WO2017039673A1

WO2017039673A1 - Improved method for preparative and commercial scale reverse phase liquid chromatography

Info

Publication number: WO2017039673A1
Application number: PCT/US2015/048356
Authority: WO
Inventors: Lou BELLAFIORE; Kevin Henretta; Ed Glicken
Original assignee: Hygeia Industries, Inc.
Priority date: 2015-09-03
Filing date: 2015-09-03
Publication date: 2017-03-09

Abstract

Methods and apparatus are provided for separating compounds from natural and synthetic feedstocks using reverse phase liquid chromatography. Reverse phase separation is conducted on short media beds in a multi-stage process. The present technology relates separating compounds from natural and synthetic feedstocks using reverse phase liquid chromatography, that can be done on a commercial scale. Most commercial scale chromatographic separation of natural and synthetic oils uses normal phase media with highly flammable and toxic solvents. The present technology uses primarily alcohols and water as solvents, which help provide a safer workplace, safer products and reduced environmental impact.

Description

IMPROVED METHOD FOR PREPARATIVE AND COMMERCIAL SCALE REVERSE PHASE LIQUID CHROMATOGRAPHY

BACKGROUND

[0001] Liquid chromatography is a technique that can be used to separate a mixture into its component parts. Liquid chromatography generally relies upon introducing a liquid mixture into a mobile phase that carries the mixture into a column containing a stationary phase. The component parts of the mixture are separated in the column through interactions with the mobile and stationary phases and move through the column at different rates. The eluent stream from the column contains the separated components. In liquid-solid column chromatography, the mobile phase is liquid and the stationary phase is on, or part of, a solid support

[0002] Normal phase liquid chromatography is typically used in an "adsorption" mode of separation with the retention and release of mixture components from a polar stationary phase media, using non-polar solvents and polar modifiers. Normal phase chromatography was the first preparative chromatography technique developed at the turn of the century and remained popular due to the low cost of normal phase media. It is now recognized as undesirable for processing materials designed for human consumption and in general for its processing solvents' toxicity to operators, toxic residues in products, flammability, and environmental concerns.

[0003] Reverse phase liquid chromatography is typically used in a partition mode for the separation of mixture components, using a non-polar stationary phase, and can be used at commercial scale with non-toxic food grade alcohol and water. The separation of components is generally achieved primarily as a result of interactions of non-polar components with the non-polar stationary phase and a partitioning of a mixture across the column bed, using a mobile phase of alcohol and water, to control the degree of resolution between eluting components. BRIEF SUMMARY

[0004] The present technology relates to separating compounds from natural and synthetic feedstocks using reverse phase liquid chromatography, that can be done on a commercial scale.

[0005] Most commercial scale chromatographic separation of natural and synthetic oils uses normal phase media with highly flammable and toxic solvents. The present technology uses primarily alcohols and water as solvents, which help provide a safer workplace, safer products and reduced environmental impact.

[0006] Systems and processes are disclosed herein that provide multi-step, high throughput reverse phase liquid chromatography.

[0007] There are numerous applications for the systems and processes disclosed herein, including the production of highly concentrated and purified dietary supplement and pharmaceutical materials from a wide range of natural and synthetic feedstocks.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

[0008] Specific examples have been chosen for purposes of illustration and description, and are shown in the accompanying drawings, forming a part of the specification.

[0009] Figure 1 illustrates one example of a system for carrying out methods of reverse phase liquid chromatography of the present technology.

[0010] Figure 2 illustrates a second example of a system for carrying out methods of reverse phase liquid chromatography of the present technology.

[0011] Figure 3 illustrates one example of a metering control system that can be used in the system of Figure 2.

[0012] Figure 4 illustrates one example of a solvent separation system that can be used to remove solvent from the product of reverse phase liquid chromatography of the present technology.

[0013] Figure 5 illustrates a UV trace from Step 1 and Step 2 of Example 2. [0014] Figure 6 illustrates an elution profile for Step 1 of Example 2.

[0015] Figure 7 illustrates an analysis trace of the Step 1 Output of Example 2.

[0016] Figure 8 illustrates an analysis trace of the Step 2 Output of Example 2.

DETAILED DESCRIPTION

[0017] The reverse phase liquid chromatography of the present technology can be used to separate compounds from natural and synthetic feedstocks and purify natural and synthetic oil extracts.

[0018] For example, the reverse phase liquid chromatography of the present technology can be used to produce purified tocotrienols, tocopherols and isomers thereof or crude stocks enriched in tocotrienols or tocopherols. Tocopherol is used widely as an antioxidant with applications in the cosmetic, pharmaceutical, nutraceutical, food and fine chemical industries. The four main isomers of tocopherol (alpha, beta, delta, and gamma) are generally referred to individually and as a group as "Vitamin E." Tocotrienols are structurally very similar to tocopherols and also have four main isomers (alpha, beta, delta, and gamma). Tocotrienols have been shown to provide a significant increase in antioxidant activities over tocopherols. Recent studies have indicated that tocotrienols, and to a somewhat lesser extent tocopherols, can provide certain health benefits, such as reduction in serum cholesterol levels and prevention of coronary heart disease, due to their antioxidant properties. Generally, tocotrienols and tocopherols occur together in natural feedstocks, and are produced together as a product of various methods of synthesis. The reverse phase liquid chromatography of the present technology can be used to produce discrete separations of each of the isomers of tocotrienols and tocopherols. Accordingly, in some examples discussed herein, the selected components, which are the target components desired to be produced through elution, comprise tocotrienols, tocopherols and isomers thereof. Additionally, in some examples of the present technology the tocotrienols and the tocopherols elute as separate groups. [0019] Reverse phase liquid chromatography systems and processes of the present technology generally include use of a stationary phase and a mobile phase.

