Adjustable height and width aperture for capillary photodetector cell

Adjustable aperture for photoelectric monitoring of a light transmitted through a capillary, wherein the aperture includes a device for providing a light beam having a predetermined limited extent in a vertical direction, a selectively operable device for limiting the extent of the light beam in a horizontal direction transverse to the vertical direction so that the extent in the horizontal direction can be selectively varied, and a device for positioning a capillary having longitudinal and diametral directions so that the diametral direction of the capillary lies in the vertical direction of the light beam and so that the beam of light passes through the capillary in a diametral dimensional thereof. The adjustable aperture can be used in combination with capillary zone electrophoresis and high performance liquid chromatography apparatus. Methods are provided for analyzing samples of one or more compounds by capillary zone electrophoresis or high performance liquid chromatography using the adjustable aperture.

BACKGROUND OF THE INVENTION 
The present invention relates to adjustable height and width apertures for 
on-column capillary detector cells used for capillary methods of 
separation, and in particular to adjustable height and width apertures for 
on-column photo-detector cells for capillary zone electrophoresis (CZE), 
also known as high-performance capillary electrophoresis (HPCE). 
Electrophoresis is a separation technique in which the fractionation of the 
components of a mixture is achieved by the migration of the components 
through a solution under the influence of an electric field. The 
individual components move through the solution at varying rates in 
response to the electric field's influence. The differences in migration 
rates are generally a function of the charge and volume of a component. 
Electrophoresis is the dominant separation method used in the study of 
DNA, proteins and other biological substances. 
Similar to chromatography, the term "theoretical plate number" is used to 
describe the separation efficiency of electrophoresis. As the number of 
theoretical plates a mixture passes through increases, the degree of 
separation of the mixture's components consequently increases. As the 
number of theoretical plates a mixture passes through over a unit period 
of time increases, the rate of separation of the mixture's components 
consequently increases. 
CZE is a recently developed form of electrophoresis in which a sample 
solution is introduced into a fine tube (50-75 micron inside diameter) 
filled with a buffered liquid with separation occurring as a result of the 
differential movement of sample components toward one of two electrodes by 
which an electric field is applied to the contents of the tube. Some major 
problems relative to electrophoresis methods, such as dissipation of heat 
and suppression of convection, have been greatly improved by using fine 
capillary tubes. CZE can generate 10.sup.5 to 10.sup.6 theoretical plates 
within 30 minutes. This separation technique has been successfully applied 
to analyze a variety of samples including proteins, amino acids, 
nucleosides, inorganic ions and neutral molecules. 
Until the present invention, suitable means for detecting sample components 
resolved by CZE have not been well developed. The problem is that for a 
typical CZE peak having a retention time of 500 seconds and 250,000 
theoretical plates, the peak width is four seconds. In a capillary with a 
50 micron inside diameter and a linear velocity of one mm per second, four 
seconds corresponds to only eight nanoliters in volume. Stated another 
way, CZE requires the detection of a series of segments of separated 
sample components occupying at best 4 mm long segments of the capillary so 
that the sample volumes to be tested for the presence of the component is 
at best several nanoliters. Making matters more difficult, the segments 
will only be separated by one mm or even less and the series of separated 
sample component segments will be passing through the capillary at a rate 
of one mm a second. To prevent overlap in the detection of the segments, a 
detector cell is required having small volume and high sensitivity. 
On-column detectors have been used to meet these requirements, employing 
fluorescent, electrochemical, ultraviolet (UV) and visible (VIS) 
absorption spectrophotometric detection methods, such as those methods 
disclosed in Walbroehl et al., J. Chromatogr., 315, 135 (1984) and Terabe 
et al., Anal. Chem., 56, 111 (1984). UV detectors, though less sensitive 
than fluorescence detectors, are still the most widely used because of 
their relative versatility. A section of the capillary downstream of the 
region where separation occurs is passed through a spectrophotometer 
detector cell. Light is transmitted through the capillary, and the sample 
component is identified by its characteristic absorption pattern. 
While it has been possible in the past to construct on-column UV detector 
cells, the prior art was not successful when reducing cell volume to 
maintain detector sensitivity. This occurs when a light beam having a 
thickness dimension greater than the inside diameter of the capillary is 
transmitted through the capillary. The light passing outside of the inner 
diameter of a capillary creates a high background and drowns out the 
absorption signal. 
