Abstract:
An apparatus and related method for optical calibration of spectrophotometers is described. The apparatus is a calibration plate including one or more cuvettes filled with solutions of interest. The cuvettes are sealed to prevent evaporation. The cuvettes also possess a compressible component to allow for expansion of the solution and a bubble control apparatus to ensure that the compressible component does not intersect the beam path. A piece of neutral density glass is optionally included in the apparatus to track optical changes of the solutions over time.

Description:
CROSS-REFERENCE TO RELATED APPLICATION  
       [0001]     The present invention relates to U.S. patent application Ser. No. 10/021,112, entitled “PHOTOMETRIC CALIBRATION OF LIQUID VOLUMES” filed Dec. 12, 2001, now U.S. Pat. No. 6,741,365 issued on May 25, 2004, and owned by a common assignee. The contents of the related application are incorporated herein by reference. 
     
    
     FIELD OF THE INVENTION  
       [0002]     The present invention relates to a device and method to calibrate spectrophotometers. More particularly, the present invention is a calibration plate with one or more references of selectable absorbance characteristics.  
       BACKGROUND OF THE INVENTION  
       [0003]     Many analysis methods used in biology, chemistry, biotechnology, pharmaceutical and other industrial and research laboratories require accurate measurement and/or calibration of small volumes of liquids. These small volumes can range from nanoliters to microliters. Small volumes of liquid are dispensed from liquid delivery devices, such as pipettes, either sequentially or simultaneously into one or more vessels, such as cuvettes.  
         [0004]     In the interest of evaluating a large number of samples of liquid in a desired period of time, multiple charges of liquid samples may be dispensed into a plurality of vessels and analyzed simultaneously or sequentially. Preferably, the plurality of vessels is contained in one or more microtiter plates during testing. A microtiter plate contains a large number of individual wells and, for photometric measurements, a transparent base. Microtiter plates enable the ability to perform more research on a shorter time scale. As a result, they have become the standard analysis platform. The wide use of microtiter plates has resulted in the creation of entire classes of supporting equipment, including various types of analytical instrumentation, such as spectrophotometers capable of measuring individual wells within a plate.  
         [0005]     Under the photometric analysis method, the liquid sample under test is contained in the sample vessel and subjected to a light beam of a spectrophotometer. The amount of light from the incident light beam that passes through the vessel and the sample to an opposing detector of the spectrophotometer is dependent upon the characteristics of the liquid sample and the beam path length. It is important that the spectrophotometer provide accurate readings to ensure the accuracy of the liquid sample evaluation.  
         [0006]     The Beer-Lambert law defines one useful equation to determine an important characteristic of a sample. Specifically, Beer-Lambert states that the absorbance of light by a liquid sample under test equals the path length traversed by the light beam multiplied by the molar absorptivity of the chromophore in the liquid sample and the concentration of the chromophore in the liquid sample. Knowing the molar absorptivity of the chromophore, and the path length of light, the absorbance measurement provided by the spectrophotometer enables calculation of the particular concentration of the chromophore in the liquid sample. If the path length is not known, or known with sufficient accuracy, the concentration calculated will be approximate, and possibly outside a permitted error range. In addition, if the spectrophotometer is out of specification, the absorbance values obtained from the measurement will be in error and the calculated concentration also in error. It is therefore important to maintain an accurate calibration of the spectrophotometer.  
         [0007]     There are several types of spectrophotometers used to measure sample light absorbance. The conventional spectrophotometer transmits a light beam horizontally across the sample vessel. One type of specialized spectrophotometer transmits a light beam vertically through the sample vessel. In horizontal beam spectrophotometers, specific or fixed path lengths can generally be established, as the transmission path length is a function of the fixed cross section of the sample vessel rather than the volume of the sample in the vessel. However, the light transmission and detection method associated with a horizontal beam spectrophotometer is not suitable for use with a microtiter plate arrangement because a filled microtiter plate cannot be properly inserted into the horizontal beam path.  
         [0008]     Vertical beam spectrophotometers generally measure solution samples in microtiter plate wells. In one form, the vertical beam spectrophotometer transmits through the sample to a detector on the opposing side of the sample, either from above the sample down through it, or from below up through the sample. The path length for the sample is potentially—and likely—variable from one sample to the next in any given microtiter plate, since the path length is directly dependent upon the volume of sample solution in the well. Other factors that contribute to variations in path length are the ionic strength of the sample solution and the surface characteristics of the plate material, which together define the curvature characteristics of the solution meniscus. All of these factors result in an undefined path length and thus imprecision in vertical beam measurements. Nonetheless, vertical beam measurements are desirable in that multiple samples may be measured more quickly than is possible using a horizontal beam spectrophotometer.  
