Abstract:
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.

Description:
BACKGROUND 
     Description of Related Art 
       [0001]    Liquid chromatography (LC) is performed in order to analyze the contents of chemicals in a liquid solution.  FIG. 1  shows that ultra-violet (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. 
         [0002]    An example of a prior art system used for liquid chromatography (LC) is shown in  FIG. 2 . Standard UV light sources, such as a mercury (Hg) lamp  20 , suffer from short lifespan, long warm-up time, and unstable light output.  FIG. 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. 
         [0003]    New light sources may have been proposed that are more stable and produce less noise compared to standard UV light sources. Among these, light-emitting diodes (LEDs) 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. 
         [0004]      FIG. 3  is a block diagram of an LC detection system that uses an LED  30  as a light source. For LC, 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 . However, 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. Therefore, 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  FIG. 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 . 
         [0005]    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. 
         [0006]    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. However, commercial flow-cell based detectors may have introduced considerable dead volume for capillary columns. Furthermore, low-volume flow cells (few nLs) may be expensive and suffer from clogging problems due to salt deposition. 
         [0007]    What is needed is a system that can provide narrow focusing of a light beam down to the internal diameter of the capillary column. 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. However, the use of a slit in front of the capillary column also reduces the light throughput as shown in  FIG. 3 . A decrease in light intensity from the light source may decrease the S/N ratio of the detector. 
       BRIEF SUMMARY 
       [0008]    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. 
         [0009]    These and other embodiments of the present invention will become apparent to those skilled in the art from a consideration of the following detailed description taken in combination with the accompanying drawings. 
     
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
         [0010]      FIG. 1  is a diagram showing the operation of a UV detection system where UV light is passed through a capillary column. 
           [0011]      FIG. 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. 
           [0012]      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. 
           [0013]      FIG. 4  is a first schematic diagram of a first embodiment of the invention showing the major hardware elements of a capillary LC system. 
           [0014]      FIG. 5  is a second schematic diagram of the first embodiment of the invention showing more construction detail of a capillary LC system. 
           [0015]      FIG. 6  is a graph of overlaid spectra of light output with (orange) and without (blue) a filter. 
           [0016]      FIG. 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. 
           [0017]      FIG. 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. 
           [0018]      FIG. 9  is a graph showing separations using integrated nano-flow pumping system and LED detector. 
           [0019]      FIG. 10  is a block diagram of the components in a second embodiment of the invention. 
           [0020]      FIG. 11  is a block diagram of the components in a third embodiment of the invention. 
       
    
    
