Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/62—Detectors specially adapted therefor
  • G01N30/74—Optical detectors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
  • G01N21/03—Cuvette constructions
  • G01N21/05—Flow-through cuvettes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
  • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
  • G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
  • G01N21/03—Cuvette constructions
  • G01N2021/0346—Capillary cells; Microcells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2201/00—Features of devices classified in G01N21/00
  • G01N2201/02—Mechanical
  • G01N2201/022—Casings
  • G01N2201/0221—Portable; cableless; compact; hand-held
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2201/00—Features of devices classified in G01N21/00
  • G01N2201/06—Illumination; Optics
  • G01N2201/062—LED's
  • G01N2201/0621—Supply
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2201/00—Features of devices classified in G01N21/00
  • G01N2201/06—Illumination; Optics
  • G01N2201/069—Supply of sources
  • G01N2201/0693—Battery powered circuitry

Definitions

• LC Liquid chromatography
• Figure 1 shows that ultraviolet (UV) light 10 from a light source may be transmitted through a liquid 12 disposed within a capillary column 14 to a UV detector 16.
• the UV light may be absorbed by compounds in the liquid 12, leaving an intensity of light on the detector 16 that can be interpreted to detect and quantify compounds.
• FIG. 1 An example of a prior art system used for liquid chromatography (LC) is shown in figure 2.
• Standard UV light sources such as a mercury (Hg) lamp 20, suffer from short lifespan, long warm-up time, and unstable light output.
• Figure 2 shows that the light source may have to be split using a lens 22, resulting in a reduced light output of the source 20.
• the light source 20 may be split in order to go through a sample material 24 and a reference material 26 and the remaining light is then detected by a sample photocell 27 and a reference photocell 28 respectively.
• New light sources may have been proposed that are more stable and produce less noise compared to standard UV light sources.
• LEDs light-emitting diodes
• They have gained interest due to their long life, high stability, bright output and low power requirement. Additionally, they are small in size and more compact compared to standard light sources. Considering the nearly monochromatic behavior of LEDs, a monochromator is not required.
• An LED-based detector may be fabricated without using expensive optical lenses.
• FIG 3 is a block diagram of an LC detection system that uses an LED 30 as a light source.
• the UV range is desirable because many compounds that may be analyzed by LC exhibit absorption in that range.
• the prior art shows that light from a flat window LED 30 may be directly focused onto a flow-through cell and detection achieved using a signal photodiode 32.
• the detector 32 suffered from high noise, high detection limits and limited linearity.
• the system may have also suffered from high stray light levels.
• One problem with the LC detection system may be that silicon photodiodes 32 may be more sensitive at higher wavelengths than the UV, which may be evident from a photodiode sensitivity plot.
• any light emission from an LED 30 at wavelengths higher than the UV may lead to significant stray light in the system.
• the system shown in figure 3 still required a beam splitter 33 which sent a portion of the light from the LED 30 to a reference diode 36. The other portion of the light was sent through a slit 34 and through a fused silica tubing 35.
• the detector 32 and the reference diode 36 were coupled as inputs to an amplifier 37.
• Another prior art system used a hemispherical lens LED as a light source with a photomultiplier tube for on-capillary detection. This system may have suffered from a high level of stray light, which compromised the linear range and detection limits of the system.
• Capillary columns have gained popularity in LC work, but a detector is needed that can fulfill the detection requirements for such columns.
• a prior art LC detection system using an Hg pen-ray lamp-based detector for on-capillary detection achieved good performance.
• commercial flow-cell based detectors may have introduced considerable dead volume for capillary columns.
• low-volume flow cells (few nLs) may be expensive and suffer from clogging problems due to salt deposition.
• One method of narrow focusing of light in order to eliminate stray light may be to use slits equal to or less than the capillary column internal diameter.
• the use of a slit in front of the capillary column also reduces the light throughput as shown in figure 3.
• a decrease in light intensity from the light source may decrease the S/N ratio of the detector.
