Patent Application
20080230479
The present application claims priority under 35 U.S.C. §119 to Japanese Patent Application No. 2007-73475, filed Mar. 20, 2007. The content of the application is incorporated herein by reference in its entirety.
The present invention relates to a liquid chromatograph control apparatus and method.
Thin layer chromatography and liquid chromatography are different from each other in the constitution and shape of the instrument, solvent feed method, etc., but are essentially identical with each other in the principle that a sample mixture is moved for being separated into its respective components. So, it is known that there is correlativity between the retardation factor of a sample in thin layer chromatography and the elution time of the sample in liquid chromatography in the case where the same stationary phase is used. That is, in general, if the Rf (retardation factor) value of thin layer chromatography is larger, the elution time in liquid chromatography tends to be shorter.
So, attempts are being made to effectively use the result obtained by performing highly simple and rapid thin layer chromatography for performing liquid chromatography.
For example, JP2001-124755A describes a method for deciding a protocol, comprising the steps of
a) selecting a library of compounds to be purified,
b) performing a TLC and/or an analytical HPLC on a representative sample of the library, which sample comprises less than ten percent of the library,
c) determining a correlated preparative HPLC method depending on how the representative sample elutes off the analytical HPLC column and/or the sample moves on a TLC plate, and
d) purifying all or substantially all of the library,
wherein the correlated preparative HPLC method is determined based on a correlation between three or more zones of retention times if analytical HPLC is performed and/or three or more zones of retention factors if TLC is performed, such that if substantially all of the compounds in the representative sample fall within a particular zone, a correlated preparative HPLC protocol can be used to purify compounds that fall within the zone.
Further, JP3423707B1 describes a liquid chromatograph control apparatus comprising: a measured value storage that stores a measured retardation factor (Rf) value of a sample, which is obtained when components of the sample are separated on thin layer chromatography using an eluent containing a plurality of ingredients at a specified mixture ratio, in association with the specified mixture ratio, a rate-of-change-in-Rf-value storage that stores a rate of change in the retardation factor (Rf) value of the sample with respect to variation in mixture ratio of ingredients of the eluent, a mixture ratio calculator that determines a mixture ratio of the eluent at which a specified retardation factor value (RfO) of the sample is obtained, based on the measured retardation factor (Rf) value corresponding to the specified mixture ratio stored in the measured value storage and on the rate of change in the retardation factor (Rf) value stored in the rate-of-change-in-Rf-value storage, and a mixture ratio controller that outputs a control signal to control the mixture ratio of the eluent fed into a column so that the retardation factor (Rf) value of the sample can be equivalent to the specified retardation factor value (RfO), based on calculated results by the mixture ratio calculator.
The samples to be separated or analyzed by using liquid chromatography include a wide variety of organic compounds in general. So, according to the method described in JP3423707B1 for obtaining the mixture ratio of an eluent at which a specified retardation factor value is obtained using a retardation factor Rf and the rate of change in the retardation factor, it is not always possible to obtain the rate of change in Rf value corresponding to the Rf value obtained as a result of performing thin layer chromatography, and it is difficult to obtain all-inclusive measured data.
Further, in the method described in JP2001-124755A, the liquid chromatography performing condition is decided zone by zone. So, the performing condition is not always appropriate for a sample having continuous Rf values obtained by thin layer chromatography.
The present invention has been created in view of the above-mentioned problems of the prior art. The object of this invention is to provide a liquid chromatograph control apparatus and method for allowing liquid chromatography to be performed according to a simply set appropriate sequence of solvent mixing ratios.
To achieve the aforesaid object of this invention, an embodiment proposes a liquid chromatograph control apparatus for controlling the solvent mixing ratios of liquid chromatography, comprising a storage means for storing the correspondence relationship between the plural discrete Rf values obtained by thin layer chromatography performed on respective samples at a preset solvent mixing ratio and the appropriate sequences of solvent mixing ratios of liquid chromatography for the respective samples; and an arithmetic means for obtaining, by interpolation, the appropriate sequence of solvent mixing ratios corresponding to any Rf value falling between the stored Rf values adjacent to each other.
Further in this invention, another embodiment proposes a liquid chromatograph control apparatus for controlling the solvent mixing ratios of liquid chromatography, comprising a storage means for storing the correspondence relationship between the plural discrete Rf values obtained by thin layer chromatography performed on respective samples at a preset solvent mixing ratio and the appropriate sequences of solvent mixing ratios of liquid chromatography for the respective samples; an arithmetic means for obtaining, by interpolation, the appropriate sequence of solvent mixing ratios corresponding to any Rf value falling between the stored Rf values adjacent to each other; and an image processing means for imaging the thin layer plates obtained by performing thin layer chromatography, to obtain Rf values from the images.
