Patent Application
20080047899
The above noted and other features of the invention will be better understood from the following detailed description, in which:
In
The step 15 of running an enhanced isocratically modified gradient profile includes the substeps 18 of determining the enhancement isocratic curve starting solvent strength, the step 20 of running a gradient profile to the enhancement isocratic curve starting solvent strength, the step 22 of starting an isocratic gradient profile or isocratic segment, the step 24 of determining the isocratic gradient profile or isocratic segment end point during the isocratically modified gradient profile chromatographic run and the step 26 of stopping the isocratic gradient profile or isocratic segment at the isocratic gradient profile or isocratic segment end point and resuming the standard gradient profile. In this specification, the phrases “isocratic curve” or “isocratic segment”—mean a curve or segment in which the concentration does not significantly change. While the change in concentration that is significant varies with the solvents used and at times with the circumstances, generally the change should not cause a change in retention factor greater than 0.1 and preferably greater than 0.05.
In this process, if an enhanced isocratically modified gradient profile is used, the overall time of the enhanced isocratically modified gradient profile is preferably adjusted to be larger than the regular isocratic gradient profile or isocratic segment time period by the length of the time the isocratic gradient profile or isocratic segment runs. In this specification, the phrases “regular isocratic gradient profile” and “isocratic segment time period” mean a time period set for a normal isocratic run within which it is expected that a peak of the target component or closest impurity will occur. However, the chromatographer may alter this if desirable. This definition is also applicable to any two components that are being separated from each other whether one is considered a target component or not.
In this specification, the phrase “enhanced isocratically modified gradient profile” means a chromatographic gradient profile that has an isocratic curve, isocratic plateau or isocratic segment over a portion that starts before or very early in the elution of a component that is to be identified, separated or purified and continues until just before or sufficiently after the elution of the component that is to be identified, separated or purified for the desired identification, separation or purification. An isocratic gradient profile or isocratic segment is a solvent solution used in liquid chromatography in which the strength of the solution does not vary in a manner that significantly degrades the separation of a target component or target components of the sample during a chromatographic run. The change in separation should not prevent a target component from being clearly identified and commercially purified. In any event the solvent strength should not change by more than ten percent.
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In the preferred embodiment, the relationship between solvent concentration and retention factors is determined by forming a first order linear equation using the two retention factors determined by the TLC runs as terms and the percentage concentration corresponding to the retraction of factors. This is done by standard gradient profile fitting to arrive at an equation in the form of the percentage concentration equals M multiplied by the retention factor plus a constant C. Using this relationship, the percentage concentration is determined for the preferred retention factor which in the preferred embodiment is 0.333. However retention factors in the vicinity of three generally provide a sufficiently good separation to be used.
If the percentage concentration is calculated to be less than zero, it is set to zero. If it is calculated to be more than 100 percent, it is set to 100 percent. Although in the preferred embodiment, a linear equation is obtained from the two relationships in standard algebraic manner, the information could be stored in tabular form in a computer or graphically used in the same manner. There are many mathematical devices for expressing such a relationship when you have two unknown and two known relationships. For example, the corresponding solvent can be calculated from a simple proportionality based on the linear relationship.
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To supply solvent to the pump array 74, the pumping system 52 includes a plurality of solvent reservoirs and manifolds, a first and second of which are indicated at 70 and 72 respectively, a pump array 74 and a motor 76 which is driven under the control of the controller 58 to operate the pump array 74. The controller 58 also controls the valves in the pump array 74 to control the flow of solvent and the formation of gradients as the motor 76 actuates pistons of the reciprocating pumps in the pump array 74 simultaneously to pump solvent from a plurality of pumps in the pump array 74 and to draw solvent from the solvent reservoirs and manifolds such as 70 and 72. Valves in the pump array 74 control the amount of liquid, if any, and the proportions of liquids from different reservoirs in the case of gradient operations that are drawn into the pump and pumped from it. The manifolds communicate with the reservoirs so that a plurality of each of the solvents such as the first and second solvents in the solvent reservoir manifolds 70 and 72 respectively can be drawn into the pump array 74 to permit simultaneous operation of a number of pumps. In some embodiments, the controller 58 may provide a signal on a conductor 90 to cause solvent to flow from a large source of solvent into individual reservoirs that are low on solvent. In some embodiments, the controller 58 stops the run when a low level signal is received or causes a read-out display 92 to indicate a low solvent level.
