Background of the Invention A well-known need exists within industries involved with macromolecules to characterize molecules created by or used in various processes. Application areas where this need is of particular interest include quality control laboratories, research laboratories and manufacturing operations in the plastics, pharmaceutical, biotech and chemicals industries.

Typical measurements to characterize molecules include determination of the molecular weight distributions and polydispersity when different molecular weights are included in the sample, molecular sizes (hydrodynamic radii, radii of gyration), concentrations and conformation information, including shape information and molecular branching information.

The most common technique for molecular characterization is liquid chromatography (LC), which involves the preparation of the sample for the particular detection method to be used and the detection method itself. In this well-known analytical technique, a flowing mixture of a solvent and the substances to be tested (a mixture of molecules with various molecular weights and other molecular characteristics) is passed through specifically selected chromatography columns which result in the component molecules being separated in time in the flowing solvent according to their size or according to some other molecular characteristic.

The flowing fluid then passes through the detector. In one type of detector, a laser beam is directed through the fluid, and the light scattered by molecules of the sample is analyzed to provide molecular characterization. A detector of this type is disclosed in U.S. Patent No.

5,305,073 issued April 19, 1994 to Ford, Jr.

Examples of uses of liquid chromatography are the analyses of proteins, commercial resins, natural and synthetic polymers, nucleic acids, plasticizers, plant and animal metabolites, lubricants, dyes, petroleum residues, pharmaceuticals, amino acids, pigments,

polysaccharides, pesticides, herbicides, fungicides, surfactants, lipids, explosives, and other materials.

The hydrodynamic radius of a complex molecule is related to the force required to move the molecule through a liquid at a given speed and is defined as the radius of a sphere which requires the same force as the complex molecule to move at the same speed. It is known that hydrodynamic radius can be determined from a scattered light intensity fluctuation measurement of a sample containing the molecules of interest. In particular, hydrodynamic radius can be determined from fluctuations in scattered light intensity in a direction perpendicular to a light beam. The rate of such fluctuations is indicative of hydrodynamic radius. Prior art techniques have involved passing a laser beam having a wavelength in the visible range through a sample and measuring the fluctuations in scattered light intensity with a photomultiplier. Suitable lasers in the visible wavelength range are typically complex and expensive. As a result, systems using a visible wavelength laser and a photomultiplier are complex and expensive.

It is desirable to provide methods and apparatus for measuring the hydrodynamic radius of molecules which overcome the drawbacks of prior art systems.

Summarv of the invention According to a first aspect of the invention, an instrument for measuring hydrodynamic radius of molecules is provided. The instrument comprises a scattering cell, conduits for passing a liquid sample through the scattering cell, a semiconductor laser for directing a light beam through the scattering cell along an optical axis so that the light beam is scattered by the molecules of the sample and produces scattered light, and a detector assembly comprising an avalanche photodetector for detecting fluctuations in intensity of the scattered light in a direction perpendicular to the optical axis. The instrument further comprises a correlator for determining a correlation function representative of a characteristic decay time of the fluctuations in scattered light intensity and a processor for determining hydrodynamic radius of the sample molecules from the correlation function.

The detector assembly preferably further includes an active quench circuit for operating the avalanche photodetector in a region of high quantum efficiency. The active quench circuit includes means for pulsing the avalanche photodetector following detection of a photon, so that the avalanche photodetector is reset to a non-conducting state. The active

quench circuit may further include means for sensing a conducting state of the avalanche photodetector and means responsive to the conducting state for pulsing the avalanche photodetector until it returns to the non-conducting state.

The detector assembly may further comprise a mask defining a slit having a long dimension perpendicular to the optical axis and optics for focusing an image of a coherence region of the light beam on the slit. The detector assembly may further comprise an optical fiber for coupling light passing through the slit to the avalanche photodetector.

The semiconductor laser preferably has an output wavelength greater than about 650 nanometers and, in a preferred embodiment, has an output wavelength of about 800 nanometers. The semiconductor laser preferably has an output power level of about 100 milliwatts or greater.