• the present invention is a system and method for performing UV LED-based absorption detection for capillary liquid chromatography for detecting and quantifying compounds in a liquid, wherein a simplified system eliminates the need for a beam splitter and a reference cell by using a stable UV source, and power requirements are reduced, resulting in a portable and substantially smaller system with relatively low detection limits.
• Figure 1 is a diagram showing the operation of a UV detection system where UV light is passed through a capillary column.
• Figure 2 is a diagram showing a prior art detection system for generating a sample light source and a reference light source using a beam splitter and an Hg light source.
• FIG. 3 is a diagram showing an alternative prior art detection system that uses a UV LED as a light source, but which still uses a beam splitter to create a reference source but which suffers from reduced UV light intensity.
• Figure 4 is a first schematic diagram of a first embodiment of the invention showing the major hardware elements of a capillary LC system.
• Figure 5 is a second schematic diagram of the first embodiment of the invention showing more construction detail of a capillary LC system.
• Figure 6 is a graph of overlaid spectra of light output with (orange) and without (blue) a filter.
• Figure 7 is a graph of the effect of software smoothing on digitization and dark RMS noise without filter and on the total RMS noise with 0.5 s filter.
• Figure 8 shows two graphs that illustrate S/N ratio enhancement of the first embodiment, where signals were obtained (A) without smoothing and (B) with 4200 data points per 0.1 s smoothing.
• Figure 9 is a graph showing separations using integrated nano-flow pumping system and LED detector.
• Figure 10 is a block diagram of the components in a second embodiment of the invention.
• Figure 1 1 is a block diagram of the components in a third embodiment of the invention. DETAILED DESCRIPTION
• on-column detection may refer to when packed bed material terminates before the end of the column so that the last part of the column is actually empty. But there may also be situations in which the column has packed bed material all the way to the end of the column and a capillary has to be added in order to perform detection in the capillary portion. Accordingly, the embodiments of the invention should all be considered to include both configurations to be within the scope of all embodiments, where detection is taking place on-column in an area of the column that does not contain packed bed material, or within a capillary that has been added to the very end of the column where the packed bed material ends.
• a first embodiment is an LED-based UV absorption detector with low detection limits for use with capillary liquid chromatography.
• an LED light source may be selected.
• the LED output wavelength may change with changes in drive current and junction temperature. Therefore, LEDs should be driven by a constant current supply, and heating of the system should be avoided.
• the quasi-monochromaticity of the LED source contributes to stray light in the system, leading to detector non-linearity.
• the detection system should be protected from any LED light outside the desired absorption band by employing a filter in the system.
• On-column capillary detection may be preferred for capillary columns, since narrow peak widths are obtained by eliminating extra-column band dispersion, and peak resolution is maintained.
• the short-term noise in the detector may determine the detection limits and may be generally reduced by performing integration, smoothing, and/or using low-pass RC filters.
• the first embodiment shows that UV LED-based absorption detectors have great potential for miniaturization for field analysis. Further optimization of the detector design and reduction in the noise level may lead to better detection limits for small diameter capillary columns.
• the first embodiment resulted in a hand-portable 260 nm LED based UV absorption detector specifically for capillary LC on-column detection.
• the system is relatively small, light-weight and has very low power consumption compared to the prior art.
• FIG 4 is a first schematic diagram for introducing the elements of the first embodiment of the invention.
• the elements include a UV-based LED 40, a first ball lens 42, a band-pass filter 46 that is tuned to the LED 40 light source, a second ball lens 48, a slit 50 comprised of razor blades, a capillary column 52 that may have an inner diameter (ID) of approximately 150 ⁇ and an outer diameter of approximately 365 ⁇ , and a silicon photodiode detector 54.
• ID inner diameter
• the scale of the elements of the invention are not shown in figure 4.
• the UV light from the second ball lens 48 may be converging much more sharply than shown.