Stationary Phase

[0020] In order to be used effectively in large-scale preparative liquid chromatography, the stationary phase is preferably robust, available at the necessary scale, and manufactured using a reproducible production method. Examples of suitable stationary phase media for use in reverse phase liquid chromatography of the present technology include particlulate, single bed, and membrane forms of silica, alumina, zeolites, polystyrene/divnylbenzene, polymethacrylate, and cellulose, having a porous, non-polar retention surface.

[0021] In examples where the reverse phase liquid chromatography is being used to separate one or more isomers of tocotrienols or tocopherols, the stationary phase preferably provides selectivity related to the hydrophobic and/or aromatic differences between the tocotrienols and the tocopherols to provide class separation. For example, stationary phase media containing either alkyl silanes and/or an aromatic character can be used to elute tocotrienols as a group separate from tocopherols. The stationary phase media can include spherical or irregular particles that average at least 10 microns in size, or larger. For example, the stationary phase media can have a size that averages within the range of between about 10 microns to about 200 microns.

Mobile Phase

[0022] Usable mobile phases are determined by the limitations in the solubility of the end products and the crude material, and generally include a solvent combined with water to form a mobile phase in which the end products and the crude stock are soluble. The water is preferably a food quality, filtered and de-ionized water. Suitable solvents for use with the present technology can include any solvents commonly used for reverse phase chromatography. Preferred solvents include ethanol, and isopropyl alcohol.

[0023] The linear velocity of the mobile phase will also affect the separation and the amount of time required to elute the desired products. The use of a precision pumping system (Hygeia Industries, Glenview, IL.) permits accurate flow rate control throughout the separation process. This allows some automation of the process and guarantees a uniform flow rate to elute products in a more reproducible fashion. The linear velocity or flow rate of the mobile phase should remain within acceptable chromatography norms. Chromatography theory shows that linear velocities should generally not be less than 10 cm/hour to avoid separation deterioration due to diffusion. The upper limits of linear velocity is generally dictated by the pressure limits of the columns, valves, tubing, and pumps used to deliver the mobile phase. However, even without pressure limitations, flow rates should not be more than 1000 cm/hour as inadequate chemical interaction between the products and the stationary phase surface may occur. In systems and processes of the present technology, flow rates having a linear velocity in the range of about 45cm/hr to about 720cm/hr are preferred.

[0024] In examples where the reverse phase liquid chromatography is being used to separate one or more isomers of tocotrienols or tocopherols, the mobile phase can be composed of alcohol with water content in the range from 0% to about 60%. While higher water concentrations can be used, the solubility of these compounds is reduced, thus compromising the process loading and throughput.

Reverse Phase Liquid Chromatography

[0025] Reverse phase liquid chromatography processes of the present technology are multi-step processes, providing for elution through at least two columns in series. Specifically, the processes include eluting a crude feedstock through at least a first column in the first step, to produce a first eluent referred to herein as the first step output, and eluting the first step output through at least a second column in the second step to produce a second eluent referred to herein as the product. Although certain examples are described herein with reference to a single column in the first step and a single column in the second step, it should be understood that any number of columns can be used in parallel in each step, and that the eluent from any number of columns in the first step can be combined and used as the first step output for elution in the second step. It should also be understood that at least some of the same equipment can be used for both of the elution steps. For example, the first step column and the second step column can be the same physical column, used once for the first step and then again for the second step after appropriate regeneration of the stationary phase therein.

[0026] Figure 1 illustrates one example of a system 100 for carrying out methods of reverse phase liquid chromatography of the present technology. System 100 includes a first step liquid chromatography column 102 and a second step liquid chromatography column 104.

[0027] System 100, as shown, also includes a first step feedstock tank 106 and a first step mobile phase tank 108. The first step feedstock tank 106 can include one or more inlets 110, through which the first step feedstock components 112 can be added to the first step feedstock tank 106. Components of the first step feedstock can include, for example, initial crude feedstock, water, and solvent. In some examples, the components of the first step feedstock can be combined in amounts such that the first step feedstock contains water in an amount equal to or greater than an amount of water in the first mobile phase. Additionally, or alternatively, the components of the first step feedstock can be combined in amounts such that the amount of water in the first step feedstock is lower than an amount of water in the second mobile phase. The first step feedstock tank 106 can include at least one outlet 114, through which the first step feedstock 116 can exit the first step feedstock tank 106. The first step mobile phase tank 108 can include one or more inlets 118, through which the components of the first step mobile phase 120 can be added to the first step mobile phase tank 108. Components of the first step mobile phase, as discussed above, can include, for example, water and solvent. The first step mobile phase tank 108 can include at least one outlet 122, through which the first step mobile phase 124 can exit the first step mobile phase tank 108. Each of the first step feedstock tank 106 and a first step mobile phase tank 108 can include an agitator 114, which can be used to mix the components within each tank.

[0028] Methods of the present technology can include a step of preparing the first step liquid chromatography column 102 using a first stationary phase and a quantity of first mobile phase 124 obtained from the first step mobile phase tank 108. The first stationary phase can be used to form a first bed 126 in the first step liquid chromatography column 102. The first bed 126 of the first stationary phase can have a height of up to about 25cm. In some examples, the first bed 126 of the first stationary phase can have a height of up to about 15cm, including about 5cm and about 10cm.

