Organic compounds -- part of the class 532-570 series – Organic compounds – Fatty compounds having an acid moiety which contains the...
Reexamination Certificate
2000-02-17
2002-06-04
Carr, Deborah D. (Department: 1621)
Organic compounds -- part of the class 532-570 series
Organic compounds
Fatty compounds having an acid moiety which contains the...
Reexamination Certificate
active
06399803
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to adsorptive separation of triglycerides. More specifically, this invention relates to a novel process for separating a first triglyceride comprising a docosahexaenoic acid residue from at least one other triglyceride using a chromatographic separation zone having a stationary phase which comprises metal ions that are capable of coordinating with a double bond of a fatty acid residue of the first triglyceride to form a metal complex with the fatty acid residue.
BACKGROUND OF THE INVENTION
Docosahexaenoic acids (“DHA”) are 22-carbon, naturally-occurring, unbranched fatty acids containing 6 carbon—carbon double bonds. It is well known that many triglycerides comprising a DHA residue (and particularly triglycerides comprising two DHA residues) have beneficial nutritional and pharmaceutical properties. For example, such compounds may be used to treat cardiovascular and inflammatory diseases. They also may be added to infant formula to promote the development of brain and retina functions.
As used herein, a “triglyceride” is an ester of three fatty acids and glycerol, and has the general chemical formula: CH
2
(OOCR
1
)CH(OOCR
2
)CH
2
(OOCR
3
), wherein OOCR
1
, OOCR
2
, and OOCR
3
are each fatty acid residues. Each residue may be saturated (i.e., all the bonds between the carbons are single bonds) or unsaturated (i.e., the residue contains one or more carbon—carbon double or triple bonds), and the double bonds may each have a cis or trans configuration. To illustrate, a triglyceride having two 4, 7, 10, 13, 16, 19-DHA residues (i.e., a fatty acid residue containing 22 carbons and 6 carbon—carbon double bonds between the 4th & 5th, 7th & 8th, 10th & 11th, 13th & 14th, 16th & 17th, and 19th & 20th carbons, counting from the carbonyl group of the residue, the carbon of the carbonyl group being the first carbon counted) and one palmitic acid residue (i.e., a saturated fatty acid residue comprising 16 carbons) may have the following Formula (I) (without taking into consideration whether the double bonds have cis or trans configurations):
Likewise, a triglyceride having a 4, 7, 10, 13, 16, 19-DHA residue, a 4, 7, 10, 13, 16-docosapentaenoic acid (“DPA”) residue (i.e., a fatty acid residue containing 22 carbons and 5 carbon—carbon double bonds between the 4th & 5th, 7th & 8th, 10th & 11th, 13th & 14th, and 16th & 17th carbons, counting from the carbonyl group of the residue, the carbon of the carbonyl group being the first carbon counted), and a palmitic acid residue may have the following formula (II) (without taking into consideration whether the double bonds have cis or trans configurations):
Unsaturated fatty acid residues of triglycerides are sometimes identified in the literature by an omega (“&ohgr;”) number. This nomenclature is used herein. The omega number denominates the position of the first double bond, when counting from the terminal methyl group of the fatty acid residue. For example, in Formula (II), the DHA residue is an &ohgr;-3 fatty acid residue, and the DPA residue is an &ohgr;-6 fatty acid residue. Often, fatty acid residues (and particularly the DHA residues) having the most beneficial cardiovascular effects are &ohgr;-3 fatty acid residues, although other fatty acid residues (such as arachidonic acid and &ggr;-linolenic acid, which are both &ohgr;-6 fatty acid residues) also have proven to be beneficial as well.
Sources of triglycerides containing at least one DHA residue include, but are not limited to, modified vegetable oils, marine animal oils (e.g., seal and whale blubber), fish oils (e.g., menhaden oil, salmon oil, mackerel oil, cod oil, herring oil, sardine oil, capelin oil, and tuna oil), marine algae, and human milk. Such sources, however, typically contain many other categories of compounds. These compounds include various other triglycerides containing a wide variety of fatty acid residues (e.g., the &ohgr;-6 DPA residue in Formula (II)) having varying degrees of nutritional or pharmaceutical value. It is therefore often desirable to separate a triglyceride containing at least one DHA residue (especially a triglyceride containing 2 DHA residues) from other compounds in the source before the triglyceride is used for nutritional or pharmaceutical purposes.
