Abstract:
A chromatography system including a chromatography column connected to receive a sample and separate different compounds in the sample, a flow-through detector that outputs a signal indicating the presence of compounds in the sample leaving the column, a fraction collector that directs fractions of the sample from the column to separate fraction wells, and an electronic controller that receives inputs from the detector and controls the fraction collector. The electronic controller monitors the signal from the detector over time and initiates a cut from one well to another at the fraction collector when the signal over time experiences a shoulder where the slope of the signal over time does not change in sign but does change by more than a predetermined amount.

Description:
BACKGROUND  
         [0001]    The invention relates to chromatographic separation and fractionation.  
           [0002]    A chromatography column can be used to separate different compounds in a sample being flushed through the column in a solvent. The different compounds in the sample are delayed in passage through the column to different extents and exit the column at different times. A fraction collector directs a first part of the sample leaving the column to one fraction-collecting well, the second part to a second well and so on. A detector can be used to detect absorption or another characteristic of the sample during the processing of the sample downstream of the column and upstream of the fraction collector in order to detect the presence of different compounds in the stream from the column and to decide when to begin and terminate collection, and when to advance the fraction collector to a new well.  
           [0003]    In a chromatography system available from the Biotage Inc. under the Parallex Flex and Horizon designation, sampling can be initiated and terminated based upon the UV absorbance values or based upon the slopes of these values over time. A fraction cut can be triggered by collected volume (i.e., when a well has been filled to capacity) and by detection of a valley between peaks based upon detection of a change in slope from a negative value to a positive value.  
         SUMMARY  
         [0004]    In one aspect, the invention features, in general, a chromatography system including a chromatography column connected to receive a sample and separate different compounds in the sample, a detector that outputs a signal indicating the presence of compounds in the sample leaving the column, a fraction collector that directs fractions of the sample from the column to separate fraction wells, and an electronic controller that receives inputs from the detector and controls the fraction collector. The electronic controller monitors the signal from the detector over time and initiates a cut from one well to another at the fraction collector when the signal over time experiences a shoulder where the slope of the signal over time does not change in sign but does change by more than a predetermined amount, and the slope is negative.  
           [0005]    In another aspect, the invention features, in general, a chromatography system including a chromatography column, a flow-through detector, and a fraction collector as already described. The system also includes an electronic controller that monitors the signal from the detector over time and initiates a cut from one well to another at the fraction collector when the slope is positive and experiences a shoulder as determined by a positive slope at one point in time that is less than a slope threshold, and a positive slope at a later time that is greater than X times the slope threshold, where X is greater than one (preferably greater than 1.5 and most preferably between 1.5 and 2.5).  
           [0006]    In another aspect, the invention features, in general, a chromatography system including a chromatography column, a flow-through detector, and a fraction collector. The system also includes an electronic controller that monitors the signal from the detector over time and initiates a cut from one well to another at the fraction collector when the signal is less than a slope detection disable threshold and the signal over time experiences a specified change in slope condition.  
           [0007]    In another aspect, the invention features, in general, a chromatography system including a chromatography column, a flow-through detector, and a fraction collector. The system also includes an electronic controller that monitors the signal from the detector over time and identifies valleys in the signal from the detector over time. The system also includes a user interactive display and input device that permits the user to select whether or not to collect during valleys in the signal over time, and the controller either ends collection after identifying a valley or causes a fraction cut at a set time.  
           [0008]    In another aspect, the invention features, in general a chromatography system including a chromatography column, a flow-through detector, a fraction collector, and an electronic controller that monitors the signal from the detector over time. The system also includes a user interactive display and input device that permits the user to select to operate in a custom mode in which the user determines setpoint conditions for initiating and ending collection or to operate in one of a set of preset collection modes in which the setpoint conditions for initiating and ending collection are preset.  
           [0009]    Preferred embodiments of the invention may include one or more of the following features. In preferred embodiments the electronic controller determines if the signal is in a valley by the slope being positive at a set time delay past the cut when the slope is negative. The controller can cause the fraction collector to move to a new well after the set time delay. Alternately the controller can end collection after the set time delay. The system includes a user interactive display and input device that permits the user to enter the slope threshold, the slope disable detection threshold, the set time delay, and a threshold value for the signal for the controller to initiate and stop collection. The detector emits a signal measuing characteristics of the compound. For example, a UV absorbance detector emits a signal measuring the UV absorbance.  
           [0010]    Embodiments of the invention may include one or more of the following advantages. The detection based on UV slope and changes in UV slope allows for more fine tuned control of the fractionation process, leading to higher purity in the chemical compounds collected and higher efficiencies. The user can enter various data to control the stop/start and cut point decision analysis, or can alternatively rely on preselected modes of operation.  