[0029] Methods of the present technology can also include a step of loading the first step chromatography column 102 with the first step feedstock 116. This step can preferably be conducted at a linear velocity in the range of about 45cm/hr to about 720cm/hr. Additionally, the loading level of the first step feedstock can be in proportion to the amount of first stationary phase in the first step chromatography column 102. For example, the step of loading the first step chromatography column 102 with the first step feedstock can be conducted at a loading level of up to about 20g of the first step feedstock per lOOg of the first stationary phase. A pump 128, which could alternatively be a pressurized canister, can be used to selectively load mobile phase 124 and first step feedstock 116 into the first step liquid chromatography column 102.

[0030] Methods of the present technology can next include a step of eluting the selected components away from the first step feedstock in the first step liquid chromatography column 102 to form a first step output stream 130. Use of a gradient elution (increasing the amount of solvent in the blend over time) of the mobile phase rather than an isocratic elution (constant level of solvent through the run) of the mobile phase can be used to improve the separation of the end products. This first step of eluting can include introducing the mobile phase 124 into the first step chromatography column 102 in a same direction as the loading of the first step feedstock 116, and can preferably be conducted at a linear velocity in the range of about 45cm/hr to about 720cm/hr. The first step output stream 130 contains all of the selected components, and preferably contains only low levels of any lipophilic compounds that also exist in the initial crude feedstock.

[0031] A first step byproduct stream 132 containing highly lipophilic components, such as glycerides, sterols and squalene, can also be generated, such as when the method includes performing a first step flush in which introducing pure alcohol, such as isopropyl alcohol (IP A), onto the first step chromatography column 102, in the opposite direction as the loading and elution. [0032] System 100, as shown, also includes a second step feedstock tank 134 and a second step mobile phase tank 136. Each of the second step feedstock tank 134 and a second step mobile phase tank 136 can include one or more inlets 138, through which the components of the second step feedstock and the second step mobile phase can be added to the respective tanks. Components of the second step feedstock can include, for example, first step output 130 and water 140.

[0033] Methods of the present technology can include a step of combining the first step output stream 130 with water 140 to form the second step feedstock 146. It should be noted that the first stream output 130 can be obtained from one or more first step chromatography columns 102, and that multiple first step outputs 130 can be combined in the second step feedstock 146.

[0034] It has been found that the addition of water 140 to the first step output 130 creates a second step feedstock that forms and maintains a focused product band on the second bed 152 of the second step chromatography column 104 during loading, and that if the step 1 output 130 is loaded directly into the column without the addition of water 140 the material spreads across the second bed 152 instead of maintaining a focused band. A lack of focused component retention of the second step feedstock can reduce separation efficiency and compromise product purity.

[0035] Components of the second step mobile phase can be the same or different than the components of the first step mobile phase, and, as discussed above, can preferably include water 140 and solvent 142. The second step feedstock tank 134 can include at least one outlet 144, through which the second step feedstock 146 can exit the second step feedstock tank 134. The second step mobile phase tank 136 can include at least one outlet 148, through which the second step mobile phase 150 can exit the second step mobile phase tank 136. Like the first step feedstock tank 106 and a first step mobile phase tank 108, each of the second step feedstock tank 134 and a second step mobile phase tank 136 can include an agitator 114, which can be used to mix the components within each tank.

[0036] Methods of the present technology can include a step of preparing the second step liquid chromatography column 104 using a second stationary phase and a quantity of second mobile phase 150, which can be obtained from the first step mobile phase tank 136. The second stationary phase can be the same or different from the first stationary phase, and can be used to form a second bed 152 in the second step liquid chromatography column 104. The second bed 152 of the second stationary phase can have a height of up to about 25cm. In some examples, the second bed 152 of the second stationary phase can have a height of up to about 15cm, including about 5cm and about 10cm.

[0037] Methods of the present technology can also include a step of loading the second step chromatography column 104 with the second step feedstock 146. This step can preferably be conducted at a linear velocity in the range of about 45cm/hr to about 720cm/hr. A pump 154, which could alternatively be a pressurized canister, can be used to selectively load second mobile phase 150 and second step feedstock 146 into the second step liquid chromatography column 104.

[0038] Methods of the present technology can next include a step of eluting the selected components away from the second step feedstock in the second step liquid chromatography column 104 to form a second step output stream 156. Use of a gradient elution (increasing the amount of solvent in the blend over time) of the mobile phase rather than an isocratic elution (constant level of solvent through the run) of the mobile phase can be used to improve the separation of the end products. This second step of eluting can include introducing the second mobile phase 150 into the second step chromatography column 104 in a same direction as the loading of the second step feedstock 146, and can preferably be conducted at a linear velocity in the range of about 45cm/hr to about 720cm/hr. The second step output stream 156 contains all of the selected components. The separation of selected components in the second step output stream 156 is dependent upon the chromatographic conditions and the timing of fraction collection of the second step output stream 156 during the elution.

[0039] In some examples where multiple first step outputs 130 are combined in the second step feedstock 146, the second step chromatography column 104 can be used to concentrate multiple first step output streams 130, to provide a concentrated second step output stream 156 containing the selected components of the first step output stream 130, without additional component separation. Such examples can also allow recycling of mobile phase.

[0040] A second step byproduct stream 158 can also be generated, such as when the method includes performing a second step flush in which introducing pure alcohol, such as isopropyl alcohol (IP A), onto the second step chromatography column 104, preferably in the opposite direction of the previous loading and elution.