Further in this invention, yet another embodiment proposes a liquid chromatograph control apparatus for controlling the solvent mixing ratios of liquid chromatography, comprising a storage means for storing the correspondence relationships between the plural discrete Rf values obtained by thin layer chromatography performed on respective samples respectively containing plural components at a preset solvent mixing ratio and the appropriate sequences of solvent mixing ratios of liquid chromatography for the respective samples, with the separation degree between any two adjacent components of the plural components developed by thin layer chromatography as a parameter; a selection means for selecting a correspondence relationship stored in the storage means in reference to the separation degree between the corresponding two adjacent components of the plural components; and an arithmetic means for obtaining, by interpolation, the appropriate sequence of solvent mixing ratios corresponding to any Rf value falling between the stored Rf values adjacent to each other in the selected correspondence relationship.
Further in this invention, a further embodiment proposes a liquid chromatograph control apparatus for controlling the solvent mixing ratios of liquid chromatography, comprising a storage means for storing the correspondence relationship between the plural discrete Rf values obtained by thin layer chromatography performed on respective samples respectively containing plural components at a preset solvent mixing ratio and the appropriate sequences of solvent mixing ratios of liquid chromatography for the respective samples, and further for storing the correction factor values corresponding to the respective degrees of separation between any two adjacent components of the plural components developed by thin layer chromatography, for correcting the correspondence relationship; and an arithmetic means for correcting the correspondence relationship stored in the storage means by the correction factor value corresponding to the separation degree between the corresponding two adjacent components of the plural components and further for obtaining, by interpolation, the appropriate sequence of solvent mixing ratios corresponding to any Rf value falling between the stored Rf values adjacent to each other in the corrected correspondence relationship.
Further in this invention, an alternate embodiment proposes a liquid chromatograph control apparatus for controlling the solvent mixing ratios of liquid chromatography, comprising a storage means for storing the correspondence relationships between the plural discrete Rf values obtained by thin layer chromatography performed on respective samples respectively containing plural components at a preset solvent mixing ratio and the appropriate sequences of solvent mixing ratios of liquid chromatography for the respective samples, with the separation degree between any two adjacent components of the plural components developed by thin layer chromatography as a parameter; a selection means for selecting a correspondence relationship stored in the storage means in reference to the separation degree between the corresponding two adjacent components of the plural components; an arithmetic means for obtaining, by interpolation, the appropriate sequence of solvent mixing ratios corresponding to any Rf value falling between the stored Rf values adjacent to each other in the selected correspondence relationship; and an image processing means for imaging the thin layer plates obtained by performing thin layer chromatography, to obtain the distances between the developed plural components and Rf values from the images.
Further in this invention, an embodiment proposes that the selection means is a manual selection means for manually selecting a correspondence relationship stored in the storage means in reference to the separation degree between the corresponding two adjacent components of the plural components.
Further in this invention, another embodiment proposes that the selection means is an automatic selection means for automatically selecting a correspondence relationship stored in the storage means in reference to the separation degree between the corresponding two adjacent components of the plural components.
Further in this invention, yet another embodiment proposes a liquid chromatograph control apparatus for controlling the solvent mixing ratios of liquid chromatography, comprising a storage means for storing the correspondence relationship between the plural discrete Rf values obtained by thin layer chromatography performed on respective samples respectively containing plural components at a preset solvent mixing ratio and the appropriate sequences of solvent mixing ratios of liquid chromatography for the respective samples, and further for storing the correction factor values corresponding to the respective degrees of separation between any two adjacent components of the plural components developed by thin layer chromatography, for correcting the correspondence relationship; an arithmetic means for correcting the correspondence relationship stored in the storage means by the correction factor value corresponding to the separation degree between the corresponding two adjacent components of the plural components and further for obtaining, by interpolation, the appropriate sequence of solvent mixing ratios corresponding to any Rf value falling between the stored Rf values adjacent to each other in the corrected correspondence relationship; and an image processing means for imaging the thin layer plates obtained by performing thin layer chromatography, to obtain the distances between the plural components and Rf values from the images.
Further in this invention, a further embodiment purposes that the degree of separation is expressed with the distance between adjacent components as an indicator, with the difference between the inverse numbers of the Rf values of adjacent components as an indicator, or with the quotient of the Rf values of the substances corresponding to adjacent components as an indicator.
