Micro-polarimeter for high performance liquid chromatography

• US 4,498,774 A
• Filed: 04/27/1983
• Issued: 02/12/1985
• Est. Priority Date: 07/22/1981
• Status: Expired due to Fees

• Current Assignee: Iowa State University Research Foundation Incorporated (Iowa State University)
• Original Assignee: Iowa State University Research Foundation Incorporated (Iowa State University)
• Inventors: Steenhoek, Larry E., Kuo, Jeng-Chung, Woodruff, Steven D., Yeung, Edward E.
• Primary Examiner(s): Marcus, Michael S.
• Application Number:
  US06/489,031
• Time in Patent Office: 657 Days
• Field of Search: 356/364, 356/365, 356/368, 356/410, 356/411, 422/70, 436/161
• US Class Current: 356/368
• CPC Class Codes: G01N 2030/746 detecting along the line of flow, e.g. axial
• CPC Class Codes: G01N 21/05 Flow-through cuvettes G01N21/09 takes precedence; handling fluid samples G01N1/10
• CPC Class Codes: G01N 21/21 Polarisation-affecting properties G01N21/19 takes precedence
• CPC Class Codes: G01N 30/74 Optical detectors measurement of intensity, velocity, spectral content, polarisation, or phase of infrared, visible or ultraviolet light G01J

Abstract

A micro-polarimeter interfaced with a system for high performance liquid chromatography, for quantitatively analyzing micro and trace amounts of optically active organic molecules, particularly carbohydrates. A flow cell with a narrow bore is connected to a high performance liquid chromatography system. Thin, low birefringence cell windows cover opposite ends of the bore. A focused and polarized laser beam is directed along the longitudinal axis of the bore as an eluent containing the organic molecules is pumped through the cell. The beam is modulated by air gap Faraday rotators for phase sensitive detection to enhance the signal to noise ratio. An analyzer records the beams'"'"'s direction of polarization after it passes through the cell. Calibration of the liquid chromatography system allows determination of the quantity of organic molecules present from a determination of the degree to which the polarized beam is rotated when it passes through the eluent.

Claims

1. An apparatus for determining trace and micro levels of selected species of optically active molecules in a non-optically active eluent which is cycled through a calibrated system for high performance liquid chromatography, comprising:

a flow cell with flat ends through which the eluent is cycled along a central bore, the bore having an input opening in one end of the cell and an output opening in the other end, and wherein the volume of the flow path between the openings is sufficiently small to allow substantially only one species of the selected molecules to be in the volume at any time;

transparent, flat, cell windows which are mounted substantially strain free over the openings, and which allow light to pass without substantial scattering, reflection or refraction;

a laser adapted to irradiate said input opening along the longitudinal axis of the central bore and to output at least two milliwatts when energized, and wherein the operating wave length of the laser will not be substantially absorbed by the molecules or the eluent so that thermal lensing is prevented;

a polarizer positioned between the laser and flow cell so that a beam from the laser will be polarized before entering the input opening;

a lens focusing the laser beam positioned between the laser and the polarizer so that the laser beam will be polarized and collimated while passing through the flow cell without substantially being reflected off the flow cell walls;

means for aligning said laser and lens whereby said laser and said lens in combination will have a beam pointing stability of at least 0.5 milliradians;

an analyzer positioned after the exit opening so that the polarization of the laser beam after the beam has passed through the flow cell can be determined, said polarizer and analyzer being selected so that an extinction ratio of at least 10¹⁰ is achieved between the combination thereof;

means for isolating said polarizer, analyzer and flow cell from vibrations which would effect their relative angular position on the order of 10^-3°

;

means for modulating the laser beam at a frequency in a substantially noise free region of the environment, and which is positioned between the polarizer and the analyzer in the path of the laser beam; and

means for filtering light, adapted to pass only light centered around the operating wave length of the laser beam and positioned in the path of the laser beam after the beam exits the analyzer so that the filtered, modulated beam can be converted to an electrical signal containing information on the direction and degree to which the polarized laser beam was rotated when it passed through the flow cell.

2. The device of claim 1 wherein said modulating means includes a Faraday rotator having air as a medium and a means for driving said rotator in response to a periodic electric signal.

