Science Together AZURA® SMB systems Chromatography for continuous separations 1 AZURA® SMB systems Simulated moving bed chromatography (SMBC) By repeated use of the SMB process each partial is increasingly applied as a separation technique fraction can be separated into a further fraction in the pharmaceutical industry, production of fine – down to binary substance mixtures. Typically, chemicals and in the field of bioengineering. SMB the SMB process is set up in advance for a two SMB chromatography is a HPLC technique for the separation is a method in process chromatography that en-component mixture. Following this, both sub-of binary mixtures with high productivity and puritiy. ables substance mixtures to be continuously sepa-stances can be immediately extracted in pure rated and extracted in two fractions. form. What is the difference between batch LC and SMBC? 1 2 3 Batch chromatography (single-column) SMB chromatography (multi-column) Work faster, save space Reduce your costs Keep your sample concentrated Unlimited number of fractions Two fractions, no waste Recovery typically below 80 % Recovery up to 100 % EITHER high purity OR high yield High purity AND high yield Isocratic or gradient Isocratic High solvent consumption Can be as low as 10 % of batch consumption Get higher productivity and pu-Save up to 90 % of the solvent Gain nearly undiluted product rity than with comparable batch and reduce the solid phase and minimize concentration Very diluted product Product concentration comparable with input concentration (feed) systems – even with a smaller costs up to 80 % efforts. system. The SMB process is ideally suited for two-com-SMB chromatography can also be used for the ponent separations (Fig. 1a). For the task of sep-separation of more than two peaks (mulit-com-arating and collecting multiple fractions, classi-ponent mixtures). Therefore it is possible to “split cal batch LC might the better option (Fig. 1b). the chromatogram” at a certain point (Fig. 1 c). KNAUER offers system solutions for continuous separation tasks as well as for batch separations. RAFFINATE EXTRACT PRODUCT RAFFINATE EXTRACT Visit us online or take a look at page 20 and 21 B s iom ule olec for more information. B ule s iom s ule olec Small molec ules Small molec Fig. 1a: Basic binary mixture for a Fig. 1b: Typical multi-component Fig. 1c: Multi-component mixture, SMB separation. mixture for classical batch chroma- can be separated in two different tography. fractions with SMB. 2 www.knauer.net/smb 3 AZURA® SMB systems PurityChrom® MCC Intuitive and highly functional control software This SMB system is optimized for separation tasks requirements of every separation the SMB system on a scale of several hundred grams. The standard can be freely configured via valve switch configuration consists of four AZURA® assistants (e.g. closed-loop, open-loop, 3-zone) and is up-ASM 2.1L with seven multi-position valves and gradable with detectors and flow meter. See table Flexibility four AZURA® pumps P 4.1S as well as our us-for available configurations of the AZURA® SMB Up to 8 columns at Gram scale er-friendly software PurityChrom® MCC including Lab. Individual configuration is available on re-max. 130 bar Flow rates up to required IT hardware. Depending on the special quest. 50 ml/min and columns up to 30 mm ID allow to increase your through- put up to several Available configurations for AZURA® SMB Lab Multi-hundred grams. position valve Max. Number Max. Standard valves flow rate** of columns pressure** Art. No. Description for flexible zone Ultra high pressure SMB 10 ml/min 6 300 bar A29002 definition and low stainless steel (sst) maintenance costs 50 ml/min 8 130 bar A29001* Stainless steel 8 130 bar A29000* Biocompatible (PAEK, ceramic) * Standard configuration ** The maximum operating parameters for flow and pressure depend on the specific columns and customer application. Small footprint Biocompatible Temperature control The AZURA® SMB Fully biocompatible Columns can be systems require little version available. heated or cooled space on the lab bench. Perfect solution for the (requires additional continuous purification equipment). of biomolecules like proteins. 4 www.knauer.net/smb 5 AZURA® SMB systems PurityChrom® MCC Intuitive and highly functional control Wide field of applications Kilogram scale software Up to 8 columns at Flow rates up to max. 100 bar 500 ml/min allow very high throughput on a The AZURA® SMB Pilot is designed for the sepa-The SMB standard configuration consists of four kilogram scale ration of binary mixtures on a hundred gram to AZURA® pumps P 2.1L and seven 8-port multi-po-kilogram scale and is typically used with columns sition valves integrated into four AZURA® assis-up to 50 or 100 mm ID. Its special emphasis is put tants ASM 2.1L. Our user-friendly software on the continuous operation mode and highest PurityChrom® MCC and the required IT hardware productivity. are also included. We offer several variations of the standard system configuration. Available configurations for AZURA® SMB Pilot Max. Number Max. flow rate** of columns pressure** Art. No. Description 8 100 bar A29504 Low dead volume 100 ml/min (1/16", stainless steel) Biocompatible Multi-position valve 8 100 bar A29502 Stainless steel Fully biocompatible version Standard valves for flexible Temperature control made of titanium and PAEK zone definition and low 500 ml/min 8 100 bar A29501* Stainless steel Columns can be on request maintenance requirements heated or cooled (requires additional * Standard configuration equipment) ** The maximum operating parameters for flow and pressure depend on the specific columns and customer application. 