           [0011]    Other advantages and features of the invention will be apparent from the following description of a particular embodiment and from the claims. 
       
    
    
     DESCRIPTION OF DRAWINGS  
       [0012]    [0012]FIG. 1 is a block diagram of an automatic chromatography system.  
         [0013]    FIGS.  2 - 4  are representations of interactive screen displays on a monitor of the FIG. 1 system.  
         [0014]    [0014]FIG. 5 is a graph of UV absorbance versus time illustrating various conditions for initiating and terminating collection and for making a fraction cut based upon the values of UV absorbance, the slope of UV absorbance over time, and the change in slope of UV absorbance over time. 
     
    
     DETAILED DESCRIPTION  
       [0015]    Referring to FIG. 1, there is shown chromatography system  10  including source of solution  12 , sample introduction port  14 , pump  16 , chromatography column  18 , flow-through UV detector  20 , fraction collection system  22 , waste  24 , fraction collection wells  26 , controller  28  (e.g., a personal computer), monitor  30 , and keyboard user input  32 .  
         [0016]    In operation, a solution  12 , including a sample introduced therein at port  14 , is pumped by pump  16  through chromatography column  18 . The different compounds in the sample are delayed in passage through the column  18  to different extents and exit the column at different times. The solution, with the sample compounds therein at least partially separated from each other, passes through UV detector  20 , which provides a signal indicating the absorbance of UV light by the solution passing therethrough to controller  28 . The solution then continues to fraction collection system  22 . Controller  28  controls fraction collection system  22  to direct the solution to waste  24  or a well  26  and to move from one well  26  to another, based upon the UV absorbance signals from UV detector  20 . Controller  28  takes as inputs the UV absorbance signals at one or two times per second. Other data rates can be used.  
         [0017]    A user can specify the operation of the fraction collection system in response to the UV absorbance signals via a graphical interactive user interface provided by monitor  30  and keyboard  32 . Referring to FIG. 2, there is shown menu  34 , which is displayed on monitor  30 . Menu  34  includes eight selection buttons  36 - 50  to permit the user to select the “collect all” mode with button  36 , one of six automatic operation modes with buttons  38 - 48 , or a custom user mode of operation with button  50 . In the “collect all” mode, fraction collection system  22  continuously collects all solution from cartridge  18  and moves from one collection well  26  to another based upon the volume discharged.  
         [0018]    In the threshold low, threshold medium or threshold high modes, selected by buttons  38 ,  40  and  42 , the fraction collection system  22  begins directing solution from cartridge  18  to a collection well  26  after controller  28  determines that the UV signal from detector  20  has gone above a UV absorbance value threshold T v  and will continue to discharge the solution to a collection well  26  as long as the sensed UV absorbance remains above T v . In these modes the fraction collection system  22  also moves from one collection well  26  to another based upon the volume discharged. In the three modes, the values for T v  are different, with the lowest being in the threshold low mode and the highest being in the threshold high mode. These modes of operation are considered value-based collection, because collection is based on the absorbance value. When the solution is not being directed to the wells  26  for collection, it is instead directed to waste  24 .  
         [0019]    The threshold low button  38 , the threshold medium button  40 , and the threshold high button  42  can be replaced by a single threshold button and box (not shown) in which the use could enter the desired threshold value.  
         [0020]    In the slope low, slope medium or slope high modes, selected by buttons  44 ,  46  and  48 , the fraction collection system  22  begins directing solution from cartridge  18  to a collection well  26  after controller  28  determines that two conditions have been met: (1) the UV signal from detector  20  has gone above a slope enable threshold T s , and (2) the slope of the UV signal from detector  20  (i.e., the change in the absorbance value from one reading at the detector to the next) has gone above a slope threshold S. Fraction collection system  22  will continue to discharge the solution to a collection well  26  as long as the sensed UV absorbance remains above T s , and the slope remains above S. In these modes the fraction collection system also moves from one collection well  26  to another based upon the volume discharged. In the various modes, the values for S are different, with the lowest being in the slope low mode and the highest being in the slope high mode. These modes of operation are considered slope-based collection, because collection is based on the rate of change of the absorbance values. When the solution is not being directed to the wells  26  for collection it is instead directed to waste  24 .  
