Title of Invention

A COST-EFFECTIVE CHROMATOGRAPHIC PROCEDURE FOR ENHANCED YIELD

Abstract

A procedure for chromatography wherein the sample solution is prepared in a solvent or mixture of solvents having elution strength greater than the mobile phase, comprising: > equilibrating the stationary phase with a weak mobile phase; > injecting the sample solution in the column; > elution wherein mobile phase is changed from a weak mobile phase to a strong mobile phase using a gradient (initial gradient); and > further elution in either isocratic mode with strong mobile phase or in gradient mode having lesser slope than the initial gradient.

Full Text

FIELD OF THE INVENTION The present invention relates to a chromatography procedure which can be used when the sample solvent has elution strength greater than the mobile phase (strong sample solvent). BACKGROUND AND PRIOR ART Preparative chromatography is often used for purification of compounds. In chromatography, the mixture of compounds ('sample') is applied to a column packed with a packing material ('stationary phase'). A fluid or mixture of fluids ('mobile phase') is then passed through the column. Differential interaction of different components from the sample with the stationary phase leads to elution of the components from the column at different times. And thus, separation of different components from the sample is achieved. Prior to injecting the sample into the column, the stationary phase is equilibrated with the mobile phase. The sample, generally, is dissolved in a solvent or a mixture of solvents ('sample solvent') and then injected into the column. In preparative chromatography, the chromatographic conditions are optimized such that the cost of separation is minimized and the productivity is maximized. Some of the parameters that affect the separation cost and productivity include cycle time, loadability, choice of stationary phase, mobile phase flow rate, mobile phase composition and yield. The cycle time is the time between two successive injections. The loadability is the amount of sample injected into the column per unit volume or mass of the stationary phase. The yield is the fraction of the desired components) from the sample that is obtained in pure form after elution from the column. Generally, solvent contributes significantly to the overall separation cost. Solvent consumed per unit mass of the purified desired component is a good indicative of this contribution. In many cases, the compounds that need to be isolated have low solubility in the mobile phase. If the sample solution is prepared in the mobile phase, in overloaded conditions in preparative chromatography, the loading volumes become high. One alternative is to prepare the sample solution in a solvent or mixture of solvents wherein solubility of the sample is higher compared to that in the mobile phase. This leads to decrease in loading volume and time, and therefore, decrease in cycle time as well as decrease in the solvent consumption during loading. But, at the same time, elution strength of this solvent is significantly higher than that of the mobile phase. Effect of such sample solvents on band profiles in preparative chromatography has been studied by Jandera and Guiochon (J. Chromatogr. A, 588: 1-14, 1991). It was observed that sample solvent with a higher elution strength than the mobile phase leads to serious deformations and to the splitting of sample component bands. Wewers et al. (in Preparative Chromatography in Fine Chemicals and Pharmaceutical Agents, Schmidt-Traub, H. (Ed.) Wiley-VCH, Weinheim) have reviewed such separations and recommend use of mobile phase as sample solvent for sample preparation. Wheat et al. (US20040035789) have disclosed a method and device wherein use of sample solvent with elution strength greater than that of the mobile phase is possible. It was shown that injecting sample in strong mobile phase to form a sample-containing strong mobile phase, diluting this to form sample-containing weak mobile phase and injecting this to the column improves the chromatographic performance. Here, a special device is required for the dilution of sample-containing strong mobile phase. Two different devices, first, a fitting device that is placed in front of the column, and second, an integral dual inlet device that is placed at the inlet of the column, have been suggested. One of these devices needs to be used for the improvement in the chromatographic performance. Thus, according to this invention, a special device is required. Moreover, although sample is immediately injected in the column after dilution to form sample- containing weak mobile phase, there is a possibility that the sample will precipitate or crystallize and adversely affect the chromatographic performance. The present invention relates to a chromatography procedure which can be used when the sample solvent has elution strength greater than the mobile phase (strong sample solvent). The procedure consists of initial equilibration of the stationary phase with a weak mobile phase, injection of the sample dissolved in strong sample solvent, and elution with a mobile phase wherein the mobile phase composition is changed using a gradient from the weak mobile phase to a strong mobile phase. To obtain full advantage of the invention, the elution may be carried out in two stages; a gradient from weak mobile phase to strong mobile phase, which is followed by either isocratic elution with the strong mobile phase or gradient elution having lesser slope as compared to the gradient slope in the first stage. Here, the gradient slope is the ratio of difference between the elution strength of the mobile phase during the gradient to the gradient time. The elution strength of the mobile phase can be measured by the content of the stronger eluting component in the mobile phase. OBJECTS OF THE INVENTION The main object of the present invention is to obtain a cost-effective chromatographic procedure for enhanced yield. Another main object of the present invention is to use sample solution prepared in a solvent(s) having