Further in this invention, an embodiment proposes that the correspondence relationships are obtained for respective types of columns and are stored to allow selection for each type of columns.
Further in this invention, another embodiment proposes that the correspondence relationships are obtained for respective solvents and are stored to allow selection for each solvent.
Further in this invention, an alternative embodiment proposes that each of the correspondence relationships stored in the storage means is the direct relationship between Rf values and appropriate sequences of solvent mixing ratios.
Further in this invention, yet another embodiment proposes that each of the correspondence relationships stored in the storage means is a correspondence relationship between the function values of Rf values and appropriate sequences of solvent mixing ratios. A further aspect of the invention proposes that the function values of Rf values are the inverse numbers of Rf values.
Further in this invention, yet another embodiment proposes that the appropriate sequences of solvent mixing ratios stored in the storage means are appropriate time sequences of solvent mixing ratios.
Further in this invention, yet another embodiment proposes that each of the appropriate sequences of solvent mixing ratios stored in the storage means is an appropriate sequence of the mixing ratio index values obtained with said preset solvent mixing ratio (solvent concentration) of thin layer chromatography as 1.
Further in this invention, yet another embodiment proposes a liquid chromatograph control method comprising the step of performing thin layer chromatography on respective samples at a preset solvent mixing ratio for obtaining Rf values, performing liquid chromatography for obtaining the appropriate sequences of solvent mixing ratios of liquid chromatography for the respective samples, and storing the correspondence relationship between the plural discrete Rf values obtained by thin layer chromatography performed on respective samples at a preset solvent mixing ratio and the appropriate sequences of solvent mixing ratios of liquid chromatography in a storage means; the step of performing thin layer chromatography on a desired sample to obtain an Rf value before performing liquid chromatography on the sample; the step of obtaining, by interpolation, the appropriate sequence of solvent mixing ratios corresponding to the obtained Rf value from the respective appropriate sequences of solvent mixing ratios of the Rf values on both sides of the obtained Rf value and adjacent to each other stored in the storage means; and performing liquid chromatography according to the obtained appropriate sequence of solvent mixing ratios.
Further in this invention, an embodiment proposes that the Rf values of thin layer chromatography are obtained from the images of thin layer plates obtained by an imaging means.
Further in this invention, yet another embodiment proposes liquid chromatograph control method, comprising the step of performing thin layer chromatography on the respective samples respectively containing plural components at a preset solvent mixing ratio to obtain Rf values and the degrees of separation between developed plural components, performing liquid chromatography for obtaining the appropriate sequences of solvent mixing ratios of liquid chromatography for the respective samples, for the respective degrees of separation between developed plural components, and storing the correspondence relationships between the plural discrete Rf values obtained by thin layer chromatography performed on respective samples at a preset solvent mixing ratio and the appropriate sequences of solvent mixing ratios of liquid chromatography in a storage means with the separation degree between any two adjacent components of the developed plural components as a parameter; the step of performing thin layer chromatography on a desired sample to obtain an Rf value and the separation degree between any two adjacent components of the developed plural components before performing liquid chromatography on the sample; the step of selecting a correspondence relationship stored in the storage means in reference to the obtained degree of separation; the step of obtaining, by interpolation, the appropriate sequence of solvent mixing ratios from the respective appropriate sequences of solvent mixing ratios of the Rf values on both sides of the obtained Rf value and adjacent to each other in the selected correspondence relationship; and the step of performing liquid chromatography according to the obtained appropriate sequence of solvent mixing ratios.
Further in this invention, yet another embodiment proposes a liquid chromatograph control method, comprising the step of performing thin layer chromatography on respective samples respectively containing plural components at a preset solvent mixing ratio to obtain Rf values and degrees of separation between developed plural components, performing liquid chromatography to obtain the appropriate sequences of solvent mixing ratios of liquid chromatography for the respective samples of each Rf values, for the respective degrees of separation between developed plural components, obtaining correction factor values corresponding to the respective degrees of separation for correcting the correspondence relationship in reference to the correspondence relationship for a certain degree of separation, and storing them in a storage means; the step of performing thin layer chromatography on a desired sample to obtain an Rf value and the separation degree between any two adjacent components of the developed plural components before performing the liquid chromatography on the sample; the step of reading the respective appropriate sequences of solvent mixing ratios of the Rf values on both sides of the obtained Rf value and adjacent to each other and the respective correction factor values from the storage means; the step of correcting the respective appropriate sequences of solvent mixing ratios by the correction factor values, and obtaining, by interpolation, the appropriate sequence of solvent mixing ratios corresponding to the obtained Rf value from the corrected appropriate sequences of solvent mixing ratios of the adjacent Rf values; and the step of performing liquid chromatography according to the obtained appropriate sequence of solvent mixing ratios.
