Figure 1. Core-Shell Particle Technology

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1 Purification of Chlorogenic cid from Green Coffee using Kinetex Core-Shell Technology in xia Preparative Formats Zeshan qeel Technical Magician Zeeshan loves to collect watches and the ack to the Future Trilogy. e has twin boys which drive him crazy! e is an pple Fanboy for life and he likes being in the lab more than anywhere else. Zeshan qeel, J Preston, J.T. Presley, and Marc Jacob Phenomenex, Inc., 4 Madrid ve., Torrance, C 95 US Introduction igh performance PLC/UPLC core-shell material is the latest technological advancement in chromatographic media. When used under analytical conditions, core-shell particles show improved efficiency and performance over fully porous particles of equivalent particle size.,2 With the recent commercialization of a lower pressure 5 µm core-shell media, it is now possible to offer core-shell media in a preparative format (>2 mm ID) that is compatible with standard prep LC systems. In this technote, we will demonstrate that this new 5 µm coreshell particle size, available in a variety of bonded phases, can be packed efficiently in preparative formats with internal diameter greater than 2 centimeters. We will highlight the advantage of such product for the isolation of antioxidant chlorogenic acid from Green Coffee. in regards to column efficiency. xia preparative columns (>2 mm ID) were introduced in 26. The xia PLC columns incorporate a patented ydraulic Piston Compression technology that increases bed density and eliates media bed collapse as a source of premature column failure Figure 2. Unlike traditional pre-packed preparative column packing methods, the xia packing method is completely automated and computer monitored which results in improved efficiencies, peak symmetries, and overall column performance. Figure. Core-Shell Particle Technology Results and Discussion Core-shell particles are composed of an inner solid core surrounded by a layer of fully porous silica as shown in Figure. Those particles were developed more than 5 years ago by C. orvath and reintroduced in 29 by Phenomenex due to the advantage they offer over fully porous material Figure 2. xia Patented Packing Technology The packing piston head is integrated into the column and locked by the piston retainers, so the pressure is never released. Media packed under ideal pressure Media is never allowed to relax, eliating voids and dramatically improving reproducibility, column-to-column. For additional technical notes, visit Page of 8

2 Results and Discussion (cont d) One investigation performed by F. Gritti and G. Guiochon analyzes the Van Deemter equation for fully porous and coreshell analytical columns and evaluates the contributions of the,, and C coefficients to plate height. They detered that core-shell media significantly reduces band broadening by affecting ddy dispersion (), and longitudinal diffusion (), while mass transfer (C) was detered to be a or component as depicted in Figure 3a. The high column performance is explained essentially by a more homogeneous column packing. 