Numerous methods have been used alone or in combination to isolate (or at least concentrate) and recover specific fatty acids and their derivatives (i.e., esters, amides, triglycerides, etc.) from various naturally occurring sources. These processes include fractional crystallization at low temperatures, molecular distillation, urea adduct crystallization, extraction with metal salt solutions, super critical fluid fractionation on countercurrent columns, and stationary bed chromatography.
A stationary bed chromatographic system may employ a non-polar stationary phase (i.e., a reverse phase chromatographic system) or a polar stationary phase (i.e., a normal phase chromatographic system). Use of a reverse phase chromatographic system is disclosed in T. Nakahara, T. Yokochi, T. Higashihara, S. Tanaka, T. Yaguchi, & D. Honda, “Production of Docosahexaenoic and Docosapentaenoic Acids by Schizochytrium sp. Isolated from Yap Islands,”
JAOCS
, vol. 73, no. 11, pp. 1421-26 (1996). Nakahara et al. report isolating four triglycerides containing DHA residues from a marine micro algae (i.e., Schizochytrium sp.) by a reverse-phase, high-performance liquid chromatographic process using an acetone/acetonitrile mobile phase and an octadecylsilane (“ODS”) stationary phase. This stationary phase separates the triglycerides based on the strength of the Van der Waals forces between the stationary phase and the fatty acid residues of the triglycerides. Such a stationary phase tends to be costly relative to other conventional stationary phases (e.g., silica gel) used in normal-phase chromatography.
Others have disclosed using high performance liquid chromatography which employs a stationary phase comprising silver ions. Separation using such a column is based on the principle that a metal ion (e.g., a silver ion) will coordinate reversibly with electrons of a &pgr; orbital of a double bond between carbon atoms of an unsaturated fatty acid residue to form a metal complex with the unsaturated fatty acid residue. The strength of the complex tends to be dependent on, for example, the chain length of the fatty acid residue, the number of carbon—carbon double bonds in the residue, the positions of the double bonds, and whether the double bonds have cis or trans configurations. Although this technique has been used to separate triglycerides from mixtures of triglycerides, the types of triglycerides which have been separated by this technique have been limited to date. Application of silver ion high performance liquid chromatography to separate triglycerides has been discussed in, for example, I. Elfman-Borjesson, S. V. D. Hark, & M. Harrod, “Gradients of n-Heptane and Acetonitrile in Silver-Ion High-Performance Liquid Chromatography Analyses of cis and trans Bonds in Lipids,”
JAOCS
, vol. 74, no. 9, pp. 1177-80 (1997). A separate discussion of such an application may be found in P. Laakso & P. Voutilainen, “Analysis of Triacylglycerols by Silver-Ion High-Performance Liquid Chromatography—Atmospheric Pressure Chemical Ionization Mass Spectrometry,”
Lipids
, vol. 31, no. 12, pp. 1311-21 (1996). An additional separate discussion of such an application may be found in G. Dobson, W. W. Christie, & B. Nikolova-Damyanova, “Review: Silver Ion Chromatography of Lipids and Fatty Acids,”
J. Chrom. B
, vol. 671, pp. 197-222 (1995). A further separate discussion of such an application may be found in B. Nikolova-Damyanova, “Silver Ion Chromatography and Lipids,”
Advances in Lipid Methodology—One
, Ch. 6, pp. 182-237 (W. W. Christie, ed., The Oily Press, Ayr, Scotland, 1992).
SUMMARY OF THE INVENTION
This invention provides for a reliable and cost-effective process that may be used to separate a triglyceride comprising at least one DHA residue (and particularly triglycerides compri
Corley David G.
Doom James P.
Duffin Kevin L.
Zeller Samuel G.
Carr Deborah D.
Omegatech, Inc.
Ross P.C. Sheridan
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