Optics: measuring and testing – Sample – specimen – or standard holder or support – Fluid containers
Reexamination Certificate
2000-08-22
2003-04-01
Font, Frank G. (Department: 2877)
Optics: measuring and testing
Sample, specimen, or standard holder or support
Fluid containers
C356S244000, C385S012000, C385S125000, C250S227110, C250S295000
Reexamination Certificate
active
06542231
ABSTRACT:
BRIEF DESCRIPTION OF THE INVENTION
This invention relates to a fiber-coupled liquid sample analyzer with a liquid flow cell that guides light by total internal reflection (called a light pipe), and more particularly to an analyzer used for light absorption measurements that are typically made in high performance liquid chromatography (HPLC), capillary liquid chromatography (&mgr;LC), capillary electrophoresis (CE), capillary electrochromatography (CEC), super critical liquid chromatography (SFC), flow injection analysis (FIA) and related techniques.
BACKGROUND OF THE INVENTION
Liquid chromatography is a widely accepted method of determining the absorbence of substances and their concentrations. The absorbence of a solution is determined according to Beer's Law:
A=&egr;bc
=log(1
/T
)=log(
P
0
/P
)
The symbol “A” represents the solution absorbence, “&egr;” is the molar absorptivity, “c” is the concentration, “b” is the path length, “T” is the transmission, “P
0
” is the incident light power, and “P” is the transmitted light power. Depending on the type of chromatography, e.g., CE compared to HPLC, there can be slightly different system design criteria and functional requirements. However, in general, all absorbence measurement systems for the various forms of liquid chromatography desire several common performance characteristics including (1) high sensitivity or a large signal-to-noise ratio (S/N) so as to observe low concentrations of an analyte, (2) low dispersion so as to maintain resolution between all analytes eluting from the separation media, and simultaneously, (3) a large linear dynamic range so that high and low concentrations can be determined with a single calibration of the absorbence.
Typically, HPLC systems with 10
4
to 10
5
linear dynamic range can measure less than one hundred micro AU (absorbence units) of absorbence. Noise levels are at about 20-40 micro AU. The resolution, on the other hand, is affected by the performance of the separation column and dispersion within the system. The chromatographer specifies the column depending on the materials to be analyzed. The other factor affecting resolution is dispersion. Compounds separated by the column pass through the detector. A plot of absorbence as a function of time will give a fairly Gaussian-shaped peak. Dispersion of the analyte within the solvent causes chromatographic peaks to broaden, reducing the absorbence signal. In addition, the distance between the borders of two peaks can become overlapped. Therefore, it is important to keep dispersion to an absolute minimum.
In general, fast separations using high flow rates are desired to reduce data acquisition times. However, this can also reduce the chromatographic resolution. Thus, in some instances, it is better to reduce the amount of analyte and reduce the flow rate. In that vein, chromatography has continued to reduce the volumes and flow rates, particularly in the area of Proteomics, or protein separation and quantification. Further, this is attractive where only small samples are available.
From a system point of view, to achieve high sensitivity and resolution, it is necessary to increase the signal as well as decrease the noise. A longer path length flow cell equates to a larger absorbence signal for a given sample concentration. Further, a longer path length implies that a lower concentration solution can be measured with equivalent incident light power, thus increasing the sensitivity of a HPLC method. Measuring lower concentrations is important for high purity drug synthesis, purity analysis, and chemical quality control.
In most instances, when achieving higher sensitivity, commercial absorbence measurement systems sacrifice dynamic range or vice versa. Noise sources, which affect the detection of absorbence changes, include short term and long-term noise sources. Short term noise arise from pressure induced index changes, light power fluctuations, electronic noise from the sensor and sensor electronics, fluctuations of light scattering from optical components such as lenses, mirrors, or gratings, high frequency jitter in the opto-mechanical components that cause the spectrum to move back and forth across the pixels of the array sensor, and background optical noise from external sources. Long-term noise arise from temperature fluctuations that cause optical alignments and coupling to vary, thermo-mechanical drift of the optical components, uncompensated drift in the detection electronics, and baseline shifts from the refractive index sensitivity of the flow cell. Clearly, the longer the path from the separation column to the flow cell the greater the dispersion resulting in a lower signal amplitude, broader signal, and lower resolution. Obviously, increasing the signal through a longer path flow cell is of no consequence if an offsetting increase in noise is simultaneously obtained.
Enhancement of the absorbence signal is clearly obtained with a longer path length flow cell that has low RI (refractive index) sensitivity, which results in drift of the baseline absorbence. The absorbence signal is further increased if the flow cell or light pipe is located near the separation column reducing the dispersion. Short-term noise caused by pressure induced index changes is typically reduced by flow restriction or pulse damping apparatus. Long-term noise from temperature drift is reduced by passive or active thermal control. The former case is usually the most cost effective, if it can be implemented. Matched diodes are usually used to obtain a signal and reference with equivalent drift that is subtracted out. Cooling sensors are often used in high sensitivity systems but results in added cost and complexity that is usually prohibited in a standard HPLC system. Often, in HPLC systems light sources must be judiciously isolated from the polychrometer to minimize thermally induced drift of the optical components. In most instances, the polychrometer is designed with costly materials and/or tight tolerances to account for these thermo-mechanical issues.
Normally, LC detector systems are made with bulk optics, that is, macro-scale lens, mirrors, and gratings where the light is transmitted between these components through free space. For instance, see U.S. Pat. Nos. 4,375,163, 4,848,904, 4,568,185, 5,495,186, 4,687,917, and 4,637,041. Generally, the light sources, relay optics, and spectrometer are integrated into a monolithic “optical bench”. Alternatively, it is possible to transmit the light through optical fibers. This approach is attractive for several reasons that will be discussed herein. Fiber-optic absorbence systems are commercially available from Ocean Optics, Inc., Dunedin, Fla., and from Carl Zeiss, Jena, Germany. (See U.S. Pat. No. 5,159,404) However, these systems are not suitable for the HPLC applications described herein since they lack the dynamic range, they are typically less than 10
4
AU's, and/or do not operate with a single grating over the desired spectral bandwidth, which is 190-800 nm for HPLC. In addition, the absorbence cells available with such systems are not designed with the functional, and performance needs of modem HPLC. What is critical to HPLC and other LC applications to obtain high dynamic range, sensitivity, and resolution is the system integration. There is interplay between all the components of the system including the pump, degasser, autosampler, injector valves, column, flow cell, spectrometer, and electronics that must be optimized.
It was previously difficult to use fiber optics in HPLC systems because the fiber would solarize or photo-darken when illuminated with ultra-violet (UV) light. Recently, “non-solarizing” silica-based fiber has become available in the form of high-OH fiber. In this fiber, OH is in-diffused during the fabrication and compensates defects of the fiber that lead to solarization. However, the OH also out-diffuses over time and thus solarization reappears. Heraeus-Amersil, Germany has developed a new non-solarizing preform for silica-based optical fibers that
Font Frank G.
Nguyen Sang H.
Thermo Finnegan LLC
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