2 Combining 5 μm core-shell and xia technology results in efficiency N that can reach up to 25, plates/meter for a 2.2 mm ID prep column Figure 3b. The Kinetex core-shell family of particles are designed such that the shell volume to particle size ratio is maintained throughout the particle size. This means that regardless of particle size (.3,.7, 2.6, and 5 µm), the retention and the selectivity remains constant, allowing for excellent scalability of methods from particle and/or system platforms. Kinetex core-shell 5 μm particles are available in eight (8) different phases for unique selectivities (phases currently available are C8, X-C8, VO C8, C8, Phenyl- exyl, iphenyl, PFP and ILIC). Six phases are seen in Figure 4. Prior to the scale-up of preparative conditions, we screened 6 RP core-shell phases to evaluate the selectivity in order to choose the best phase for the isolation of pure chlorogenic acid from green coffee extract (the green coffee extract was obtained from ChromaDex ). The results using a 2 gradient are represented in Figure 4. Under. % TF conditions, the best separation was obtained on a Kinetex 5 µm X-C8 column. It is interesting to note that with the C8 phase, the selectivity was reversed between chlorogenic acid and impurity. Figure 3a. Core-Shell Particles Impact on and roadening and Column fficiency x 4.6 mm Fully Porous Luna C8(2) x 4.6 mm Core-Shell Kinetex C8 Reduced plate height (h) 5 5 x 4.6 mm Luna C8(2) ddy dispersion Longitudinal diffusion Solid-liquid mass transfer Reduced plate height (h) 5 5 x 4.6 mm Kinetex C8 ddy dispersion Longitudinal diffusion Solid-liquid mass transfer Reduced velocity ( ) Reduced velocity ( ) Van Deemter quation: = d p + /µ + C d e 2 µ Figure 3b. Naphthalene peak efficiency for a Kinetex 5 µm X-C8 xia preparative column Response - Millivolts N=2,26 plates/meter pp ID 2455 Column: Kinetex 5 µm X-C8 xia Packed Dimensions: 5 x 2.2 mm Mobile Phase: Water/ cetonitrile (5:5) Injection Volume: µl Flow Rate: 25 ml/ Temperature: mbient Detection: 254 nm Instrument: Shimadzu LC 2 Injection Volume 2 ml Sample:. Uracil 2. cetophenone 3. Toluene 4. Naphthalene Page 2 of 8

3 Figure 4. Screening of Green Coffee xtract on Various Kinetex Phases C8 X-C8 O CO Si O 6 C8 4 2 C D F G pp ID D C X- C F G pp ID O O O Chlorogenic cid O O VO C C + D iphenyl C D F G VO C8 iphenyl F G pp ID pp ID C C D Phenyl-exyl C D F G C8 Phenyl- exyl F G pp ID pp ID Conditions for all columns: Columns: Kinetex 5 µm C8 Kinetex 5 µm X-C8 Kinetex 5 µm VO C8 Kinetex 5 µm C8 Kinetex 5 µm iphenyl Kinetex 5 µm Phenyl-exyl Dimensions: x 4.6 mm Mobile Phase: :. % TF in Water :. % TF in cetonitrile Gradient: Time () % Flow Rate:.5 ml/ Temperature: 25 C Detection: 33 nm Instrument: gilent Sample:. Chlorogenic cid Others: ntioxidants from green coffee Packing Material Kinetex C8 Phase Characteristics Particle Size (µm).3,.7, 2.6, 5 Pore Size (Å) Surface rea (m 2 /g) Carbon Load (%) p Range * Kinetex X-C8.7, 2.6, * pplications Type of Compounds Loading Small Molecules Peptides Proteins Chiral Oligonucleotides cids Polar ydro-phobic ases vailable Surface rea Kinetex VO C8.7, 2.6, Kinetex C8.7, 2.6, * Kinetex Phenyl-exyl.7, 2.6, * Kinetex iphenyl.7, 2.6, * Key: est Suited Very Good *Columns are p stable from.5- under isocratic conditions. Columns are p stable under gradient conditions. For additional technical notes, visit Page 3 of 8

