial dispersion relative to solution. Because the rate of radial dispersion of liposomes is slow, and T is always less than 0.01, convection is the primary influence
ANALYTICAL CHEMISTRY, VOL. 80. NO. 8. APRIL 15. 1988 195
fi
sampls LODO
open ,me 23rm
OOrn
nm
175sm
LIPOSOMES rn SOLUTIONS 0
K"L1Ld
1
LIPOSOMES SOLUTION
SlrnDI.
COlYrn"
LOOP
Tme
Flaw 4. Effect of indMdual FIA system components on sample peak asymmetries was tested using a minimum of connecting tubing. A 95-cm sample iwp was used in all wnflguratims. followed by 5.3 cm of pecomponent connecting tubing associated with a "T", and 5.8 cm of postcomponent crmnecting tubing to the cwene. The knmed tubing hclujed an addinonai 1.1 cm of sbabht tubing and me ccdumn required an additional 3.9 cm of connecting tubing. on liposome concentration profiles (26). The slope of the profiles that we see for liposomes is accurately described by the mathematical reconstruction performed by Vanderslice (26): liposome concentration rises sharply and then falls exponentially. The fact that peak asymmetry of solution decreases with large tubing lengths indicates that radial redistribution is significant in the solution profile where values for r approach 0.8, the value a t which radial redistribution in straight tubing is complete (21). A t the same time, peak asymmetry of liposomes is unaffected which c o n i i i the small rate of radial diffusion of liposomes. According to theory developed by Taylor for flow of solutions in open-bore tubing (27), molecular diffusion is the only cause of radial diffusion in laminar flow conditions. If the residence time of the solution, t, is such that
t , 2 R2/3.S2D, then the molecular diffusion between velocity strata is fast enough for significant radial dispersion to occur. This condition is usually met in FIA where the radius of tubing is typically 0.5 mm and molecules =e small. From the diffusion coefficients of CF in solution and of liposomes, the residence times in the system must be greater than 8.6 and 865 s, respectively, for radial diffusion to be significant. The experimental residence times in 100 cm of tubing are 38 s for solution and 32 s for liposomes, indicating that in the experiments using open-bore tubing, the solution, but not liposomes, has achieved sufficient radial redistribution to approach an averaging of velocities within the velocity profile. Influence of Knitted Delay Tubing on Flow Profiles. Increasing radial mixing by replacing straight tubing with knitted tubing of the same length reduces dispersion of CF solution by about 45%. The knitted tubing does not have as dramatic an effect on liposome dispersion. Dispersion of liposomes in the knitted tubing decreases by 23%, which suggests that radial convection of liposomes is less stimulated by centrifugal flow induced in the knitted tubing. For solution, peak asymmetry in the knitted tubing was not significantly different from open tubing of the same length (Figure 4). This is not surprising since solution profiles were expected to have approached radial equilibration in long lengths of tubing. Residence time decreased from 38 to 30.6 s in the knitted tubing, as shown in Figure 5. The residence time for liposomes is less in the knitted tube compared to straight tubing (from 32.4 to 28.4) but was accompanied by a significant reduction in peak asymmetry (from 4.15 to 3.22). Thus the knitted tube does effect mixing of liposomes, but to a smaller
OOC" Tube
235.
OD*" Tubs 175Frn
Xn,llee T"bB
Co8"mn
F p r e 5. Conditions for examining sample retention times in the various system components were as described for Figure 4.