Moreover, the prior art did not set a reasonable width of the light beam to 
avoid overlap in the detection of component segments. Such designs are: 
Yang, J. High Resolut. Chromatogr Chromatogr. Commun., 4, 83 (1981), which 
discloses an on-column UV detector constructed by stripping the polymer 
coating of a capillary and placing the capillary in the light path of a 
detector. Terabe et al., Anal. Chem., id., disclose a UV detector with a 
0.05 by 0.75 mm slit. Walbroehl et al., J. Chromatogr., id., disclose a 
100 micron pinhole as the aperture of a UV detector cell. Spino et al., J. 
Lig. Chromatogr., 10, 1603 (1987) disclose a detector cell fabricated by 
glueing a capillary and two razor blades onto a cell block, producing an 
aperture about 6 mm by the capillary inner diameter. Kientz et al., J. 
High Resolut. Chromatogr. Chromatogr. Comm., 11, 294 (1988) disclose a 
cell aperture made by drilling a 0.4 mm diameter hole in the outer holder 
of the capillary. Foret et al., Electrophoresis, 7, 430 (1986) disclose 
an on-column detector fabricated from optical fibers. None of the 
disclosed devices use aperture dimensions that maximize signal to noise 
ratio and at the same time prevent overlap in the detection of separated 
sample components. 
Optimum detector performance is a function of three important aspects of 
the design of on-column UV detector cell apertures for CZE. First, light 
should only pass through the inner diameter of the capillary. When a large 
amount of light passes through the rim of the capillary, the signal 
becomes very sensitive to the refractive index changes of the solution as 
well as the distance between the capillary and the photodetector, and the 
signal to noise ratio and linear range of detection will be reduced. 
Second, the dimension of the aperture corresponding to the portion of the 
capillary segment selected for detection should be minimized to prevent 
overlap in the detection of separated component segments and should be 
adjustable to meet different detection requirements that vary with the 
samples to be separated. Finally, installation and removal of capillaries 
should be convenient and accurate. 
A photodetector cell aperture capable of meeting these requirements would 
be highly desirable. 
SUMMARY OF THE INVENTION 
The above requirements are addressed by the present invention. 
One aspect of the present invention provides an adjustable aperture for 
photoelectric monitoring of light transmitted through a capillary. The 
adjustable aperture combines a means for providing a beam of light having 
a predetermined limited extent in a vertical direction with a selectively 
operable means for limiting the extent of the light beam in a horizontal 
direction transverse to the vertical direction so that the extent of the 
light beam in the horizontal direction can be varied, and a means for 
positioning a capillary having longitudinal and diametral directions so 
that a diametral direction of the capillary lies in the vertical direction 
of the light beam and so that the light beam passes through the capillary 
in a diametral direction thereof. 
In preferred embodiments of the present invention, the means for providing 
a beam of light having a limited extent in a vertical direction is capable 
of providing a beam of light having a limited extent in a vertical 
direction between about 10 and about 100 microns. In more preferred 
embodiments of the present invention, the diametral direction of the 
capillary includes an inner diametral dimension and the means for 
providing a beam of light having a limited extent in a vertical direction 
is capable of providing a beam of light having a limited extent in the 
vertical direction less than the inner diametral dimension of the 
capillary. 
Another aspect of the present invention provides the adjustable aperture of 
the present invention in combination with a capillary zone electrophoresis 
apparatus or a high performance liquid chromatography apparatus. 
In still yet another aspect of the present invention, methods are provided 
for analyzing samples containing one or more compound by capillary zone 
electrophoresis or high performance liquid chromatography using capillary 
devices having photodetectors with adjustable apertures which are adjusted 
so as to maximize the resolution or the sensitivity of the photodetector 
with respect to the sample components. 
Other objects, features and advantages of the present invention will be 
more readily apparent from the detailed description of the preferred 
embodiments set forth below taken in conjunction with the accompanying 
drawings.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
The adjustable-width aperture of the present invention can be used with 
photocell detector systems for any method of capillary separation, 
including, but not limited to High Performance Liquid Chromatography 
(HPLC) and Capillary Zone Electrophoresis (CZE). As used in this 
disclosure, the term "light" refers broadly to radiation in the infrared 
and ultraviolet regions of the electromagnetic spectrum, as well as to 
visible light. The portion of the electromagnetic spectrum selected for 
detection is that portion convenient for the identification of the 
materials to be separated by the capillary. The photocell selected for use 
with the invention will be responsive to that portion of the 
electromagnetic spectrum to be detected. For materials conventionally 
separated by HPLC and CZE, the ultraviolet portion, the visible portion, 
and the combined ultraviolet-visible portion of the electromagnetic 
spectrum typically are used to detect and identify such materials. 
