Abstract:
A multiple-vial, rotating, sample container assembly for Raman spectroscopy comprises a container with two or more receptacles formed therein, which are suitable for positioning two or more vials inside the sample measurement area of a spectrometer. The openings are located such that when the container is rotated, the vials inside the holder are alternately positioned in the laser beam path, and the Raman scattering from each sample material is co-collected during the same measurement period. The rotation of the container (RPM) is sufficiently fast so that the material in each vial is measured many times during a sampling period, thereby ensuring a high degree of reproducibility in measuring the combination of vials. For a quantitative or peak comparison method, one vial contains a reference material. This material may be pure (100% of a compound), a dilution of the material in a solvent (such as water), or a combination of materials. Another vial contains the sample, or material to be evaluated.

Description:
[0001]    This application claims priority to U.S. Provisional Patent Application Ser. No. 61/720329, titled, “Multiple-Vial, Rotating Sample Container Assembly for Raman Spectroscopy,” filed Oct. 30, 2012, the disclosure of which is incorporated herein by reference in its entirety. 
     
    
     FIELD OF INVENTION 
       [0002]    The present invention relates generally to Raman spectroscopy, and in particular to a multiple-vial, rotating, sample container assembly allowing for the co-collection of Raman spectra from two or more materials. 
       BACKGROUND 
       [0003]    Raman spectroscopy is an analytic instrumentation methodology useful in ascertaining and verifying the molecular structures of materials. Raman spectroscopy relies on inelastic scattering, or Raman scattering, of monochromatic light, resulting in an energy shift in a portion of the photons scattered by a sample. From the shifted energy of the Raman scattered photons, vibrational modes characteristic to a specific molecular structure can be ascertained. In addition, by analytically assessing the relative intensity of Raman scattered photons, the concentration of a sample can be quantitatively determined. 
         [0004]    Typically, a sample is illuminated with a laser beam. Light from the illuminated spot is collected by lenses and analyzed. Wavelengths close to the laser line due to elastic Rayleigh scattering are blocked or filtered out, while chosen bands of the collected light are directed onto a detector. 
         [0005]    The Raman effect occurs when light impinges upon a molecule and interacts with the electron cloud and the bonds of that molecule. For the spontaneous Raman effect, which is a form of light scattering, a photon excites the molecule from its ground state to a virtual energy state. The energy state is referred to as virtual since it is temporary, and not a discrete (real) energy state. When the molecule relaxes, it emits a photon and it returns to a different rotational or vibrational state. The difference in energy between the original state and this new state leads to a shift in the emitted photon&#39;s frequency away from the excitation wavelength. 
         [0006]    If the final vibrational state of the molecule is more energetic than the initial state, then the emitted photon will be shifted to a lower frequency in order for the total energy of the system to remain balanced. This shift in frequency is known as a Stokes shift. If the final vibrational state is less energetic than the initial state, then the emitted photon will be shifted to a higher frequency, which is known as an Anti-Stokes shift. Raman scattering is an example of inelastic scattering because of the energy transfer between the photons and the molecules during their interaction. 
         [0007]    The pattern of shifted frequencies is determined by the rotational and vibrational states of the sample, which are characteristic of the molecules. The chemical makeup of a sample may thus be determined by an analysis of the Raman scattering. 
         [0008]    For quantitative Raman analysis, normalization of the scattered spectra using a constant or standard peak is recommended. See U.S. Pharmacopeial Convention (USP) Monograph 1120, “Raman Spectroscopy Theory and Practice,” the disclosure of which is incorporated herein by reference in its entirety. Several approaches to providing such normalization are known. 
         [0009]    One approach is to mix an excipient or solvent with the sample. The excipient or solvent is ideally selected to exhibit a Raman spectroscopic peak which essentially remains unchanged (i.e., constant intensity) as the analyte concentration in the sample varies. However, mixing materials with the sample in one container presents numerous problems. Raman spectroscopy results may vary due to inaccuracies in dispensing and mixing of the different materials. Additionally, potential undesired chemical reactions between the materials may occur, altering the sample&#39;s composition and/or concentration. 
         [0010]    Another approach to providing a reference Raman spectroscopic peak is to pass the excitation laser beam through a reference window comprising or impregnated with the reference material. Raman scattered photons are collected from both the sample and the reference window. However, this approach also has known deficiencies. Raman scattered photons typically comprise less than one part per million of the optical return from an excitation laser beam, and consequently already exhibit a low Signal to Noise Ratio (SNR). Passing Raman scattered photons from the analyte through a filter attenuates the optical signal, reducing the SNR even further. This requires a more sensitive detector, and/or more sophisticated signal processing, to achieve a sufficiently strong signal. 
