Anal. Chem. 1990, 62, 2484-2490
2484
Separations of Chemicafly Different Particles by Capillary Electrophoresis Harlan K. Jones' and Nathan E. Ballou*
Chemical Sciences Department, Pacific Northwest Laboratory, Richland, Washington 99352
Fast, welk8solved separatkms of mlxtwes of chemically different latex particbs (dmerent numbers of attached carboxylate or sulfate orolqw) have been "My achieved bycaplllsryebhphds. 6estexped"talco"of column brdde dtsmetc# and length, butfer concentratbn and pH, and app#ed voltage were Menttfied for the separations. Electrophoretlc migraUon tbnes and moMIltles were determlned. The separations were charactetlzed as to numbers of theoretkal plates, selectiv#lee, and reeoknlon at Werent pH and applied vdtage vslws. M"of theoreikal plates ranged from about 400 to 3000. SslectCttbs ranged from about 0.1 to 0.4, and lbookffkn valws ranged from 0.8 to 2.3. Electrophoretlc n?obWRies varled especially at the hlgher applied voltages.
INTRODUCTION Improved particle separations techniques are required in biotechnology, environmental and health sciences, and industry in order to attain higher resolution and faster separations as well as to obtain separations of chemically different particles. A variety of separation techniques already exist and are well characterized (1-15). Recent work has shown separations of polystyrene nanospheres according to particle sizes by capillary electrophoresis (16). Separations required treatment of capillary walls with surfactant; separations were postulated as being due to capillary wall-particle interactions rather than electrophoretic mechanisms. Separation of particles according to their chemical identities by capillary electrophoresis (CE) is reported in this work. Micrometerand nanometer-size latex particles were separated according to numbers and kinds of functional groups attached to the particles. Capillary electrophoresis has developed rapidly since Jorgenson's pivotal work (17) in the early 1980s and has been widely applied in the fields of biochemistry and molecular biology. Such molecules as amino acids, small peptides, proteins, and oligonucleotides have been separated under conditions that provide numbers-of-theoretical-plates in the hundreds of thousands and more. Separation can be accomplished in tens of minutes and less. Because of its effectiveness, the technique is now sometimes referred to as high-performance capillary electrophoresis (HPCE). The recent review by Ewing (18) discusses the development of, applications for, and literature on CE. In addition to providing desired separations, CE also directly supplies electrophoretic mobility data from which zeta potential values of particles under given experimental conditions can be calculated (19, 20)* THEORY Particle separations are obtained in CE as a consequence of differences in their electrophoretic mobilities. Particle electrophoretic mobilities are determined from experimentally
'Present address: Merrell Dow Research Institute, 2110 E. Galbraith Rd., Cincinnati, OH 45215-6300. 0003-2700/90/0362-2484$02.50/0
measured particle migration times, electroosmoticflow rates, and electric field strengths. Expressions relating these parameters to given experimental conditions are given by Jorgenson (17, 21) and Ewing (18). From these, the following equation for electrophoretic mobility is obtained where pp is particle electrophoretic mobility, L is the total length of capillary column over which the potential V is applied, 1 is the distance between point of injection and the on-column detector, ti is the migration time for a given analyte particle, and t, is the migration time of the neutral marker (neutral marker migrates at the electroosmotic flow rate). The electrophoretic mobility of a particle is basic to the separation mechanism of electrophoresis performed in free solution, and it is central to the optimization process, which seeks higher numbers of theoretical plates and improved resolution between neighboring components. Resolution, R,, according to Giddings (221, is proportional to the product of the square root of the number of theoretical plates, N, and a selectivity term. If one assumes Gaussian peak shape and employs peak width at half-height in place of variance, the following useful expression for N is obtained (23):
N = (8 In
~ ) ( X / W ~ , ~ ) ~
In electrophoresis, the specified selectivity is the relative difference in electrophoretic mobilities between two close lying peaks or ApJii,,, where ACL,is the mobility difference between the two components and pP is the average electrophoretic mobility. The CE resolution expression combines the number of theoretical plates with selectivity in the following way:
(3) The resolution will improve by increasing N, increasing the relative difference in mobilities, or if possible, increasing both terms simultaneously. In this paper, all three approaches will be considered in optimization studies involving variations in capillary diameter and length, buffer conditions, and applied electrical field gradients. EXPERIMENTAL SECTION Apparatus. The apparatus we used for all CE experiments was built at our laboratory and is similar to the simple system described by Ewing (18). After preliminary experimentation,the dimensions of fused-silica capillary columns (Polymicro Technologies, Inc., Phoenix, AZ) chosen for the studies reported here were 55 cm long, 75 ym i.d., and 193 pm 0.d. In all cases, the column distance between high-voltage anode and the on-column detector window was 40 cm, and the distance from detector window to grounded cathode was 15 cm. Electrophoresis in the capillary was driven by a high-voltagedc (0-50 kV) power supply (Glassman High Voltage, Inc., Whitehouse Station, NJ). An analog microampere current meter (Assembly Products, Inc., Chesterland, OH) for monitoring current was positioned between the platinum wire ground cathode and ground. The on-column capillary detector used was an Isco CV4 variable-wavelength UV-vis absorbance detector (Isco, Inc., Lincoln, NE),and the W wavelength most often used for particle detection was 254 nm. For detection of the neutral marker, acetone, the Isco CV4 was set at 190 nm. The detector signal was simultaneouslyrecorded 0 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990
Table I. Properties of Polystyrene Latex Particles no. of functional groups per particle 1.29 X 1.35 X 1.36x 3.97 x 3.18 X 4.96 X 6.20 x
lo2 lo2 104 104
lo6 lo6 107
functional group
particle diameter, pm
solids
sulfate sulfate carboxylate carboxylate carboxylate carboxylate carboxylate
0.030 f 0.005 0.079 f 0.009 0.070 f 0.005 0.100 f 0.003 0.200 f 0.007 0.500 f 0.004 1.16 f 0.030
8.4 10.7 2.5 2.5 2.5 2.5 2.5
%
Table 11. Initial and Final Latex Sample Concentrations final sample particle initial latex concn, % solids final latex concn, size, pm particles/mL (by weight) particles/mL 0.030 0.070 0.079 0.100 0.200 0.500 1.16
5.66 X 1.32 x 3.95 x 4.55 x 5.68 X 3.62 X 2.91 X
10l6 1014 1014 1013 10l2 10" 1O'O
0.10 0.021 0.044 0.010 0.010 0.010 0.042
7.07 x 1.10 x 1.64 X 1.89 X 2.37 X 1.51 x 4.86 X
1013 10'2 10l2 10" 1O'O 109 lo8
Table 111. Phosphate Buffer pH, Concentration, and Conductivity pH
NaOH
6.64 7.21 8.49 10.71
2.5 4.5 3.0 1.0
concn,mM conductivity, KH2P04 NazHP04 pW/cm 7.5 7.5 3.75 5.0
992 1178 744 1029
by a strip-chart recorder (Linear Instruments Corp., Reno, NV) and integrated by an HP 3396A integrator (Hewlett-Packard,Palo Alto, CA). Particle Samples and Chemical Reagents. Latex particles with different numbers of attached carboxylate or sulfate functional groups were used. Particles with known numbers of milliequivalents of functional group per gram of latex were obtained from Polysciences, Inc., Warrington, PA (carboxylate),and Interfacial Dynamics Corp., Portland, OR (sulfate). Particles were supplied in specified particle size ranges. From the given numbers of milliequivalents per gram, particle sizes, and density of the particles (1.05 g/cm3), the numbers of functional groups per particle were calculated. See Table I. The narrow particle size distributions indicate the narrow range expected for the distributions in the calculated number of functional groups per particle. The concentrations of latex samples used in these CE experiments are the result of approximately 240-fold dilutions of the original latex dispersions. The initial and final sample percent solids and particle number concentrations are listed in Table 11. The concentrations were calculated from percent solids, particle diameter, and particle density of 1.05 g/cm3. Phosphate buffers were used exclusively in this study. Composition,concentrations, and conductivities of the buffers are listed in Table 111. The Na2HP04,KH2P04,and NaOH for preparing buffer standards were purchased from Sigma Chemical Co., St. Louis, MO, and the deionized water was purified with a milli-Q filtration system (Millipore Corp., Bedford, MA). The neutral marker used for measuring electroosmotic migration times was acetone (High Purity Chemical, Portland, OR). P d u r e . Injection was accomplished by gravity feed. Before each experiment,the capillary column was removed from a 1.5mL glass buffer vial at the high-voltage end and manually raised to the sample vial, which could be adjusted to several heights (0-30 cm) above the level of both the high-voltage and ground buffer reservoirs. All reservoirs, regardless of whether they contained buffer only or buffer and sample, held 1.5-mL volumes. The vials were capped with a septum, and electrode wire and capillary column were threaded through a small hole in the septum cap