         [0009]     For a horizontal beam spectrophotometer, a common calibration approach used to determine the optical response for a given solution on a given instrument is to either create a series of dilutions of the solution of interest, or measure the solution in different, but known path lengths. In this approach, the ability to use a known path length allows for highly accurate determinations of the optical response of a solution and direct comparison of results from one spectrophotometer to another, or from one solution to another.  
         [0010]     One method presently used to calibrate horizontal and vertical beam spectrophotometers involves the measurement of light absorption through a known stable glass filter referred to as a Neutral Density (ND) filter. These filters are pieces of gray-tinted sheet glass that have nearly flat absorbance characteristics over a broad range of wavelengths. ND filters are commonly sent to reference laboratories for certified measurement, which provides results that are traceable to national standards. Once standardized, the ND filters are measured in the horizontal or vertical beam spectrophotometer and the absorbance results are compared to the reference laboratory results as a gauge of accuracy of the instrument.  
         [0011]     Although this is a common calibration method, a method that relies on ND filters alone gives little or no information about various optical effects such as: 1) out of band transmission (light passing from the source through the wavelength selection device and the sample to the detector, but outside of the desired wavelength range), 2) wavelength selection accuracy, 3) bandpass of the wavelength selection device, or 4) the shape of the transmission curve of the bandpass selection device. Therefore, reliance exclusively on instrument calibration using ND filters can lead to inaccurate absorbance results, especially when comparison is made between different instruments.  
         [0012]     One alternative method for calibrating vertical beam spectrophotometers involves the testing of samples of reference concentrations. Solutions containing different concentrations of the specific dye or chromophore are dispensed into different wells of a microtiter plate. Measurements of optical response are then conducted on the solution-filled wells. This method has several sources of error that limits its usefulness. First, the solution may not obey the Beer-Lambert law exactly, but may slightly deviate from a linear relationship between the concentration of dye and the resulting absorbance of the solution. Second, in order to provide quantitative results, highly accurate control over the amount of liquid dispensed into each well is required. Third, the exact dimensions of the wells in the microtiter plate must be known. Fourth, any meniscus present at the surface of the solution can add to the overall error since it directly affects the path length of light through the solution.  
         [0013]     Therefore, what is needed is an apparatus and related method for calibrating vertical beam spectrophotometers. The apparatus should be configured and arranged to be compatible with the arrangement of vertical beam spectrophotometers. The apparatus and method should allow the spectrophotometer operator to account for out of band transmissions, wavelength selection accuracy, the bandpass selection characteristics of the particular spectrophotometer, and the shape of the transmission curve of the bandpass selection. Further, the apparatus and method should resolve deviations in the linear relationship between the concentration of any reference dye in a sample under test and the resulting absorbance of the sample, be independent of the sample volume used to calibrate the spectrophotometer, allow accurate control over the path length, and eliminate meniscus errors in the light beam path.  
       SUMMARY OF THE INVENTION  
       [0014]     The present invention is an apparatus and related method for calibrating spectrophotometers. The apparatus is configured and arranged to be compatible with the arrangement of vertical beam spectrophotometers, but may also be used with horizontal beam spectrophotometers. The apparatus and method enable a spectrophotometer operator to account for out of band transmissions, wavelength selection accuracy, the bandpass selection characteristics of the particular spectrophotometer, and the shape of the transmission curve of the bandpass selection. Further, the apparatus and method enables resolution of deviations in the linear relationship between the concentration of any reference dye in a sample under test and the resulting absorbance of the sample. The apparatus and related method provide accurate control over the path length, independent of the sample volume used to calibrate the spectrophotometer. Finally, the present invention eliminates meniscus errors associated with vertical beam measurements. While the description of the present invention will be directed to its advantageous use in a vertical beam spectrophotometer, it may also be used to calibrate horizontal beam spectrophotometers as well.  
         [0015]     These and other features are achieved in the present invention through the arrangement of a calibration plate having one or more sealed calibration cuvettes, each cuvette containing a solution having one or more chromophores of selectable reference concentrations. The present invention provides the operator with the ability to calibrate a vertical beam spectrophotometer at wavelengths that correspond exactly with the solutions to be tested, rather than using the broad spectral response of ND glass. For example, if a specific dye is used as an absorbance indicator in an assay, a set of solutions with increasing concentration of the specific dye may be made and inserted into the cuvettes in the calibration plate. The calibration plate is then used to calibrate the optical response of the vertical beam spectrophotometer over the expected absorbance range of the specific dye. The potential exists for incorporating more than one dye in each cuvette, so long as the multiple dyes do not significantly overlap in their absorbance responses.  