elution strength greater than that of mobile phase. Yet another object of the present invention is to elute the sample with strong mobile phase by step gradient, followed by isocratic mode or a gradient mode. Still another object of the present invention is to use the chromatographic procedure in reverse phase chromatography or normal phase chromatography. Still another object of the present invention is to obtain a chromatographic procedure which results in less consumption of the solvent. Still another object of the present invention is to obtain a chromatographic procedure which provides no risk of sample precipitation or crystallization during sample loading on column. STATEMENT OF THE INVENTION The present invention relates to a cost-effective chromatographic procedure for enhanced yield wherein sample solution is prepared in a solvent(s) having elution strength greater than that of mobile phase, said procedure comprising steps of (a) equilibrating stationary phase with a weak mobile phase followed by injecting the sample solution in equilibrated column; and (b) eluting the sample with strong mobile phase by step gradient, followed by isocratic mode or a gradient mode. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a cost-effective chromatographic procedure for enhanced yield wherein sample solution is prepared in a solvent(s) having elution strength greater than that of mobile phase, said procedure comprising steps of: a) equilibrating stationary phase with a weak mobile phase followed by injecting the sample solution in equilibrated column; and b) eluting the sample with strong mobile phase by step gradient, followed by isocratic mode or a gradient mode. In another embodiment of the present invention, the chromatography is reverse phase chromatography or normal phase chromatography. In yet another embodiment of the present invention, the solvent(s) used as the mobile phase are selected from a group comprising heterocyclic aromatic compounds, aliphatic compounds, ketones, cyanides, alcohols and mixtures of one or more thereof. In still another embodiment of the present invention, the solvent(s) used as the mobile phase are selected from a group comprising acetonitrile, acetone, tetrahydrofuran, methanol, ethanol, n-propanol, isopropanol, ethyl acetate, chloroform, dichloromethane, dichloroethane, diethylether, dioxane, TBME and mixtures of one or more thereof. In still another embodiment of the present invention, the solvent is acetonitrile. In still another embodiment of the present invention, the sample solution is prepared in the solvent(s) selected from a group comprising heterocyclic aromatic compounds, aliphatic compounds, ketones, cyanides, alcohols and mixtures of one or more thereof. In still another embodiment of the present invention, the sample solution is prepared in the solvent(s) selected from a group comprising acetonitrile, acetone, tetrahydrofuran methanol, ethanol, n-propanol, isopropanol, ethyl acetate, chloroform, dichloromethane , dichloroethane, diethylether, dioxane, TBME and mixtures of one or more thereof. In still another embodiment of the present invention, the solvent is methanol. In still another embodiment of the present invention, the procedure results in less consumption of the solvent. In still another embodiment of the present invention, the procedure provides no risk of sample precipitation or crystallization during sample loading on column. BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS FIG. 1 is an illustration of possible elution strength of the mobile phase as a function of time according to the present invention FIG. 2 contains chromatograms that illustrate distortion of band profile due to strong sample solvent at high injection volumes with sample concentration 1 mg/ml. FIG. 3 contains chromatograms that illustrate distortion of band profile due to strong sample solvent at high injection volumes with sample concentration 25 mg/ml. FIG. 4 contains chromatograms that illustrate improvement in band profile according to the present invention at high injection volume with sample concentration 1 mg/ml. FIG. 5 contains chromatograms that illustrate improvement in band profile according to present invention at high injection volume with sample concentration 25 mg/ml. In still another embodiment of the present invention, the term 'elution strength' is related to the velocity at which components from the sample move on the column. 'Higher' and 'lesser' elution strengths are relative terms. In a higher elution strength mobile phase or a solvent, sample components will move on the column at higher velocity (and elute earlier) than that in lesser elution strength mobile phase or solvent. As used herein, the terms 'weak' and 'strong' mobile phase are also relative. A weak mobile phase refers to a mobile phase with lesser elution strength than a strong mobile phase, and vice a versa. As used herein, 'good-solvent' refers to solvent which has high solubility for the sample components and 'anti-solvent' refers to solvent that has poor solubility for the sample components. The present invention provides a procedure for chromatography, which improves the chromatography performance. This performance is improved by achieving one or more of reduction in cycle time, increase in loading and increase in yield. The overall effect is decrease in separation cost and/or increase in productivity. The present invention has been illustrated using a reversed phase chromatography. Those skilled in the art will appreciate that this invention may be implemented on other types of chromatography systems including normal phase chromatography, hydrophobic interaction chromatography or ion exchange chromatography. According to the present invention, the sample is dissolved in a solvent or a mixture of solvents wherein solubility of the sample components is higher compared to the mobile phase. This solvent may be different than the solvent components that constitute the mobile phase. If the sample solvent has same components as in the mobile phase, the anti-solvent content in the sample solvent can