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into the buffer or sample solution. The cap reduced buffer evaporation. Immersion of the capillary in the sample reservoir was timed by a stopwatch, then the capillary was returned to the high-voltage buffer reservoir. Injection times varied from 10 to 15 s; the injection height difference was maintained at 16.5 cm. Under these conditions volumes injected ranged from 25 to 37.5 nL. Following injection, electrophoresiswas initiated by manual activation of the Glassman HV power supply, Hp 3396A integrator, and strip chart recorder. The particle mixtures were analyzed at four buffer pH conditions (6.64,7.21,8.49, and 10.71) with an applied potential of 30 kV. In addition, at pH 10.71, the sample mixtures were run at 20,25,30, and 35 kV. Because of the different wavelengths used to detect particles (254 nm) and the neutral marker acetone (190 nm), measurements of electroosmotic flow rates were not made in the particle separation runs. Instead, separate quadruplicate measurements of electroosmotic flow rates were made for each of the four different pH conditions and the four different applied voltage conditions studied. The precision of the electroosmotic flow rate measurements had an average relative standard deviation of 1.69%. Column Conditioning. After the installation of a new capillary column, the column was flushed with fresh buffer by a syringe infusion pump (Hanard Apparatus, Millis, MA) for approximately 30 min. The column then was connected to the power supply, the experimental running potential was applied, and the stability of the current across the column was monitored. After a stable current level was achieved for 2-5 min, the potential was turned off, an injection made, and a CE experiment run. After each experiment,the column was flushed by infusion with fresh buffer for approximately 15 min. The buffer reservoirs were replenished with fresh buffer every fourth experimental run. RESULTS AND DISCUSSION System/Operating Parameters. Optimal capillary column inner diameter (i.d.) is critical for both molecular and particle separations. We tested 200-, loo-, and 75-pm-i.d. capillary columns before selecting the 75-pm-i.d., 193-pm-0.d. (outside diameter) capillary columns for these separation studies. The laboratory-built CE instrument was thermostated only by the ambient air temperature (24 "C); at the buffer concentrations used in this study and the applied potentials required to resolve the synthetic latex mixtures prepared for analysis, the 75-pm-i.d. column was able to dissipate heat adequately without thennostating. Overheating and boiling occurred occasionally in the 100-pm column and often in the 200-pm column, which interrupted the flow of electrolyte and charged latexes. The 75-pm column provided an increase in resistance and a decrease in current for a constant applied potential and column length, which eliminated the overheating and boiling problems. Reduction of total column length and distance from injection to detector also proved crucial to reducing peak width and improving resolution between components in the latex mixture. The migration time of each component was also reduced by shortening each of the two column distance parameters. The final column specifications for the 75-pm-i.d. column used in these experiments was 40 cm from the high-voltage electrode (injection point) to detector and a total length of 55 cm. At 30 kV, the column had an electrical field gradient of about 545 V/cm. Other types of CE particle separations may require column inner diameter and length parameters that vary from those described above. For many of the CE biotechnology applications, sample introduction by electromigration appears to be more often used than hydrodynamic flow (gravity feed). Jorgenson (21) compared the electromigration method to hydrodynamic flow sample introduction and showed that sample components are introduced differentially by electromigration due to the difference in net charge on the components. This differential in rates of introduction was experimentally verified with the particles of different net charges and sizes used in these studies. Hydrodynamic sample introduction, on the other
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990
A
Table IV. Migration Times for Latexes in Seven-Component Mixture at pH 8.49 and pH 10.71 and an Applied Potential of 30 kV
migration
particle
order
size, km
1 2 3 4 5 6 7
0.030 0.079 0.070 0.100 0.200 0.500 1.16
I " " " " " " 0
2
4
6 6 1 Time (min)
0
1
2
migration time, min pH 8.49 pH 10.71 1.86 2.06 2.44 3.05 5.23 10.6
1.58 1.72 1.84 2.11 2.57 4.57 6.63
Table V. Electroosmotic Migration Times, Particle Migration Times, and Electrophoretic Mobilities at Four pH Conditions and an Applied Potential of 30 kV
PH
0
2
4
6
8 1 0 1 2
Time (min)
Flgure 1. uecbopherograms of sevemponent latex mixtue at four pH values: (A) 6.64, (B) 7.21, (C) 8.49, (D) 10.71. Peaks 1, 2, 3, 4, 5, 6, and 7 cwespond to particles of 0.030-, 0.079-, 0.070-, 0.100-, 0.200-, 0.500-, and 1.16-pm diameter, respectively.