The scattering cell is preferably configured to produce a substantially laminar flow of the liquid sample in the coherence region. This avoids the effects of turbulence on the measurement of hydrodynamic radius.

According to another aspect of the invention, a molecular characterization detector is provided for detecting fluctuations in scattered light intensity from molecules in a liquid sample. The molecular characterization detector comprises a scattering cell, conduits for passing a liquid sample through the scattering cell, a semiconductor laser for directing a light beam through the scattering cell along an optical axis so that the light beam is scattered by molecules of the sample and produces scattered light, and a detector assembly comprising an avalanche photodetector for detecting fluctuations in intensity of the scattered light in a direction perpendicular to the optical axis.

According to a further aspect of the invention, a method is provided for detecting fluctuations in scattered light intensity from molecules in a liquid sample. A liquid sample is passed through a scattering cell. A light beam from a semiconductor laser is directed through the scattering cell along an optical axis, so that the light beam is scattered by molecules of the sample and produces scattered light. Fluctuations in scattered light intensity in a direction perpendicular to the optical axis are detected with a detector assembly comprising an avalanche photodetector.

Brief Description of the Drawings For a better understanding of the present invention, reference is made to the

accompanying drawings, which are incorporated herein by reference and in which: Fig. 1 is a block diagram of an instrument for measuring hydrodynamic radius of molecules in accordance with an embodiment of the invention; Fig. 2 is an enlarged end view of the scattering cell of Fig. 1; Fig. 3 illustrates a coherence region viewed by the detector assembly of Fig. 1; Fig. 4 is a schematic diagram of an example of the active quench circuit of Fig. 1; Fig. 5 is a graph of avalanche photodetector voltage as a function of time; and Fig. 6 is a graph of received photons as a function of time.

Detailed Description A schematic diagram of an example of an instrument for measuring hydrodynamic radius of molecules in accordance with the invention is shown in Fig. 1. A scattering cell 10 contains a sample for measurement of hydrodynamic radius. The sample is in liquid form and flows continuously through an enclosed chamber 11 of scattering cell 10 from an inlet conduit 12 to an outlet conduit 14. The liquid sample is typically received from a liquid chromatography column. The liquid sample is typically a solvent containing a group of complex molecules to be measured. The scattering cell 10 includes a transparent window/lens 16 at one end and a transparent window/lens 18 or a window at the other end. The chamber 11 of scattering cell 10 preferably has a small volume, on the order of about 10 microliters, and may be cylindrical in shape. In a preferred embodiment, the chamber 11 of scattering cell 10 has a length of about 3 millimeters and a diameter of about 2 millimeters.

A laser 20 directs a laser beam 22 along an optical axis 24 through the chamber 11 of scattering cell 10. The laser beam 22 passes through window/lens 16, through the liquid sample, and through window/lens 18. The laser beam output from the scattering cell 10 may be detected for other measurements, as described in the aforementioned Patent No. 5,305,073, and is preferably intercepted by a beam dump 26 that is constructed to minimize reflections.

The laser 20 is preferably a semiconductor laser having an output wavelength greater than about 650 nanometers and a power level in the range of about 100 to 500 milliwatts. In a preferred embodiment, the laser 20 comprises a type SDL-53 11-G1 having a typical output wavelength of 800 nanometers. The liquid sample must be substantially transparent to the laser wavelength. The laser beam 22 that passes through the scattering cell 10 is preferably polarized, but is not required to be polarized. In a preferred embodiment, the laser 20

generates a polarized beam. The window/lens 16 focuses the laser beam 22 to its smallest diameter at a coherence region 30 in the scattering cell 10. The laser beam 22 may be focused by window/lens 16, by laser optics, or by combinations thereof as known in the art.