4 Results and Discussion (cont d) fter screening and before purification, we optimized the method and used a Kinetex 5 µm X-C8 5 x 4.6 mm ID (Internal diameter) column with a 25 gradient as depicted in Figure 5. We also increased the loading up to 5 mg on-column. (Figure 6). Figure 5. nalysis of Crude Green Coffee xtract The separation was then scaled-up to a 2.2 mm ID xia preparative column packed with same core-media and the loading was increased accordingly to mg of green coffee extract (scaling factor of 2 from 4.6 to 2.2 mm ID) as represented in Figure 7. Fractions of ml were collected, analyzed, and the fractions containing chlorogenic acid with purity greater than 94 % at 33 nm, were pooled together (Fraction 3-5). Fraction overlays are shown in Figure 8 and the overlay includes reference standard obtained from ChromaDex, Inc. nalysis of the pool shows a purity greater than 99 % and contained approximately 5 mg of pure product for a recovery of 42 % in one run (Figure 9). Scaling parameter and basic equations 8 6 Column Column pp ID 23 L L Column: Kinetex 5 µm X-C8 Dimensions: 5 x 4.6 mm Part No.: F-465- Mobile Phase: :. % TF in Water :. % TF in cetonitrile Gradient: 5 to 2 % in 25 hold 2 at 2 % Flow Rate:.5 ml/ Temperature: 25 C Detection: 33 nm Instrument: gilent Injection Volume µl Sample: 5 mg/ml in water Figure 6. Loading xperiments on nalytical Column 3.5 mg mg.5 mg 2.5 mg 5 mg On Column Column: Kinetex 5 µm X-C8 Dimensions: 5 x 4.6 mm Part No.: F-465- Mobile Phase: :. % TF in Water :. % TF in cetonitrile Gradient: 5 to 2 % in 25 hold 2 at 2 % Flow Rate:.5 ml/ Temperature: 25 C Detection: 33 nm Instrument: gilent Injection Volume Varied to µl Sample: 5 mg/ml in water pp ID 239 d d 2 Scaling Factor SF SF = (d 2 /d ) 2 Flow Rate F F 2 = F x SF Loading m m2 = m x L 2 L x SF Notes: - Recommended to keep particle size and length constant - If column length is increased, loading can be increased proportionally and retention time as well as backpressure will also increase. - With scale up, change in hardware and flow cell may be required Page 4 of 8

5 Figure 7. Preparative Purification of Chlorogenic cid Figure 9. Purity for Pooled Fractions Fraction 3,4,5, Chlorogenic cid purity > 99 % Volts Volts.4 Figure 8. Purification Fractions Overlay Volts uv Column: Kinetex 5 µm X-C8 Dimensions: 5 x 2.2 mm Part No.: F-465-P-X Mobile Phase: :. % TF in Water :. % TF in cetonitrile Gradient: 5 to 2 % in 25 Flow Rate: 3 ml/ Temperature: 25 C Detection: 33 nm Instrument: Shimadzu LC 2 Injection Volume 2 ml Sample: 5 mg/ml in water Standard Fraction 3 Fraction 4 Fraction 5 Fraction 6 Impurity pp ID 238 Chlorogenic cid pp ID Conclusion Conditions: Same as figure 5 Instrument: Shimadzu Proence LC With the release of Kinetex core-shell 5 µm media, it is now possible to perform lab-scale purifications and as a result take advantage of core-shell technology for both analytical and preparative purposes. Combination of Kinetex core-shell 5 μm particle and xia patented packing technologies result in preparative columns with high efficiency in the range of 25-6 % greater than columns packed with fully porous media of equivalent particle size. Scale up from