Further in this invention, an alternate embodiment proposes that the Rf values of thin layer chromatography and the degrees of separation between developed plural components are obtained from the images of thin layer plates obtained by an imaging means.
Further in this invention, an embodiment propose that the degree of separation as a parameter is expressed with the distance between adjacent components as an indicator, with the difference between the inverse numbers of the Rf values of adjacent components as an indicator, or with the quotient of the Rf values of the substances A and B corresponding to adjacent components as an indicator.
Further in this invention, another embodiment proposes that the correspondence relationships are obtained for respective types of columns and are stored to allow selection for each type of columns.
Further in this invention, yet another embodiment proposes that the correspondence relationships are obtained for respective solvents and are stored to allow selection for each solvent.
Further in this invention, a further embodiment proposes that each of the correspondence relationships stored in the storage means is the direct relationship between Rf values and appropriate sequences of solvent mixing ratios.
Further in this invention, yet another embodiment proposes that each of the correspondence relationships stored in the storage means is a correspondence relationship between the function values of Rf values and appropriate sequences of solvent mixing ratios. Furthermore, a further aspect of the invention proposes that the function values of Rf values are the inverse numbers of Rf values.
Further in this invention, an embodiment proposes that the appropriate sequences of solvent mixing ratios stored in the storage means are the appropriate time sequences of solvent mixing ratios.
Further in this invention, another embodiment proposes that each of the appropriate sequences of solvent mixing ratios stored in the storage means is an appropriate sequence of the mixing ratio index values obtained with said preset solvent mixing ratio (solvent concentration) of thin layer chromatography as 1.
As described before, thin layer chromatography and liquid chromatography are substantially identical with each other in the principle of moving a sample mixture for separating it into respective components. So, there is correlativity between the retardation factor of a sample in thin layer chromatography and the elution time of the sample in liquid chromatography if the stationary phase is the same.
Therefore, according to this invention, the plural discrete Rf values of thin layer chromatography performed on respective samples at a preset solvent mixing ratio and appropriate sequences of solvent mixing ratios of liquid chromatography for the respective samples are obtained beforehand, and their correspondence relationship is stored. Before liquid chromatography is performed on a sample of interest, highly simple and rapid thin layer chromatography is performed to obtain an Rf value, and the appropriate sequence of solvent mixing ratios corresponding to the Rf value is obtained, by interpolation, from the stored correspondence relationship. So, the result obtained by performing highly simple and rapid thin layer chromatography can be effectively used for accurately performing liquid chromatography.
According to an aspect of the invention the separation degree between any two adjacent components of the plural components developed by thin layer chromatography can be taken into account for obtaining a correspondingly appropriate sequence of solvent mixing ratios. So, liquid chromatography can be appropriately performed even if there is a non-intended sample existing near the intended sample to be separated.
In this case, the separation degree between any two adjacent components of the plural components can be expressed with the distance between adjacent components as an indicator, or with the difference between the inverse numbers of the Rf values of adjacent components as an indicator, or with the quotient of the Rf values of the substances corresponding to adjacent components.
Since the results obtained by performing thin layer chromatography can be obtained by processing the images of thin layer plates, the appropriate sequences of solvent mixing ratios of liquid chromatography can be simply and rapidly obtained. Thus, the series of operations can be automated.
In this invention, if the correspondence relationships are obtained for respective types of columns and stored to allow selection for each type of columns, or obtained for respective solvents and stored to allow selection for each solvent, an appropriate sequence of solvent mixing ratios of liquid chromatography can be obtained simply and rapidly for each Rf value, each degree of separation, each type of columns, or each solvent.
In this invention, each of the correspondence relationships can be a direct correspondence relationship between Rf values and appropriate sequences of solvent mixing ratios, or a correspondence relationship between the function values of Rf values and appropriate sequences of solvent mixing ratios, for example, a correspondence relationship between the inverse numbers of Rf values and appropriate sequences of solvent mixing ratios.