         [0021]    In the custom UV mode, selected by button  50 , the user can adjust the decisions for starting and stopping collection and for making cuts between collection wells based upon the detected UV absorbance. Selecting button  50  causes display screen  52  shown in FIG. 3, and completion of screen  52  causes display of screen  54  shown in FIG. 4. FIG. 5 is a graph of UV absorbance A[n] versus time, where A is UV absorbance, and time is in units of datapoint number n. In screens  52  and  54  the user selects values for various parameters, many of which are shown on FIG. 5, and enters the values into respective boxes  56 - 66 . These include box  56  for value threshold T v , box  58  for slope threshold S, box  60  for slope enable threshold T s , box  62  for slope detection disable threshold T sd , box  64  for valley threshold slope limit T sl , and box  66  for slope cut count limit C c . The user also selects either button  68  or button  70  to specify whether or not samples should be collected during valleys in the UV absorbance data. The set points for these parameters and collection conditions entered by the user are summarized in the following table.  
                                                   Set points (Symbol)   Explanation                   1. Value Threshold (T v )   This is the value level minimum for value-           based collection       2. Slope Threshold (S)   This is the threshold slope that starts slope-           based collection.       3. Slope Enable Threshold   This is the value level minimum to consider       (T s )   slope-based collection       4. Slope Detection   The threshold value for valley and shoulder       Disable Threshold   collection. Above this value valleys and       (T sd )   shoulders below are ignored; they are           considered and cut.       5. ValleyThreshold   This is the slope trigger value for valley and       SlopeLimit (T s1 )   shoulder collection, typically much smaller           than S.       6. SlopeCutCountLimit   This adjusts sensitivity to turning off valley       (C c collection, )   with higher values resulting in higher frac-           tions of the valleys being collected            Collection Conditionals            7. ValleySlope   These are Boolean values for slope-based       8. ValleyThreshold   and threshold collection modes indicating           whether to collect valleys in fractions or to           send them to waste.                  
 
         [0022]    The conditions for starting collection, making a fraction cut (move from one well  26  to another), and ending collection are set forth below.  
                                     Further Definitions                                Value of the absorbance at time n is A[n], where n is the sample (i.e.,       datapoint) number.       Change in absorbance, slope, is dA[n] = A[n] − A[n−1].       Time (sample number) at which downward shoulder cut was made is n ds .       K is the time delay in number of samples.                  
 
         [0023]    [0023]                                         Starting conditions for fraction collection                                    Slope collection mode: dA[n] &gt; S and A[n] &gt; T s .           Value collection mode: A[n] &gt; T v .                        
         [0024]    [0024]                                     Fraction cutting conditions during fraction collection                                Upward shoulder cut: cut at time n+k, when A[n+k] &lt; T sd  and dA[n] &gt;and       dA[n] &lt; T s1  and dA[n+k] &gt; 2*T s1 .       Downward shoulder cut: cut at time n, when A[n] &lt; T sd  and dA[n] &lt; 0 and       dA[n]&gt; −T s1 .       Valley Cut: cut at time n, when dA[n ds ] &lt; 0 and dA[n ds ] &gt;       −T s1  and dA[n] &gt; 0, n−n ds  &gt; C c .                    
         [0025]    [0025]                                     Stopping conditions for fraction collection                                Slope collection mode: Stop at n: (1) if A[n] &gt; T sd  or (2) if valley slope is       false and dA[n ds ] &lt; 0 and dA[n ds ] &gt; −T s1  and dA[n] &gt; 0 and n−n ds &gt; C c .       Value collection mode: Stop at n: (1) if A[n] &lt; Tv or (2) if valley       threshold is false and dA[n ds ] &lt; 0 and dA[n ds ] &gt; −T s1  and dA[n] &gt; 0 and       n−n ds &gt; C c .                    
         [0026]    These conditions will be described with reference to FIG. 5.  
         [0027]    Value Collection Mode  
         [0028]    If a user wishes to operate in a value collection mode, he will set T v  to the desired set point in box  56  (FIG. 3). Fraction collection system  22  will waste the solution while the absorbance value A[n] is below T v  and will direct the solution to a collection well  26  while A[n] is greater than T v . Thus, as indicated in the “starting condition for fraction collection” table above, collection will start when A[n]&gt;T v . Thereafter, collection system  22  will automatically advance from well  26  to another well  26  after a specified volume has been discharged into a well  26 . If desired, the user can also specify fraction cutting based upon slopes when the slope values indicate the existence of different compounds. To do this the user must enter values for T sd  in box  62 , T sl  in box  64 , and C c  in box  66  (FIG. 4).  