analytical sub-2 μm to preparative columns in 8 different bonded phases is now feasible and readily available. From green coffee extract, we were able to recover pure chlorogenic acid with purity of 99 % at 33 nm and a recovery of 4 % in one purification run using an xia preparative column 5 x 2.2 mm ID. This can be scaled up further to a large format such as 25 x 3 mm ID for increased throughput and yield. References. F. Gritti and G. Guiochon LC-GC, 22, 3, F. Gritti and G. Guiochon J. Chromatogr., 23, 23, pp ID Conditions: Same as figure 5 Instrument: Shimadzu Proence LC For additional technical notes, visit Page 5 of 8

6 Kinetex Ordering Information 5 μm Minibore Columns (mm) ULTR Cartridges Phases 3 x 2. 5 x 2. x 2. 5 x 2. 3/pk VO C N N D-4633-N F-4633-N J-9298 iphenyl N N D-4627-N J-929 X-C8-465-N -465-N D-465-N J-8782 C8-46-N -46-N D-46-N F-46-N J-8782 C8-468-N D-468-N J-8784 Phenyl-exyl -463-N D-463-N J-8788 for 2. mm ID 5 μm Midore Columns (mm) ULTR Cartridges Phases 5 x 3. x 3. 5 x 3. 3/pk VO C Y D-4633-Y F-4633-Y J-9297 iphenyl Y D-4627-Y F-4627-Y J-928 X-C8-465-Y D-465-Y F-465-Y J-8775 C8-46-Y D-46-Y F-46-Y J-8775 C8-468-Y D-468-Y J-8777 Phenyl-exyl -463-Y D-463-Y J-878 for 3. mm ID 5 μm nalytical Columns (mm) ULTR Cartridges Phases 5 x 4.6 x x x 4.6 3/pk VO C D F G J-9296 iphenyl D F G J-927 X-C D-465- F-465- G-465- J-8768 C8-46- D-46- F-46- G-46- J-8768 C D-468- F-468- G-468- J-877 Phenyl-exyl D-463- F-463- G-463- J-8774 for 4.6 mm ID 5 μm Semi-Preparative Columns (mm) SemiPrep Cartridges*** Phases 5 x 25 x x C8 F-46-N G-46-N J-9278 iphenyl F-4627-N G-4627-N J-928 for mm ID 5 μm xia Packed Preparative Columns (mm) PRP Cartridges* Phases 5 x 2.2 x x x x 2.2 VO C P-X D-4633-P-X F-4633-P-X G-4633-P-X J-934 iphenyl P-X D-4627-P-X F-4627-P-X G-4627-P-X J-9272 X-C8-465-P-X D-465-P-X F-465-P-X G-465-P-X J-945 C8-46-P-X D-46-P-X F-46-P-X G-46-P-X J-945 C8-468-P-X D-468-P-X F-468-P-X G-468-P-X J-925 Phenyl-exyl -463-P-X D-463-P-X F-463-P-X G-463-P-X J-947 ILIC D-466-P-X F-466-P-X G-466-P-X J-9277 for 2.2 mm ID 5 μm xia Packed Preparative Columns (mm) PRP Cartridges** Phases 5 x 3 x 3 5 x 3 25 x 3 5 x 3 VO C U-X D-4633-U-X F-4633-U-X G-4633-U-X J-935 iphenyl F-4627-U-X J-9273 X-C8-465-U-X D-465-U-X F-465-U-X G-465-U-X J-924 C8-46-U-X D-46-U-X F-46-U-X G-46-U-X J-924 C8-468-U-X D-468-U-X F-468-U-X G-468-U-X J-927 Phenyl-exyl -463-U-X D-463-U-X F-463-U-X G-463-U-X J-926 for 3 mm ID ULTR Cartridges require holder, Part No.: J-9 * PRP Cartridges require holder, Part No.: J-8223 ** PRP Cartridges require holder, Part No.: J-8277 *** SemiPrep Cartridges require holder, Part No.: J-928 Page 6 of 8

7 Kinetex Ordering Information 2.6 μm Minibore Columns (mm) ULTR Cartridges Phases 3 x 2. 5 x x 2. x 2. 5 x 2. 3/pk VO C N N D-4725-N F-4725-N J-9298 F N N D-4723-N F-4723-N J-9322 iphenyl N N D-4622-N F-4622-N J-929 X-C N N C-4496-N D-4496-N F-4496-N J-8782 C N N C-4462-N D-4462-N F-4462-N J-8782 C N N C-4497-N D-4497-N F-4497-N J-8784 ILIC -446-N -446-N C-446-N D-446-N F-446-N J-8786 Phenyl-exyl N N C-4495-N D-4495-N F-4495-N J-8788 for 2. mm ID 2.6 μm Midore Columns (mm) ULTR Cartridges Phases 3 x 3. 5 x x 3. x 3. 5 x 3. 