hand, should not discriminate against sample components in this way. Consequently, it was the method chosen for this study. Calculations of numbers of particles introduced by hydrodynamic flow can be illustrated for the case of 0.100-pm latex particles. The volume of sample introduced into the capillary column is calculated by the method of Jorgenson (21). A 10-s injection at a height of 16.5 cm above the high-voltage and ground buffer reservoirs has a volume of 25 nL. This compares to a total column volume of 2.43 pL, or approximately 1% of the total column volume. Samples of the 0.100-pm latex particle suspensions injected into the capillary contain 0.0104% solids. Density of the polystyrene latex is 1.05 g/cm3. From theae numbers the total number of particles loaded onto the capillary column in a 25-nL injection volume is calculated to be about 4.7 X lo6. The Isco CV4detector has not reached its limit of detection with this sample load, and it has the capability to detect at least an order of magnitude fewer particles while maintaining a good signal to noise ratio. Applied voltage and pH were the two parameters studied in the search for and establishment of conditions for electrophoretic separation of particles. Seven-component and two-component latex mixtures were used for this purpose. Components of the seven-member group are given in Table I. Members of the two-component group were 0.079-pm sulfate and the 0.100-pm carboxylate latex particles. The seven-component latex mixture was used to study the preliminary limits of CE as a particle separation method. The two-component mixture served to demonstrate migration times, numbers of theoretical plates, and resolution between two typical submicrometer size latex particles. Four pH conditions a t constant voltage were chosen for CE analysis of the seven-component and two-component mixtures. An optimal pH was identified among the four pH conditions
electroosmotic particle particle electrophoretic migration time, size, migration mobility, cm2/(V s) km time, min min
6.64
1.07
7.21
1.00
8.49
0.99
10.71
0.83
0.079 0.100 0.079 0.100 0.079 0.100 0.079 0.100
2.02 2.82 1.71 2.45 2.02 2.27 1.71 1.98
-5.33 x 10-4 -7.00 x 10"' -5.09 X 10"' -7.26 X lo4 -6.26 X 10"' -6.92 X lo4 -7.63 X 10"' -8.61 X 10"'
tested. In the second parametric study, the effect of variation of applied voltage at constant pH was determined for each of the two latex mixtures. pH Studies. All CE separations where pH was varied were run at 30 kV (545 V/cm). The series of electropherograms in Figure 1 shows steady improvement in resolution of the seven-component mixture as the pH of the phosphate buffer is increased from 6.64 to 10.71. In parts A and B of Figure 1, we observe that the seven latex standards are poorly resolved at pH 6.64 and 7.21. The severely broadened peaks suggest possible particle aggregation or interaction with the untreated fused-silica capillary walls. It is to be noted that latex particle-capillary wall interactions have been reported for surfactant-treated silica capillary walls at pH values of 5.80 and 6.46 (16). The manufacturer stated that the pK, of the carboxyl groups on the latex particles is relatively high, approximately 5, while the pK, of the sulfate groups is low at approximately 2. As a result the latexes with sulfate groups attached to the polystyrene surface will be more stable in acid conditions than the latexes with carboxylate groups bonded to their surface. At the four pH conditions used in this study, the latex particles were all observed to remain in suspension. When the buffer pH is raised to 8.49, we observe improved resolution (Figure 1C) among the seven components of the mixture; at pH 10.71 (Figure lD), all seven latex groups are resolved. The first cluster of three peaks contains the 0.030-pm sulfate latex, the 0.079-pm sulfate latex, and the 0.070-pm carboxylate latex, which migrate past the detector in increasing order of number of functional groups per particle. See Tables I and IV. The best resolved electropherogram appears to result from the CE separation done at pH 10.71; it also has the shortest separation time, 7 min, for resolution of the entire mixture (Figure 1D). The two-component mixture was analyzed at the above four pH conditions and at an applied potential of 30 kV. The electropherograms in Figure 2 show the changes in migration times, peak widths, and relative resolution of the two latex standards a t the four pH conditions. Listed in Table V are the electroosmotic and particle migration times and the respective electrophoretic mobilities, calculated from eq 1. As
ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990
2487
4000
*P
N(79nm latex) N(100nm latex)
B+
C
D
,lil
01 6
I 7
8
1
0
1
1
PH Flgure 4. Numbers of theoretical plate for two-component latex
mixture as a function of pH.
ik
u 0
1
2
3
.