be less than that in the mobile phase. Prior to injection of the sample on the column, the stationary phase may be equilibrated with weak mobile phase. After the equilibration, the sample dissolved in strong sample solvent is injected on the column. The elution is then started wherein the mobile phase is changed from a weak mobile phase to a strong mobile phase using a gradient. The gradient may be a continuous gradient such as linear gradient or a step gradient. This gradient may be followed by either isocratic elution by a strong mobile phase or another gradient elution with lesser slope. Some of the examples of the elution strength during equilibration and elution as function of time are shown in FIG. 1. The initial gradient time may be kept short to reduce the run time, and therefore, cycle time to improve the chromatographic performance. For example, the initial gradient time can be less than 10% of the total run time. The present invention can provide several advantages in chromatography where sample has limited solubility in the mobile phase, and therefore, sample is dissolved in a strong solvent. In preparative chromatography, large amount of sample is required to be loaded on the column. In traditional chromatography, distortion of the sample bands occurs under this overloaded condition. In such a case, if the chromatographic separation is carried out according to the present invention, the band distortion is minimized, which leads to improvement in the chromatographic performance. Because of this improvement, purity of the product obtained after the chromatographic separation is improved. Alternately, the chromatographic conditions can be modified in one or more ways including increase in the loading (amount of sample per unit mass or volume of the stationary phase per cycle), increase in the strong solvent in the mobile phase and increase in the pure product cut during elution. The increase in the strong solvent in the mobile phase leads to decrease in the cycle time. The increase in the pure product cut leads to increase in yield. Thus, the present invention provides decrease in separation cost and/or increase in productivity. Additional advantage of the present invention is that it does not require any modification of the traditional chromatography system and there is no risk of sample precipitation or crystallization during sample loading on column. The advantages of the present invention are illustrated in the non-limiting examples below. The invention is further elaborated with the help of following examples. However these examples should not be construed to limit the scope of the invention. EXAMPLES Example 1 Distortion of the Band Profile due to a Strong Sample Solvent a) A sample mixture was prepared by dissolving ascomycin containing 5% of an analog impurity in acetone at a concentration of 1 mg/ml. This sample was injected on a column with 4.6 mm internal diameter and 150 mm length, which was packed with reversed phase C18-bonded silica. The temperature during chromatography was maintained at 55°C. The column was equilibrated with 55% acetonitrile and 45% water containing 0.1% trifluoroacetic acid at a flow rate of 1.5 ml/min. Different volumes of sample solution were injected on the column. The column was eluted with 55% acetonitrile and 45% water containing 0.1% trifluoroacetic acid in isocratic mode at a flow rate of 1.5 ml/min. The elution was monitored using a UV detector at 210 nm. Effect of sample volume on the band profile of ascomycin and its analog impurity is shown in FIG. 2. The band profiles (peak shapes) were good at 20 jxl injection volume. Distortion of the peaks was observed at 50 and 75 |al injection volume. The resolution between ascomycin and its analog impurity was also lost at these high injection volumes. b) A similar experiment was carried out wherein the sample concentration was 25 mg/ml. All other conditions were same as above. FIG. 3 shows the effect of sample volume on the band profile of ascomycin and its analog impurity at 25 mg/ml concentration. Distortion of the peaks was observed at 50 and 75 |_tl injection volume. Example 2 Improvement in Band Profile Using Chromatography Procedure of the Present Invention a) The 50 and 75 |xl injections in example la were repeated using same conditions except that the equilibration was at using a weak mobile phase and short gradient was introduced in the beginning of the elution. The acetonitrile and water content in the mobile phase during equilibration and loading is given in Table 1. The mobile phase also contained 0.1% TFA at all times. The band profiles under these conditions are shown in FIG. 4. Comparison of FIG. 2 and 4 shows that there is significant improvement in band profiles at high injection volumes (50 and 75 |il). Also, the resolution between ascomycin and its analog impurity is increased at these high injection volumes. b) The chromatographic conditions from example 2a were also applied to 25 mg/ml sample concentration. The band profiles under these conditions are shown in FIG. 5. Comparison of band profile in FIG. 3 and FIG. 5 shows that the chromatography according to the present invention has led to better band profiles and higher resolution. Example 3 Effect of Strong Solvent in Mobile Phase on Chromatography Performance when the sample is dissolved in a Strong Sample Solvent Ascomycin with purity 95% and containing 5% of a polar impurity was dissolved in methanol at a concentration of 150 mg/ml. This was used as a load for the HPLC. This sample was injected on a column packed with reversed phase C18-bonded silica. The column internal diameter was 4.6 mm and length was 250 mm. The mobile phase consisted of acetonitrile and water. The acetonitrile content in the mobile phase was varied from 44 to 50% (Samples 3 to 6 in Table 2). During each HPLC run, the column was equilibrated with mobile phase containing acetonitrile same as that used during elution. 