Field amplified polarity switching sample injection in capillary zone electrophoresis

An improved electroinjection method of introducing a sample having positive and negative ions into a capillary column for CE or CZE process by introducing a short plug of a low concentration buffer before introducing the sample into the column which results in increasing amount of charged ions of the sample introduced in the capillary column due to increasing the electric field at the injection point. A method for efficiently introducing both positive and negative ions is also shown.

FIELD OF THE INVENTION 
The invention relates to methods for electric separation and detection of 
sample components by differential electrokinetic migration in a narrow 
capillary tube, and more particularly to methods for introducing sample 
into the capillary. 
BACKGROUND OF THE INVENTION 
Capillary electrophoresis (CE) or specifically capillary zone 
electrophoresis (CZE) is a known efficient separation technique useful for 
fast, high resolution and efficient separation of charged species in 
solutions. High resolution in CZE technique requires the introduction of a 
small volume sample, i.e., in the range of 5 to 50 nl. 
There are a number of methods for injecting such volume of sample into the 
capillary column. The two principal sample injection techniques are 
electrokinetic and hydrostatic injection as described in the following 
articles: Jorgenson, J. W. and Lukacs, K. D., Science, 1983, v. 222, p. 
266-272, Wallingford R. A., and Ewing A. G. Anal. Chem., 1987, v 59, p. 
678-681. 
Electrokinetic injection is performed through the pumping activity of 
electroosmosis and electrophoresis. Samples are introduced at one end of a 
buffer filled capillary and, stimulated by an applied high-voltage 
electric field, move towards the other end of the capillary tube. Samples 
are usually injected at the positive high-voltage end due to 
electroosmotic flow that carries solutes, regardless of charge, toward the 
negative electrode. This injection technique, while simple and easily 
controlled, can affect the introduced quantities of different sample 
components since some sample species will have higher electrokinetic 
mobilities than other species, (see Huang, X., et al., Anal. Chem., 1988, 
v. 60, p. 375-377). 
Hydrostatic injection is characterized by physically introducing samples in 
the capillary and might be referred to as suction, pressure, or gravity 
injection. In this method one end of the buffer filled capillary tube is 
removed from the buffer reservoir and introduced into a sample solution 
which is raised vertically above the capillary outlet. This height 
difference between the level of the sample solution and the buffer 
reservoir yields a hydrostatic pressure which siphons sample into the 
capillary. Alternatively, for automatically introducing a sample into the 
capillary, a vacuum can be applied to the end of the capillary tube. 
However, hydrostatic injection increases the zone broadening effect due to 
laminar flows generated during the injection, and influences the 
separation efficiency in zone electrophoresis. Comparison of 
electrokinetic and hydrostatic injection techniques indicate that 
electrokinetic introduction is preferably because it is simpler and 
produces less zone broadening. 
DISADVANTAGE OF THE PRIOR ART 
In conventional electro-injection in CZE, the buffer solution that is used 
for sample preparation and the buffer solution inside the capillary column 
have the same concentrations. The number of ions injected into the column 
under this condition is rather limited, especially due to the fact that 
negative ions can not move against the electric fields, assuming the 
column wall is negatively charged. Preparing samples in a low conductivity 
solution, e.g., H.sub.2 O, and injecting the sample solution 
electrokinetically into the column, one achieves a field enhancement at 
the injection point. The injected amount of positive ions will be 
proportional to this enhancement factor. However, if the injection is 
accomplished by changing the capillary column input directly from the high 
conductivity buffer to the low conductivity sample solution, the buffer 
boundary at the input end of the capillary column is disturbed and the 
electric field at the injection point might not get amplified properly. In 
addition, the negative ions, not only will not be enhanced, but will be 
repelled from the column by this high electric field. 
SUMMARY OF THE INVENTION 
The foregoing disadvantage of the prior art sample introduction methods for 
capillary zone electrophoresis are overcome by the present invention. 
According to the invention, an improved electrokinetic injection technique 
is used for introducing a sample electrically into the capillary column by 
injecting a short plug of low conductivity buffer before sample 
introduction, for a short period of time which is relatively short in 
comparison to analysis time. The analysis time is a time interval between 
the sample introduction into the capillary columns and complete detection 
of its components. The short plug of the low conductivity buffer is 
introduced into the capillary column by hydrostatic or electroinjection. 