In order to obtain an accurate measurement of hydrodynamic radius, liquid flow through chamber 11 of scattering cell 10 should be relatively free of turbulence, at least in coherence region 30. As discussed below, the coherence region 30 is a small volume along optical axis 24 where the measurement of fluctuations in scattered light intensity is made. A view of one end of scattering cell 10 along optical axis 24 is shown in Fig. 2. In order to achieve a laminar flow that is relatively free of turbulence, the scattering cell 10 may be provided with a circular passage 40 connected to inlet conduit 12. Passages 42, 43 and 44 extend radially inwardly from circular passage 40 to one end of chamber 11. This ensures that the liquid sample is introduced into chamber 11 relatively uniformly. The outlet end of the scattering cell 10 may have a similar configuration, so that the liquid sample is exhausted from chamber 11 relatively uniformly. In a preferred embodiment, three radial passages are used at the inlet, and three radial passages are used at the outlet. It will be understood that different inlet and outlet configurations and different numbers of inlet and outlet passages may be utilized. The scattering cell 10 is designed to achieve a laminar flow in coherence region 30 that is relatively free of turbulence. Typically, coherence region 30 is located at or near the midpoint of the axis of chamber 11. The flow rate through scattering cell 10 is preferably less than about 2 milliliters per minute to provide measurements of hydrodynamic radius that are independent of flow rate.

Referring again to Fig. 1, the instrument for measuring hydrodynamic radius includes a detector assembly 50 for detecting fluctuations in intensity of scattered light from the liquid sample in a direction perpendicular to optical axis 24 and perpendicular to the direction of polarization of laser beam 22. In particular, the detector assembly 50 detects light scattered from molecules in the liquid sample in the coherence region 30. The detector assembly 50 provides an output to a correlator 200.

Optics, which may include a lens 52 and a lens 54, focuses an image of coherence region 30 on a slit 58 formed in a mask 60. The mask 60 may, for example, be fabricated of 302 stainless steel 0.0005 inch thick. As shown in Fig. 3, slit 58 has a long dimension perpendicular to optical axis 24 and is oriented such that light scattered from molecules in coherence region 30 in a direction perpendicular to optical axis 24 is incident on slit 58. In a

preferred embodiment, slit 58 has a width w of 18 micrometers. In this example, light beam 22 has a diameter of about 18 micrometers in coherence region 30. Thus, the coherence region 30 from which scattered light is detected has a length of 18 micrometers and a diameter of 18 micrometers. Preferably, the width of slit 58 and the diameter of light beam 22 in the coherence region 30 are approximately equal. Furthermore, the width of slit 58 and the diameter of light beam 28 should be as small as is practical in order to permit use of a relatively large area detector, so as to collect a relatively large amount of scattered light and obtain a large signal-to-noise ratio.

The detector assembly 50 further includes an optical fiber 70 having one end 72 positioned to intercept scattered light passing through slit 58. In a preferred embodiment, optical fiber 70 is an Amphenol type 907-11025-10001 having a core diameter of 62.5 micrometers and a cladding of 125 micrometers. End 72 of optical fiber 70 is preferably spaced from slit 58 by a distance d of about 2 millimeters. In a preferred embodiment, the distance d is adjustable. The scattered light collected by optical fiber 70 is output through a lens 76 and is focused on an avalanche photodetector 80.

The avalanche photodetector 80 is controlled by an active quench circuit 82. The avalanche photodetector and its operation are selected to provide high quantum efficiency at the wavelength of laser 20. The avalanche photodetector is a highly sensitive device in which one photon results in multiple electrons at the output. The avalanche photodetector is biased beyond breakdown, such that a single received photon initiates conduction. The avalanche photodetector is operated in the Geiger mode as a single photon detector. The active quench circuit 82 applies a voltage pulse that temporarily biases the avalanche photodetector below breakdown and stops conduction, effectively resetting the device for reception of another photon. Operation of the active quench circuit 82 is described in more detail below.