6 www.knauer.net/smb 7 AZURA® SMB systems Add a batch system into your SMB Upgrade your system and perform simple separa-Flow rate Material Art. No. tion tasks or estimate your separation para meters directly without buying an additional batch LC sys-up to Stainless steel A29601 tem. The Batch-Upgrade kits include a detector 100 ml/min and an injection valve. Biocompatible A29600 up to Stainless steel A29603 500 ml/min Save space and time with our SMB oven or multi-column stands Biocompatible A29602 Easy access to columns, less bench space required Art. No. Standard installation and familiarization - including system installation, instruction to system and Europe A0000SMBEU software as well as general considerations for SMB operation. International A0000SMBIN Up to 80°C A29902 High-accuracy mass flow meters for highest process stability Full control over the Number of column temperature mini-CORI- Position Monitors Monitor- FLOW ing level Field of application TM 1 Column Process stability + Process evaluation; Up to eight KNAUER standard systems columns (max. 50 mm ID, Process stability; Process evaluation; demanding 250 mm length) 2 Column; feed pump feed flow ++ separations; expensive feed; low feed flow 4 At every pump Pump flow ++++ Production process; complete control over system stability Description Art. No. A29900 A29901 One A29800 mini CORI-FLOW™ M13 for AZURA® SMB Lab, incl. accessories Description Art. No. Four A29801 Oven for AZURA® SMB Lab and AZURA® SMB Pilot systems One A29802 (8 KNAUER columns with up to 250 × 50 mm inner dimension) A29900 mini CORI-FLOW™ M14 for AZURA® SMB Pilot, incl. accessories Four A29803 Column holder for 8 SMB columns with up to 250 × 50 mm A29901 Column holder for 8 SMB columns with 8, 16 mm ID One mini CORI-FLOW™ M13 Hastelloy for AZURA® SMB Lab, incl. accessories A29805 and up to 250 mm length or 20 mm ID and 150 mm length A29902 8 www.knauer.net/smb 9 AZURA® SMB systems When to use which column hardware solution? KNAUER offers stationary phases for nearly any application in normal- or reversed phase mode as well as for special purification tasks in ion exclusion and ligand exchange mode for example. Nearly all KNAUER KNAUER offers purpose-built SMB sets. Additional y to the quality test of every single column, SMB sets phases are also available in larger particle sizes for easy upscaling to SMB processes. are tested for reproducibility to ensure the appropriateness for SMB processes. dAX preparative column hardware • Dynamic axial compression for an extended lifetime • No formation of void volume at the column inlet possible For a stable SMB process, a set of identical and robust HPLC columns is essential as in every of the four zones the separation has to be identical to reach a continuous process. It is recommended to order one additional column in a SMB set (needed columns + 1) to have one column with exactly the specifications of the SMB set for determination of the parameters needed for process layout. dAX Hardware The particle size of the separation material should be 15 µm or larger to ensure a robust purifica-Extremely high life time Special demand tion process. Excellent Reproducibility for extended More cost-intensive column life time While the stationary phase always depends on the application, the recommended column dimensions are mainly dependent on the amount of sample that must be purified. Refill possible Column Loadability* compared to Recommended An overview of all KNAUER dimension analytical 250 x 4 mm column flow rate [ml/min] Standard preparative column hardware columns can be found on www.knauer.net/columns 250 x 4 mm 1 x (analytical reference) 1 • Flanged or standard threaded • Without axial compression 250 x 8 mm 4 x 4 250 x 16 mm 16 x 16 Standard flanged 250 x 20 mm 25 x 25 Hardware • Simple All classical 250 x 30 mm 56 x 55 • cost-effective prep 250 x 50 mm 156 x 150 • versatile applications * The mass and volume loadabilities always depend on the specific sample and application as well as on the stationary phase filled in the HPLC column. The chart only gives a first clue and can be higher or lower than the No refill given numbers. Scale-up factor (SF) calculation: SF = ID²(preparative)/ID²(analytical) option 10 www.knauer.net/smb 11 AZURA® SMB systems Software for multi-column chromatography (MCC) SMB Parameter Wizard System visualization Insert your calculated adsorption The system visualization is everything you parameters. The SMB Parameter need to control the SMB system. The