In addition, since the electroosmotic velocity of the bulk solution is much 
slower than the electrophoretic velocity of sample ions under the enhanced 
field, one can inject and concentrate both positive and negative ions into 
the column by switching the polarity of the electrodes at the proper time. 
For example, one can first inject positive ions for a time period of 
t.sub.1, then switch the polarity to inject negative ions for time period 
t.sub.2. When t.sub.2 =t.sub.1 /2 the number of positive and negative ions 
injected will be roughly equal for the reasons described below. 
Furthermore, one can also achieve selected charge discrimination by 
injecting either positive or negative ions only. We call this Field 
Amplified Polarity Switching Injection (FAPSI). 
Experiment data was obtained with an electrolyte solution containing 100 mM 
2-N-(morpholino)ethanesulfonic acid (MES) and histidine (HIS). For field 
amplified sample injection (FASI), a stock solution contained 2.1 mg 
PTH-Arginine and 2.0 mg PTH-Histidine in 10 ml of H.sub.2 O was made. The 
sample solution was further diluted down to about 10.sup.-4, 10.sup.-5 or 
10.sup.-6 M, respectively, and injected after inserting a small plug of 
water in the column by application of a voltage of 3 kV in 10 sec. By 
initial introduction of the short water plug a high electric field 
intensity is built up at the column inlet from the beginning of the 
injection. A hundred fold enhancement in the sensitivity was confirmed 
experimentally at low sample concentration. 
For field-amplified polarity-switching injection (FAPSI), a 2.8 mg 
PTH-Aspartic acid and 3.2 mg PTH-Glutamic acid were added into the 
previous stock sample solution with PTH-Arginine and PTH-Histidine. A 
.+-.5 kV switchable power supply was used for injection. Several different 
time programming injection experiments were tried, and the enhancement in 
the sensitivity for both positive and negative ions was confirmed. 
Gravity-assisted injection was also performed using the above materials.

DETAILED DESCRIPTION 
Technical Background 
Consider a capillary column with length L filled with buffering medium 
having different concentration in two or more regions as shown on FIG. 1. 
In a useful and substantially accurate model of a capillary system, the 
total resistance R of the column will be: 
EQU R=(.rho..sub.1 x+.rho..sub.2 (1-x))L/A (1) 
Where .rho..sub.1, .rho..sub.2 are the resistivities of buffer with 
concentration C.sub.1 over the length of xL, and C.sub.2 over the length 
of (1-x)L respectively, and A is the cross section of the column, x is 
fraction of the capillary length occupied by the media with concentration 
C.sub.1 ; i.e., 0.ltoreq.x.ltoreq.1. 
For a column filled with buffering medium of a single concentration in the 
capillary column, i.e., x=1 or x=0, we have R=R.sub.1 =.rho..sub.1 L/A, or 
R=R.sub.2 =.rho..sub.2 L/A. Substituting the resistivities in Eq (1) with 
the resistances of the column with straight buffer gives the total 
resistance of aforementioned system. 
EQU R=R.sub.1 x+R.sub.2 (1-x) (2) 
If the voltage V is applied across the column, the electric current I will 
be: 
EQU I=V/R=V/(R.sub.1 x+R.sub.2 (1-x)) (3) 
Since the electric field E is a product of the current density and 
resistivity, the local field E.sub.1 and E.sub.2 in the two regions with 
different concentrations are 
EQU E.sub.1 =.rho..sub.1 I/A=IR.sub.1 /L, (4) 
and 
EQU E.sub.2 =.rho..sub.2 I/A=IR.sub.2 /L (5) 
Substituting into Eq (3), Eq (4) and (5) respectively the expression result 
in 
EQU E.sub.1 =E.sub.0 R.sub.1 /(R.sub.1 x=R.sub.2 (1-x)) (6) 
and 
EQU E.sub.2 =E.sub.0 R.sub.2 /(R.sub.1 x+R.sub.2 (1-x)) (7) 
Where E.sub.0 =V/L is the field strength of a uniform system, filled with 
buffer 1 or buffer 2. 
FIG. 2 element 20 shows a plot of Eq (6) and element 21 shows the plot of 
Eq (7) for R.sub.1 /R.sub.2 =2. 