Further, in this invention, each of the appropriate sequences of solvent mixing ratios stored in the storage means can be a time sequence of solvent mixing ratios per se, or a sequence of the mixing ratio index values obtained with said preset solvent mixing ratio (solvent concentration) of thin layer chromatography as 1. Meanwhile, the time sequence of solvent mixing ratios can be of either the stepwise control method (stepwise elution method) or the concentration gradient control method (gradient elution method).
This invention is explained below in detail in reference to the drawings showing embodiments.
Symbols 1a and 1b denote solvent tanks for storing the solvents to be mixed for forming the mobile phase of liquid chromatography. In the solvent tanks 1, plural solvents different in polarity must be placed in the case where stepwise control (stepwise elution method) or concentration gradient control (gradient elution method) is performed for purification.
Symbols 2a and 2b denote pumps for sucking and sending the solvents stored in the solvent tanks 1a and 1b to the column, detector and fraction collector described later. In this embodiment, two pumps are installed in correspondence to the solvent tanks 1a and 1b for feeding the solvents using a high pressure gradient method, and downstream of these pumps 2a and 2b, a mixer 3 is installed.
Symbol 4 denotes a column packed with the material used as the stationary phase of liquid chromatography.
Symbol 5 denotes a detector for allowing the liquid from the column 4 to pass through it for detecting whether or not a sample is contained in the mixed solvent (mobile phase). The detector 5 can be, for example, an absorbance detector, fluorescence detector or differential refractometer detector, etc.
Symbol 6 denotes a fraction collector, and the fraction collector 6 consists of a test tube rack 8 supporting many test tubes 7 arranged on it, a fractionation nozzle 10 with a valve 9 provided above the test tube rack 8 and capable of moving along the arranged test tubes 7, and a waste liquid tank 11. The valve 9 is a switching valve for switching between the fractionation to the test tube rack 8 and the liquid feed to the waste liquid tank 11.
Symbol 12 denotes a control apparatus, and the control apparatus 12 is a computer application apparatus consisting of a controller 13, a display 14, a pointing device 15 such as a mouse, etc.
Symbol 16 denotes a thin layer plate obtained by performing thin layer chromatography, and symbol 17 denotes a substance developed on the thin layer plate 16. Thin layer plates 16 are illuminated by an illuminator 19 and imaged by a camera 18, and the images are applied to an image processing means 20 constituting the controller 13.
Further, the controller 13 contains, as described later in detail, a storage means 21 for storing the correspondence relationships between the plural discrete Rf values obtained by thin layer chromatography performed on respective samples at a preset solvent mixing ratio and the appropriate sequences of solvent mixing ratios of liquid chromatography for the respective samples, an arithmetic means 22 for obtaining, by interpolation, the appropriate sequence of solvent mixing ratios corresponding to any Rf value falling between the stored Rf values adjacent to each other, and a control means 23 for controlling the pumps 2a and 2b, mixer 3, etc. according to the obtained appropriate sequence of solvent mixing ratios.
The correspondence relationship stored in the storage means 21 is explained below.
At first, in this invention, at a preset solvent mixing ratio, for example, at a solvent mixing ratio of 1:1 for two solvents, thin layer chromatography is performed on many samples using the same stationary phase as that of liquid chromatography, and from the results, Rf values are obtained, for example, as shown in
In this invention, the correspondence relationship between Rf values and appropriate sequences of solvent mixing ratios as shown in
In the above constitution, when liquid chromatography is performed on a sample of interest, highly simple and rapid thin layer chromatography is performed beforehand to obtain an Rf value, and the value is entered into the arithmetic means 22. The arithmetic means 22 that has received the Rf value obtains, by interpolation, the appropriate sequence of solvent mixing ratios for the Rf value from said correspondence relationship stored in the storage means 21.
That is,
Subsequently the control means 23 can control the pumps 2a and 2b, mixer 3, etc. based on the appropriate sequence of solvent mixing ratios obtained as described above, for performing liquid chromatography according to the appropriate sequence of solvent mixing ratios.
As the interpolation used for obtaining the appropriate sequence of solvent mixing ratios in this invention, any adequate method such as interpolation using a straight line or using a curved line can be applied.