[0041] Figures 2 and 3 illustrate a second example of a system 200 for carrying out methods of reverse phase liquid chromatography of the present technology, with Figure 2 showing the first step and Figure 3 showing the second step. Similar to the example of system 100, system 200 includes a first step liquid chromatography column 202 and a second step liquid chromatography column 204. The system 200 also includes in-line mixing, which can be used for solvent blending and/or product stream adjustment, and can provide a more consistent and efficient process.

[0042] Referring to Figure 2, system 200, as shown, also includes a first step feedstock tank 206 and a first blending module 208.

[0043] The first step feedstock tank 206 can be used to store and combine the first step feedstock 210. The first step feedstock tank 206 can include at least one outlet 212, through which the first step feedstock 210 can exit the first step feedstock tank 206. Components of the first step feedstock 210 can include, for example, initial crude feedstock, water, and solvent.

[0044] The first blending module 208 can be used to store and blend the components of the first step mobile phase 214. The first blending module 208 can be connected to holding tank for the components of the first step mobile phase. For example, as shown, solvent tank 216 can have at least one outlet 218 through which it can be connected to, and provide solvent 220 to, first blending module 208. A first solvent proportioning mechanism 222 can be used to control and meter desired amounts of solvent 220 into the first blending module 208. Also as shown, water tank 224 can have at least one outlet 226 through which it can be connected to, and provide water 228 to, first blending module 208. A first water proportioning mechanism 230 can be used to control and meter desired amounts of water 228 into the first blending module 208.

[0045] A first proportion control 232 can be used to measure the amounts of solvent and water in the first mobile phase exiting the first blending module. In some examples, the proportion control 232 can perform spectral (e.g., near-infrared) measurement of the first step mobile phase. The first proportion control 232 can be operatively connected to the first solvent proportioning mechanism 222 and the first water proportioning mechanism 230, and can provide feedback to each to control the metering of the solvent and the water. One example of a system that can provide proportioning mechanisms and proportion control can be a blending module as described in U.S. Patent Nos. 7,072,742 and 7,515,994, and is available from Hygeia Industries Inc., IL.

[0046] Methods of the present technology using system 200 can include a step of forming the first mobile phase 214 by combining solvent and water in the first blending module 208 at a predetermined ratio. The formation of the first step mobile phase can include metering an amount of solvent 220 into the first blending module 208 with the solvent proportioning mechanism 222, and metering an amount of water 228 into the first blending module 208 with the first water proportioning mechanism 230. The formation of the first step mobile phase can also include using the proportion control 232 to measure the amounts of solvent and water in the first mobile phase 214 exiting the first blending module 208, and providing feedback from the proportion control to the solvent proportioning mechanism 222 and the water proportioning mechanism 230 to control the metering of the solvent 220 and the water 228.

[0047] Methods using system 200 can also include a step of preparing the first step liquid chromatography column 202 using a first stationary phase and a quantity of first mobile phase 214 obtained from the first blending module 208. The first stationary phase can be used to form a first bed 234 in the first step liquid chromatography column 202. The first bed 234 of the first stationary phase can have a height of up to 15cm. [0048] Methods using system 200 can further include a step of loading the first step chromatography column 202 with the first step feedstock 210, which can be performed in the same manner as described above with respect to system 100. A pump 236, which could alternatively be a pressurized canister, can be used to selectively load mobile phase 214 and first step feedstock 210 into the first step liquid chromatography column 202.

[0049] Methods using system 200 can next include a step of eluting the selected components away from the first step feedstock in the first step liquid chromatography column 202 to form a first step output stream 238. Use of a gradient elution (increasing the amount of solvent in the blend over time) of the mobile phase rather than an isocratic elution (constant level of solvent through the run) of the mobile phase can be used to improve the separation of the end products. This first step of eluting can include introducing the mobile phase 214 into the first step chromatography column 202 in a same direction as the loading of the first step feedstock 210, and can preferably be conducted at a linear velocity in the range of about 45cm/hr to about 720cm/hr. The first step output stream 238 contains all of the selected components, and preferably contains only low levels of any lipophilic compounds that also exist in the initial crude feedstock.

[0050] A first step byproduct stream 240, containing highly lipophilic components, such as glycerides, sterols and squalene, can also be generated, such as when the method includes performing a first step flush in which introducing pure alcohol, such as isopropyl alcohol (IP A), onto the first step chromatography column 202, in the opposite direction as the loading and elution.

[0051] Referring to Figure 3, system 200, as shown, can include a second blending module 242 that can be connected to a source of first step output 238, such as first step output tank 244, a source of water 246, such as second water tank 248, or alternatively first water tank 224, and a source of solvent 220, such as solvent tank 216. As shown, first step output tank 244 has at least one inlet 250, through which first step output 238 can be added to the first step output tank 244, and at least one outlet 252, through which first step output tank 244 can supply first step output 238 to the second blending module 242. Additionally, second water tank 248 has at least one inlet 254, through which water 246 can be added to the second water tank 248, and at least one outlet 256, through which second water tank 248 can supply water 246 to the second blending module 242.

[0052] Second blending module 242 can be used to separately form the second step feedstock 258 and second mobile phase 260. A pump 262, which could alternatively be a pressurized canister, can be used to selectively load second mobile phase 260 and second step feedstock 258 into the second step liquid chromatography column 204. Alternatively a third blending module can be provided, along with one or more additional pumps, and the third blending module can be used to form the second step feedstock 258 or the second mobile phase 260 while the other is formed in the second blending module 242.