While the absolute value of the electric field in the regions of buffer 1 
and 2 will depend on the length of the buffer regions, the ratio between 
them will remain constant, and only depends on their resistivities, which 
in general are inverse proportional to the concentrations so 
##EQU1## 
The Eq (8) indicates that the ions inside the lower concentration region 
will experience higher electric field and hence will move faster than the 
ions inside the higher concentration region. Once these faster ions pass 
the concentration boundary, they will experience lower electric field, 
slow down, and stack into higher concentration. As a consequence, this 
concentration boundary is a stationary boundary with respect to the 
electroosmotic flow. 
For analyzing the electroosmotic flow property in a mixed concentration 
system, a double layer model is used. In this well known model (see 
Gordon, M. J., et al. Science, 1988, v. 242, p. 225), the electroosmotic 
mobility is proportional to the zeta potential, i.e., the dielectric 
constant, at the silica/water interface. This zeta potential is 
proportional to the product of the charge on the interface and the Debye 
length, a characteristic distance beyond which the electric field of a 
charged particle is shielded by particles having charges of the opposite 
sign. Since the Debye length is inversely proportional to the square root 
of the ionic strength, or the concentration, as the concentration 
increases, the electroosmotic mobility decreases. 
In columns with a single phase buffer, the electric field strength is 
uniformly distributed along the column. Consequently, the electroosmotic 
velocity, which is equal to the electroosmotic mobility times the electric 
field strength, is also a constant. 
If concentration is nonuniform, the electric field strength will be 
nonuniform also according to Eqs. (6) and 7). In addition, the 
electroosmotic mobility is larger in the lower concentration region, which 
further enhances the difference in the local electroosmotic velocities 
between the two regions. 
However, the bulk solution has to move with a single averaged velocity. The 
difference of the local electroosmotic velocities and this bulk velocity 
will generate a hydrostatic pressure across the local regions. 
The high osmotic velocity of the input section is then balanced by the 
hydrostatic pressure which drives fluid back along the axis of the column, 
while the fluid in the second section is driven forward along the axis, 
relative to the lower osmotic flow. The boundary now behaves as a soft 
wall between the two regions with different concentrations. If the leading 
buffer has a slower electroosmotic velocity than the bulk velocity, this 
"soft wall" will be pushed forward by the trailing buffer, if the leading 
buffer has a faster electroosmotic velocity, it will pull the "soft wall" 
along. 
If the resistance to laminar flow is less than the resistance to osmotic 
flow, the average velocity v.sub.b is: 
EQU v.sub.b =xv.sub.e1 +(1-x)v.sub.e2 (9) 
where v.sub.e1, v.sub.e2 are the local osmotic velocities in the two 
concentration regions 1 and 2, respectively. Equation (9) shows that the 
boundary moves with a weighted average of electroosmotic velocities. 
The local electroosmotic velocity is 
##EQU2## 
where v.sub.eoj is the electroosmotic velocity is a column filled with 
pure buffer j. Substitution of Eq. (10) into Eq. (9), yields: 
##EQU3## 
where .DELTA.v.sub.eo =v.sub.eo1 -v.sub.eo2 is the difference in the 
osmotic velocities of pure systems. Eq. (1) shows that the average osmotic 
velocity of the system is not only weighted over the lengths of their 
components but also weighted over their partial resistances. 
If we now dissolve samples in a lower concentration buffer, and inject them 
electrokinetically into the column, the electric field at the injection 
point will be much stronger than the electric field in the capillary 
column. If the injection buffer has the same composition as the capillary 
column buffer, the electric field ratio is from Eq. (8): 
##EQU4## 
where E.sup.(i), E.sup.(c) and C.sub.b.sup.(i), C.sub.b.sup.(c) are the 
electric fields and buffer concentrations at the injection point and in 
the column, respectively. 
While the electrophoretic velocity for ion species i at the injection point 
is proportional to the enhanced field, v.sub.ep =.mu..sub.ep rE.sub.o, the 
average electroosmotic velocity v.sub.eo of the bulk solution in a mixed 
buffer system changes insignificantly, v.sub.eo .apprxeq..mu..sub.eo 
E.sub.o. For r&gt;&gt;1, the ions are injected into the capillary column faster 
than the neutral solution. 