Time (min)
Fbure 2. Electropherograms of twotomponent latex mixture at four pH values: (A) 6.64, (B) 7.21, (C) 8.49, (D) 10.71. Peaks 1 and 2 correspond to particles of 0.079- and 0.100-pm diameter, respectively. 0
0.4
6
+
0.1
7
8
9
1
0
1
1
Figure 5. Resolution values of two-component latex mixture as a function of pH.
‘2 0.2 d
6
7
PH
0.3
0
9
8
9
1
0
1
1
PH Figure 3. Relative differences of mobilities of two-component latex mixture as a function of pH.
stated earlier, the relative mobility difference between two components determines the selectivity of the CE separation. Figure 3 shows how changing pH affects the relative mobility difference between the 0.079-pm and 0.100-pm latexes. The data show that their relative mobility difference increases to about 0.35 at pH 7.21 and drops abruptly to 0.10 at pH 8.49. Within experimental error, it remains at that value when the pH is raised to 10.71. The second term in eq 3 affecting resolution is the square root of N (number of theoretical plates). It is to be remembered that the expression for N (eq 2) is based on the assumption of a Gaussian peak. However, the peaks in the experimentalelectropherograms frequently exhibited skewness that limits the accuracy with which N can be calculated. It is nevertheless useful to examine trends in N as shown by the experimental data. Calculationsof N were accomplished by first converting the measured temporal widths of the peaks to spatial widths in
order to correct for different zone velocities at the detector. This is discussed by Huang et al. (24). In Figure 4, calculated values of N are plotted versus pH for both the 0.079-pm and 0.100-pm latex standards. For both latexes the trend is an increase in N as pH increases, but the slope for the 0.100-pm latex is greater than that of the 0.079-pm latex. At pH 10.71, both latexes reach their highest observed N value, about 3000 theoretical plates for the 0.100-pm latex and about lo00 plates for the 0.079-pm latex. Since the resolution between two neighboring peaks is the product of App/ppand Nf2/4 (eq 31, the optimal pH for their separation will occur when both A p , / b and Nf2are maximized. The results show opposite trends for App/pp and N as pH is increased. The plot of R, versus pH in Figure 5 shows a general decrease in R, as pH increases. A comparison of the shape of the R, versus pH curve with the App/pp versus pH curve (Figure 3) shows a similarity that serves to illustrate the predominant influence of App/pp czn R,. Quantitative calculations of R, are limited because of the leading edge skewness of the second peak in each electropherogram. However, the general trend is for lower resolution a t higher pH values. At the two lower pH values the peaks exhibit baseline resolution, which corresponds to resolution values greater than one, as is shown by the calculated values of R,. Cause of the leading edge skewness is presently unknown, but it may be the result of overloading or localized electric field differences. This will be examined in future studies. Applied Voltage Studies. The effect of variations of applied voltage on separations was examined for both the seven- and two-component latex mixtures. For the sevencomponent mixture, the applied potential was varied from 25 to 35 kV at 5-kV intervals while the pH was held constant
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990
2
B
’ 1 5
ly 6
4
LJ-
0
2
4
6
8
1
0
1
2
1
4
Time (min) Figure 8. Electropherograms of seven-component latex mixture at three applied voltages: (A) 25 kV, (6)30 kV, (C) 35 kV. Peaks 1, 2, 3, 4, 5, 6, and 7 correspond to particles of 0.030-, 0.079-, 0.070-, 0.100-, 0.200-, 0.500-, and 1.16-pm diameter, respectively.
at 10.71. The series of three electropherograms in Figure 6 demonstrates the effect that increasing applied potential has on migration times, peak widths, and resolution. A t 25 kV and 30 kV (Figure 6A,B), the initial three-component cluster corresonding to the 0.030-, 0.070-, and 0.079-pm latexes is resolved into three identifiable peaks. At 35 kV, only two peaks appear in the initial cluster. In contrast, aa the applied potential is i n c r d from 25 kV to 35 kV, the 0.5- and 1.6-pm latex peaks become noticeably narrower and the particles require less time to migrate past the detector window. For example, at 25 kV the 1.16-pm latex has a peak width at half-height of approximately 2 min and a migration time of 12.6 min. After increasing the applied potential to 35 kV, width at half-height decreases to 0.257 min and migration time is reduced 4.1 min. The result of increasing the field has a similar effect on the 0.500- and 0.200-pm latexes, though not as dramatic. Such resolution behavior suggests application of potential programming to take advantage of the resolving power of CE for smaller-diameter particles (0.200 pm) at the higher applied potentials. A more detailed study of effects of applied potential on particle migration, peak efficiency, and resolution was carried out with the two-component latex particle mixture (i.e., 0.079-pm sulfate and 0.100-pm carboxylate particles). The pH was again held constant at 10.71 while the applied potential was increased from 20 to 35 kV at 5-kV intervals. Figure 7 shows the electropherogramsof the two-component mixture at each of the four applied potentials. As applied potential increases, migration times for each component are shorter, and peak widths become much narrower. Table VI lists for each latex particle the electroosmotic and electrophoretic migration times and particle electrophoretic mobilities as calculated from eq l . In Figure 8, as applied po-
0
1
2
3
4
5
Time (min) Figwe 7. Electropherograms of twocomponent latex mixture at four applied voltages: (A) 20 kV, (6)25 kV, (C) 30 kV, (D) 35 kV. Peaks 1 and 2 correspond to particles of 0.079- and 0.100-um diameter, respectively.