0.36 ml of the load solution was injected on the column. The elution was carried out with mobile phase containing desired acetonitrile content. After the run, the column was washed with 3 column volumes of acetonitrile. The flow rate was 1 ml/min during equilibration, elution and washing. During elution, fractions were collected, which were analyzed for purity. The yield was calculated based on the product content in the fractions that when pooled, will give product with greater than 99.5% purity. Table 2 shows the effect of strong solvent (acetonitrile) in the mobile phase on run time, yield and solvent consumption per unit mass of purified product. As the acetonitrile content increases from 44 to 50%, the run time decreases from 160 to 76 min as well as the acetonitrile consumption decreases from 1400 to 1120 L/Kg. However, at the same time, the yield decreases significantly from 94 to 63%. * L of acetonitrile per Kg of purified ascomycin Example 4 Improvement in Chromatography Performance Using the Procedure of Present Invention at High Content of Strong Solvent in Mobile Phase As shown in sample 6 (TABLE 2), at high content (50%) of strong solvent (acetonitrile) in the mobile phase, the chromatographic separation was adversely affected leading to yield of 63%. The elution was modified according to the present invention while keeping all other conditions same as in sample 6 (TABLE 2). The mobile phase ratio was kept as given in Table3. The yield in this case was increased to 92% with corresponding significant decrease in solvent consumption per unit mass of the purified product (see Table 2). Example 5 Effect of Sample Loading on Chromatography Performance when the sample is dissolved in a Strong Sample Solvent Ascomycin with purity 95% and containing 5% of a polar impurity was dissolved in methanol at a concentration of 150 mg/ml. This was used as a load for the HPLC. This sample was injected on a column packed with reversed phase C18-bonded silica. The column internal diameter was 4.6 mm and length was 250 mm. The mobile phase consisted of acetonitrile and water in the ratio of 44:56. During each HPLC run, the column was equilibrated with mobile phase containing 44% acetonitrile. Different volumes of the load solution were injected on the column according to the loading values given in Table 4. The elution was carried out with mobile phase containing 44% acetonitrile. After the run, the column was washed with 3 column volumes of acetonitrile. The flow rate was 1 ml/min during equilibration, elution and washing. During elution, fractions were collected, which were analyzed for purity. The yield was calculated based on the product content in the fractions that when pooled, will give product with greater than 99.5% purity. As the loading was increased from 10 to 19 g/L, the yield reduced from 94 to 66%. Example 6 Improvement in Chromatography Performance Using the Procedure of Present Invention at High Sample Loading As shown in sample 10 (TABLE 4), at high loading (19 g/L), the chromatographic separation was adversely affected leading to yield of 66%. The elution was modified according to the present invention while keeping all other conditions same as in sample 10. The mobile phase ratio was kept as given in Table3. The yield in this case was increased to 85% (see Table 4). From the above examples, it is clear that the procedure of the present invention significantly improved chromatographic performance by allowing use of higher organic content in the mobile phase and/or by allowing increased sample loading without significant loss in the yield. Overall, this is expected minimize the separation cost and maximize the productivity in chromatographic separations. We Claim: 1. A cost-effective chromatographic procedure for enhanced yield wherein sample solution is prepared in a solvent(s) having elution strength greater than that of mobile phase, said procedure comprising steps of: a) equilibrating stationary phase with a weak mobile phase followed by injecting the sample solution in equilibrated column; and b) eluting the sample with strong mobile phase by step gradient, followed by isocratic mode or a gradient mode. 2. The cost-effective chromatographic procedure as claimed in claim 1, wherein the chromatography is reverse phase chromatography or normal phase chromatography. 3. The cost-effective chromatographic procedure as claimed in claim 1, wherein the solvents) used as the mobile phase are selected from a group comprising heterocyclic aromatic compounds, aliphatic compounds, ketones, cyanides, alcohols and mixtures of one or more thereof. 4. The cost-effective chromatographic procedure as claimed in claim 3, wherein the solvents) used as the mobile phase are selected from a group comprising acetonitrile, acetone, tetrahydrofuran, methanol, ethanol, n-propanol, isopropanol, ethyl acetate, chloroform, dichloromethane, dichloroethane, diethylether, dioxane, TBME and mixtures of one or more thereof. 5. The cost-effective chromatographic procedure as claimed in claim 4, wherein the solvent is acetonitrile. 6. The cost-effective chromatographic procedure as claimed in claim 1, wherein the sample solution is prepared in the solvent(s) selected from a group comprising heterocyclic aromatic compounds, aliphatic compounds, ketones, cyanides, alcohols and mixtures of one or more thereof. 7. The cost-effective chromatographic procedure as claimed in claim 6, wherein the sample solution is prepared in the solvent(s) selected from a group comprising acetonitrile, acetone, tetrahydrofuran methanol, ethanol, n-propanol, isopropanol, ethyl acetate, chloroform, dichloromethane , dichloroethane, diethylether, dioxane, TBME and mixtures of one or more thereof. 8. The cost-effective chromatographic procedure as claimed in claim 7, wherein the solvent is methanol. 9. The cost-effective chromatographic procedure as claimed in claim 1, wherein the procedure results in less consumption of the solvent. 10. The cost-effective chromatographic procedure as claimed in claim 1, wherein the procedure provides no risk of sample precipitation or crystallization during sample loading on column. 11. The cost-effective chromatographic procedure as herein described with reference to examples and figures.