While in the preferred embodiment, arrays of pumps, columns and detectors are used, any type of pump, column or detector is suitable. A large number of different liquid chromatographic systems are known in the art and to persons of ordinary skill in the art and any such known systems may be adaptable to the invention disclosed herein with routine engineering. While two solvents are disclosed in the embodiment of
To process the effluent, the collector system 56 includes a fraction collector 80 to collect solute, a manifold 82 and a waste depository 84 to handle waste from the manifold 82. One or more fraction collectors 80 communicate with the column and detector array 54 to receive the solute from the columns, either with a manifold or not. A manifold 82 may be used to combine solute from more than one column and deposit them together in a single receptacle or each column may deposit solute in its own receptacle or some of the columns each may deposit solute in its own corresponding receptacle and others may combine solute in the same receptacles. The manifold 82 communicates with the column and detector array 54 to channel effluent from each column and deposit it in the waste depository 84. The fraction collector 80 may be any suitable fraction collector such as that disclosed in U.S. Pat. No. 3,418,084 or the above-identified FOXY fraction collector.
With this arrangement, the chromatographic run progresses in the manner discussed above in connection with
For example, in one simple technique, only readily available simple equipment is needed. This technique may be broken into five steps, which are: (1) preparing the developing container; (2) preparing the TLC plate; (3) spotting the TLC plate; (4) developing the TLC plate; and (5) visualizing the spots. These five steps are described below:
Firstly, the developing container can be a specially designed commercially obtained chamber or an ordinary jar with a lid or a beaker with a watch glass on the top. Typically, solvent is poured into the container to a depth of just less than 0.5 cm. To aid in the saturation of the TLC chamber with solvent vapors, part of the inside of the beaker may be lined with filter paper. The container is covered, swirled gently, and allowed to stand while a TLC plate is prepared.
Secondly, TLC plates may be 5 cm×10 cm sheets. The more samples that are to be run on a plate, the wider it needs to be. A mark is made on the plate 0.5 cm from the bottom of the plate. A line is drawn across the plate at the 0.5 cm mark. This is the origin for the sample spots. The samples may be identified under the line in pencil. Enough space is left between the samples so that they do not run together.
Thirdly, about one mg of the sample may be dissolved in a few drops of a volatile solvent such as hexanes, ethyl acetate, or methylene chloride. A few drops of solvent is added to obtain the desired concentration for each of the two runs, with the number of drops selected to maintain a significant difference. In each case, the container is swirled until the samples are dissolved. For each of the two runs, the solution is applied to the TLC plate with a 1 microliter microcap or drawn-out pipette.
Fourthly, the prepared TLC plate is placed in the developing beaker, the beaker is covered with the watch glass, and left undisturbed on your bench top. It is run until the solvent is about half a centimeter below the top of the plate. The TLC plate is placed in the developing container. The solvent rises up the TLC plate by capillary action. The plate is removed from the beaker when the solvent is near the top of the plate and a line is marked across the plate at the solvent front with a pencil. The solvent is permitted to evaporate completely from the plate. If the spots are colored, they are simply marked with a pencil.
Fifthly, if samples are colored, they are marked before they fade by circling them lightly with a pencil. If they are not colored, they are visualized with a UV lamp, and marked with a pencil. The retention factors for components of interest in the samples are determined with a ruler alone or specialized optical equipment may be utilized to read the distance that the solvent front has moved on the TLC plate as compared to the distance the component and close impurities have moved. Equipment is available in which the plates are read automatically by scanning, and the retention factors calculated and utilized for purifying or separating or identifying components. However, in the preferred embodiment, the retention factors are utilized as described above with respect to
Although a preferred embodiment of the invention has been described with some particularity, it is to be understood that the invention may be practiced other than as specifically described. Accordingly, it is to be understood that, within the scope of the appended claims, the invention may be practiced other than as specifically described.