[0053] A second step proportioning and control system can be used to measure and control amounts of first step output 238, water 246 and solvent 220 that are added to second blending module 242. As shown, the second step proportioning and control system includes a first step output proportioning mechanism 264, a second water proportioning mechanism 266, a second solvent proportioning mechanism 268, and a second proportion control 270 that is operatively connected to each of the proportioning mechanisms to provide feedback thereto and control of the metering thereof. Proportioning and control systems suitable for use with the first step of system 200 are also suitable for use with the second step.

[0054] When second blending module 242 is being used to form the second step feedstock 258, first step output proportioning mechanism 264 can be used to control and meter desired amounts of first step output 238 into the second blending module 242, and second water proportioning mechanism 266 can be used to control and meter desired amounts of water 246 into the second blending module 242. Second proportion control 270 can be used to measure the amounts of first step output 238 and water in the second step feedstock 258 exiting the second blending module 242. The second proportion control 270 can provide feedback to each relevant proportioning mechanism to control the metering of the first step output 238 and the water 246 into the second blending module 242. [0055] Accordingly, methods of the present technology using system 200 can include a step of forming the second step feedstock 258 by combining the first step output stream 238 and water 246 in the second blending module 242 at a predetermined ratio. The formation of the second step feedstock 258 can include metering an amount of the first step output stream 238 into the second blending module 242 with the first step output stream proportioning mechanism 264, and metering an amount of water 246 into the second blending module 242 with the second water proportioning mechanism 266. The formation of the second step feedstock 258 can also include using the second proportion control 270 to measure the amounts of the first step output stream 238 and the water 246 in the second step feedstock 258 exiting the second blending module 242, and providing feedback from the second proportion control 270 to the first step output stream proportioning mechanism 264 and the second water proportioning mechanism 266 to control the metering of the first step output stream 238 and the water 246.

[0056] When second blending module 242 is being used to form the second mobile phase 260, second solvent proportioning mechanism 268 can be used to control and meter desired amounts of solvent 220 into the second blending module 242, and second water proportioning mechanism 266 can be used to control and meter desired amounts of water 246 into the second blending module 242. Second proportion control 270 can be used to measure the amounts of solvent and water in the second mobile phase 260 exiting the second blending module 242. The second proportion control 270 can provide feedback to each relevant proportioning mechanism to control the metering of the solvent 220 and the water 246 into the second blending module 242.

[0057] Accordingly, methods of the present technology using system 200 can include a step of forming the second mobile phase 260 by combining the solvent 220 and water 246 in the second blending module 242 at a predetermined ratio. The formation of the second mobile phase 260 can include metering an amount of the solvent 220 into the second blending module 242 with the second solvent proportioning mechanism 268, and metering an amount of water 246 into the second blending module 242 with the second water proportioning mechanism 266. The formation of the second mobile phase 260 can also include using the second proportion control 270 to measure the amounts of the solvent 220 and the water 246 in the second mobile phase 260 exiting the second blending module 242, and providing feedback from the second proportion control 270 to the second solvent proportioning mechanism 268 and the second water proportioning mechanism 266 to control the metering of the solvent 220 and the water 246.

[0058] When performing the second step of reverse phase liquid chromatography using system 200, methods of the present technology can include a step of preparing the second step liquid chromatography column 204 using a second stationary phase and a quantity of second mobile phase 260. The second stationary phase can be the same or different from the first stationary phase, and can be used to form a second bed 272 in the second step liquid chromatography column 204. The second bed 272 of the second stationary phase can have a height of up to 15cm.

[0059] Methods of the present technology can also include a step of loading the second step chromatography column 204 with the second step feedstock 258. Loading of the second step chromatography column 204 can be done in the same manner as described above for loading the second step chromatography column 104 of system 100.

[0060] Methods of the present technology can next include a step of eluting the selected components away from the second step feedstock in the second step liquid chromatography column 204 to form a second step output stream 274. Use of a gradient elution (increasing the amount of solvent in the blend over time) of the mobile phase rather than an isocratic elution (constant level of solvent through the run) of the mobile phase can be used to improve the separation of the end products. This second step of eluting can include introducing the second mobile phase 260 into the second step chromatography column 204 in a same direction as the loading of the second step feedstock 258, and can preferably be conducted at a linear velocity in the range of about 45cm/hr to about 720cm/hr. The second step output stream 274 contains all of the selected components.

[0061] A second step byproduct stream 276 can also be generated, such as when the method includes performing a second step flush in which introducing pure alcohol, such as isopropyl alcohol (IP A), onto the second step chromatography column 204, in the opposite direction as the loading and elution.

[0062] Systems for performing reverse phase liquid chromatography of the present technology preferably include temperature control, for maintaining the temperature at or near a desired set point when performing the reverse phase liquid chromatography. Preferably, the temperature can be maintained within the range of the desired set point plus or minus two degrees. More preferably, the temperature can be maintained within the range of the desired set point plus or minus one degree. It has been found that the temperature affects efficiency of the reverse phase liquid chromatography process, and can affect such aspects of the process as load homogeneity, viscosity, and time-based fractionation. In some examples, the desired set point can be within the range of 25°C to about 80°C. In examples where the initial crude feedstock is red palm extract, the desired set point can be in the range of 30°C to about 50°C, including for example about 35°C, about 40°C, and about 45°C.

