Patent Publication Number: US-6040190-A

Title: Method for determining the concentration C of an absorbent homogeneously distributed in a carrier

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application is a continuation of application Ser. No. 08/458,844, filed Jun. 2, 1995, now abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     The invention relates to a method for determining the concentration c of an absorbent homogeneously distributed in a carrier, in which the absorption a(λ) of the absorbent is measured while the value of spectral absorption ε(λ) is known, using Lambert-Beer&#39;s law a(λ)=ε(λ).c.d as a basis. 
     In a variety of applications it is necessary to determine the concentration c of a substance by means of an absorption measurement. Departing from Lambert-Beer&#39;s law 
     
         a(λ)=ε(λ).c.d 
    
     the concentration c is computed from the absorption measurement a(λ), the spectral absorption curve ε(λ) and the film thickness d being known. 
     On the other hand it is often necessary in such applications that the film thickness d be calibrated or at least checked at regular intervals, as any error would have its direct effects on the computed value calculated of the concentration c. Such calibrations or checks are usually performed with the use of a medium whose concentration and spectral absorption behavior are fully known. 
     Series production of such a calibrating medium therefore requires extreme precision in the fabrication process, which cannot always be achieved at a reasonable cost. Instead, every batch is carefully measured before leaving the production plant, and the target values obtained are noted on an enclosed data sheet. In this instance the responsibility for adequate calibration of the equipment rests with the user. The disadvantage of this method is that a number of new sources of error are introduced, such as enclosing a wrong data sheet in the instance of different batches, reading errors, wrong input data, etc. 
     DESCRIPTION OF THE PRIOR ART 
     The known procedures and measuring devices described below, which utilize Lambert-Beer&#39;s law to obtain the concentrations of individual sample components by measuring the absorption of the sample, are characterized by the disadvantages mentioned above, necessitating in every instance knowledge and regular calibration of the exact film thickness of the sample. 
     In AT-E 56 271, for example, a method for determining the concentrations of haemoglobin derivatives in whole blood is described, which differs from conventional multi-component analyses in that sample turbidities due to leucocytes, excess blood lipids, erythrocytes, etc., can be taken into account. 
     A similar type of multi-component analysis is disclosed in DE-A 42 03 587. The components in a sample are determined on the basis of the absorption capacity at fixed wavelengths in the absorption spectrum of the sample. In this context a hypothetical matrix is prepared beforehand for the purpose of determining concentrations, using a combination of reference spectra for a number of components whose concentrations are known. The concentrations of the components to be measured are computed with the use of this matrix, which will permit quantitative analysis to be performed at great precision within a very short time. 
     In the method described in CH-A 637 767, finally, the concentrations of substances are measured by adding a first indicator to the sample, which will respond to a change in the concentration of a substance to be measured by a spectral change, and by further adding a reference indicator, which will change the measurement light but will not be changed by the concentration of the substance to be measured. As mentioned before, all known methods require accurate knowledge of the measured film thickness. 
     SUMMARY OF THE INVENTION 
     It is an object of the invention to propose a method for determining the concentration of an absorbent distributed homogeneously in a carrier, which will avoid the above disadvantages and which, in particular, will not necessitate accurate knowledge of the film thickness of the sample to be measured. 
     In the invention this is achieved by employing, in the absence of a known value for the film thickness d of the carrier, an absorbent whose measured absorption a(λ) deviates from Lambert-Beer&#39;s law, in particular in the region of higher concentration, and by assuming n≧2 model components of the absorbent, so that 
     
         a(λ)=(ε.sub.1 (λ).c.sub.1 +ε.sub.2 (ε).c.sub.2 + . . . ε.sub.n (λ).c.sub.n).d, 
    
     and by measuring the absorption a(λ i ) for at least n wavelengths, and by computing the spectral absorption values ε i  (λ i ) and concentrations c i  of the model components, and, further, by determining the concentration c from the functional relationship between the concentration c and concentrations c i  of the model components c=f(c i ), which relationship is obtained by calibrating measurements. 
     With the method of the invention it is also possible of course to determine the film thickness d of the carrier without knowing the concentration c of the absorbent distributed in the carrier. 
     In preferred variants of the invention the proposal is put forward that the classes of substances indicated in the table below, or rather, their typical representatives are used as absorbents. 
     