Table I. Peak Overlap and Numbers of Theoretical Plates in System Components componenta
% peak overlap
straight tubing 3.0 cm 3.0b cm 28.1 cm 37.1 cm 48.1 cm 58.1 em 105.6 em
knitted tubing column
76 88 71 63 64 71 58 71 78
no. of theoretical plates' liposomes solution
0.9b
1.3d
l.ld 2.3d 3.1' 2.gd
2.7d
5.7' 7.3' 5.0'
#Sampleloop was 95 cm of straight tubing. 'Liposome sample loop was 95 cm of knitted tubing. 'Theoretical plates were calculated according to Foley et sl. (1983) for modified Gaussian peaks. * 1% error associated with N due to 1.00 < PA < 2.76. *lo%error associated with N due to 2.77 < P A < 4.00. extent than its effect on solution. The knitted tube significantly improves overlap of liposomes and solution peaks compared to open tubing as shown in Table I. In the knitted tubing, overlap of the two samples is 71% compared to 58% for long lengths of straight tubing. That the knitted tubing is less effective at inducing mixing of the liposomes appears to be the result of the small diffusion coefficient for liposomes and is predicted by the theoretical relations for radial dispersion in open helical tubing as presented by Tijssen (25). In theory, radial dispersion, D,, can be separated into diffusive and convective components where radial convection is the result of redirected flow in a helix and is described as secondary flow. Secondary flow, or D., contributes to radial dispersion by D,= D, f D, In helical coils, theory has been developed which describes the relationship between the extent of secondary flow and velocity. A velocity parameter, DeSe2, is inversely proportional to D , but allows segregation of data into groupings within which empirically derived inverse relationships between D, and Dmexist. No such theory has been developed for knitted tubing, but we have calculated the numher of theoretical plates based on the theoretical treatment for exponentially modified Gaussian profiles as described by Foley and Dorsey (28)
N
= 41.7(tR/W0J2/PA f 1.25
Although this treatment has been developed for use in HPLC with small sample volumes, our data tested favorably against their criteria for modified Gaussian peaks (29). The results are shown in Table I. There is a significant difference in theoretical plate heights for CF solution and liposomes in long
796
9
ANALYTICAL CHEMISTRY, VOL. 60, NO. 8, APRIL 15, 1988
lengths of open tubing as compared to the knitted tubing. The knitted tubing increased the number of theoretical plates for liposomes to 3.1 compared to 2.3 in straight tubing, and for solution it increased the number of theoretical plates to 7.3 from 5.7. Influence of the Packed Bead Reactor on Peak Profiles. We also examined the packed bead column for mixing effectiveness by comparing flow profiles of liposomes compared to CF solution. The effect of packed bead columns on inducing mixing in FIA systems is well established (20). Figures 4 and 5 compare the flow profile data for liposomes and solution in the various system components. There is no significant difference in dispersion of liposomes (approximately 1.34) or solution (approximately 1.24) in the knitted tubing as compared to the column; peak asymmetry, however, is noticeably affected in both samples. In the case of liposomes, peak asymmetry is somewhat lower in the column (2.90)than in the knitted tubing (3.14), indicating better radial mixing. The knitted tubing has the opposite effect on peak asymmetry for solution, and this effect contributes to the similarity between solution and liposome profiles through the column. Although the number of theoretical plates for liposomes in the column is calculated to be slightly less than in the knitted tubing, the column has a greater effect on mixing of liposomes than it has on solution, and as a result, profile overlap of solution and liposomes on the column is increased to 78% (Table I). This overlap is greater than that resulting from only 3 cm of connecting tubing indicating that differences develop in the 95-cm sample loop prior to flow into the downstream components. Experiments were performed to try to minimize this effect by knitting the sample loop. Influence of Knitted Sample Loop. A simple approach to reducing profile differences is to knit the liposome sample loop to induce radial mixing and to use a straight sample loop for injection of CF solution to discourage radial mixing. Experiments were performed in which the sample loop was either knitted or straight for both CF solution and liposomes in system components. The results are shown in Figure 2B. From the figure, it can be seen that knitting the injection loop induces enough radial mixing of liposomes to result in a substantial increase in similarity between liposome and solution profiles. As shown in Table I, when the knitted sample loop was used for liposome injection, the peak overlap increased to 88% as compared to the 76% overlap seen when only straight injection loops were used. It is interesting to note that when a knitted sample loop is used for injection of CF solution, dispersion is 1.0, indicating that the flow cell design is not encouraging dilution of the sample zone. The dispersion which is due to the straight sample loop alone is 1.1and is a noticeable effect. Liposome Stability in the FIA System. Stability of liposomes to disruption and leakage of contents was also examined. For these experiments, we prepared liposomes containing 25 mM CF, at which concentrationthe fluorescence of CF is partially quenched (30). Entire fluorescent peaks were collected after flowing liposomes through the system. Leakage was determined by measuring the fluorescence of the sample in the absence and then in the presence of 1-0-n-octyl-P-Dglucopyranoside, a detergent. The detergent disrupts the liposomes, which results in maximum fluorescence intensity, and the ratio of initial liposome fluorescence to maximum fluorescence is used to determine the remaining intraliposomal concentration of CF. No leakage from liposomes was found to occur in the glass bead column, or in the Teflon tubing, even at flow rates of 2 mL mi&. Under conditions where the system had been previously washed with very large volumes of buffer, or was allowed to contain buffer solution for several days, the first three liposome injections resulted in detectable
leakage of dye which decreased with each injection, after which no more leakage was detected. It appears that relatively small amounts of liposomal lipid are necessary to saturate the hydrophobic sites in the system, and the subsequent slow rate of phospholipid transfer results in this saturation being a relatively stable condition. Effect of Liposomes on Solution Flow Profiles. The addition of liposomes to a solution of CF did not alter the flow profile of the solution. This was tested in the straight tubing by mixing liposomes prepared in the absence of CF (and therefore invisible to the fluorescence detector) with CF solution. Other investigators, studying the behavior of red blood cells in flow injection systems, found that the heterogeneous nature of red blood cells causes unpredictable mixing in the flow streams resulting in unresolved dilutional errors in the sample (6). We observe no such effect from liposomes. This is explained by the differences in size and shape of liposomes and red blood cells. It has been pointed out that red blood cells are effective mixers for the exchange of blood gases and that the discus shape of these structures encourages this mixing. Liposomes, on the other hand, are spherical structures. Also, red blood cells are much larger than liposomes. Typically, red blood cells are about 7.5 pm in diameter and 2 km thick compared to liposomes which are approximately 0.1 pm in diameter. Even though liposomes in aqueous solution constitute a heterogeneousmixture, liposome behavior more closely resembles solution behavior, and the results are predictable and reproducible.