i
o’lol €
0.05 1
300
400
500 600 E (V/cm)
700
Flgure 8. Relative differences of mobilities of two-component latex mixture as a function of electric field.
Table VI. ElectroosmoticMigration Times, Partiole Migration Times, and Electrophoretic Mobilities at Four Applied Potentials applied electroosmotic particle electrophoretic electrophoretic voltage, migration time, size, migration time, mobility, kV wn min cmz/ (V8 ) min
20
1.68
25
1.18
30
0.83
35
0.58
0.079 0.100 0.079 0.100 0.079 0.100 0.079 0.100
3.80 4.32 2.65 3.01 1.71
1.98 1.14 1.40
-6.09 x lo-’ -6.67 X lo-’ -6.89 X 10” -7.56 x 10-4 -7.63 X lo-‘ -8.61 X lo-‘ -8.84 X lo-’ -10.5 X lo-’
tential is increased at constant pH, the relative mobility difference is shown to increase. The data show that the
ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990
u,
-4a
3500
7
3000
-
2500
-
0
2*o
79nm latex lWnm latex
2480
I
p.
g.-
2000 -
.L
2
8
1500 -
5
e
I:
$
0
v
=
1000
-
300
400
500
600
700
E (Vlcm) Flgurr 0. Numbers of theoretical plates for twotomponent latex mixture as a function of electric field.
electrophoretic mobility for both latexes grows more negative with increasing applied potential. One explanation for the change in electrophoretic mobility with increasing field strength is a change in the viscosity of the aqueous buffer as a result of Joule heating. An increase in Joule heating at the higher field strengths will cause a decrease in the viscosity of the buffer, resulting in greater electrophoretic mobilities. Another possible explanation for changing electrophoretic mobilities with changing applied potentials can be related to the relaxation effect, or distortion of the ionic atmosphere surraunding the particle as it migrates through the buffer medium under high field conditions (19,20). At high fields, the particle will not be at the center of ita ionic atmosphere. Differences in motion of the counterions that form the ionic atmosphere around the particle and the latex particle result in shearing of counterions from part of the surface of the charged particle, which is then more directly exposed to the electric field. This then results in an increase in the electrophoretic velocities and mobilities of the particles. A plot of N versus E is s h o w in Figure 9. As we have seen, IV1f2 is the second important parameter in the resolution equation (eq 3). As electric field is increased from 364 to 636 V/cm, the 0.079-pmparticle remains at a relatively constant N value of approximately lo00 theoretical plates. In contrast, the 0.100-pmlatex has an N value of about 1300 theoretical plates at 364 V/cm, reaches a maximum of 3000 plates at 545 V/cm, and then drops to 2500 plates at 636 V/cm. Using the experimental values of mobility difference and the number of theoretical plates obtained at the four different potential gradients, we calculate resolution, R,, and plot it against E in Figure 10. R, has a calculated value of approximately 0.8 for the 364 and 455 V/cm cases, and >1 for the higher field cases of 545 and 636 V/cm. Some error in the calculation of R, may be partly due to deviations from ideal Gaussian peak shape and the use of a peak integrator that assumes Gaussian peak shape. Another source of error that may cause higher than expeded resolution values may be found in the values of electrophoretic mobilities measured at very high field strengths. Figure 11 graphically shows the variation of electrophoreticmobility with applied potential. As applied potential decreases, the mobilities of both latexes appear to be approaching a plateau. Their relative mobility difference appears to be approaching a constant value as well (see Figure 8). Further work is required to better define and understand the variation in electrophoretic mobility with applied potential. CONCLUSION Fast and effective separations of chemically different particles in the micrometer- and nonometer-size range can be
I
0.5 I 300
500
400
700
500 600 E (Vlcm)
Figure 10. Resolution values of twozomponent latex mixture as function of electric field. -2
+0
79 nm Latex 100 nm Latex
-4
0
e
-6 -
0
4
e4
E
0
+
P
E
-8
0
3.