• the diameter of the second ball lens 48 may be more than 10 times larger than the inner diameter of the capillary column 54. Accordingly, it should be understood that figures 3 and 4 are provided to show the physical order of the components of the invention without showing the actual sizes.
• first and second ball lens 42, 48 are shown in figure 4, a different type of lens may be substituted and should still be considered to fall within the scope of the first embodiment. It may also be possible to provide the functionality of the first two ball lenses using a single lens and obtain the desired focusing effect. It should also be understood that the values to be given for all aspects of the first embodiment are approximate only and may vary up to 50% without departing from the desired functionality of the first embodiment.
• Figure 5 is a cut-away profile view of the first embodiment shown with more construction detail. This system shown in figure 5 should not be considered as limiting of the invention, but as a demonstration of the principles of the first
• Figure 5 shows an LED 40 having a first ball lens 42.
• the LED 40 may be disposed within an LED holder 44.
• the LED 40 may be manufactured as integrated with the first ball lens 42, or it may be attached or disposed adjacent to the first ball lens 42 after manufacturing.
• the LED 40 may be selected from any desired bandwidth of UV light that is appropriate for the compounds being analyzed in the capillary column 52.
• the first embodiment uses a 260 nm LED 40, but this
• wavelength of UV light may be changed as desired.
• a commercially available 260 nm UV LED 40 with a first ball lens 42 was used as a light source.
• the LED 40 was mounted on the LED holder 44.
• the LED holder 44 was threaded into a black lens tube and held tight with the help of retaining rings.
• the first ball lens 42 may be 6 mm in diameter, or any appropriate size to focus the light from the LED 40.
• the first embodiment includes a band-pass filter 46 disposed after the first ball lens 42.
• the band-pass filter 46 may be a 260 nm band-pass filter used to reduce stray light from reaching a detector from the LED 40 and/or any surrounding light.
• the value of the band-pass filter may be adjusted as needed in order to be optimized for the LED 40 light source.
• a 260 nm band-pass filter may be positioned in between the LED 40 and the second ball lens 42 in the black threaded tube.
• the function of the second ball lens 48 may be to receive the UV light that is focused by the first ball lens 42 and focus the UV light even further. It is desirable to focus the UV light so that the light sent into the capillary column 52 may be equal to or smaller than the width of the inside diameter (ID).
• the focusing of the UV light source may not be equal to or smaller than the ID of the capillary column 52 in the first embodiment.
• a fused silica ball lens may have a 3 mm diameter for the second ball lens 48 and may be mounted on a 3 mm ball lens disk and may be disposed at the LED focal point.
• the second ball lens mount may be centered on a mount, which may be threaded into the black lens tube containing the LED 40 and the band-pass filter 46.
• one or more slits 50 are disposed after the second ball lens 48 and in front of the capillary column 52.
• the slits 50 may be provided by razor blades or any other appropriate device.
• the slits may be approximately 100 ⁇ in width.
• the UV light that passes through the capillary column 52 is positioned so that it strikes a UV detector 54.
• the UV detector 54 may be any UV sensitive device.
• the UV detector 54 may be a silicon photodiode.
• the photodiode 54 may be disposed on a diode holder with external threads.
• a black cap may be built to thread into the diode holder. This black cap may have a V-shaped groove to hold the capillary column 52 in the center, a central hole to allow light passage, and grooves on opposite sides of the hole to hold the slits in place.
• a pair of razor blades may be used to fabricate the adjustable slit or slits 50.
• the slits 50 may be disposed on opposite sides of the central hole in the cap covering the outer diameter of the capillary column longitudinally.
• an operational amplifier may be used to receive the current from the photodiode and convert it into voltage values.
• An analog-to-digital converter may be used to record the voltage output with a computer or other recording device. It should be understood that a low pass RC filter may be used at the input to the analog-to-digital converter.
• the LED 40 and silicon photodiode detector 54 may require 6 V and 12 V DC power, respectively, for operation.