     DETAILED DESCRIPTION 
       [0021]    Reference will now be made to the drawings in which the various embodiments of the present invention will be given numerical designations and in which the embodiments will be discussed so as to enable one skilled in the art to make and use the invention. It is to be understood that the following description illustrates embodiments of the present invention, and should not be viewed as narrowing the claims which follow. 
         [0022]    Before beginning, it should be understood that 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. 
         [0023]    A first embodiment is an LED-based UV absorption detector with low detection limits for use with capillary liquid chromatography. In a first aspect of the first embodiment, 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. 
         [0024]    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. 
         [0025]    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. 
         [0026]    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. 
         [0027]      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 μm and an outer diameter of approximately 365 μm, and a silicon photodiode detector  54 . 
         [0028]    The scale of the elements of the invention are not shown in  FIG. 4 . The UV light from the second ball lens  48  may be converging much more sharply than shown. Furthermore, 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  FIGS. 3 and 4  are provided to show the physical order of the components of the invention without showing the actual sizes. 
         [0029]    In addition, it should be understood that any drawings of the convergence of UV light caused by the first and second ball lenses  42 ,  48  is not being shown to scale and is for illustration purposes only. 
         [0030]    It may be possible to provide the same functionality as the components of the detection system listed above by substituting other components that provide the same function. For example, while a first and second ball lens  42 ,  48  are shown in  FIG. 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. 
         [0031]      FIG. 5  is a cut-away profile view of the first embodiment shown with more construction detail. This system shown in  FIG. 5  should not be considered as limiting of the invention, but as a demonstration of the principles of the first embodiment. Accordingly, specific values given for size, shape, weight, power, sensitivity or any other characteristics of components of the first embodiment are for example only and may vary from the values given. 
         [0032]      FIG. 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. 
         [0033]    In the first embodiment, 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 . 
         [0034]    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. 
         [0035]    In the first embodiment, 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. 
         [0036]    Another element of the first embodiment may be the use of a second ball lens  48  disposed after the band-pass filter  46 . 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). While it is preferred, 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. 
         [0037]    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 . 
         [0038]    With increased light throughput through the capillary column  52  and received by the detector, it was experimentally determined that light intensity incident on the detector may be up to three orders of magnitude higher than prior art capillary LC designs. 
         [0039]    To reduce stray light reaching the detector  54 , 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 μm in width. 
         [0040]    The combination of the band-pass filter  46  and the slits  50 , stray light was experimentally reduced to a value of 3.6%, which is very low compared to prior art systems that may operate at reductions of stray light to a value 30.5%. Prior art designs may have either used special UV index photodiodes for higher wavelength stray light elimination which apparently were not very effective, or may have used a slit (100 μm width) in front of 250 μm ID hollow capillary connected to the end of a commercial column (1 mm ID). This led to reduced light throughput through the capillary column  52 . Furthermore, band broadening due to a connection between a larger diameter tube to a smaller diameter tube impairs detection sensitivity. 
         [0041]    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. 
         [0042]    In the first embodiment, 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. 
         [0043]    In the detector  54 , 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. 
         [0044]    The examples to follow show experimental values for the first embodiment only and should not be considered as limiting performance thereof. Data points were sampled at a rate of 1 KHz to 42 KHz. These data points were then smoothed at a 10 Hz rate to reduce the noise level in the detection system. These values should not be considered as limiting, but serve to illustrate the principles of the embodiments of the invention. The data point sampling rate and data smoothing rate may be adjusted in order to optimize results for the detection system being used. 
         [0045]    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. However, it should be understood that 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. 
         [0046]    An integrated stop-flow injector with an injection volume of 60 nL was used in these experiments, unless otherwise specified. A 150 μm ID×365 μm OD Teflon-coated capillary column  52  was used in all experiments. The absorbance values, where reported, were calculated by taking the common logarithm of the inverse of the transmittance values. The transmittance was calculated by dividing the sample signal by the reference signal obtained by recording the baseline. 