The total amount of ion species injected into the column is given by 
##EQU5## 
where A is the cross sectional area of the capillary and t is the 
injection time. To calculate the total amount of ions injected into the 
column and the plug length, knowledge of v.sub.eo, E.sup.(i) and E.sup.(c) 
with respect to the injection time t is required. For a short injection 
time, they could be assumed constant, and Eq. (12) gives 
EQU N.sub.i =C.sub.i.sup.(i) A(.mu..sub.eo +r.mu..sub.epi)E.sub.o t.(13) 
Since the electrophoretic velocity exceeds the electroosmotic velocity at 
the injection point, some of the injected ions would pass the boundary and 
move into the low field region. Inside the capillary column, the injected 
ions will now be distributed into the two regions with difference 
concentrations. In the region limited by the electroosmotic flow, ions 
have the same concentration as in the original sample solution. In the low 
field region, the ion concentration is enhanced by the same factor r, 
i.e., C.sub.i.sup.(c) rC.sub.i.sup.(i). Equation (13) can be rewritten as 
EQU N.sub.i =C.sub.i.sup.(i) AX.sup.(i) +C.sub.i.sup.(c) AX.sup.(c)(14) 
where X.sup.(i) =.mu..sub.eo E.sub.o t and X.sup.(c) =.mu..sub.epi E.sub.o 
t are the plug lengths of the sample ions in low and high buffer 
concentration regions, respectively. Eventually, all sample ions will 
migrate into the high concentration region and stack into narrow bands 
according to their electrophoretic mobilities. 
For the sample injection using pure water, the theoretical enhancement 
factor might be several hundreds if a 10 mM buffer was used. We have 
obtained an enhancement factor of about ten when the capillary column was 
directly switched from the high conductivity buffer reservoir to the low 
conductivity aqueous sample solution. 
The lower field enhancement may be explained by the perturbation of 
electric field in the region close to the boundary. Injection of short 
plug of low concentration buffer or water prior the sample introduction 
maintains high electric field at the injection point and an enhancement of 
a hundred were obtained experimentally. 
Field-amplified sample injection works for positive ions only. The negative 
ions, will be pushed in the opposite direction by the high electric field. 
However, since the electroosmotic velocity of the bulk solution is much 
slower than the electrophoretic velocity of the sample ions under the 
enhanced field, we have discovered that it is possible to inject and 
concentrate both positive and negative ions into the columns by switching 
the polarity of the electrodes at the proper time. We call this technique 
Field-Amplified Polarity-Switching Injection (FAPSI). 
In FAPSI, samples of both positive and negative ions are prepared in the 
low conductivity buffer. As shown in FIG. 3(b), we can inject a large 
amount of positive ions under a positive high voltage with respect to the 
outlet end of the column for a time period t.sub.1. To obtain maximum 
enhancement, we can introduce a short plug of a low conductivity buffer 
into the column end 31 before sample injection. A short plug, 32, of low 
conductivity buffer, x.sub.w, will be injected electroosmotically into the 
column and establishes a concentration boundary. Most of the positive ions 
will stack after the boundary into the high concentration region. The 
lengths of the low conductivity region, x.sub.n, inside the column after 
t.sub.1 are 
EQU x.sub.n =v.sub.eo t.sub.1 +x.sub.w (15) 
We then switch the voltage to the opposite polarity and cause the 
electroosmotic flow to migrate in the other direction as shown in FIG. 
3(c). Since the electric field at the injection end of the column is high, 
the negative ions which now have a very high electrophoretic velocity 
overcome the slow electroosmotic flow and migrate into the column. This 
reverse polarity voltage can last for t.sub.2 such that the positive ions 
will also remain inside the column. 
The plug length of low conductivity region is 
EQU x.sub.n =v.sub.eo (t.sub.1 -t.sub.2)+x.sub.w 
The amount of positive and negative ions injected into the column are 
EQU N.sup.(+) =C.sub.i.sup.(+) A(v.sub.eo +.mu..sub.epi.sup.(+) 
rE.sub.o)(t.sub.1 -t.sub.2) 
and 
EQU N.sup.(-)=C.sub.i.sup.(-) A(.mu..sub.epi.sup.(-) rE.sub.o 
-v.sub.eo)t.sub.2, 
respectively, where r is the field enhancement factor. For t.sub.2 =t.sub.1 
/2 and .mu..sub.ep.sup.(-) .apprxeq..mu..sub.epi.sup.(+), we have 
N.sup.(+) =N.sup.(-), if r&gt;&gt;1. 