In the correspondence relationship between Rf values and appropriate sequences of solvent mixing ratios stored in the storage means 21, each of the appropriate sequences of solvent mixing ratios can be a time sequence of solvent mixing ratios as shown in
At a preset solvent mixing ratio, for example, at the solvent mixing ratio of 1:1 (50%), thin layer chromatography was performed on respective samples, to obtain Rf values, and liquid chromatography was performed at the same solvent mixing ratio, to obtain the measured elution times shown in
For this reason, in the correspondence relationship between plural discrete Rf values and appropriate sequences of solvent mixing ratios stored in the storage means 21, if the intervals between the plural Rf values are linearly set, the differences between the respective appropriate sequences of solvent mixing ratios corresponding to the Rf values adjacent to each other become small when the Rf values become large as shown in
However, if the measured results of
On the other hand,
So, in normal phase liquid chromatography, it is effective to keep the mixing ratio of a highly polar solvent lower so that it can take more time in passing through the column. On the contrary, in the case where a non-intended substance does not exist near an intended substance to be separated from a sample, liquid chromatography can be accomplished in a shorter period of time for shortening the time and for decreasing the amount of the solvent used.
This suggests that it is desirable to set the sequence of solvent mixing ratios of liquid chromatography, considering not only the Rf values of thin layer chromatography but also the separation degree between any two adjacent components of the plural components developed by thin layer chromatography.
So, as one embodiment of this invention, the correspondence relationships between the plural discrete Rf values obtained by thin layer chromatography performed on respective samples containing plural components at a preset solvent mixing ratio and the appropriate sequences of solvent mixing ratios of liquid chromatography for the respective samples is stored in the storage means 21, with the separation degree between any two adjacent components of the plural components developed by thin layer chromatography as a parameter.
For example, in
In the constitution described above, the controller 13 has a selection means (not shown in the drawing) for selecting the correspondence relationship between the plural discrete Rf values and the appropriate sequences of solvent mixing ratios of liquid chromatography for the respective samples, corresponding to the separation degree between any two adjacent components of the plural components obtained by thin layer chromatography and entered into the controller 13, from the correspondence relationships stored in the storage means 21, and the arithmetic means 22 obtains, by interpolation, the appropriate sequence of solvent mixing ratios corresponding to the entered Rf value from the selected correspondence relationship.
Thus, the appropriate sequences of solvent mixing ratios can be set into the control means, considering not only the Rf values of thin layer chromatography but also the separation degree between any two adjacent components of the plural components developed by thin layer chromatography, to perform liquid chromatography.
On the other hand, as the separation degree between any two adjacent components of the plural components, the difference between the inverse numbers of the Rf values of the adjacent components can also be used as an indicator. That is, if the indicator of the separation degree is X and the Rf values of the substances A and B are RfA and RfB, then the following is the indicator indicating the separation degree.
X=(1/RfA)−(1/RfB)
In the above, as the case of (a), if the RfA value is 0.10 and the RfB value is 0.15, then we have X=3.34. Further, as the case of (b), if the RfA value is 0.45 and the RfB value is 0.5, then we have X=0.2. That is, even in both the cases where the difference between Rf values is 0.05, said indicator X can have values showing clear differences.
Considering the actual relationship between Rf values and elution times, in the case of (a), the elution times are greatly different, and therefore separation is easy. On the contrary, in the case of (b), the elution times are not so different, and therefore it is evident that separation is difficult. If the appropriate sequences of solvent mixing ratios considering not only the Rf values of thin layer chromatography but also the separation degree between any two adjacent components of the plural components developed by thin layer chromatography are set into the control means, liquid chromatography can be effectively performed.
That is, since the indicator X is large in the case of (a), the appropriate sequence of solving mixing ratios of liquid chromatography can be set to assure shorter elution times, and since the indicator X is small in the case of (b) on the contrary, the appropriate sequence of solvent mixing ratios of liquid chromatography can be set to assure longer elution times. Therefore, even if the separation degree between any two adjacent components of the plural components developed by thin layer chromatography is different, liquid chromatography can be performed according to an appropriate sequence of solvent mixing ratios.
Further, for the separation degree between any two adjacent components of the plural components, the quotient of the Rf values of the adjacent components can also be used as the indicator. That is, if the indicator indicating the separation degree is Y and the Rf values of substances A and B are RfA and RfB, then the following is the indicator indicating the separation degree.
Y=Rf
A
/Rf
B (or Y=RfB/RfA)
In the above, as described before, in the case of (a), if the RfA value is 0.10 and the RfB value is 0.15, then we have Y=0.67. Further, in the case of (b), if the RfA value is 0.45 and the RfB value is 0.5, then we have Y=0.9. That is, even in both the cases where the difference between Rf values is 0.05, said indicator Y can have values showing clear differences.