• the detector 54 required 0.139 Amp current, and could operate for approximately 25 hours using a 4 Amp-hour 12 V DC battery, as well as operate from line power with an AC to DC adapter.
• the detection system of the first embodiment may operate using a DC power source and therefore may be portable not only because of the DC power requirements, but because of the relatively small dimensions of the detection system.
• Detector noise was determined over 1 -min measurements of baseline data.
• a hollow fused silica capillary was connected to a nano-flow pumping system and filled with water. The baseline was then recorded for approximately 1 min, and the peak-to- peak absorbance was calculated. This gave the peak-to-peak (p-p) noise.
• Short term noise (RMS) was calculated as the standard deviation of the recorded baseline.
• the LED 40 was turned off and the dark noise was measured as the standard deviation in the baseline.
• digitizer noise the positive and negative terminals of the A/D converter were shorted.
• Detector drift was determined by flowing water through the capillary at 300 nL/min and recording the baseline for 1 h, followed by measuring the slope of the baseline.
• embodiment is fixed window averaging.
• smoothing techniques that may be used include but should not be considered as limited to, smoothing by averaging over a sliding window of fixed width, smoothing using an exponentially weighted moving average, and smoothing using a causal or non-causal filter constructed to whiten the baseline noise process.
• Solutions of different concentrations of sodium anthraquinone-2-sulfonate (SAS), adenosine-5- monophosphate (AMP), DL-tryptophan (DLT) and phenol were made in HPLC grade water. Solutions were made to flow, under nitrogen pressure, through a capillary inserted into the detector 54. Baseline data were recorded before and after each concentration experiment by flowing water through the capillary. Baseline corrected maximum absorbance unit (AU) values were plotted against molar concentrations (M) to determine the linearity of the detector 54. Detection limits for flow-through experiments were reported as 3 times the standard deviation in the baseline. Log-log plots were used to determine the sensitivity of the detector 54.
• SAS sodium anthraquinone-2-sulfonate
• AMP adenosine-5- monophosphate
• DLT DL-tryptophan
• phenol phenol
• Isocratic separations of phenols were performed using a PEGDA monolithic capillary column 52 (16.5 cm ⁇ 150 ⁇ ID) using the integrated system.
• the pretreated capillary column 52 was filled with monomer mixture and subjected to UV-initiated polymerization for 5 min. After polymerization, the monolithic column was washed with methanol followed by water for at least 6 h to remove unreacted compounds.
• the monomer mixture composition was: DMPA (0.002 g), PEGDA 700 (0.2 g), dodecanol (0.15 g), decanol (0.15 g), decane (0.2 g) and tergitol 15-S-20 (0.3 g).
• the phenolic compounds were dissolved in HPLC water and the mobile phase was 80/20 % (v/v) acetonitrile/water mixture. Separations were performed at 350 nL/min.
• the UV LED-based absorption detector 54 may be much smaller than an earlier described Hg pen-ray lamp-based detector. For on-capillary column detection, absorbance values may be small, so noise reduction may be important to obtaining good detection limits. A bright light source LED 40 may increase the photocurrents used to calculate absorbances without proportional increase in noise. A single wavelength (260 nm) detector 54 was fabricated instead of a multi-wavelength detector in order to reduce the cost and size of the detection system.
• the LED 40 had an integrated fused silica first ball lens 42 (6.35 mm diameter), which focused the light beam down to a 1 .5-2.0 mm spot at the focal point (15-20 mm), this was still too broad for the capillary column 52 dimensions (0.075 to 0.20 mm ID). Therefore, the second fused silica ball lens 48 (3 mm diameter) was placed at the focal point of the LED 40 to obtain improved focusing of the light.
• the first and the second ball lenses 42, 48 may be constructed of any appropriate material.
• the LED 40 was selected to emit light with a bandwidth of ⁇ 5 nm; however, with a spectrometer, it was determined that the LED emitted light at higher wavelengths as well. The additional wavelengths of light may have contributed significantly to the stray light of the system.