         [0011]    The background section of this document is provided to place embodiments of the present invention in technological and operational context, to assist those of skill in the art in understanding their scope and utility. Unless explicitly identified as such, no statement herein is admitted to be prior art merely by its inclusion in the Background section. 
       SUMMARY 
       [0012]    The following presents a simplified summary of the disclosure in order to provide a basic understanding to those of skill in the art. This summary is not an extensive overview of the disclosure and is not intended to identify key/critical elements of embodiments of the invention or to delineate the scope of the invention. The sole purpose of this summary is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later. 
         [0013]    According to one or more embodiments described and claimed herein, a multiple-vial, rotating, sample container assembly for Raman spectroscopy comprises a container with two or more receptacles formed therein, which are suitable for positioning two or more vials inside the sample measurement area of a spectrometer. The openings are located such that when the container is rotated, the vials inside the holder are alternately positioned in the laser beam path, and the Raman scattering from each sample material is co-collected during the same measurement period. The rotation of the container (RPM) is sufficiently fast so that the material in each vial is measured many times during a sampling period, thereby ensuring a high degree of reproducibility in measuring the combination of vials. For a quantitative or peak comparison method, one vial contains a reference material. This material may be pure (100% of a compound), a dilution of the material in a solvent (such as water), or a combination of materials. Another vial contains the sample, or material to be evaluated. 
         [0014]    In one embodiment, by using a series of samples with known concentrations of the material of interest, a calibration curve may be constructed using the ratio of Raman peaks. Using this calibration relationship, an unknown sample may be tested and the concentration determined by measuring the sample using the same device and reference standard. Either Raman peak heights or peak areas may be used in this determination. 
         [0015]    One embodiment relates to a Raman spectroscopy system. The system includes an excitation laser source operative to selectively generate an excitation laser beam in a fixed position; an optical system operative to collect Raman scattered photons from material excited by the laser beam; a detector positioned and operative to detect Raman scattered photons collected from the material; a data processor operative to analyze the spectra of Raman scattered photons detected by the detector; and a rotating container having at least two receptacles formed therein, each receptacle operative to hold a vial containing material to be analyzed by the Raman spectroscopy system, the receptacles arranged to alternately pass each vial over the excitation laser beam as the container rotates. 
         [0016]    Another embodiment relates to a method of performing Raman spectroscopy on two or more different materials simultaneously. An excitation laser source operative to selectively generate an excitation laser beam in a fixed position is provided. At least two materials, each in a vial disposed in a rotating container, are also provided. The container is rotated such that each vial is alternately illuminated by the excitation laser beam as the container rotates. Raman spectroscopy is performed on an optical signal generated by the excitation laser alternately illuminating each material as the container rotates. 
         [0017]    Yet another embodiment relates to a non-transient computer readable media storing program instructions operative to control a Raman spectroscopy system. The system includes an excitation laser source operative to selectively generate an excitation laser beam in a fixed position, and at least two materials, each in a vial disposed in a rotating container. The program instructions are operative to cause a controller to control mechanical means to rotate the container such that each vial is alternately illuminated by the excitation laser beam as the container rotates; and perform Raman spectroscopic analysis on an optical signal generated by the excitation laser alternately illuminating each material as the container rotates. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0018]    The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. However, this invention should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Like numbers refer to like elements throughout. 
           [0019]      FIG. 1  is a perspective view of a multiple-vial, rotating, sample container assembly. 
           [0020]      FIG. 2  is a perspective view of a multiple-vial, rotating, sample container assembly depicting the containers both in and out of the assembly. 
           [0021]      FIG. 3  is a perspective view of the bottom of the multiple-vial, rotating, sample container, with a laser path illustrated. 
           [0022]      FIG. 4  is a section view of the multiple-vial, rotating, sample container assembly and Raman spectrometer and optical system. 
           [0023]      FIG. 5  is a representative Raman spectrograph of two different concentrations of sample material and a common reference material. 
           [0024]      FIG. 6  is graph of a calibration curve obtained using the multiple-vial, rotating, sample container assembly. 
       