3/pk VO C Y D-4725-Y F-4725-Y J-9297 F Y D-4723-Y F-4723-Y J-932 iphenyl Y D-4622-Y F-4622-Y J-928 X-C Y Y C-4496-Y D-4496-Y F-4496-Y J-8775 C Y Y C-4462-Y D-4462-Y F-4462-Y J-8775 C Y Y C-4497-Y D-4497-Y F-4497-Y J-8777 ILIC -446-Y F-446-Y J-8779 Phenyl-exyl Y D-4495-Y F-4495-Y J-878 for 3. mm ID 2.6 μm nalytical Columns (mm) ULTR Cartridges Phases 3 x x x 4.6 x x 4.6 3/pk VO C D F J-9296 F D F J-932 iphenyl D F J-927 X-C C D F J-8768 C C D F J-8768 C C D F J-877 ILIC C-446- D-446- F-446- J-8772 Phenyl-exyl C D F J-8774 for 4.6 mm ID.7 μm Minibore Columns (mm) ULTR Cartridges Phases 3 x 2. 5 x 2. x 2. 5 x 2. 3/pk VO C N D-4726-N F-4726-N J-9298 F N D-4722-N F-4722-N J-9322 iphenyl N D-4628-N F-4628-N J-929 X-C N N D-4498-N F-4498-N J-8782 C N N D-4475-N F-4475-N J-8782 C N N D-4499-N F-4499-N J-8784 ILIC N N D-4474-N J-8786 Phenyl-exyl -45-N D-45-N F-45-N J-8788 for 2. mm ID.7 μm Midore Columns (mm) ULTR Cartridges Phases 3 x 3. 5 x 3. x 3. 3/pk X-C Y Y D-4498-Y J-8775 C Y D-4475-Y J-8775 C Y Y D-4499-Y J-8777 ILIC Y J-8779 for 3. mm ID.3 μm Minibore Columns (mm) Phases 3 x 2. 5 x 2. C8-455-N -455-N ULTR Cartridges require holder, Part No.: J-9 If Kinetex analytical columns do not provide you with at least equivalent separations to a competing column of the same phase, particle size, and dimensions, return the column with comparative data within 45 days for a FULL RFUND. For additional technical notes, visit Page 7 of 8

8 ustralia t: +6 () f: +6 () ustria t: +43 ()-39-3 f: +43 ()-39-3 Luxembourg t: +3 () f: +3 () Mexico t: f: Terms and Conditions Subject to Phenomenex Standard Terms and Conditions, which may be viewed at Trademarks Kinetex and Luna are registered trademarks, xia Midore, and are trademarks of Phenomenex. gilent is a registered trademark of gilent Technologies, Inc. Shimadzu and Proence are registered trademarks of Shimadzu Corporation. ChromaDex is a registered trademark of ChromaDex, Inc. xia column and packing technology is patented by Phenomenex. U.S. Patent No. 7, 674, 383. Kinetex VO is patented by Phenomenex. U.S. Patent Nos. 7,563,367 and 8,658,38 and foreign counterparts. 25 Phenomenex, Inc. ll rights reserved. elgium t: +32 () (French) t: +32 () (Dutch) f: +3 () beinfo@phenomenex.com Canada t: + (8) f: + (3) info@phenomenex.com China t: +86 () f: +86 () chinainfo@phenomenex.com Denmark t: f: nordicinfo@phenomenex.com Finland t: +358 () f: nordicinfo@phenomenex.com France t: +33 () f: +33 () franceinfo@phenomenex.com Germany t: +49 () f: +49 () anfrage@phenomenex.com India t: +9 () f: +9 () indiainfo@phenomenex.com Ireland t: +353 () f: eireinfo@phenomenex.com Italy t: f: italiainfo@phenomenex.com The Netherlands t: +3 () f: +3 () nlinfo@phenomenex.com New Zealand t: +64 () f: +64 () nzinfo@phenomenex.com Norway t: f: nordicinfo@phenomenex.com Puerto Rico t: + (8) 54-PLC f: + (3) info@phenomenex.com Spain t: f: espinfo@phenomenex.com Sweden t: +46 () f: nordicinfo@phenomenex.com United Kingdom t: +44 () f: +44 () ukinfo@phenomenex.com US t: + (3) f: + (3) info@phenomenex.com ll other countries Corporate Office US t: + (3) f: + (3) info@phenomenex.com Phenomenex products are available worldwide. For the distributor in your country, contact Phenomenex US, International Department at international@phenomenex.com TN337465_W Page 8 of 8