         [0047]    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. For dark noise measurements, the LED  40  was turned off and the dark noise was measured as the standard deviation in the baseline. To determine 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. 
         [0048]    Software smoothing was performed to reduce the noise level. However, it should be understood that the smoothing function may be performed in hardware at a faster rate and may be substituted for the software smoothing. Although we can use a variety of smoothing techniques, the smoothing technique used in the first embodiment is fixed window averaging. Other 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. 
         [0049]    Using a 150 μm ID capillary column  52  and 5.35 pmol injections of uracil in solution, the S/N ratio was determined at different smoothing rates, and the best smoothing rate was used for further work. The effect of RC filters (time constants of 0.5 s and 1 s) on short-term noise was also studied with and without performing any smoothing. A black ink-filled capillary was used for stray light assessment in the system. The stray light level was measured by dividing the voltage signal obtained for black-ink conditions by the voltage signal obtained with a water-filled capillary column  52 , multiplied by 100. 
         [0050]    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 . Calibration data for phenol were also obtained by making injections on a PEGDA monolithic column, and baseline corrected peak areas were plotted against concentrations. Elution conditions were as described in the next paragraph. Detection limits were determined as 3 times the standard deviation in the baseline area obtained from blank injections (n=4) and calculated within the analyte peak zone. 
         [0051]    Isocratic separations of phenols (i.e., phenol, catechol, resorcinol and pyrogallol) were performed using a PEGDA monolithic capillary column  52  (16.5 cm×150 μm 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. 
         [0052]    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. 
         [0053]    Although 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. 
         [0054]    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. 
         [0055]    A 260 nm band-pass filter with a FWHM of 20 nm was used during experimentation. The overlaid spectra in  FIG. 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 . 
         [0056]    Due to the reported inherent stability of the LED  40 , a reference cell was eliminated from the design of the first embodiment. Elimination of the reference cell also resulted in elimination of a beam splitter in the first embodiment, which elimination increases the UV light throughput through the capillary column  52 . Since no other optical lenses except the first and second ball lenses  42 ,  48  were used, complex alignment of optical elements and transmission losses from multiple surfaces were also avoided in the first embodiment. 
         [0057]    A feature of the first embodiment that is part of the functionality of the detector  54  is to perform processing of the detection data. In the experimental use of the first embodiment, 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 μV as shown in  FIG. 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  FIG. 7  and the minimum RMS and p-p noise levels obtained were 15 μV and 95 μV, respectively. 
         [0058]    As can be seen from the data, 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. To further reduce the total noise level, two low-pass filters (time constant=0.5 s and 1 s) were applied to the input of the A/D converter. The unaveraged RMS noise level dropped to 2.42 mV and 2.3 mV, respectively. 
         [0059]    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 (Io) (5.7 μAU) 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 μAU). 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. 
         [0060]    As can be seen in  FIG. 7 , 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  FIG. 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 μA) was three orders of magnitude higher than previous work (nA range). 
         [0061]    Stray light may cause negative deviations from true absorbance values. When the slit  50  width was adjusted to be equal to the internal diameter of the capillary column  52  (i.e., 150 μm), the stray light level was measured to be 17.3%. By visual inspection, it was found that a significant level of light reached the detector  54  through the curved capillary column  52  wall. Therefore, the slit  50  width was reduced to 100 μm, 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). Therefore, the output voltage signal intensity was not compromised at all by a reduction in the slit  50  width. 
         [0062]    During experimentation, 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). 
         [0063]    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. 
         [0064]    Considering that anthraquinone and anthracene exhibit similar molar absorptivities, 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 μm ID) than a non-referenced LED detector reported earlier. For DLT, 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 μm ID. Thus, the variations in detection limits for the various compounds is consistent with the variations in molar absorptivities at 260 nm. 
         [0065]    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. The RSDs in peak areas (n=3) for the three compounds ranged from 0.4-2.6%. These areas were calculated by injecting different concentrations of each compound three times into a hollow capillary column using water as carrier fluid at 600 nL/min. 
         [0000]    
       