[0063] Temperature control can be provided through the use of one or more temperature control mechanisms, such as heat exchanger. System 200 as illustrated in Figures 2 and 3 includes temperature control mechanisms 278, 280, 282 and 284. Temperature control mechanism 278 is operatively connected to the first step feedstock tank 206, and can be used to control the temperature of the first step feedstock 210. Temperature control mechanism 280 is operatively connected to the first step liquid chromatography column 202, and can control the temperature therein. Temperature control mechanism 282 is operatively connected to the second blending module 242, and can be used to control the temperature of the contents therein, such as to control the temperature of the second step feedstock 258. Temperature control mechanism 284 is operatively connected to the Temperature control mechanism 284 is operatively connected to the second step liquid chromatography column 204, and can control the temperature therein.

[0064] Methods of the present technology can include controlling temperature by controlling temperature within at least one of the first step liquid chromatography column 202 and the second step liquid chromatography column 204. Alternatively, or additionally, methods of the present technology can include controlling temperature by controlling temperature of the feedstocks. In one such example, temperature control can include controlling temperature of the first step feedstock 210 to maintain the first step feedstock 210 within a range of a first desired set point plus or minus two degrees, and controlling temperature of the second step feedstock 258 to maintain the second step feedstock 258 within a range of a second desired set point plus or minus two degrees. The second desired set point may be the same or different than the first desired set point, but is preferably the same for process consistency.

Mobile Phase Recycling

[0065] One of the most significant operating cost associated with liquid chromatography is related to the cost of solvent used for product preparation, loading and elution. The solvents are generally re-usable when they have released the selected components dissolved therein to the stationary phase in the column, as in Step 1 and Step 2, forming a component-free output solvent. As the component-free output solvent and mobile phase elute from the first or second column, they can be directed to process solvent and mobile phase delivery tanks, either manually, or as part of an automated process.

[0066] Accordingly, methods of the present technology can include recycling at least one of a component-free output solvent or a mobile phase from a process step for re- introduction to the process. Process steps can include any relevant step in the process, including for example, column preparation, equilibration, and feedstock loading.

Separation of Solvent from Selected Components in Step 2 Output

[0067] The second step output stream (e.g., 156 of Figure 1 or 274 of Figure 3) will contain solvent, in the form of the mobile phase, as well as highly purified concentrations of the selected components. Methods of the present technology can include a step of removing solvent from the second step output stream. While, generally, the selected components can be separated from the solvent by any suitable solvent removal technique, it has been found that an oil/water separation technique can be used successfully when the selected components are highly hydrophobic. One such example is when the selected components comprise tocotrienols and tocopherols. [0068] Figure 4 illustrates an example of a solvent separation system 300 that can be used to remove mobile phase from the second step output stream to recover at least one selected component in a concentrated form. Typically, when the one or more selected components are highly hydrophobic, such as tocotrienols and tocopherols, the concentrated form is an oil. In such examples, the one or more selected components can be separated from the mobile phase by adding water to the second step output stream.

[0069] In the illustrated example, a settling tank 302 has at least one inlet 304 located at the bottom thereof, through which water and second step output stream can be added to the settling tank 302. Second step output stream can be placed into settling tank 302 before or after water is added to the settling tank 302. The water will tend to cause the selected components to come out of solution and rise to the surface, forming product layer 306. Accordingly, when the second step output stream and water are allowed to settle in the settling tank for a period of time, known as the settling period, product layer 306 will tend to form on top of the water layer 308 in the settling tank 302. The product layer can be removed through a product layer outlet 310, preferably located in the settling tank 302 at a height determined to be at the bottom of the product layer 306. If the oil level is below outlet 310, additional water can be pumped into inlet 304, after settling, to lift the oil layer to the desired height to remove. The water layer 308 can be removed through a water layer outlet 312, preferably located at the bottom of the settling tank 302. The amount of water added to the settling tank 302 is preferably a volume that produces the desired component oil layer separation within an hour, creating product layer 306. This addition is typically in the range of 1-1 OX volume of water added, relative to second output stream volume.

[0070] In some examples, temperature control, such as reducing or elevating the temperature within solvent separation system 300, can be used to control the separation process. In one example, chilling the water and second step output stream in the settling tank 302, such as to a temperature in the range of about 1°C to about 10°C, can facilitate the formation of the product layer 306. Temperature control can be implemented in any suitable manner, including using a jacketed vessel and heat exchanger(s) for temperature control of the separation process. [0071] Methods of solvent removal from output streams can also include using centrifugation, after water addition, to accelerate the separation process. Temperature control can be used during centrifugation, and can facilitate separation, as described above.

Example 1: Commercial Application for Natural or Synthetic Feedstocks

[0072] The following process can be conducted at a commercial scale.

STEP 1 - Remove highly retained lipophilic compounds from intial crude feedstock.

• Prepare the initial crude feedstock for loading

o Dissolve quantity of initial crude feedstock that equates to 20% of the weight of stationary phase in the packed column into a suitable solvent.

Use heat and mixing as needed. Solvent can be alcohols, acetone, or other water miscible solvents,

o Add an amount of water that brings the total water content to above the water content of the mobile phase for elution.

• Pre-condition a reverse phase separation bed (column, membranes, etc), sized for the scale of production required, with at least 2.5 bed volumes of the mobile phase for Step 1. Mobile phase make-up can consist of a solvent and water, and can vary for different extracts.

• Load the prepared first step feedstock solution onto the bed, using a pump or pressurized canister, at a linear velocity in the range of 45cm/hr to 720cm/hr. Linear velocity should be fast enough for time efficiency but slow enough for optimum feedstock retention.