After injection of both positive and negative ions, the inlet end of the 
capillary column is connected from low conductivity reservoir 33 back to 
the high conductivity buffer reservoir 34. The polarity of the high 
voltage is also switched, via switch 35, back to the normal setting 
connected to the high negative voltage and the normal separation process 
starts as shown in FIG. 3(d). 
PREFERRED EMBODIMENT OF THE INVENTION 
A FAPSI method in CZE were performed using a CZE system developed by the 
inventors. A schematic diagram of the system is shown in FIG. 4. As shown 
in FIG. 4, apparatus includes a capillary column 30 with inlet end 31 and 
outlet end 41. 
A high concentration electrolyte is supplied to capillary column 30 from a 
reservoir 34 at the inlet end 31. A reservoir 37 at the outlet end 41 of 
the capillary column 30 collects the electrolyte of after it has passed 
through the column. Two more reservoirs may be used for sample 
introduction. One reservoir 36 is filled with low concentration 
electrolyte or water. The other reservoir 33 is filled with the sample 
solution prepared in the low concentration electrolyte or water. 
A high voltage is applied between the inlet and the outlet ends of the 
column, causing the electrolyte to move from one end to the other end. The 
system is supplied with a power supply 44 providing three high voltages; 
two for injection, i.e., .+-.5 kV dc, the other for separation, i.e., 30 
kV dc. 
The ground end of the power supply for injection is connected to a wire 38, 
preferably platinum, in the reservoir at the outlet end of the column. The 
high voltage end of the injection power supply 44 which can be switched 
between .+-.5 kV, is connected to a wire 39, preferably platinum, in the 
reservoir filled with low concentration electrolyte 36 or the reservoir 33 
filled with the sample solution. This power supply 44 is connected to the 
system only during sample introduction. 
The ground end of the power supply 44 for separation is connected to a wire 
40, preferably platinum, in the reservoir filled with high conductivity at 
the inlet end 31 of the column 30. The high voltage end of the power 
supply 44, which is set at -30 kV, is connected to a platinum wire 38 
connected to the reservoir at the outlet end 41 of the column 30 through 
switch 35. 
Electrophoresis may be carried out in any capillary column, as is well 
known in the art, such as a 75 .mu.m i.d., 360 .mu.m o.d., fused-silica 
capillary column (Polymicro Technologies, Phoenix, Ariz.) of 100 cm 
length. Any suitable column detector, such as on-column UV 
high-performance liquid chromatography absorption detector 42 (TASCO, 
Tokyo, Japan) may be used. The distance from the injection point to 
detection point is adjustable. For these experiments it was held at 75 cm. 
Electroinjection was used for introducing a sample into the column. 
In addition to the optical signal the electrophoresis current may be 
monitored by measuring the voltage drop across a resistor 43 in series 
with the capillary column. 
SAMPLE INTRODUCTION PROCEDURES 
For introduction of positive ions only the column was filled with 100 mM 
MES/HIS buffer. The inlet end 31 of the capillary column 30 was 
transferred from the 100 mM MES/HIS buffer to the buffer reservoir 
containing H.sub.2 O. A short plug of H.sub.2 O was injected into the 
column 30 by applying +5 kV with respect to the outlet end 41 of the 
column 30 for 30 sec or by gravity injection at 5" high for 10 sec. The 
positive sample ions were then injected electrically into the column by 
transferring the inlet end of the column from the H.sub.2 O reservoir to 
the fourth reservoir 36 containing the sample dissolved in the water. A 
potential of +5 kV with respect to the outlet end of the column was then 
applied for 10 sec. causing a small plug of positive ions to 
electromigrate into the column. The inlet end 31 of column 30 was 
transferred back to 100 mM MES/HIS buffer and the separation is started 
with 30 kV applied to that the inlet end 31 is positive with respect to 
the outlet end 41 of the column 30. 