• the overlaid spectra in figure 6 show the light output from the LED 40 with and without the filter, confirming that the filter successfully eliminated the light from higher wavelengths.
• the LED 40 position was optimized to obtain the best focus at the center of the capillary column 52.
• the short-term RMS noise of the detector 54 was found to be 8 mV without the use of signal smoothing and low pass filter.
• the dark RMS noise without smoothing was calculated to be 6.95 mV.
• Software smoothing reduced the dark RMS noise level to 74.4 ⁇ as shown in figure 7.
• the dark voltage values were the same in a lighted and dark room, confirming that the capillary column 52 did not act as a light guide.
• Digitizer noise can contribute significantly to the minimum noise obtainable with a detector.
• the digitizer RMS and p-p noise were found to be 2.4 mV and 7.7 mV, respectively.
• the effect of software smoothing on the digitizer RMS noise was studied as shown in figure 7 and the minimum RMS and p-p noise levels obtained were 15 ⁇ and 95 ⁇ , respectively.
• dark current noise which includes noise from the photodiode and amplifier, and digitizer noise both contributed to the total baseline noise, and both were effectively reduced using software smoothing of the first embodiment.
• the unaveraged RMS noise level dropped to 2.42 mV and 2.3 mV, respectively.
• the effect of software smoothing on the S/N ratio was also studied and, while it was found that the effect of smoothing on the signal intensity for peak widths in the chromatogram was negligible, the RMS noise level was reduced to a level of 0.18 mV in the voltage corresponding to intensity of incident light (lo) (5.7 ⁇ ) without the use of a filter. With a 0.5 s filter and 4200 data points per 0.1 s smoothing, the RMS noise further dropped to 0.14 mV (4.4 ⁇ ). Thus, the LED detector RMS noise was an order of magnitude lower (-10-6 AU) than previous detectors and other UV LED detectors ( ⁇ 10-5 AU). The detector 54 drift was found to be very low (10-5 AU per h), which may be negligible over a peak width and may present no problems for the duration of a typical chromatogram.
• the RMS noise level decreased as the number of data points averaged per 0.1 s was increased from 100 to 2400; however, further decrease in the noise level after 2400 data points smoothing was not significant.
• the S/N ratio for uracil increased from 14 (without smoothing) to 408 (with 4200 data points smoothing) as shown in figure 8. Due to the reported and observed low drift and inherent light stability of the LED, a reference cell was not included, simplifying the detector 54 design of the first embodiment without compromising its performance.
• the photodiode signal through the capillary column 52 (an average of 70 ⁇ ) was three orders of magnitude higher than previous work (nA range).
• the slit 50 width was adjusted to be equal to the internal diameter of the capillary column 52 (i.e., 150 ⁇ )
• the stray light level was measured to be 17.3%.
• the slit 50 width was reduced to 100 ⁇ , which reduced the stray light level to 3.6%.
• a decrease in light intensity was compensated for by increasing the driving current on the LED 40 (13.3 mA).
• the output voltage signal intensity was not compromised at all by a reduction in the slit 50 width.
• the LED 40 was operated at only half of its maximum operating current.
• the maximum absorbance of the detector 54 with the capillary was calculated to be 1 .4 AU, which is higher than the value obtained with the Hg pen-ray lamp detector (0.94 AU).
• the linearity of a UV absorption detector may be compromised by improper focusing of the light source on the ID of the capillary column 54.
• Limits of detection depend on detector 54 short-term noise and the test analyte molar absorptivity. For the experiment, selection of test analytes was based on molar absorptivities and relevant previous LED detector work. The detector 54 gave a linear response up to the highest concentration tested, confirming that stray light was low in the system. The linear dynamic range was three orders of magnitude for all of the test analytes.
• the limit of detection at a S/N ratio of 3 was found experimentally to be 24.6 nM (7.63 ppb) or 1 .5 fmol for SAS. This detection limit may be five times lower than a prior art pen-ray Hg lamp-based detector.