• Introduce mobile phase into the bed, in the same direction as the loading, at a linear velocity in the range of 45cm/hr to 720cm/hr. The water content should be high enough to allow isolation of the target class of compounds but low enough to allow timely elution.

• Collect and fractionate eluent to generate a Step 1 Output. This fraction

should contain all selected components with low levels of any lipophilic compounds that also exist in the starting feedstock. • Step 1 Flush - Introduce a solvent that has stronger dissolving power than the mobile phase solvent into the bed, in the opposite direction as the loading and elution, at a linear velocity in the range of 360 to 1440cm/hr, and continue cleaning solvent until at least 3 bed volumes has been flushed through the column. Collect eluent into a dedicated container.

STEP 2 - High purity extraction of selected components from feedstocks with reduced or no highly lipophilic compounds

• Adjust Step 1 Output with water to create a Step 2 Feedstock that has 1 -5% more water than the Step 2 mobile phase. Use elevated temperature (35°C to 50°C) and agitation to facilitate dissolution as needed. Ensure a uniformly solubilized solution before proceeding.

• Pre-condition a reverse phase separation bed (column, membranes, etc), sized for the scale of production required, with at least 2.5 column volumes of the mobile phase (see below) for Step 1.

• Load multiple combined collections of Step 2 Feedstock onto bed, using a pump or pressurized canister, at a linear velocity in the range of 45cm/hr to 720cm/hr. Linear velocity must be fast enough for time efficiency but slow enough to facilitate feedstock focusing on the bed.

• Introduce mobile phase into the bed, in the same direction as the loading, at a linear velocity in the range of 45cm/hr to 720cm/hr. Mobile phase make-up consists of a solvent and water, and it varies for different extracts. The water content should be high enough to allow isolation of the target class of compounds but low enough to allow timely elution.

• Collect and fractionate eluent to generate Step 2 Output containing selected components.

• Step 2 Flush - Introduce a solvent that has stronger dissolving power than the mobile phase solvent onto the column, in the opposite direction as the loading and elution, at a linear velocity in the range of 360 to 1440cm/hr, and continue cleaning solvent until at least 3 bed volumes has been flushed through the bed. Collect eluent into a dedicated container. [0073] Step 1 and Step 2 as described above can be repeated as desired.

[0074] Solvent can be removed from the Step Output by any suitable method, including, for example, wiped film evaporation, or oil/water separation as described above.

Example 2: Red Palm Oil Extract as Feedstock

[0075] The following process was conducted at a laboratory scale to isolate tocotrienols and tocopherols from red palm oil extract.

STEP 1

• The red palm oil extract feedstock was prepared for loading by:

o Dissolving lOOmg of feedstock into 0.5 to 1ml of SDA3C alcohol, using elevated temperature (35°C to 50°C) and mixing as needed, o Adding water to bring the total water content to >15%.

• The first step column was prepared by pre-conditioning a cleaned 4.6mm

diameter x 5cm height chromatography column, packed with stationary phase (C8-bonded spherical silica 40μηι) with mobile phase (2.5ml of 85.5/14.5 SDA3C/water) at 540cm/hr. The stationary phase used had a narrow particle size range, centered on a diameter in the 30 to 50 micron range, and 120A pore size.

• The red palm oil extract solution was loaded onto the column, using a pump, at 180cm/hr.

• Mobile phase (85.5/14.5 SDA3C/water) was introduced onto the column, in the same direction as the loading, at a linear velocity of 180cm/hr.

• Fractionate eluent was collected as follows:

o First 0.25ml to dedicated container - Front End

o Following 2.25ml to dedicated container - Step 1 Output

*Note that Step 1 Output can be separated into multiple streams for different applications and target compound profiles.

• The step 1 column was then flushed by introducing pure isopropyl alcohol

(IP A) onto the column, in the opposite direction as the loading and elution, at a linear velocity of 360cm/hr, and continuing introduction of IP A until 5ml had been flushed through the column. The 5ml eluent was collected into a dedicated container.

STEP 2

• Second step feedstcock was prepared by diluting Step 1 Output with 10% extra water, using elevated temperature (35°C to 50°C) and agitation to facilitate dissolution as needed.

• The second step column was prepared by pre-conditioning a cleaned 4.6mm diameter x 10cm height chromatography column, packed with stationary phase (ΙΟμηι C18-bonded silica) with mobile phase (2.5ml of 79.0/21.0

SDA3C/water) at 540cm/hr. The stationary phase had a narrow particle size range, centered on a diameter in the 10 to 20 micron range, and 120 A pore size.

• The second step column was loaded by loading up to 3.5 combined collections of Step 2 Feed onto the second step column, using a pump or pressurized canister, at 180cm/hr.

• Mobile phase (79/21 SDA3C/water) was loaded onto the column, in the same direction as the loading, at a linear velocity of 180cm/hr.

• Fractionate eluent was collected as follows:

o First 1.5ml to dedicated container - Front End

o Following 5ml to dedicated container - Product

*Note that Product can be separated into multiple streams for different applications and target compound profiles.

• The second step column was then flushed by introducing pure SDA3C onto the column, in the opposite direction as the loading and elution, at a linear velocity of 360cm/hr, and continuing to introduce SDA3C until 5ml had been flushed through the column. The eluent was collected into a dedicated container.

[0076] Step 1 and Step 2 as described above can be repeated as desired. [0077] Figure 5 illustrates a UV trace from Step 1 and Step 2, showing highly loaded conditions. Fraction cuts are taken within the large flat peak.