For introduction of negative ions only, the column was filled with 100 mM 
MES/HIS buffer. The inlet end 31 of the capillary column 30 was 
transferred from the 100 mM MES/HIS buffer to the buffer reservoir 36 
containing H.sub.2 O. A short plug of H.sub.2 O was injected into the 
column 30 by applying +5 kV with respect to the outlet end of the column 
30 for 30 sec or by gravity injection at 5" for 10 sec. The negative 
sample ions were injected electrically into the column 30 by transferring 
the inlet end 31 of the column 30 from the H.sub.2 O reservoir to the 
fourth reservoir 33 containing the sample dissolved in the water. A -5 kV 
potential with respect to the outlet end of the column was then applied 
for 10 sec. causing a small plug of negative ions to electromigrate into 
the column 30. The inlet end 31 of column 30 was then transferred back to 
100 mM MES/HIS buffer, starting the separation with a +30 kV applied at 
the inlet end 31 with respect to the outlet end 41 of the column 30. 
For injecting both positive and negative ions the column was filled with 
100 mM MES/HIS buffer. The inlet end of the capillary column 30 was 
transferred from the 100 mM MES/HIS buffer to the buffer reservoir 36 
containing H.sub.2 O. A short plug of H.sub.2 O was injected into the 
column 30 by applying +5 kV with respect to the outlet end 41 of the 
column 30 for 30 sec or by gravity injection at 5" for 10 sec. The 
negative sample ions are injected electrically into the column 30 by 
transferring the inlet end 31 of the column 30 from the H.sub.2 O 
reservoir to the fourth reservoir 33 containing the sample dissolved in 
the water. A potential of -5 kV with respect to the outlet end of the 
column 30 was then applied for 20 sec. causing a small plug of negative 
ions to electromigrate into the column. Next, the positive sample ions 
were injected electrically into the column 30, with the inlet end 31 of 
column 30 still in the sample reservoir, switching the polarity of the 
power supply 44 to +5 kV with respect to the outlet end 41 of the column 
30 for 10 sec. (Although some negative ions inside the column 30 are 
caused to migrate out of the column 30 from the inlet end 31, a small plug 
of positive ions electromigrate into the column 30.) The inlet end 31 of 
column 30 is then transferred back to 100 mM MES/HIS buffer and the 
separation is started by applying 30 kV positive at the inlet end 31 with 
respect to the outlet end 41 of the column 30. 
FIG. 5 is a capillary electropherogram of two positive ions and two 
negative ions using the method of this invention: Positive-PTH-Arginine 
(peak 1), PTH-Histidine (peak 2), and Negative-PTH-Aspartic Acid (peak 3), 
PTH-Glutamic acid (peak 4). 
For comparison, a electropherogram of the same sample ions using 
conventional electroinjection, where the sample is prepared in the 
electrolyte that has the same concentration as the electrolyte inside the 
capillary column 30 and no polarity switching during injection, is shown 
in FIG. 6. The detector sensitivity in FIG. 6 is set at sixth four times 
the sensitivity setting in FIG. 5. Although one can also obtain signals 
from both positive and the negative ions in conventional electroinjection, 
the number of ions injected is much smaller than in FAPSI, especially for 
negative ions. 
FIG. 7 and FIG. 8 are two electropherograms of the same sample solution 
prepared in H.sub.2 O obtained using the charge discrimination injection 
described earlier. They show a clear charge discrimination against either 
positive or negative ions. We observe only the negative ions signals in 
FIG. 7 and only the positive ion signals in FIG. 8. 
FIG. 9 shows the enhancement in the signals of PTH-Arginine ions using FASI 
with a water plug in front of the sample compared with using conventional 
electroinjection and FASI without water plug. An order of magnitude of 
improvement in detection limit is obtained between FASI without water plug 
and conventional electroinjection. Another order of magnitude of 
improvement in detection limit is obtained between FASI with and without 
water plug. The trace 90 is conventional electroinjection of PTH-Arginine 
without a water plug. Trace 91 is for FASI without a water plug. Trace 92 
is for FASI with a water plug in front of the sample according to our 
invention. These results show an improvement in the detection limit for 
PTH-Arginine from 10.sup.-5 M to 10.sup.-7 M.