Referring to FIGS. 1 and 3, an adjustable height and width aperture 
according to one embodiment of the present invention has an aperture body 
1 formed from metal plates 12a and 12b having confronting sides 13a and 
13b spaced apart by shims 4a and 4b with an opening 6 between the two 
shims. The aperture body has a front face 8 and a rear face 10. The rear 
face has a circular depression 14, in which is situated a washer 16 and 
spring 18. A base member 20 is affixed to the rear face of the aperture 
body, retaining the washer and spring in the circular depression. The base 
member has a channel 22 in alignment with the aperture body opening to 
permit the passage of light therethrough from light source 24. The spring 
has an open interior that also permits light to pass to the aperture body 
opening. 
The washer is provided with a plurality of slits 26a and 26b of differing 
width. As shown in FIG. 2, the confronting sides of the metal plates have 
bevels 28a and 28b along the front face of the aperture body including the 
aperture body opening. The front face to rear face dimension of the metal 
plates is thicker than that of the shims between the plates so that the 
beveled confronting sides of the metal plates in combination with the 
shims forms V-shaped grooves 30a and 30b on the front face of the aperture 
body on opposite sides of the aperture body opening 6. Capillary 32 is 
positioned in the V-shaped grooves and held in place by capillary retainer 
34, which is fastened to the aperture body by screw 36. The capillary 
retainer has an opening 38 in alignment with the aperture body opening and 
of sufficient width to permit the beam of light emerging from the 
capillary to pass undisturbed to photocell detector 40. 
The circular depression is positioned on the rear face of the aperture body 
and the edge of the washer protrudes from the side of the aperture body 
and is rotatable in the depression on the spring. The position of the 
circular depression also fixes the location of the washer so that each 
washer slit is capable of alignment with the aperture body opening by 
rotation of the washer. This arrangement permits interchangeability of the 
washer slits by rotation of the washer. 
The washer with slits serves as a selectively operable means for limiting 
the extent of a beam of light in a horizontal direction. Beams having a 
horizontal extent between about 0.2 and about 2.0 mm are preferred. 
Accordingly, the narrowest slit 26 may be about 0.2 mm wide, whereas the 
widest slit 26 may be about 2.0 mm wide. Shorter horizontal dimensions are 
used when maximum resolution is desired. Broader horizontal dimensions are 
used when greater sensitivity is desired. 
The capillary positioned in the V-shaped grooves has a longitudinal 
direction and diametral directions transverse to the longitudinal 
direction. The capillary has inner diametral dimensions defined by the 
inner wall surface of the capillary. The body opening 6 has a vertical 
dimension defined by the distance between the confronting sides 13a and 
13b of the metal plates defined by the thickness of the two shims 4a and 
4b, and a horizontal dimension defined by the distance between the two 
shims 4a and 4b. The vertical dimension of the body opening 6 defined by 
the thickness of the two shims 4a and 4b is typically between about 10 and 
100 microns. More preferably, the vertical dimension of the body opening 6 
defined by the thickness of the two shims 4a and 4b is less than the inner 
diametral dimension of the capillary. The V-shaped grooves position the 
capillary in alignment with body opening 6 so that a diametral direction 
of the capillary lies in the vertical dimension of the body opening. 
The light beam to be detected originates at the light source 24 and passes 
through the channel in the base member and the spring. The light beam next 
passes through the washer slit 26b which is in alignment with the aperture 
body opening. That slit limits the extent of the horizontal direction of 
the light beam to the width of the slit. The horizontally limited light 
beam passes through the aperture body opening 6, which limits the extent 
of the vertical direction of the light beam to the vertical dimension of 
the aperture body opening. The light beam, the extent of which has been 
limited horizontally and vertically, then passes through the capillary and 
the channel and the capillary retainer to the photocell detector. 
As used herein, horizontal and vertical direction references are made from 
the frame of reference of the photocell detector and the capillary 
separation device to which it is attached and correspond to the 
longitudinal and diametral directions of the capillary, respectively, 
which for various reasons may not be strictly aligned with the earth's 
horizon. 