    
    
     DETAILED DESCRIPTION 
       [0025]      FIG. 1  depicts a multiple-vial, rotating, sample container assembly  10  for performing Raman spectroscopy of a desired sample and a reference material simultaneously. The assembly  10  comprises a multiple-vial, rotating, sample container  12 . The container  12  is preferably formed from a material with low Raman activity, such as black Delrin®. A first cylindrical hole is formed in the container  12 , forming a receptacle  14  for a corresponding first vial  16 . Also formed in the container  12  is a second cylindrical hole, forming a receptacle  18  for a corresponding second vial  20 . The vials  16 ,  20  are made from a suitable material, such as borosilicate glass, quartz, or clear plastic, for Raman measurements. 
         [0026]    The receptacles  14 ,  18  and corresponding vials  16 ,  20  are preferably distinct, such as being of different diameters, as depicted in  FIG. 1 . Of course, the receptacles  14 ,  18  could be differentiated in other ways—for example, one hole could be square, triangular, or another shape unique from the other hole, with the vials  16 ,  20  correspondingly shaped. However, since cylindrical vials  16 ,  20  are common and available in a variety of sizes, the preferred embodiment features cylindrical receptacles  14 ,  18 , distinguished by size.  FIG. 2  is a view of the vials  16 ,  20  outside of the container  12 , as well as within it. 
         [0027]    The receptacles  14 ,  18 , and corresponding vials  16 ,  20  are preferably differentiated to reduce errors in performing calibrated Raman spectroscopy. By establishing a standard protocol—for example, reference material is always placed in the smaller vial  20 , and sample material is always placed in the larger vial  16 —more consistent results may be expected, and sample and reference materials are handled and stored consistently. However, differentiation of the receptacles  14 ,  18  is not a critical feature of the present invention, and in other embodiments, holes of the same size and shape may be formed in the multiple-vial, rotating, sample container  12 . 
         [0028]      FIG. 3  depicts a bottom view of the multiple-vial, rotating, sample container assembly  10 . As the container  12  rotates, a fixed excitation laser beam  22  traces out a circular path  24  on the bottom surface  13  of the container  12 . The laser beam incident path  24  passes repeatedly over each hole; Hence, as the container  12  rotates, the excitation laser beam  22  will enter the corresponding vials  16 ,  20 , exciting materials contained therein for Raman spectroscopy. The present invention is not limited to holding two vials  16 ,  20 . In other embodiments (not shown in the figures), the sample container  12  may hold three, four, or more vials, with each being arranged and configured to lie within the path  24  traced by an excitation laser beam  22  as the container  12  rotates during Raman spectroscopy. 
         [0029]      FIG. 4  is a section view of the multiple-vial, rotating, sample container assembly  10 , including a representative optical system  30 . Each receptacle  14 ,  18  is formed by a hole extending, at a constant diameter, to nearly, but not completely, the bottom surface  13  of the container  12 . At the bottom of the receptacle  18 , a through-hole  28 , of a smaller diameter, extends through the bottom surface  13  of the container  12 , leaving an annular rim  26 . The annular rim  26  supports the corresponding vial  20 , while the through-hole  28  allows for excitation of material in the vial  20  by laser beam  22 , and the collection of Raman scattered photons from the material within the vial  20 . Receptacle  14  is constructed similarly; the corresponding parts are not numbered in  FIG. 4  for clarity. 
         [0030]      FIG. 4  depicts a representative optical system  30  of a Raman spectrometer  32 . A laser source  34  generates an excitation laser beam  22 . The excitation beam  22  is reflected by a dichroic mirror  36 , and directed toward the container  12  so as to trace a circular path  24  when the container  12  rotates (see  FIG. 3 ). The excitation beam  22  passes through an assembly  38  of lenses. The collimated excitation beam  22  has a small diameter compared to the lenses  38 . It passes through the center of the lenses  38  where the excitation beam  22  is normal to the lens surfaces and experiences little refraction, thus remaining substantially collimated. Additionally, the excitation beam  22  has a very small “dot” of cross-section area, and the lenses  38  do little to focus or otherwise optically alter the excitation beam  22 . 
         [0031]    The lens assembly  38  has a fixed focus point configured to lie within a vial  16 ,  20  when the corresponding hole  28  is positioned over the optical system  30 . As one non-limiting example, the lens assembly  38  may comprise a two-element inverse Galilean Telescope lens system, comprising anti-reflection coated quartz elements. At the focal point of the lens assembly  38 , Raman scattering may be modeled as a point source optical phenomenon, with isotropic emission. Raman scattered photons are collected from the focal point as an optical signal, the envelope of which is depicted in  FIG. 4 . This optical signal passes through the dichroic mirror  38 , and is focused by lenses to a point, where it passes through a spectrometer aperture slit  40 , which isolates the interior of the spectrometer  32  (in particular, the detector  46 ) from extraneous photons. In one embodiment, a laser rejection dichroic filter  42  blocks photons at the wavelength of the excitation laser beam  22 . This removes most non-Raman scattered photons (e.g., Rayleigh scattered photons), which have the same wavelength as the excitation laser beam  22 , from the optical signal, thus enhancing the signal to noise ratio (SNR) of the Raman spectroscopy signal. 