         
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
             
             
               
                   
                   
               
               
                   
                 Peak area (AU) 
                   
               
             
          
           
               
                 Analytes 
                 Concentration range 
                 Regression equation 
                 R 2   
                 Sensitivity d   
                 LOD 
               
               
                   
               
               
                 SAS 
                 24.6 nM-50.4 μM 
                 y = 498.09x + 9 × 10 −6   
                 1 a   
                 0.9968 
                 24.9 nM 
               
               
                 AMP 
                 87.9 nM-22.5 μM 
                 y = 185.44x + 5 × 10 −6   
                 0.9999 b   
                 1.0138 
                 87.9 nM 
               
               
                 DLT 
                  299 nM-0.61 mM 
                 y = 54.744x + 5 × 10 −6   
                 1 c   
                 0.9855 
                  299 nM 
               
               
                   
               
               
                   a For n = 12; 
               
               
                   b For n = 9; 
               
               
                   c For n = 12; 
               
               
                   d Sensitivity was obtained using log (AU) vs log (M) plots. 
               
             
          
         
       
     
         [0066]    Since the detector  54  is specifically designed for on-column detection, the detector performance was tested under LC conditions using phenol and compared with the flow-through experiments as shown in Table 2. The detector linearity was excellent under both conditions, and the detection limits were found to be similar. Hence, the detector performance was not compromised when used under actual LC conditions. 
         [0000]    
       
         
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 2 
               
             
             
               
                   
                   
               
               
                   
                 Peak area (AU) 
                   
               
             
          
           
               
                 Method 
                 Concentration range 
                 Regression equation 
                 R 2   
                 LOD 
               
               
                   
               
             
          
           
               
                 Flow- 
                 1.95 μM-1.33 mM 
                 y = 14.252x + 
                 1 
                 1.95 μM 
               
               
                 through 
                   
                 2 × 10 −5   
               
               
                 On- 
                 1.70 μM-1.33 mM 
                 y = 3118.9x + 
                 0.9997 
                 1.96 μM 
               
               
                 column 
                   
                 0.0121 
               
               
                   
               
             
          
         
       
     
         [0067]    Capillary LC is performed by the first embodiment of the invention. Accordingly, 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. 
         [0068]    It is also noted that 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. 
         [0069]    Application of the capillary LC system was demonstrated using phenolic compounds as shown in  FIG. 9 . Good resolution was obtained for all analytes in an isocratic mode. Baseline stability under LC experiments was remarkable, confirming the low drift exhibited by this detector. The retention times in min and peak widths in s (tR/wb1/2) of the compounds were found to be: phenol (11.79/10.4), catechol (12.98/13.4), resorcinol (14.03/12.8) and pyrogallol (14.85/16.6). The reproducibility of peak retention times ranged from 0.1-0.2% (n=4). The column efficiencies (N/m) and minimum plate heights (μm) for the retained compounds were: phenol (156,838/6.4), catechol (113,118/8.8), resorcinol (144,673/6.9) and pyrogallol (96,963/10.4). 
         [0070]    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 μAU, 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. 
         [0071]    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 μm pathlength may be 3 times lower than the LED-based detector with 1 cm pathlength. For AMP and DLT, the detection limits were improved by a factor of 230 and 60 in comparison with the detectors with the same pathlength. Also, 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. 
         [0072]    Software smoothing was used to reduce the noise level in the detection system. Without smoothing, the total root mean square noise level was 8 mV. With the 4200 data points per 0.1 second smoothing, the noise level was reduced to 0.18 mV and when a low pass RC filter (2 Hz time constant) was employed to the input of the analog-to-digital converter, the noise further reduced to 0.14 mV (equivalent to 4.4 μAU). This is one of the lowest noise levels ever attained with capillary based detectors. Without software smoothing and using just the RC filter, the noise level was only reduced from 8 mV to 2.4 mV. Thus, low-pass filtering was clearly not enough to effectively eliminate high frequency noise from the detection system. The S/N ratio increased from 14 to 408 for 5.35 pmol uracil peaks. The noise level was up to 2 orders of magnitude smaller than the prior art in which some detectors only relied on a low pass filter. 
         [0073]    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 11×13×22 cm, have a weight of 3.3 lbs., require a regular AC power line, and have a sensitivity that is approximately 1 mAU. In contrast, the first embodiment may have dimensions that are approximately 5.2×3×3 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 μAU. 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. 
         [0074]      FIGS. 10 and 11  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 .  FIG. 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. 
         [0075]    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. 
         [0076]    In contrast,  FIG. 11  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  FIG. 10 . In other words, it may be possible to dispose the filter  46  in front of both ball lenses  42 ,  48 , and between the ball lenses, or eliminate the filter entirely and still achieve the desired focusing and filtering of the UV light from the LED  40 . 
         [0077]    Although only a few example embodiments have been described in detail above, those skilled in the art will readily appreciate that many modifications are possible in the example embodiments without materially departing from this invention. Accordingly, all such modifications are intended to be included within the scope of this disclosure as defined in the following claims. It is the express intention of the applicant not to invoke 35 U.S.C. §112, paragraph 6 for any limitations of any of the claims herein, except for those in which the claim expressly uses the words ‘means for’ together with an associated function.