         [0016]     The present invention provides further advantage in its stability as a standard. Specifically, by sealing a standard in a cuvette retained in the calibration plate, the present invention minimizes changes in the standard caused by its prolonged exposure to air. An expansion zone is maintained within the cuvette to allow for thermal expansion and contraction of the solution therein during any thermal cycling. The expansion zone may be established in a variety of ways, including the means to be described herein. An expansion zone isolator is preferably included in the cuvette to isolate the expansion zone from the portion of the cuvette exposed to the spectrophotometer beam. Options on the form of the isolator will be described herein.  
         [0017]     The cuvette is oriented in the calibration plate such that it presents to the vertically aligned light beam a fixed path length through the solution. Further, that cuvette orientation displaces any meniscus from the beam path. In effect, the present invention provides a fixed path length, defined by the cross section of the cuvette, to a spectrophotometer, whether a vertical beam type or a horizontal beam type.  
         [0018]     The calibration plate includes means for retaining one or more cuvettes. Each retained cuvette defines a test section of the plate and may include a unique chromophore solution for calibration. However, it is to be understood that a single test cuvette may be deployed in the calibration plate, and that multiple cuvettes may contain the same chromophore solutions. Each test section of the plate may be further defined by one or more transparent beam ports for the light beam to pass to and through the solution in the retained cuvette. Each port establishes a fixed path length cell based on the cuvette&#39;s cross section. The calibration plate optionally includes a test section including an ND glass filter for selectable comparison to that standard.  
         [0019]     The calibration plate of the present invention includes one or more cuvettes that allow for insertion of solutions of interest. The solutions may be user defined and unique to the user&#39;s interests, or standardized solutions available commercially. In the later case, the filled cuvettes could be referenced to a spectrophotometer in the manufacturer&#39;s facility in the manner described in the related patent. The solution-filled cuvettes function as optical standards for calibrating the spectrophotometer response, or can be used to establish a relation between a given concentration of a solution and the absorbance exhibited. The solutions retained therein, the sealed arrangement of the cuvette(s), and the arrangement of the cuvette(s) within the calibration plate provide an effective means for spectrophotometer calibration, while avoiding the deficiencies in the vertical beam spectrophotometer calibration techniques in existence.  
         [0020]     These and other advantages and aspects of the apparatus and method of the present invention will become apparent upon review of the following detailed description, the accompanying drawings, and the appended claims. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0021]      FIG. 1  is a simplified perspective view of the calibration plate of the present invention with a plurality of cuvettes and an ND glass filter shown in phantom.  
         [0022]      FIG. 2A  is a top view of a cuvette of the present invention showing the crimp top and a first expansion isolation means.  FIG. 2B  is a side view of the cuvette of  FIG. 2A   
         [0023]      FIG. 3  is a top view of a cuvette of the present invention showing the crimp top and a second expansion isolation means.  FIG. 3B  is a side view of the cuvette of  FIG. 3A .  
         [0024]      FIG. 4A  is a top view of a cuvette of the present invention showing the crimp top and a third expansion isolation means.  FIG. 4B  is a side view of the cuvette of  FIG. 4A .  
         [0025]      FIG. 5A  is a top view of a cuvette of the present invention showing the crimp top and an optional expansion means.  FIG. 5B  is a side view of the cuvette of  FIG. 5A .  
         [0026]      FIG. 6A  is a top view of the cuvette of  FIG. 5  showing the optional expansion means in a pressed form.  FIG. 6B  is a side view of the cuvette of  FIG. 6A . 
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0027]     A calibration plate  10  of the present invention is shown in  FIG. 1 . The calibration plate  10  includes a plurality of light beam ports  11  through which a light beam may pass from a top calibration plate side  12 , through one or more cuvettes  13  to a back side (not shown) of the calibration plate  10 . (Alternatively, the light beam may pass from the back side to the top calibration plate side  12 , dependent upon the operation of the particular spectrophotometer to be calibrated.) The frame of the calibration plate  10  that defines the structure of the calibration plate  10  is preferably fabricated of a rigid opaque material including, but not limited to, a metallic material, a rigid plastic material, or a combination thereof. The frame of the calibration plate  10  is preferably fabricated of two or more sections detachably connectable to one another. The cuvettes  13 , and an optional ND glass filter plate  14  are removably captured in the calibration plate  10 . Specifically, they may either be inserted into, or removed from, the calibration plate at a front end  15  thereof, or by connecting/disconnecting a top calibration plate  16  to/from a bottom calibration plate  17 .  