         [0032]    A transmission grating  44  then directs the collected, Raman scattered photons to a detector  46 . In one embodiment, the transmission grating  44  is a holographic transmission grating comprising a transparent window with periodic optical index variations, which diffract different wavelengths of light from a common input path into different angular output paths. In one embodiment, the holographic transmission grating  44  comprises a layer of transmissive material, such as dichromated gelatin, sealed between two protective glass or quartz plates. The phase of incident light is modulated, as it passes through the optically thick gelatin film, by the periodic stripes of harder and softer gelatin. In another embodiment, the transmission grating  44  comprises a “ruled” reflective grating, in which the depth of a surface relief pattern modulates the phase of the incident light. In all embodiments, the spacing of the periodic structure of the transmission grating  44  determines the spectral dispersion, or angular separation of wavelength components, in the diffracted light. In one embodiment, the detector  46  comprises a charge-coupled device (CCD) array. The detector  46  converts incident photonic energy to electrical signals, which are processed by readout electronics  48 . 
         [0033]    The spectroscopy data from the readout electronics  48  are analyzed by a signal processor  50 , such as an appropriately programmed Digital Signal Processor (DSP) or other microprocessor, also operatively connected to memory  52 . Data representing the processed Raman spectra may be stored, output to a display, transmitted across a wired or wireless network, or the like, as known in the art. In addition to analyzing Raman spectra data, the signal processor  50 —or another processor (not shown in FIG.  4 )—may additionally control the overall operation of the spectrometer  32 , including initialization, calibration, testing, control of mechanical means for rotating the container  12  (not shown), automated data acquisition procedures, user interface operations, remote communications, and the like. The memory  52  may comprise any non-transient machine-readable media known in the art or that may be developed, including but not limited to magnetic media (e.g., floppy disc, hard disc drive, etc.), optical media (e.g., CD-ROM, DVD-ROM, etc.), solid state media (e.g., SRAM, DRAM, DDRAM, ROM, PROM, EPROM, Flash memory, etc.), or the like. The memory  52  is operative to store program instructions  54  operative to implement the functionality described herein, as well as general purpose control functions for analytical instrumentation, as well known in the art. 
         [0034]    The Raman spectrometer  32  and its optical system  30  as depicted in  FIG. 4  are representative only, and the above description is provided only to enable those of skill in the art to practice embodiments of the present invention. Specific details of the spectrometer  32  and the optical system  30  are not critical features of the present invention. Any optical system  30  capable of directing an excitation laser  22  through the holes  28  of the container  12 , and collect Raman scattered photons from within vials  16 ,  20 , and any spectrometer  32  capable of performing Raman spectroscopy, may be advantageously employed in embodiments of the present invention. 
         [0035]    Not depicted in  FIG. 4  is a mechanism for spinning the multiple-vial, rotating container  12 . Rotating mechanisms are well known in the art, and any means of imparting rotary motion to the container  12  may be employed. In one embodiment, the container  12  is sized and shaped so as to be inserted into the rotating sample chamber of a Verifier™ Tri-Test 1000 (VTT-1000) analytical instrumentation system, available from Mustard Tree Instruments of Research Triangle Park, N.C. The multiple-vial, rotating container  12  is preferably rotated at a speed greater than 100 revolutions per minute (RPM). 
         [0036]    An additional benefit to rotating the vials  16 ,  20  into and out of the contact point of the excitation laser beam  22  is that the potential for deleterious thermal effects is minimized, as compared to a prior art Raman spectroscopy technique, where an excitation laser beam is concentrated on a single spot for an extended period of time. Deleterious thermal effects may include degradation of the material, phase change, oxidation/explosion, or the like. 
         [0037]    In one embodiment, the multiple-vial, rotating Raman spectroscopy assembly  10  is useful in analytically determining the concentration of analyte in a sample of material. The concentration is determined by the relationship between the size of a Raman peak characteristic of the analyte and a reference peak, the latter caused by a reference material. In a series of Raman spectroscopy runs, the concentration of analyte in a sample in one vial  16  is varied. At each concentration, Raman spectroscopy is performed of the sample and a reference material in the other vial  20 .  FIG. 5  depicts representative Raman spectra for two such runs (i.e., two different concentrations of analyte in the sample in vial  16 . 
         [0038]    Raman shifts are typically described as wavenumbers, which have units of inverse length. A wavenumber relates to frequency shift by 
         [0000]    
       