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Class              Representatives                                        
______________________________________                                    
Triphenylmethane dyes                                                     
                   rhodamine                                              
   sulforhodamine                                                         
   crystal violet                                                         
   fluorescein                                                            
  Azo dyes butter yellow                                                  
   Bismarck brown (vesuvin)                                               
  Quinonoid dyes anthraquinone                                            
   naphthoquinone                                                         
   indanthrene                                                            
  Porphyrins and phthalocyanines oxazole                                  
  Cyanines merocyanine                                                    
  Polycyclic aromatic decacyclene                                         
  Hydrocarbons perylene                                                   
   naphthacene                                                            
  Indigo dyes indigo                                                      
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     DESCRIPTION OF THE DRAWINGS 
     Following is a more detailed description of the invention as illustrated by the accompanying drawings, in which: 
     FIG. 1 shows absorption spectra of calibrating solutions with different concentrations, 
     FIG. 2 the reference spectra for a two-component system, 
     FIG. 3 a diagram of the fictitious components c 1 , c 2 , plotted over the weighed concentration c, and 
     FIG. 4 the functional relationship between the absolute concentration c and the quotient of the hypothetical concentrations c 2  and c 1 . 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Various dyes, such as sulforhodamine B, show deviations from Lambert-Beer&#39;s law, especially in the region of higher concentrations. These deviations are characterized in that the measured absorption a(λ) does not rise in direct proportion to the concentration of the absorbing substance. FIG. 1 gives the absorption curves from 520 to 640 nm for nine different concentrations of the high-purity basic substance, which are represented by different graphic symbols. 
     It is obvious even to the naked eye that the left peak seems to grow more &#34;rapidly&#34; than the right. For this reason the entire family of curves cannot be described satisfactorily by a single component 
     
         a(λ)=ε(λ).c.d 
    
     even if there is only one basic substance. This image will be completely changed if a description with n fictitious components 
     
         a(λ)=(ε.sub.1 (λ).c.sub.1 +ε.sub.2 (λ).c.sub.2 + . . . ε.sub.n (λ).c.sub.n).d. 
    
     is chosen. By means of mathematical procedures (such as Singular Value Decomposition) it is possible to compute the spectral absorption curves ε(λ) of the individual fictitious components which are orthogonal to each other. For the sake of simplicity and without implying restrictions on the general case, the example of a two-component system, i.e. n=2, is described below (see reference spectra FIG. 2). 
     For this system the concentrations of the individual components are determined by means of the known methods of multi-component analysis, the assumption being that the number of measuring wavelengths m≧n. 
     If we analyze the data in more detail we will find that the two fictitious components c 1  and c 2  do not occur independently of each other, but that quite the reverse is true; if we determine the concentrations c 1  and c 2  for the dilution series and if we enter them as functions of the weighed concentration c of the basic substance, we obtain the diagram as in FIG. 3. 
     Each concentration c 1  thus goes with a precisely defined concentration c 2 , the quotient c 2  /c 1 , for example, being independent of the actual film thickness and containing only the information on the absolute concentration c of the basic substance. The functional relationship c=f(c 2  /c 1 ) is shown in FIG. 4. 
     In practice this function could be represented by a polynomial, a table or similar means. 
     The information on the actual film thickness is contained in the computed concentrations c 1  and c 2 . Assuming that the reference spectra were obtained with the normal film thickness do being precisely known, we have a uniquely defined function c 1  =g(c), which describes the relationship between the concentration of the basic substance c and the corresponding concentration c 1 . A deviation of the computed concentration c 1  from the expected value thus is directly proportional to the deviation of film thickness from the normal value do. We have ##EQU1## with dact . . . actual film thickness 
     d o  . . . normal film thickness 
     c 1  measured . . . result of the multi-component analysis 
     c 1  expected . . . from the function c 1  =g(c) with c=f(c 2  /c 1 ) 
     This will open up the, at first sight surprising, possibility of calibrating the film thickness of a cuvette with a dye of unknown concentration. This method may be implemented as a fully automatized procedure, i.e., without any interference by the user, so that the errors mentioned at the beginning of this paper are avoided.