         [0028]     With continuing reference to  FIG. 1 , each cuvette  13  is fabricated of a transparent material, such as glass. The cuvette  13  includes a body  18  of fixed cross sectional dimensions, an expansion allowance zone  19 , a neck  20 , and a head with a sealing cap  21  that seals the contents of the cuvette  13  therein. The expansion allowance zone  19  is positioned with respect the body  18  such that when the calibration plate  10  is positioned in a vertical beam spectrophotometer, the light beam ports are positioned only over the body  18  and not over the expansion allowance zone  19 . Each cuvette  13  is preferably substantially filled with a liquid solution having a reference concentration or concentrations of one or more chromophores. Providing the calibration plate  10  with a plurality of cuvettes  13  allows for a range of absorbance values to be used to calibrate the optical response of a vertical beam spectrophotometer.  
         [0029]     The cuvettes  13  are formed in a substantially rectangular shape and formed with a flanged cuvette head to enable crimp sealing thereof. Hellma International of Plainview, N.Y., is capable of providing such a cuvette arrangement. The cuvettes  13  are placed flat in their long dimension within the calibration plate  10  when the calibration plate  10  is in use to allow the calibration solution to be sealed in the cuvette  13  for long periods of time. Unlike prior calibration cuvettes, the cuvettes  13  of the present invention are sealed by sealing cap  21  to minimize or substantially eliminate gas or fluid exchanges between the solution in the cuvettes and the external atmosphere. The sealing cap  21  is a crimp top seal applied with a crimping tool in a manner well known to those skilled in the art of crimping materials onto container tops. A nonporous film is applied to the cuvette head prior to affixing the crimp top to the cuvette  13 . The film is preferably a polymeric film such as Parafilm™, a plastic wrap material manufactured by Pechiney Plastic Packaging of Menasha, Wis. The crimp top itself is a layered liner fabricated of a combination of an interior nonmetallic material and an exterior metallic material. For example, Wheaton Corp. of Millville, N.J., provides a commercially available material that includes an Aluminum/Teflon/Grey Butyl combination suitable as the crimp top material. Inside the sealed cuvettes  13 , the expansion allowance zone  19  includes a compressible component that may be a bubble of gas, such as air. The compressible component is established in the cuvette  13  to allow for the solution to expand/contract due to thermal fluctuations.  
         [0030]     The compressible component must be held out of the light path defined by the ports  11 , or it will adversely affect the absorbance values measured. A bubble control apparatus  23  is therefore used to hold the compressible component in place in the expansion allowance zone  19 , out of the beam path near the top of the cuvette  13 . While the compressible component may be displaced within the body  18  of the cuvette  13  during transport, shaking of the calibration plate  10  in any manner commonly employed in the field of liquid analysis, and the positioning of the calibration plate  10  in a vertical orientation will move the compressible component into the expansion allowance zone  19  where it is retained in place once the calibration plate  10  is returned to a horizontal orientation by the bubble control apparatus  23 .  
         [0031]     As indicated, the ND glass filter  14  is an optional component of the calibration plate  10 . The ND glass filter  14  may be used to check the long-term stability of the solution filled cuvettes  13 . The ND glass filter  14  is preferably a gray-tinted sheet glass, which provides a non-changing, relatively flat absorbance over large portions of the visible spectrum. Unlike its application as a calibration device in and of itself, the optional ND glass filter  14  of the present invention is used mainly to test potential degradation of the solutions in the cuvettes  13 . Specifically, the operator of the spectrophotometer under calibration may track the test responses of the solution-filled cuvettes  13  versus the ND glass filter  14 , rather than using the ND glass filter  14  itself to calibrate the spectrophotometer. This is accomplished by establishing a relationship between the optical response of the ND glass filter  14  at a given wavelength, and the response of a solution-filled cuvette  13  at the same wavelength. This relation may be established by the manufacturer and passed to the user, or can be established directly by the user. The relationship can be a ratio or a simple difference of the absorbance measured at a given wavelength. As the calibration plate  10  is used in the field, a comparison may be made between the ND glass filter  14 /solution-filled cuvette  13  relationship measured on the date of manufacture and the relationship measured on the current day. The manufacturer or user can set tolerance limits, which will define whether one or more specific cuvettes  13  of a particular calibration plate  10  is out of specification and in need of recertification. Thus, the ND glass filter  14  is preferably optionally used as a standard reference point for checking the solution-filled cuvettes  13 .  