CONCLUSION The theory of concentrationprofile development in FIA has been largely based on the study of small molecules. It has been stressed that molecular diffusion coefficients have a very pronounced effect on radial and axial dispersion. This effect has been applied in FIA for the determination of diffusion coefficients of small molecules (31),and for the determination of molecular weights of larger, biologically important molecules (32). For our application, we are reducing the effect of differences in diffusion coefficients to maximize profile similarities of liposomes and small molecules. This is required for the development of an unbiased competitive assay. Although liposomes are composed of large numbers of lipid molecules and can range in size from tens to thousands of nanometers in diameter, the caveats associated with their flow behavior can also be anticipated for large enzymes which can have diffusion coefficients 10 times slower than their substrates. As demonstrated by Vanderslice (26),a &fold difference in diffusion coefficients is enough to produce radically different boluses, and for many types of analyses, this mixing problem can lead to serious experimental errors. Studying the hydrodynamic behavior of liposomes in FIA is also important since they are good models for potential FIA analytes and reagents such as virus particles and micelles. Since the difference in concentration profiles of liposomes and small molecules is due to the differences in radial diffusion, inducing sufficient radial mixing of liposomes should cause the profiles to overlap more efficiently. The system components that we chose for stimulating mixing were not effective enough to overcome the profile differences which were established in the sample loop. We found that these differences could be nearly eliminated by knitting the sample loop for the liposomes. This provides some encouragement for the development of a simple precolumn merging zones configuration for competition reactions.
CF C,,
GLOSSARY 5- (and 6)-carboxyfluorescein maximum concentration of the sample by the detector
Anal. Chem. 1988, 60, 797-001
co
D
De De-
original concentration of the sample dispersion coefficient, C,/C,, Dean number, Re/X1/2 velocity parameter
sc2
Dm D, D, PA R Re
sc tr 7
W0.1
x K
H U
molecular diffusion coefficient radial dispersion secondary radial dispersion peak asymmetry radius of open tube Reynolds’ number Schmidt number, p / p D m residence time dimensionless time unit, tDm/R2 width of peak at 10% of peak maximum ratio of helix diameter to tube diameter velocity profile factor theoretical plate height linear flow velocity
LITERATURE CITED Ruzlcka, J.; Hansen, E. H. Anal. Chlm. Acta 1988, 779, 1-58. Masoom, M.; Worsfold. P. J. Anal. Chim. Acta 1988, 779, 217-223. Mlller, J. N.; Abdullahl, G. L.; Sturley, H. N.; Gosslan, V.; McCluskey, P. Anal. Chlm. Acta 1988, 179, 81-90, (4) Worsfold, P. J.; Hughes, A.; Mowthorpe, D. J. Analyst (London) 1985, 710, 1303-1305. (5) Linares, P.; Luaue du Castro, M. D.; Valcarcel, M. Rev. Anal. Chem. 1985, 8(3),229-257. Harrow, J. J.; Janata, J. Anal. Chim. Acta 1985, 774, 115-122. Poste, G.; Kirsh, R.; Koestler, T. I n Llposome Technology;Gregoriadls, G., Ed.; CRC Press: Boca Raton, FL, 1984; Vol. 3, Chapter 1. Memon, M. H.; Worsfold, P. J. Anal. Chlm. Acta 1986, 783,179-185. Alhara, M.; Aral. M.; Tomltsugu, T. Anal. Lett. 1988, 19(19&20), 1907- 19 16. Ishll, M.; Yamada, M.; Suzuki, S. Anal. Lett. 1988, 79(15&16), 1591-1601.