         
           
             
               Δ 
                
               
                   
               
                
               w 
             
             = 
             
               ( 
               
                 
                   1 
                   
                     λ 
                     0 
                   
                 
                 - 
                 
                   1 
                   
                     λ 
                     1 
                   
                 
               
               ) 
             
           
         
       
     
         [0000]    where
       w is the wavenumber;   λ 0  is the wavelength of the excitation laser beam  22 ; and   λ 1  is the wavelength of the Raman scattered photon.       
 
         [0042]    Quantitative analysis of the concentration of analyte is determined by the following equations. First, the intensity of a sample peak is proportional to the concentration of analyte in the sample: 
         [0000]        I   S ∝[ C   S ]  (1)
 
         [0000]    However, the intensity of the reference peak is constant (k)—nothing about the reference material in vial  20  changes between Raman spectroscopy runs: 
         [0000]      I R =k   (2)
 
         [0000]    Finally, the ratio of a sample peak to the reference peak indicates the concentration of analyte in a sample: 
         [0000]    
       
         
           
             
               
                 
                   
                     
                       I 
                       S 
                     
                     
                       I 
                       R 
                     
                   
                   → 
                   
                     [ 
                     
                       C 
                       S 
                     
                     ] 
                   
                 
               
               
                 
                   ( 
                   3 
                   ) 
                 
               
             
           
         
       
     
         [0000]    where I S  is the intensity of a sample peak,
       I R  is the intensity of the reference peak,   [C S ] is the concentration of analyte in the sample, and   k is a constant.       
 
         [0046]    A series of calibration samples, comprising known concentrations of the analyte, may be measured and the ratio of the sample peak to the reference peak may be plotted, yielding the graph of  FIG. 6 . An unknown sample may then be measured, and the ratio of the two peaks determined from the calibration curve. Using the linear relationship between peak ratios and concentration, the amount of analyte in the unknown sample may be determined. 
         [0047]    Embodiments of the present invention present numerous advantages over the prior art. Raman spectra may be captured from both a sample and a reference material at the same time, without mixing the materials. Thus, chemical reactions between them are not a concern. Furthermore, the Raman signal (which is always weak, comprising only approximately 1% of all scattered photons) is not degraded by passing it through a gel filter to collect spectra from a reference material. The spinning container also minimized thermal effects potentially caused by the excitation laser beam. 
         [0048]    The present invention may, of course, be carried out in other ways than those specifically set forth herein without departing from essential characteristics of the invention. The present embodiments are to be considered in all respects as illustrative and not restrictive, and all changes coming within the meaning and equivalency range of the appended claims are intended to be embraced therein.