-10
-12
3 00
400
500 E (Vicm)
600
700
Figure 11. Electrophoretic mobilities of particles in two-component latex mixture as a functlon of electric field.
accomplished by capillary electrophoresis. Registry No. NaOH, 1310-73-2;KH2P04,7778-77-0; Na&P04, 7558-79-4. LITERATURE CITED (1) McCann, G. D.; Vandemoff, J. W.; Strickler, A.; Sacks, T. 1. Sep. P U f i f . Met)rods 1973. 2 , 153-198. (2) Snyder, R. S.; Rhodes, P. H.; Miller, T. Y.; Micale, F. J.; Mann, R. V.; Seaman, G. V. F. Sep. Scl. Technol. 1988, 27, 157-185. (3) Schmale, J. D.; Kellog, D. S., Jr.; Miller, C. E.; Schammel, P.; Thayer, J. D. A m / . MicrobEd. 1970. 19. 287-289. (4) Orban.' 'L.; Tietz, D.; Chrambach, A. Electrophoresis 1987, 8 . 471-476. (5) Hahn, E.; Wurts, L.; Tletz, D.; Chrambach. A. €/ectrophoresis 1988. 9 , 243-255. (6) Tietz, D. J . Chromatogr. 1987, 478, 305-344. (7) Seiwer, P. J . Chrmtogr. 1987, 418. 345-357. ( 8 ) Sugrue, S.; Ob,T.; Bott, S. Am. Lab. 1978, February, 98-109. (9) Karaiskakis, G.; Graff, K. A.; CaMwell, K. D.; W i n g s , J. C. Int. J . Environ. Anal. Chem. 1982, 12, 1-15. (10) W i n g s . J. C.; Chen, X.; Wahlund, K.G.; Myers, M. N. Anal. Chem. 1987. 59. 1957-1962. (11) Glddlngs, J. C. Sep. Sci. Technol. 1984-1985, 19. 831-847. (12) Caklwell, K. D. Anal. Chem. 1988, 60. 959A-971A. (13) McHugh. A. J. CRC Crit. Rev. Anal. Chem. 1984, 15. 63-117. (14) Brough, A. W. J.; Hlllman. D. E.; Perry, R. W. J . Chromatogr. 1981, 208, 175-182. (15) Tijssen, R.; Bos, J.; van KreveM, M. E. Anal. Chem. 1988, 58, 3036-3044. (16) VanOrman, 8. B.; McIntire, G. L. J . Mlcrocolumn Sep. 1989, 7. 289-293.
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2490
(17) Jorgenson, J. W.; Lukacs, K. D.Ana/. Chem. 1981, 53, 1298-1302. (18) Ewlng. A. G.; Wallingford, R. A.; Oleflrowicz, T. M. Anal. Chem. 1989, 61, 292A-303A. (19) Karger, B. L.; Snyder. L. R.; Horvath, C. An Introductbn to Separat&ns Science: John Wllev & Sons, Inc.: New York. 1973: ChaDter 17. (20) W h e m , D. H.; Loeb, A. L.; Overbeek, J. Th. G. J . colloid Interfece SCi. 1968, 22, 78-79. (21) Jorgenson,J. W.; Rose, D.J. Anal. Chem. 1988, 60, 642-648. (22) Giddings, J. C. Sep. S d . 1989, 4 , 181-189. (23) Karger. B. L.; Snyder. L. R.; tiorvath, c. An Intrcxiuctjm to separation Sclence: John Wlley 8 Sons, Inc.: New York, 1973: Chapter 5.
(24) Huang, X.; Coleman, W. F.; &re, R. N. J . Chrmtogr. 1989, 480, 95-110.
RECEIVED for review March 21. 1990. AcceDted August 15. 1990. This research was supported by the U.S.Depirtment of Energy under Contract DE-AC06-76RLO 1830. Pacific Northwest Laboratory is operated for the US.Department of Energy by Battelle Memorial Institute under Contract DE-AC06-76RLO 1830.