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(11) Harrow, J. J.; Janata, J. Anal. Chem. 1983, 55, 2461-2464. (12) Engelhardt, H.; Neue, U. D. Chromotographle 1982, 15(7), 403-408. (13) Snyder, L. R.; Klrkland, J. J. Introduction to Modern LiquM Chromatography; Wlley: New York, 1981; p 222. (14) Batzrl, S.; Korn, E. D. Biochim. Blophys. Acta 1973, 2 9 8 , 1015- 10 19. (15) Fry, D. W.; White, J. C.; Goldman, I . D. Anal. Biochem. 1978, 9 0 , 809-815. (16) Lodge, T. P.; Han, C. C.; Akcasu, A. 2. Macromolecules 1983, 76, 1180- 1183. (17) Koppel, D. E. J . Chem. fhys. 1972, 5 7 , 4814-4820. (18) Chu, 6. I n The Applicatlon of Laser Light Scattering to the Study of 6loiogical Motion; Earnshaw, J. C., Steer, M. W., Eds.; Plenum: New York, 1983; p 53. (19) Reijn, J. M.; Van der Linden, W. E.; Poppe H. Anal. Chlm. Acta 1981, 126, 1-13. (20) Deedler, R. S.; Kroll, M. G. F.; Beereen, A. J.; Van den Berg, J. H. M. J . Chromatogr. 1978, 749, 669-682. (21) Anathakrlshnan, V.; Gill, W. N.; Barduhn, A. J. J . Am. Inst. Chem. Eng. 1985, 7 7 , 1063-1072. (22) Ruzlcka, J.; Hansen, E. H. I n Flow Injection Analysis; Wiley: New York, 1981; p 20. (23) Tljssen, R. Anal. Chlm. Acfa 1980, 774, 71-89. (24) Golay, M. J. E. In Gas Chromatography; Desty, D. H., Ed.; Butterworths: London, 1958; p 36. (25) Tljssen, R. Sep. Scl. Techno/. 1978, 73,681-722. (26) Vandersllce, J. T.; Rosenfeld, A. G.; Beecher, G. R. Anal. Chlm. Acta 1988, 779, 119-129. (27) Taylor, 0.froc. R. Soc. London, Ser. A 1953, 279. 186-203. (28) Foley, J.; Dorsey, J. G. Anal. Chem. 1983, 55, 730-737. (29) Foley, J. Anal. Chem. 1987, 5 9 , 1984-1987. (30) Magln, R. L.; Weinstein, J. N. In Lbosome Technology; Gregorladls, G., Ed.; CRC Press: Boca Raton, FL, 1984; Vol. 3, Chapter 10. (31) Gerhardt, 0 . ; Adams, R. N. Anal. Chem. 1982, 5 4 , 2618-2620. (32) Trumbore, C. N.; Grehlinger, M.; Stowe, M.; Kelleher, F. M. J . Chromatogr. 1985, 322, 443-454.
RECEIVED for review September 3,1987. Accepted December 15, 1987. Certain commercial products are identified in order to adequately specify the experimental procedure. This does not imply endorsement or recommendation by the National Bureau of Standards.
Reversed-Phase Chromatographic Separation of Highly Charged Inorganic Cations and Anions Using Ion Interaction Reagents and Competing Ions A. D. Kirk* and A. K. Hewavitharana Department of Chemistry, University of Victoria, Victoria, British Columbia, Canada V8W2Y2 Reversed-phase liquid chromatographic separations of inorganlc complexes with charges to 4+ and 3- have been achieved in separation times of as little as 15 mln. For cations the standard method employs an ion interaction reagent such as sodium butanesulfonate incorporated in the eluent. For 3+ Ions and higher, peak shape Is improved and elution t h e reduced by the incorporation of a competing ion such as trlethyiammonium. The method provides faster anaiysls with better resolution on smaller samples than conventional cation exchange. Applied to anionic mixtures, hexyiammonium or octyiammonium was used as the ion interaction reagent and citrate was the most effective competlng ion. I t was possible to resolve a mlxture of Co(CN),X” lons for X- = CN-, CI-, Br-, and I- In a 12min period. The method provides a rapid and precise method of analysis and determination applicable to a wide range of preparatlve, klnetlc, and photochemical studies.
For many years, ion exchange chromatography has remained the most used separation and analysis technique in
kinetic and photochemicalstudies of coordination complexes, except for systems where NMR can be used effectively. Unfortunately ion exchange is slow, often involving elution times of two or more hours; the same is true of the occasionally used gel permeation technique. In addition, ion exchange becomes increasingly more difficult as the charge on the ions increases; for cations, triply charged ions are difficult to separate and there are only rare reports of sepaiation of quadruply charged species (1). For anions the situation is worse; there are reports of the separation of up to doubly charged anions, but it is a common observation that it is virtually impossible to even recover triple minus or more negatively charged ions from anion exchange columns. It is clear therefore that there is room here for an improved analytical capability and that such a method would be extremely important in extending dramatically the range of kinetic and photochemical systems that could be studied. The work reported here arises from the observation by Buckingham and co-workers (2-4) that a variety of cobalt cationic complexes with charges up to 3+ could be quickly separated by reversed-phase liquid chromatography using hexanesulfonate as an ion-interaction reagent. More recently
0003-2700/88/0360-0797$01.50/00 1968 American Chemical Society