Documents:

1975-CHE-2006 AMENDED CLAIMS 14-11-2013.pdf

1975-CHE-2006 EXAMINATION REPORT REPLY RECEIVED 14-11-2013.pdf

1975-CHE-2006 FORM-3 14-11-2013.pdf

1975-CHE-2006 FORM-5 14-11-2013.pdf

1975-CHE-2006 CORRESPONDENCE OTHERS 13-12-2013.pdf

1975-CHE-2006 CORRESPONDENCE OTHERS 17-02-2014.pdf

1975-CHE-2006 CORRESPONDENCE OTHERS 25-04-2014.pdf

1975-CHE-2006 CORRESPONDENCE OTHERS 29-06-2012.pdf

1975-CHE-2006 EXAMINATION REPORT REPLY RECEIVED 02-05-2014.pdf

1975-CHE-2006 FORM-1 02-05-2014.pdf

1975-CHE-2006 FORM-13 25-04-2014.pdf

1975-CHE-2006 OTHERS 02-05-2014.pdf

1975-CHE-2006 ABSTRACT.pdf

1975-CHE-2006 CLAIMS.pdf

1975-CHE-2006 CORRESPONDENCE OTHERS.pdf

1975-CHE-2006 DESCRIPTION (COMPLETE).pdf

1975-CHE-2006 DRAWINGS.pdf

1975-CHE-2006 FORM 18.pdf

1975-che-2006-abstract.pdf

1975-che-2006-claims.pdf

1975-che-2006-correspondnece-others.pdf

1975-che-2006-description(provisional).pdf

1975-che-2006-drawings.pdf

1975-che-2006-form 1.pdf

1975-che-2006-form 26.pdf

1975-che-2006-form 3.pdf

1975-che-2006-form 5.pdf