Determination of Nucleotides in Fish Tissues Using Capillary Electrophoresis An-Lac Nguyen, John H. T. Luong,* and Claude Masson Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2
Capillary .kctrophore&s has been applled to quantltate nucleotide ckgrsdatlon In flrh tluues, to provlde a basis for detemrkrlng the K value, an lndicotor of f k h froshness. The three major comporndr,h o h e -te (IMP), Inoskre (HxR), and hypoxanthine (Hx) were dirtlncthrely wparated at 416 V/cm aQpUed potentlal, 100 mM CAPS buffer, pH 11. There was a good correlstbn betweem the peak area and the nueleotkk concentraton. By whrg a short dlstance (22 cm)from the same entrance to the detector, the Identtfkatkn and determlnatlon of these compounds fn each sampb were compkted wltMn 13 mln. The r d s OMalned correlated vsry w d l wtth how o b h d hy efum8tk assays. ThecapIhry wascompktdyregrrmwated with 1 N NaOH, to dlssoclate all bound materlak from the capwSry watl, mainly catlons In the Hsh extract. TMS provided the same d k a surface for repeated runs, resulting In reproducible electropherograms.
INTRODUCTION Capillary electrophoresis (CE) is conducted in an open capillary to resolve species according to their different migration rates resulting from an applied electric field. The electrophoreticmigration provides a high resolution while the capillary format confers high speed, accurate quantitation, and ease of automation. Various aspects of this new and promising technique have been reviewed and discussed recently (1-4). The impressive resolving power of CE is a great improvement over gel electrophoresis or high-pressure liquid chromatography (HPLC). This technique has been successfully used to separate organic and inorganic ions (5,6), amino acids (7),peptides (€0,and oligonucleotides (9,10). In the past, the separation of proteins preeented some difficulty due to protein adsorption onto the capillary wall, but the problem has now been overcome (11-13). The different separation principle of CE has enabled the detection of impurities in products which appeared homogeneous by HPLC analyses (13). Therefore CE has been proposed as a technique of crm-checking quality control. The speed of CE also suggests its applicability for process control and for analyses of biological samples. In the production of specialty biochemicals such as antibodies or synthetic poly0003-2700/90/0362-2490$02.50/0
peptides, if the product level is quickly determined, corrective measures can be timely taken to optimize productivity. In the analyses of biological samples, the prerequisite is to establish conditions that provide good resolution of the substances of interest. In practice, application of CE to complex sample matrices is problematic. The interaction among the sample constituents and their interaction with the capillary wall is quite unpredictable. Reconditioning of the capillary after each run is still a trial-and-error procedure. Consequently, the reproducibility of the electropherogramcan only be obtained after a time-consuming series of trials. To date, there are only a few reports on the capillary electrophoresis of natural biological samples, to separate and quantitate the nucleotides in the organs of rats (14), and guinea pigs (15), and the polyamines in rat tissues (16). All other CE studies have been conducted on highly purified materials reconstituted in appropriate buffers. In this study, CE was applied to separate and quantitate the purine nucleotides in fiih tissue which are involved in the decomposition of adenosine 5’-triphosphate (ATP) following the death of a fish and during subsequent storage ATP ADP AMP IMP HxR Hx where ADP and AMP are adenosine diphosphate and adenosine monophosphate, IMP is inosine monophosphate, HxR is inosine, and Hx is hypoxanthine. In most fmh species, ATP degrades very quickly to IMP and this compound is reported to impart the pleasant flavor of fresh fish while the accumulation of Hx results in an off-taste. The concentrations of Hx, HxR, and IMP, and a freshness index derived from these concentrations (see definition later) have been used as indicators of fish freshness. The results obtained by enzymatic assays were also presented and compared with the CE results.
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EXPERIMENTAL SECTION Materials. Xanthine, hypoxanthine, inosine, inosine 5’monophosphate, xanthine oxidase from butter milk (XO), nucleoside phosphorylase from calf spleen (NP), and nucleotidase from Crotalus adamanteus venom (NT)were purchased from Sigma Chemical Co. (St. Louis,MO). Other reagents including 3-[cyclohexylamino]-l-propanesulfonic acid (CAPS)were products of Aldrich Chemical Co. (Milwaukee, WI). Fresh rainbow trout and frozen fillet of haddock were obtained from a local market. Preparation of Fish Extract. Tmue samples from fish fillet (5 g) were homogenized with 3 mL of 10% trichloroacetic acid. After centrifugation at 27000g,the supernatant was neutralized with 2 M sodium hydroxide. Phosphate buffer (10 mM, pH 7.8; 0 1990 American Chemical Society
