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 . 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.

[0019] 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 .

[0020] 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 _s1 , 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. 1

[0021] The conditions for starting collection, making a fraction cut (move from one well 26 to another), and ending collection are set forth below. 2

[0022] These conditions will be described with reference to FIG. 5 .

[0023] Value Collection Mode

[0024] 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]>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 _s1 in box 64 , and C _c in box 66 ( FIG. 4 ).

[0025] 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 _s1 , the trigger value, and fourth, the slope at time n+k must be greater than two times T _s1 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 _s1 set to a higher slope threshold than for the cuts indicated at dashed vertical lines 82 , 86 .

[0026] 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 _s1 , the trigger value. Downward cuts are made as soon as the −T _s1 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 _s1 set to a higher slope threshold than for the cuts indicated at dashed vertical lines 90 , 94 .

[0027] 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 _s1 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 _s1 . 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.

[0028] 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.

[0029] 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 T _v .

[0030] Slope Collection Mode

[0031] 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 .

[0032] 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.

[0033] 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.

[0034] 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.

[0035] Other embodiments of the invention are within the scope of the appended claims.