Any means of providing a beam of light having a predetermined limited 
extent in a vertical direction suitable for use with photodetector cells 
in capillary separation systems may be used in the present invention. 
Means of providing a beam of light including a body having a predetermined 
limited extent in a vertical direction with openings having a vertical 
dimension that limit the extent in a vertical direction of a beam of light 
passing therethrough are also preferred, such as the aperture body opening 
of the aperture body of the depicted embodiment. 
Any means suitable for selectively limiting the extent of a beam of light 
in a horizontal direction suitable for use with photodetection cells for 
capillary separation systems may be used with the present invention to 
selectively vary in a horizontal direction the beam of light provided. Of 
the selectively operable means for limiting the extent of a light beam in 
a horizontal direction, an element defining a plurality of selectively 
interchangeable slits of differing horizontal dimensions in combination 
with means for aligning each slit with the means for providing a beam of 
light having a predetermined limited extent in a vertical direction are 
preferred, such as the washer with slits positioned in the circular 
depression on the rear face of the aperture body of the depicted 
embodiment. 
Other means useful as the selectively operable means for limiting the 
extent of a light beam in a horizontal direction include a means having a 
tapered slot moveable relative to the aperture body. Alternatively, the 
means for limiting the beam may include a pair of bodies and a micrometer 
adjustment capable of selectively adjusting the horizontal distance 
between these bodies. Moreover, the rotatable single means defining a 
plurality of selectively interchangeable slits is preferred because it 
provides a simple means by which the horizontal dimension of a beam of 
light can be varied in a set and reproducible manner. 
Other means for positioning the capillary may be used in accordance with 
the present invention. Thus, fasteners, clamps and alignment devices such 
as positioning markers on the body may be employed, and such alone or in 
combination. When the means for providing a beam of light having a limited 
extent in a vertical direction includes a body having an opening with 
vertical dimensions that limit the extent in a vertical direction of a 
beam of light passing therethrough, such as the aperture body of the 
depicted embodiment, preferred capillary positioning means include a 
groove on the front surface of the body having an opening, the groove 
being colinear with the horizontal dimension of the opening, and means for 
retaining the capillary in the groove, such as the grooves in the front 
surface of the aperture body and the capillary retainer of the depicted 
embodiment. Even more preferred are beveled grooves, such as the beveled 
grooves of the depicted embodiment. 
The grooved means of the present invention is preferred because it provides 
a simple means providing accurate alignment of the capillary inside 
diameter in the path of the light beam in a set and reproducible manner. 
Moreover, accurate positioning of the capillary relative to the light beam 
can be provided even without particularly precise or intricate machining 
of the components. This accurate positioning eliminates as a variable the 
alignment of the capillary in the paths of the light beams when the 
results of different sample preparations are compared. 
The following examples serve to provide further appreciation of the 
invention but are not meant in any way to restrict the effective scope of 
the invention. 
EXAMPLES 
EXAMPLE 1 
The aperture of the present invention was evaluated using a Kratos SF770UV 
detector (Applied Biosystems, Ramsey, NJ, U.S.A.) by a static method in 
which a capillary was filled with test solution with no flow or applied 
voltage. The capillary was 75 micron inner diameter (I.D.) by 195 micron 
outer diameter (O.D.) fused silica (Polymicro Technology, Phoenix, AZ, 
U.S.A.). A 3.0.times.10.sup.-4 M solution of acetophenone in acetonitrile 
was used to determine signal-to-noise ratio and linear range of detection. 
The wavelength of detection was 240 nm. The aperture having a 60 micron 
vertical dimension was set at 0.95 mm horizontal dimension. 
Because no commercial cells for CZE or capillary HPLC are currently 
available with which to compare the performance of the new aperture, a 
SF770 standard flow cell was used. The capillary was taped on the surface 
of the SF770 cell and across the aperture (1.0 mm diameter), after which 
signal-to-noise ratio and linear range of detection were measured. 
The signal and noise portions of absorbance measurements for both the cell 
having the aperture of the invention and the cell having a conventional 
aperture are depicted in Table I. The absorbance measured is expressed in 
absorbance units (a.u.), which is a standardized measurement of the 
intensity of a beam of light. The unit expresses a fraction or multiple of 
the intensity of a light standard against which the measured light beam is 
compared. The data indicates that although the noise measured with the 
cell using the aperture of the invention is 2.5 times higher than that of 
the cell with the conventional aperture, the signal obtained from the cell 
using the aperture of the invention is 14.7 times higher, resulting in an 
overall improvement of signal-to-noise of nearly six-fold. 