3. The device of claim 1 further comprising an aperture positioned between the analyzer and the filter to further prevent stray light from entering the conversion means.

4. The device of claim 1 wherein:

said transparent cell windows are microscope cover slips which are mounted slightly off normal on the flow cell with silicone sealant, said laser is an argon ion laser operated at 0.5 W and 488 nm, and the distance between the polarizer and the analyzer is two meters.

5. The device of claim 1 further including a means for stabilizing the intensity of a beam from the laser and positioned between the laser and the polarizer in the path of the laser beam.

6. The device of claim 1 wherein the intensity stabilization means comprises:

a Pockels cell;

a Glan prism;

a beam splitter, wherein the Pockels cell, Glan prism and beam splitter are in the path of the laser beam between the lens and the laser, and the Glan prism is between the Pockels cell and beam splitter;

a photodetector adapted to receive the portion of the laser beam which is split from the initial path of the laser beam by the beam splitter and which is adapted to output an electrical signal if the portion of the beam thus split differs in intensity from a predetermined level; and

an operational amplifier adapted to receive the electrical signal from the photodetector and to adjust the Pockels cell in response thereto so that the intensity of the laser beam is stabilized.

7. The device of claim 1 further including a means for converting the modulated laser beam to an electric signal.

8. The device of claim 7 wherein said conversion means is a photomultiplier.

9. The device of claim 8 further comprising:

means for detecting the frequency of the converted light signal;

means for generating a first signal in response to the detection of electrical signals with a frequency equal to the modulation frequency;

means for generating a second signal in response to the detection of the amplitude and phase of the first signal at the modulation frequency; and

means for adjusting the modulation of the modulated beam in response to said second signal, and until said second signal is substantially zero.

10. The device of claim 1 wherein said adjustment means is a compensating Faraday rotator with air as a medium which is positioned between the polarizer and the analyzer in the path of the laser beam.

11. An apparatus for determining trace and micro levels of selected species of optically active molecules in a non-optically active eluent which is cycled through a calibrated system for high performance liquid chromatography, comprising:

a flow cell with flat ends through which the eluent is cycled along a central bore, the bore having an input opening in one end of the cell and an output opening in the other end, and wherein the volume of the flow path between the openings is sufficiently small to allow substantially only one species of the selected molecules to be in the volume at any time;

transparent, flat, cell windows which are mounted substantially strain free over the openings, and which allow light to pass without substantial scattering, reflection or refraction;

a laser adapted to irradiate said input opening along the longitudinal axis of the central bore and to output at least two milliwatts when energized, and wherein the operating wave length of the laser will not be substantially absorbed by the molecules or the eluent so that thermal lensing is prevented;

a polarizer positioned between the laser and flow cell so that a beam from the laser will be polarized before entering the input opening;

a lens for focusing the laser beam positioned between the laser and the polarizer so that the laser beam will be polarized and collimated while passing through the flow cell without substantially being reflected off the flow cell walls;

means for aligning said laser and lens whereby said laser and said lens in combination will have a beam pointing stability of at least 0.5 milliradians;

an analyzer positioned after the exit opening so that the polarization of the laser beam after the beam has passed through the flow cell can be determined, said polarizer and analyzer being selected so that an extinction ratio of at least 10¹⁰ is achieved between the combination thereof;

means for isolating said polarizer, analyzer and flow cell from vibrations which would effect their relative angular position on the order of 10^-3°

;

means for modulating the laser beam at a frequency in a substantially noise free region of the environment, and which is positioned between the polarizer and the analyzer in the path of the laser beam; and

means for filtering light, adapted to pass only light centered around the operating wave length of the laser beam and positioned in the path of the laser beam after the beam exists the analyzer so that the filtered, modulated beam can be converted to an electrical signal containing information on the direction and degree to which the polarized laser beam was rotated when it passed through the flow cell;

means for aligning said flow cell whereby reflections within said cell are substantially eliminated.

12. The apparatus of claim 11 wherein said means for aligning said flow cell includes a plurality of set screws adapted to engage said cell for adjustable positioning thereof.