[0078] Figure 6 illustrates an elution profile for Step 1 described above. In figure 5, the x-axis is time and the y-axis is concentration. As shown, greater than 90% of the Sterols and Squalene were excluded from the first step output, and the process cut that captured significant amounts of the tocotrienol. Additionally, highly lipophilic compounds such as triglycerides do not appear in the Figure, because they were retained on the first column bed and were eluted in flush step, rather than being components of the first step output.

[0079] Figure 7 illustrates an analysis trace of the Step 1 Output. The peak triplet represents the tocotrienols, and the two following peaks represent tocopherols, where the larger peak at the end of the trace is alpha-tocopherol.

[0080] Figure 8 illustrates an analysis trace of the Step 2 Output. Compared to the analysis trace of the Step 1 Output shown in Figure 6, the tocopherol peaks are removed. The level of tocopherol, as well as the relative levels of tocotrienols, can be fine-tuned by adjusting the fraction start and stop times in Step 2.

[0081] From the foregoing, it will be appreciated that although specific examples have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit or scope of this disclosure. It is therefore intended that the foregoing detailed description be regarded as illustrative rather than limiting, and that it be understood that it is the following claims, including all equivalents, that are intended to particularly point out and distinctly claim the claimed subject matter.

Claims

CLAIMS What is claimed is:

1. A method of isolating selected components from an initial crude feedstock using reverse phase liquid chromatography, the method comprising: preparing a first step liquid chromatography column using a first stationary phase and a first mobile phase, the first stationary phase being selected from the group consisting of non-polar and aromatic media, and the first mobile phase being capable of maintaining the solubility of the crude feedstock and the selected components; loading the first step chromatography column with a first step feedstock comprising the crude feedstock; eluting the selected components away from the first step feedstock in the first step liquid chromatography column to form a first step output stream containing the selected components; combining the first step output stream with water to form a second step feedstock; preparing a second step liquid chromatography column using a second stationary phase and a second mobile phase, the second stationary phase being selected from the group consisting of non-polar and aromatic media, and the second mobile phase being capable of maintaining the solubility of the crude feedstock and the selected components; loading the second step chromatography column with the second step feedstock; and eluting the selected components away from the second step feedstock in the second step liquid chromatography column to form a second step output stream containing the selected components.

2. The method of claim 1, wherein the selected components comprise tocotrienols, tocopherols and isomers thereof.

3. The method of claim 2, wherein the tocotrienols and the tocopherols elute as separate groups.

4. The method of claim 1, wherein the step of loading the first step chromatography column with the crude feedstock is conducted at a linear velocity in the range of about 45cm/hr to about 720cm/hr.

5. The method of claim 1, wherein the step of eluting the selected components away from the crude feedstock in the first step liquid chromatography column comprises: introducing the mobile phase into the first step chromatography column in a same direction as the loading of the first step feedstock, at a linear velocity in the range of about 45cm/hr to about 720cm/hr.

6. The method of claim 1, wherein the first step liquid chromatography column comprises a bed of the first stationary phase having a height of up to 15cm, and the second step liquid chromatography column comprises a bed of the second stationary phase having a height of up to 15cm.

7. The method of claim 1, wherein the step of loading the first step chromatography column with the first step feedstock is conducted at a loading level of up to about 20g of the first step feedstock per lOOg of the first stationary phase.

8. The method of claim 1, wherein the first and second mobile phases each comprise a solvent and water.

9. The method of claim 8, wherein the first step feedstock further comprises water in an amount equal to or greater than an amount of water in the first mobile phase.

10. The method of claim 8, wherein the amount of water in the first step feedstock is lower than an amount of water in the second mobile phase.

11. The method of claim 1 , further comprising a step of: maintaining the process temperature within a range of a desired set point plus or minus two degrees.

12. The method of claim 1, further comprising a step of: recycling at least one of a component-free output solvent or a mobile phase from a process step for re-introduction to the process.

13. The method of claim 1, further comprising a step of: forming the first mobile phase by combining solvent and water in a first blending module at a predetermined ratio.

14. The method of claim 13, further comprising steps of: metering an amount of solvent into the first blending module with a solvent proportioning mechanism; metering an amount of water into the first blending module with a first water proportioning mechanism; measuring the amounts of solvent and water in the first mobile phase exiting the first blending module with a proportion control; and providing feedback from the proportion control to the solvent proportioning mechanism and the water proportioning mechanism to control the metering of the solvent and the water.

15. The method of claim 1, further comprising a step of: forming the second step feedstock by combining the first step output stream and water in a second blending module at a predetermined ratio.

16. The method of claim 15, further comprising steps of: metering an amount of the first step output stream into the second blending module with a first step output stream proportioning mechanism; metering an amount of water into the second blending module with a second water proportioning mechanism; measuring the amounts of the first step output stream and the water in the second step feedstock exiting the second blending module with a second proportion control; and providing feedback from the second proportion control to the first step output stream proportioning mechanism and the second water proportioning mechanism to control the metering of the first step output stream and the water.

17. The method of claim 1, wherein the step of loading the second step chromatography column with the second step feedstock comprises combining multiple first step outputs in the second step feedstock.

18. The method of claim 1, further comprising a step of: removing mobile phase from the second step output stream.

19. The method of claim 18, wherein the step of removing mobile phase from the second step output stream includes adding water to the second step output stream.

20. The method of claim 18, wherein the step of removing mobile phase from the second step output stream includes controlling the temperature to facilitate formation of a product layer.