A schematic diagram of an example of the avalanche photodetector 80 and the active quench circuit 82 is shown in Fig. 4. In the example of Fig. 4, the avalanche photodetector 80 is a silicon avalanche photodiode, type C30902S-TC, supplied by EG&G. This device has a nominal breakdown voltage at a temperature of 0°C of about 170 to 230 volts and is biased at a voltage about 20 volts above the breakdown voltage when in a non-conducting state. At this bias voltage, the avalanche photodetector has a quantum efficiency of about 30% to 50%. The anode of avalanche photodetector 80 is coupled through a resistor 100 and a capacitor 102 to an inverting input of a high speed comparator 104. When a photon is detected, the avalanche

photodetector 80 begins to conduct, which raises the voltage on the non-inverting input of comparator 104. A transistor 110 coupled to the inverting input of comparator 104 limits the voltage rise. The output of comparator 104 falls, causing a transistor 112 to conduct and raising the anode voltage of avalanche photodetector 80 to about 24 volts, so that the voltage across the avalanche photodetector no longer sustains an avalanche current. The falling voltage at the output of comparator 104 causes a comparator 120 to generate a negative pulse starting on the falling voltage at its input. A comparator 124 coupled to the output of comparator 120 generates a positive pulse starting on the rising edge of the output of comparator 120. The output of comparator 124 turns on a transistor 130 and a transistor 132, thereby returning the input of comparator 104 and the anode of the avalanche photodetector 80 to a low level. At the end of the pulse generated by comparator 124, the avalanche photodetector is ready to detect another photon. The entire process requires about 0.2 microseconds. If the avalanche photodetector is not fully quenched after one pulse, a second pulse is generated by comparator 120. A comparator 140 coupled to the output of comparator 120 and a line driver 142 generate an output pulse to drive a coaxial line connected to correlator 200.

A graph of avalanche photodetector voltage as a function of time is shown in Fig. 5.

The avalanche photodetector is biased at a photon detection voltage VPD that is sufficiently above the breakdown voltage VBD to achieve high quantum efficiency. In the above example for a specific detector, VPD is about 230 volts and VBD is about 212 volts. When a photon is received, the avalanche photodetector conducts current and then is reset by the active quench circuit as described above. A reset pulse 144 is approximately 50 nanoseconds in duration and is of sufficient amplitude (approximately 24 volts) to return the avalanche photodetector voltage to its non-conducting state. As noted above, two or more reset pulses may be applied to the avalanche photodetector in the event that it continues to conduct.

The correlator 200 receives pulses representative of received photons from the output of active quench circuit 82 and determines a correlation function that is representative of a characteristic decay time of the fluctuations in scattered light intensity. The correlator 200 may be implemented as a digital signal processor programmed to perform a correlation operation. A FIFO buffer may be used to buffer the pulses received from the active quench circuit 82. A graph of received photons as a function of time is shown in Fig. 6. Pulses 210 represent received photons. The correlator 200 first determines the number of photons

received in each time interval AT, where AT is typically 5 microseconds. Thus, for example, with reference to Fig. 6, two photons were received in the first time interval, one photon was received in the second time interval, no photons were received in the third time interval, etc.

The correlation function is determined from the statistics as follows: g(k#T) = #nini+k (1) where g is the correlation function, kAT is the separation between two time intervals AT labeled i and i + k, and n and n + k are the numbers of received pulses in intervals i and i + respectively.

The correlation function, representative of fluctuations in scattered intensity, is supplied to a computer 240 which may, for example, be a personal computer (PC). The computer 240 is programmed to determine the hydrodynamic radius of molecules in the liquid sample in scattering cell 10 from the correlation function. The correlation function of the fluctuations in scattered light intensity in scattering cell 10 may be expressed as follows: where D is the diffusion constant, n is the index of refraction of the fluid, X is the wavelength of laser 20 and 6 is the scattering angle. Since the correlation function g is measured and all other quantities in equation (2) except the diffusion constant D are known, the diffusion constant D may be calculated from equation (2). The diffusion constant D is used to determine the hydrodynamic radius from the following equation: <BR> <BR> <BR> <BR> <BR> <BR> kB T <BR> <BR> D = (3) <BR> <BR> <BR> 6##rh where kB is Boltzmann's constant, T is temperature in degrees Kelvin, TI is the solution viscosity and rh is the hydrodynamic radius. The hydrodynamic radius rh is determined from equation (3), since all other quantities in equation (3) are known.

While there have been shown and described what are at present considered the preferred embodiments of the present invention, it will be obvious to those skilled in the art that various changes and modifications may be made therein without departing from the scope of the invention as defined by the appended claims.