TABLE I 
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Aperture Signal (a.u.) 
Noise (a.u.) 
S/N 
______________________________________ 
60 Micron .times. 0.95 mm 
2.2 .times. 10.sup.-2 
1.0 .times. 10.sup.-3 
22 
1.0 mm diameter 
1.5 .times. 10.sup.-3 
0.4 .times. 10.sup.-3 
3.7 
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Measurement of the linear range of detection indicates that the upper limit 
of concentration is about 3.times.10.sup.-3 M for both cells regardless of 
aperture dimension. However, the lower limit of concentration is 
3.times.10.sup.-5 M for the cell using the aperture of the present 
invention, compared to a lower limit of 2.times.10.sup.-4 M for the cell 
having a conventional aperture. The aperture of the present invention 
producing a higher signal-to-noise ratio expands the linear range of 
detection by one order of magnitude. If detectors with reduced noise were 
used, the linear range and detection limit could be improved further. 
EXAMPLE 2 
Refractive index sensitivity of cells using the aperture of the present 
invention was examined by determining the baseline shift resulting when 
the capillary was alternatively filled with 100% methanol followed by a 
60:40 ratio of methanol and water. 
The tests were run as in Example 1, substituting the 100% methanol and the 
60:40 methanol-water solution for the acetophenone-acetonitrile solution. 
Absorbance was measured at 330 nm. Each test was run four times and 
statistically analyzed. For each run, the capillary was removed and 
repositioned. The baseline shift results are summarized in Table II. The 
data in Table II show that the baseline shift is about 0.02 a.u. This 
relatively large shift is due largely to the long distance (4 cm) between 
the cell and the photodetector on the SF770, which is known to cause 
refractive index sensitivity. 
TABLE II 
______________________________________ 
Absorbance (a.u.) 
Solution 1 2 3 4 - x + S.D. 
______________________________________ 
Methanol-water 
.0244 .0194 .0226 .0256 
.023 .+-. .0027 
Methanol .0016 .0031 .0013 .0014 
.0019 .+-. .0008 
Baseline Shift 
.0228 .0163 .0213 .0242 
.0212 .+-. .0034 
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Table II shows that the repeatability of absorbance measurement is good, 
with the maximum difference being 0.0062 a.u. This result indicates that 
the groove on the aperture of the present invention can reliably position 
the capillary. 
EXAMPLE 3 
In order to demonstrate the actual performance of a cell utilizing the 
aperture of the present invention, a mixture of nucleosides (AMP, CMP, 
GMP, UMP, 1 mg/ml each) was analyzed by CZE. 
The CZE apparatus described by Jorgenson et al., Science, 222, 266 
(Washington, D.C. 1986) was constructed using the cell and detector of 
Example 1. The power supply (0-30 kV) was a model PS/MJ 30P0400-11 
(Glassman High Voltage, Whitehouse Station, NJ, U.S.A.). The buffer 
solution was 0.05 M sodium dodecyl sulfate in a borate-phosphate solution 
(pH=7.0). The fused silica capillary was 50 micron I.D. by 355 micron O.D. 
The total capillary length was 84 cm, while the length from injection end 
to the detector was 60 cm. The capillary was rinsed with 0.1 M potassium 
hydroxide (20 min., about 100 microliters), water and the buffer solution 
respectively, then was conditioned under high voltage for 24 h. The test 
sample in the same buffer solution was injected at 2.0 kV for 15 seconds. 
Analysis was at 25 kV and 50 microamps. The aperture width was set at 1.4 
mm. The wavelength of detection was 267 nm with a time constant of 0.50 s. 
The electropherogram obtained using the cell with the aperture of the 
invention is depicted in FIG. 4. The separation is quite good, with peak 1 
representing GMP, peak 2 representing AMP, peak 3 representing CMP and 
peak 4 representing UMP. The column efficiency calculated from the UMP 
peak is 65,000 theoretical plates (10% peak height). 
The invention being thus described, it will be obvious that the same can be 
varied in many ways. Such variations are not to be regarded as a departure 
from the spirit and scope of the invention, and all such modifications are 
intended to be included within the scope of the following claims.