         [0032]     As illustrated in  FIGS. 2A and 2B , a first bubble control apparatus  30  is a shelf positioned adjacent to top wall  31 , spaced away from back wall  32 , and located between sidewalls  33  and  34  of the cuvette  13 . The shelf  30 , preferably fabricated of Teflon™, provides a physical barrier and holds the compressible component (bubble  19 ) in place mainly by surface tension. Alternatively, the shelf  30  may be fabricated of glass and fused to the interior of the cuvette  13 , such as on the interior of cuvette  13  between sidewalls  33  and  34  during cuvette manufacturing. For purposes of the description of this invention, a top wall is the portion of the cuvette  13  within the top calibration plate  16  that is adjacent to the light beam ports  11  when the cuvette  13  is in position in the calibration plate  10  as shown in  FIG. 1 , and a bottom wall is that portion of the cuvette  13  within the bottom calibration plate  17  that is adjacent to the underside of the calibration plate when the cuvette  13  is positioned therein. All references herein to a top wall or a bottom wall of the cuvette  13  are based on this orientation. As illustrated in  FIGS. 3A and 3B , a second bubble control apparatus  40  is a piece of tubing, preferably fabricated of silicone, spaced adjacent to the top wall  41 , away from the bottom wall  42 , and wedged into place between sidewalls  43  and  44 . It too operates as a physical barrier for entrapping the compressible component (again, in this instance, bubble  19 ). As illustrated in  FIGS. 4A and 4B , a third bubble control apparatus  50  is a bladder filled with the appropriate amount of gas to allow for solution expansion. In the device of  FIGS. 4A and 4B , the compressible component is contained within the bladder  50 , which extends within the cuvette  13  substantially from top wall  51  toward bottom wall  52 , about from sidewall  53  to sidewall  54 , and partially into neck  20 . The bladder  50  is sized to prevent its passage into the cuvette body  18 . Each one of these control mechanisms ensures that the compressible material will remain out of the light path during spectrophotometer calibration.  
         [0033]     A fourth bubble control apparatus  60  is shown in  FIGS. 5A-5B  and  6 A- 6 B. The apparatus  60  is a porous compressible or crushable material, such as foam. The material  60  wedged between cuvette top wall  61  and bottom wall  62 . It may also be spaced between sidewalls  63  and  64 , or spaced away therefrom. The bubble control apparatus  60  preferably extends into a portion of the neck  20 . It holds a majority of the necessary gas in its interior. Under pressure, it will either compress to a smaller overall volume and re-expand upon release of that pressure, if compressible, or it will be crushed to that smaller volume and remain that way if the material  60  is only crushable. A crushable material may be preferred for the purpose of making a rapid visual determination of solution expansion within the cuvette  13 . Specifically, as shown in  FIGS. 5A and 5B , under standard temperature, the solution within the cuvette  13  is of a certain volume that, in combination with the volume of the material  60 , essentially fills the cuvette  13 . When a change of temperature causes the solution in the cuvette  13  to expand, the material  60  is compressed, thereby reducing its volume, as shown in  FIGS. 6A and 6B . At all times the path length through the cross section of the cuvette  13  remains the same. It is to be understood that any of the cuvettes  13  may include a combination of any two or more of the bubble control apparatuses described herein. For example, the shelf  30  may be used to retain the bladder  50  in place, or the compressible material  60 . Alternatively, the tubing  40  may be used in place of the shelf  30  for the same purpose.  
         [0034]     A method of calibrating a vertical-beam spectrophotometer using the calibration plate  10  includes the steps of inserting the solution-filled cuvettes  13  into the calibration plate  10 , arranging the calibration plate  10  such that the compressible component is retained in the expansion allowance zone  18 , and inserting the calibration plate  10  into the spectrophotometer. The cuvettes  13  are preferably filled with one or more solutions having one or more chromophores of reference absorbance characteristics and concentrations. The spectral analysis is then performed and absorbance values for the solutions in the cuvettes  13  are obtained. Those values are then compared with the reference values. The spectrophotometer operation may then be adjusted as necessary to establish a match of measured and reference absorbance values. The measurements are preferably re-run after spectrophotometer adjustment to confirm the results. The ND glass filter  14  may be used to confirm the absorbance values for the solutions in the cuvettes  13  over time as described hereinabove.  
         [0035]     The present invention is an apparatus to calibrate the optical response of a vertical bean spectrophotometer. It allows for use of absorbance standards specific to the chromophores of interest. In effect, a chromophore commonly analyzed in assays may be used to create specific absorbance characteristics and the response thereto may be acquired using a vertical beam spectrophotometer. While the present invention has been described with particular reference to certain embodiments of the calibration plate  10  and the designs of the cuvette  13 , it is to be understood that it includes all reasonable equivalents thereof as defined by the following appended claims.