Therefore, since the indicator Y is small in the case of (a), the appropriate sequence of solvent mixing ratios of liquid chromatography can be set to assure shorter elution time, and since the indicator Y is large in the case of (b) on the contrary, the appropriate sequence of solvent mixing ratios can be set to assure longer elution times. Thus, liquid chromatography can be performed according to an appropriate sequence of solvent mixing ratios.
In the embodiments explained above, the storage means stores the correspondence relationship between the plural discrete Rf values obtained by thin layer chromatography performed on respective samples at a preset solvent mixing ratio and the appropriate sequences of solvent mixing ratios of liquid chromatography for the respective samples, or said correspondence relationship further involving the separation degree between any two adjacent components of the plural components developed by thin layer chromatography as a parameter. In a further other embodiment, the storage means can store the correspondence relationship between the plural discrete Rf values obtained by thin layer chromatography performed on respective samples respectively containing plural components at a preset solvent mixing ratio and the appropriate sequences of solvent mixing ratios of liquid chromatography, and can also store the correction factor values corresponding to the separation degrees between any two adjacent components of the plural components developed by thin layer chromatography, for said correspondence relationship.
In this case, in the case where the concentration gradient control method is applied, as described before, the correspondence relationship stored in the storage means 21 is, as shown in
The correction factor can be adequately set for the above-mentioned respective ranges of separation degrees. For example, in the case where the medium range of separation degrees is selected as the reference range, the correction factor can be set at 1 for the reference range of separation degrees.
Further, for example as shown in
In addition to the above, the correspondence relationships obtained for the respective types of columns and/or the respective solvents can be stored in the storage means 21, so that the correspondence relationships between the plural discrete Rf values obtained by thin layer chromatography performed on respective samples at a preset solvent mixing ratio and the appropriate sequences of solvent mixing ratios of liquid chromatography for the respective samples can be stored with the type of columns and/or the solvent as parameters, or that these correspondence relationships can also be stored with the separation degree between any two adjacent components of the plural components developed by thin layer chromatography as a parameter.
In the case where the storage means 21 stores those correspondence relationships using the above parameters, the controller 13 can have a selection means (not shown in the drawings). In this case, when any one of the parameters is applied to the controller 13, the selection means selects the correspondence relationship between plural discrete Rf values and appropriate sequences of solvent mixing ratios, corresponding to the applied parameter. That is, the selection means selects the correspondence relationship corresponding to the applied parameter, from the correspondence relationships stored in the storage means 21. Then, the arithmetic means 22 is used to obtain the appropriate sequence of solvent mixing ratios for the entered Rf value from the selected correspondence relationship.
As described above, if the constitution as shown in
An example of this invention is explained below.
Table 1 shows an example of the correspondence relationship between Rf values and appropriate sequences of solvent mixing ratios stored in the storage means 21. The appropriate sequences of solvent mixing ratios are for the concentration gradient control (gradient elution method), and the solvent mixing ratios (solvent concentrations) at the respective time points (1) through (6) are not stored as actual values, but as the mixing ratio index values obtained with said preset solvent mixing ratio (solvent concentration) of thin layer chromatography as 1. The solvents used for thin layer chromatography are hexane and ethyl acetate, and the solvent mixing ratio (the solvent concentration) as 1 is an ethyl acetate concentration of 30% (a solvent mixture consisting of 70% of hexane and 30% of ethyl acetate).
In this situation, if the Rf value obtained by performing thin layer chromatography is 0.28 before liquid chromatography is performed for the sample of interest, the appropriate sequence of solvent mixing ratios for the Rf value is obtained, by interpolation, from the correspondence relationship of Table 1 stored in the storage means 21.
Table 2 shows the results obtained by linear interpolation. The solvent mixing ratios at the respective time points (1) through (6) corresponding to the Rf value of 0.28 are obtained as the mixing ratio index values with the solvent mixing ratio (solvent concentration) of thin layer chromatography as 1. From the mixing ratio index values and the solvent mixing ratio (solvent concentration) of thin layer chromatography, the solvent mixing ratios at the respective time points (1) through (6) can be obtained.
Tables 3 and 4 show a particular example of the operation for selecting a correspondence relationship stored in the storage means 21 in reference to the separation degree and the operation for obtaining the quotient of the Rf values of the substances corresponding to the components adjacent to each other as the separation degree indicator.
Table 3 shows the stored appropriate sequences of solvent mixing ratios of liquid chromatography for a case of separation degree indicator≧0.8 where the separation degree is small. As in Table 1, the solvent mixing ratios (solvent concentrations) at the respective time points (1) through (6) are not stored as actual values, but as the mixing ratio index values obtained with said preset solvent mixing ratio (solvent concentration) of thin layer chromatography as 1.
