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Written by The Clever Chromatographer
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Thursday, 02 September 2010 18:18 |
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A recent article in the Journal of Separation Science entitled “Semi-preparative high-speed counter-current chromatography separation of alkaloids from embryo of the seed of Nelumbo nucifera Gaertn by pH-gradient elution” introduces a new gradient CCC method . In this study, the authors prepared and pre-equilibrated a diethyl ether/aqueous sodium phosphate buffer (pH 7.5) solvent system. The CCC column was filled by pumping a 50/50 mixture of the two phases into the column. The column was then equilibrated by pumping the aqueous mobile aqueous phase through the column prior to sample injection. After the sample was injected, the pH 7.5 buffer was gradually replaced by a pH 7.2 buffer. Amazingly, shake flask experiments had shown that the K values of the three target compounds, liensinine, isoliensinine, and nerferine changed significantly in a pH 7.5 aqueous phase compared to a pH 7.2 aqueous phase. It is worth noting that liensinine and isoliensinine are positional isomers. In this case, diminishing the pH of the aqueous phase by 0.3 pH units reduced the K values of the target compounds by a factor of four. The chromatogram resembles the classical CCC elution peaks rather than pH-zone refining “plateaus.”
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Last Updated on Thursday, 02 September 2010 18:20 |
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Written by The Clever Chromatographer
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Thursday, 05 August 2010 19:51 |
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A recent article in the Journal of Chromatography B entitled “Centrifugal Partition Chromatography as a Tool for Preparative Purification of Pea Albumin with Enhanced Yields” highlights an interesting application of CPC to the isolation of proteins. The pea albumin in question refers to a cysteine-knot toxin 
unceremoniously called PA1b. This polypeptide has been found to be toxic to cereal weevils and other pests as described in PA1b, an insecticidal pro tein extracted from pea seeds (Pisum sativum). A biphasic solvent system consisting of n-butanol and 20 mM aqueous TFA (pH 2.2) was used for the separation. Interestingly the inclusion of TFA made the target protein less soluble in the aqueous phase. The target protein had a K value of 0.3 in a butanol/water solvent system. When TFA was added the K value increased to 1.7. Water solu ble components were first eluted with the aqueous phase mobile, and the PA1b was eluted by switching to the mobile phase to butano l. Recovery yield was increased more than 50 time over (an)ion exchange chromatography preparation. Purity was assessed by Reversed Phase HPLC.(image from Dr. Ulf Diederichsen)
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Last Updated on Friday, 06 August 2010 07:29 |
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Multiple Dual-Mode Centrifugal Partition Chromatography |
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Written by The Clever Chromatographer
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Monday, 26 July 2010 04:45 |
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I recently read a review article published in an issue Journal of Liquid Chromatography and Related Technologies dedicated to Jack Cazes entitled “Centrifugal Partition Chromatography: Application to Natural Products in 1994-2009.” In addition to offering a comprehensive review of CPC natural product separations the authors included very up-to-date articles in their review. One of these articles is entitled “Multiple dual-mode centrifugal partition chromatography, a semi-continuous development mode for routine laboratory-scale purifications” found in the Journal of Chromatography B. The first thing that caught my attention was that they were looking for mycosporines (not to be confused with Mycosporin) in lichens. Lichens is a species that does not show up too often in natural products papers. The authors present a very colorful and detailed figure of the multiple dual mode method. The column is filled with lower (aqueous) phase. The column is loaded with the upper (organic) phase mobile (“ascending” in CPC language). A few milliliters of nonpolar compounds are eluted before aqueous phase is introduced in descending mode. A few milliliters of polar compounds are subsequently eluted. Then, the mode is switched back to ascending mode with the organic phase mobile. At this point, two target compounds are eluted in a larger volume. Finally, the column is extruded with organic phase being pumped in with descending mode. A lot of gymastics for a couple of compounds, but an interesting and educational application of countercurrent separation technology.
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3 HSCCCs for the price of 1 |
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Written by The Clever Chromatographer
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Sunday, 11 July 2010 13:30 |
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A recent article in Journal of Chromatography A covers an interesting CCC method termed “Multi-channel CCC.” The article entitled “Multi-channel counter-current chromatography for high-throughput fractionation of natural products for drug discovery” Essentially, a traditional three-bobbin HSCCC centrifuge was wrapped with three parallel tubes instead of just one. Each tube can be run as a separate column; each with its own solvent reservoir, pump, detector, and fraction collector. Each column is essentially identical so the results should be the same as doing three different experiments on the same instrument. In this case the authors ran three different extracts in the same hexane-ethanol-water solvent system. The centrifuge is the biggest investment in a CCC setup so maximizing its performance seems reasonable. The “peripherals” are common liquid chromatography instruments and apparatuses that can be found in abundance in most laboratories. Obviously, the full capacity of an HSCCC centrifuge cannot be realized if several tubes are wound on the bobbins instead of just one. Are three parallel chromatographic separations more desirable than three sequential chromatographic separations? In my experience processing the fractions obtained from a separation is the rate-limiting step in a chromatographic procedure and not the length of the chromatographic separation. I wonder if there is a way to plumb this system so that two or three columns can be run sequentially – basically lengthening the column by a factor of 2 or 3 respectively. In my mind, this may be a better feature than three parallel runs.
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Last Updated on Monday, 26 July 2010 04:56 |
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Written by The Clever Chromatographer
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Monday, 21 June 2010 18:48 |
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The Journal of Chromatographic Science, a journal that has not published many CCC/CPC articles in the past, had a special issue on counter-current chromatography during May/June 2009. Seven articles described high speed countercurrent chromatography separations of natural products and other molecules. The article in this issue that caught my eye describes the “Separation of Five Isomers of Dihydroxybenzoic Acid by High-Speed Counter-Current Chromatography with Dual-Rotation Elution Method.” This article represents not only an interesting application of HSCCC to separate five positional isomers of dihydroxybenzoic acid but also describes two methods of extending the chromatographic run to empty the stationary phase. The solvent system of choice was the well-known n-hexane-ethyl acetate-methanol-water formulation. The lower aqueous phase was the mobile phase in both methods. The “dual-mode” method requires that the phase being pumped into the column is changed while the head-to-tail direction is changed to tail-to-head. The effectively elutes the most highly retained solutes soon after the solvent and flow directional change. In the “dual-rotation” method involves changing the phase being pumped into the column while the direction of rotation is changed from “forward” to “reverse.” Surprisingly, the compounds continued to elute in the order of increasing partition coefficient with this method. In this solvent system 3,5 and 3,4-dihydroxybenzoic acid had overlapped peaks but the other three positional isomers were apparently baseline separated.
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Last Updated on Monday, 26 July 2010 04:56 |
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Written by The Clever CHromatographer
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Thursday, 17 June 2010 05:36 |
What do you know, there is a follow up article to the previous blog. The article in Journal of Chromatography A is entitled “Application of silver ion in the separation of macrolide antibiotic components by high-speed counter-current chromatography.” The authors describe the separation of ascomycin, tacrolimus, and dihydrotacrolimus with a hexane-methy tert-butylether- (0.10 M) aqueous silver nitrate solvent system. The contrast between the separation of the three macrolides without and with silver nitrate was quite impressive. The silver nitrate could even be recovered at the end by evaporating the solvents. Various concentrations of silver nitrate were tested with quite good results between 0.05 and 0.2 M. I must also confess that a commercial silver-ion impregnated SPE column is commercially available – Discovery Ag-Ion SPE marketed by SUPELCO.
 
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