         [0029]    An upward shoulder cut (e.g., as indicated by vertical lines  80 ,  82 ,  84 , and  86  on FIG. 5) will be made at an inflection point on the curve that indicates the presence of an overlapping compound. As described in the “fraction cutting conditions” table above, the upward shoulder cut is made at time n+k, when the specified conditions are met. First, the aborbance value A[n] must be less than T sd , because above this value slopes are not considered in order to avoid a false determination of noise at the top of a peak as a change in compound. Second, the slope dA[n] must be positive, indicating an upward portion of the curve. Third, the slope at time n must be less than T sl , the trigger value, and fourth, the slope at time n+k must be greater than two times T sl  in order to have a sufficient change in slope to indicate a different compound. Other multipliers could be used besides two. For example, the value could be another value greater than 1.0, e.g., a value between 1.5 and 2.5. The solid vertical lines  80 ,  84  indicate upward shoulder cuts that were made with T sl  set to a higher slope threshold than for the cuts indicated at dashed vertical lines  82 ,  86 .  
         [0030]    A downward shoulder cut (e.g., as indicated by vertical lines  88 ,  90 ,  92 ,  94 , and  96  on FIG. 5) will also be made at an inflection point on the curve that indicates the presence of an overlapping compound. As described in the “fraction cutting conditions” table above, the downward shoulder cut is made at time n, when the specified conditions are met. First, the absorbance value A[n] must be less than T sd , because above this value slopes are not considered to avoid a false determination of noise at the top of a peak as a change in compound. Second, the slope dA[n] must be negative, indicating a downward portion of the curve. Third the slope at time n must be greater than −T sl , the trigger value. Downward cuts are made as soon as the −T sl , trigger is passed, without the need to consider slopes at different sample times. The solid vertical lines  88 ,  92  and  96  indicate downward shoulder cuts that were made with T sl  set to a higher slope threshold than for the cuts indicated at dashed vertical lines  90 ,  94 .  
         [0031]    A “valley cut” can be made at a specified time after a downward shoulder cut has been made at time n ds  (e.g., at time  90  on FIG. 5, assuming that a lower T sl  value has been selected). The user sets the time for the valley cut by entering the value for C c  into box  66  (FIG. 4). As described in the “fraction cutting conditions” table above, the valley cut is made at time n+1, when the specified conditions are met. The first two relate to the conditions indicating that a downward shoulder cut had been made at time n ds , namely the slope at n ds  was negative but greater than −T sl . The next condition requires that the slope is positive at time n, i.e., that there is a valley between time n ds  and time n. The last condition requires that the specified time, C c , has passed since the downward shoulder trigger.  
         [0032]    For further fraction collections to be made in a valley, the valley collection “YES” button  68  (FIG. 4) must be selected. If not, collection will stop pursuant to condition (2) indicated in the “stopping conditions for fraction collection” table above. These conditions are the same as for making a valley cut, and indicate stopping collection (i.e., directing to waste  24 ) at that time.  
         [0033]    The other condition (condition (1) in the “stopping conditions for fraction collection” table above) that will cause stopping of fraction collection in the value collection mode is that the absorbance value A[n] goes below Tv.  
         [0034]    Slope Collection Mode  
         [0035]    If a user wishes to operate in a slope collection mode, he will set S and T s  to the desired set points in boxes  56  and  60  (FIG. 3). Fraction collection system  22  will waste the solution until two conditions have been met, as indicated in the “starting condition for fraction collection” table above. First, the absorbance value A[n] must be greater than T s . Second, the slope dA[n] must be greater than S. After these conditions have been met, controller  28  causes fraction collection system  22  to direct the solution to a collection well  26 . After starting collection, collection system  22  will automatically advance from one well  26  to another well  26  after a specified volume has been discharged into a well  26 .  
         [0036]    If desired, the user can also specify fraction cutting based upon slopes when the changes in slope values indicate the existence of different compounds. The fraction cutting in the slope collection mode is the same as has already been described for the value collection mode. Thus the conditions in the “fraction cutting conditions” table apply in the slope collection mode as well.  
         [0037]    The two situations for stopping collection in the slope collection mode are set forth in the “stopping conditions for fraction collection” table above. The first situation is when the absorbance value A[n] goes below the slope enable threshold T s . The second is the same as the conditions for making a valley cut, as described above.  
         [0038]    By using real-time analysis of the UV absorbance data, the system enables separation of fractions accurately and automatically. The detection based on UV slope and changes in UV slope allows for more fine tuned control of the fractionation process, leading to higher purity in the compounds collected and higher efficiencies. This is particularly useful when separating a mixture of unknown compounds, such that the operator cannot select optimal solvents and absorption characteristics ahead of time.  
         [0039]    Other embodiments of the invention are within the scope of the appended claims.