Table 4 shows the appropriate sequences of solvent mixing ratios of liquid chromatography stored for a case of 0.3<separation degree indicator<0.8 where the separation degree is medium.
Table 5 shows the Rf values of the respective substances A, B and C obtained by performing thin layer chromatography at the aforesaid preset solving mixing ratio.
In the operation for obtaining the appropriate sequence of solvent mixing ratios for the purpose of fractionation between the substances A and B and between the substances A and C by liquid chromatography, if the quotients of Rf values of the substances to be fractionated are employed as separation degree indicators, the indicator of the separation degree between the substances A and B and the indicator of the separation degree between the substances A and C are as shown below. Indicator of the separation degree between substances A and B: RfB/RfA=0.83 Indicator of the separation degree between substances A and C: RfC/RfA=0.53 Therefore, in the case where the correspondence relationships stored in the storage means 21 are selected in reference to the separation degrees, if the quotients of the Rf values of the components adjacent to each other are employed as separation degree indicators, the correspondence relationship shown in Table 3 should be selected for the separation degree indicator of 0.83 for fractionation between the substances A and B, and the correspondence relationship shown in Table 4 should be selected for the separation degree indicator of 0.53 for fractionation between the substances A and C.
If the fractionation between the substances A and B and the fractionation between the substances A and C by liquid chromatography are performed by selecting the correspondence relationships shown in Tables 3 and 4, the results are as shown in
As shown in
An example concerning the operation for correcting the correspondence relationship stored in the storage means by a correction factor is explained below. Table 8 shows the Rf values of substances A and D obtained by performing thin layer chromatography at the aforesaid preset solvent mixing ratio. The substance A is already described before.
In the operation of obtaining the appropriate sequence of solvent mixing ratios for the purpose of fractionation between the substances A and D by liquid chromatography, the quotient of the Rf values of the substances to be fractionated as the separation degree indicator is as follows.
Indicator of the separation degree between substances A and D: RfD/RfA=0.25
If this separation degree indicator is divided by the separation degree indicator for substances A and C of RfC/RfA=0.53 in the medium separation degree range as described before, to obtain the quotient as a correction factor Q, we have
Q=(RfD/RfA)/(RfC/RfA)=0.47
The correction factor Q is an indicator for the medium separation degree range.
If Q is equal to 1, the separation is as easy as the separation performed without correction.
If Q is larger than 1, the separation is more difficult than the separation performed without correction.
If Q is smaller than 1, the separation is easier than the separation performed without correction.
That is, the correction factor value of Q=0.47 for the substances A and D shows that separation is very easy.
Table 9 shows an example of correction by the correction factor Q corresponding to the separation degree between any two adjacent components of the plural components developed by thin layer chromatography. In this example, as shown in Table 9, the storage means 21 stores a correction factor sequence for elapsed times and a correction factor sequence for solvent mixing ratios at respective time points (1) through (6), and the value of Q is used to directly obtain the appropriate sequence of solvent mixing ratios. That is, the appropriate sequence of solvent mixing ratios of Table 7 is corrected by the correction factor Q stored as shown in Table 9, to obtain the appropriate sequence of solvent mixing ratios as shown in Table 10. Meanwhile, in Table 10, the values at time points (5) and (6) are written as 100%, since the calculated values are larger than 100%.
As can be seen from
Table 11 shows another example of correction by the values of correction factor Q corresponding to the separation degrees between any two adjacent components of the plural components developed by thin layer chromatography. As shown in the table, the storage means 21 stores correction factor sequences for elapsed times and correction factor sequences for solvent mixing ratios at respective time points (1) through (6) with the value of correction factor Q as a parameter.
In this example, the value of correction factor Q suitable for the separation degree concerned is selected from the values stored in the storage means, or the correction factor value suitable for the separation degree concerned is obtained, by interpolation, from the values stored in the storage means. The selected or calculated correction factor value is used to correct, for example, the appropriate sequence of solvent mixing ratios of Table 7, for obtaining a corrected appropriate sequence of solvent mixing ratios.
According to this invention, as described above, the results obtained by performing highly simple and rapid thin layer chromatography can be used for accurately performing liquid chromatography. So, this invention can be highly applied to the liquid chromatography performed for purification or analysis.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2007-73475 | Mar 2007 | JP | national |