• this detection limit is also three times lower than the non-referenced single-wavelength flow cell-based (1 cm long) detection limit reported earlier.
• the detection limits for AMP (87.9 nM or 30.5 ppb) would be 230 times lower for the same capillary column 54 dimensions (75 ⁇ ID) than a non-referenced LED detector reported earlier.
• the detection limit at an S/N ratio of 3 was found to be 299 nM (61 ppb) or 17.9 fmol. This detection limit would be 60 times lower than the referenced detector reported earlier for the same capillary dimensions (250 ⁇ ID.
• the variations in detection limits for the various compounds is consistent with the variations in molar absorptivities at 260 nm.
• the detection limits for the detector 54 are remarkable considering the fact that detection was performed on the capillary scale.
• the calibration data for SAS, AMP and DLT are listed in Table 1 .
• Capillary LC is performed by the first embodiment of the invention.
• the detection system includes a system for analyzing absorption of the UV light by at least one compound disposed in a liquid within the capillary column by analyzing the UV light that is received by the detector.
• the system for analyzing absorption may be part of the detector or may be a computer system that is coupled to the detection system for receiving data from the detector.
• the first embodiment performs on-column LC detection using a monolithic capillary column.
• Using on-column detection may improve peak shapes and increase detection sensitivity because extra-column band broadening may be reduced.
• the first embodiment may be a highly sensitive on-column detector 54 fabricated using a 260 nm UV LED 40 that can detect in the ppb range.
• the noise level of the detector 54 was remarkably reduced by the use of software smoothing and a low pass filter, i.e., 3.4-4.4 ⁇ , which is among the lowest noise levels ever attained with absorption detectors designed for capillary column 52 work.
• the low detection limits may be attributed to good light focusing, low stray light, and very low noise in the system.
• the low detection limits of the first embodiment may be obtained for the test compounds due to the good light focusing, low stray light and very low noise in the capillary LC system.
• the detection limits for SAS in the capillary format with 150 ⁇ pathlength may be 3 times lower than the LED-based detector with 1 cm pathlength.
• the detection limits were improved by a factor of 230 and 60 in comparison with the detectors with the same pathlength.
• phenol detection limits in our detector were the same under flow-through experiments and under separation conditions. Thus, detector performance was not compromised under actual liquid chromatography work. Reproducible isocratic separation of a phenol mixture was also demonstrated.
• a final comment regarding the size, weight, power requirements and portability of the first embodiment are a direct result of the uncomplicated design of the capillary LC system.
• a typical commercial system may have size dimensions of 1 1 x 13 x 22 cm, have a weight of 3.3 lbs., require a regular AC power line, and have a sensitivity that is approximately 1 mAU.
• the first embodiment may have dimensions that are approximately 5.2 x 3 x 3 cm, may have a weight of 0.2 lbs., may operate from a 12 DC power source and only use 1 .68 W, and may have a sensitivity of approximately 10 ⁇ . It should be understood that these values are approximate only and may vary up to 50% without departing from the characteristics of the first embodiment.
• Figures 10 and 1 1 are provided as a second and third embodiments of the invention. Specifically, all features and functionality of the second and third embodiments are the same as the first embodiment, with the exception of a change in the order of the first ball lens 42, the filter 46 and the second ball lens 48.
• Figure 10 illustrates in a diagram that it may be possible to position the first ball lens 42 adjacent to the second ball lens 48, and to then eliminate the filter 46 entirely.
• the filter 46 may eventually become unnecessary if the UV light source can be made more perfectly monochromatic. Filtering is performed in order to prevent any stray light from reaching the capillary column 54. If no or very little stray light is generated by the UV light source, then the filter becomes unnecessary and may be removed from the system without departing from the principles of the present invention.
• figure 1 1 illustrates in a diagram that it may be possible to position the filter 46 between the LED 40 and the first ball lens 42, and then position the second ball lens 48 adjacent to the first ball lens as in figure 10.

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