visuali-Wizard will calculate the operation zation can be freely configured. Keep track point. The parameters can easily be of all the information. Security options transferred to the SMB method file. The user management ensures the integrity of your separa- tion methods. In addition every change made through the method is protocolled by the software and saved in the result files. Our PurityChrom® software is 21 CFR part 11 compliant. SMB Operation Point Calculator Adsorption isotherms can be entered into an integrated starting point calculator. The generated values can be checked via a visual feedback very easily. The parameters will be transferred directly into the SMB Parameter Wizard. Software for multi-column chromatography (MCC) System monitor Monitor as many channels as necessary at once and keep full control of your SMB process all the time. 12 www.knauer.net/smb 13 AZURA® SMB systems Feed ZONE 3 Raffinate Column Switching ZONE 2 ZONE 4 AZURA® SMB Lab manifold, stainless steel version. Extract ZONE 1 Eluent SMB process scheme Where can a SMB separation be used? The SMB process enables the separation of bina-The bulk of the eluent is continuously circulated in Range of applications Separation and extraction … ry mixtures by means of a simulated countercurrent the system, making it necessary to replace only between the solid and liquid phases. This is ac-that small amount which is removed in the extract Pharmaceutical chemistry Chiral compound (cis-trans phytol, steroids, peptides, antibiotics, etc.) complished with a series of chromatography col-and raffinate, thereby enabling savings of up to umns arranged in a ring. An eluent flow circulates 90 % of the eluent in comparison to a batch pro-Food chemistry Fatty acids, carbohydrate mixtures (sucrose/molasses or fructose/glucose, etc.) through this ring. Two inlets (for feed and eluent) cess. Due to the simulated countercurrent, the and two outlets (extract/red and raffinate/blue) stationary phase is significantly better utilized with Biochemistry Phenylalanine, fermentation/cell culture products (citric acid, sugars, antibodies, enzymes, etc.) define four separation zones. By continuously feed-the SMB technique as compared to the batch pro-ing sample and synchronously switching the col-cess technique. The number of theoretical plates Petrochemistry C8-Hydrocarbon (xylene/toluene, etc.) umns against the eluent flow direction, a counter-might be also less important, making it possible current is achieved between the solid and liquid to use cost-effective larger particle size for the sta-phases, leading to high purity of both target frac-tionary phases. tions. The movement of the solid phase is realized by simultaneously switching seven multi-position valves (AZURA® SMB) or one central multi-position valve (former KNAUER SMB, CSEP®). 14 www.knauer.net/smb 15 AZURA® SMB systems AZURA® SMB systems System confi guration System configuration Classical SMB chromatography The standard AZURA® SMB systems consists of Due to this confi guration the SMB system can be Th f e s ou ta r p n u da m r p d A s anZU d sRA ev ® S e M n m B s ulti y - s p t oesm iti s c o o n vn a slivsets o s. f Du u e t se o t d vh e irs c y o fl n e fi x g ib u l r e afti o o r n t m h a e S ny M di B s ff e y re s nte m c sep a a n b rati e o n The standard configuration is designed to run aration of a multi-component mixture into two fou T r p h u e d m evp i s a ce nd s s ar ev e ar e r n m ang u e lti- d a po s f s oliltion v ows: alves. use m d o v d e e r s y . fl A e dxib ditlie f o o n r al m ly atn o y d th iff e ere pr n o t c s e e s p s a s rtati b o i n lit y classical SMB separations. This mode is perfect different fractions is possible, too. This is typically Th • e d T ev h i re ce e s a pu re a mpsr r(a E n x g tread a ct s f , R o a ll ffi ow na s t : e, Eluent) are mo A d Z e U s R . A A® d S d Miti B o s n y a st ll e y t m o s t a h re e p out ro pecre f s o s r s mi ta n b g ili ev ty er y for the separation of binary mixtures, like sugars used as one pre-purification step for very • Th p re la e p cedu i m nsp i s d (E e t x h tra e S ct M , R B ca y ffi cl n e a . te, Eluent) are AZ o U thR e A r ® S S M M B B sysyts e tem m o s n atre h e ou m t ap r e k r e ft o r r em g ian r g d iev n e g ry m a- or e.g. pharmaceutical racemic mixture. The sep-demanding separations. p • la T c h e e d fe iens d id p e t um h p e S is M plaB c ce y d c ole ut.side the SMB cycle. oth e e ri r S al M anB s d cys o t ne fi m o gur n t ati h o e m n fl ea x rk ibet r ilit e y g a a s rdi we n l g m l usa a b-le • T • h Fe f o e u ed p r valvu emp i s ar s p e p laced o d aut t tsi hde t e p h u e S m M p oB c utl y ectlse.. ter p ira el sa s n u d c re ro a nfi gg eu . ration flexibility as well usable • F • o T u h r v re a e lv v e al s a ve r s e p a l re a p c l e a d a ce t t d a h t te p he u p m u p o mp iu ntlle et tss. . The pressure range. • Th f re eee v d a p lv u e ms a p ire p nle la t i c s ed a not t t c h o e p nn u e m ct p i ed ntle o ts a . T m h u e lt i- fee p d p ositiu omp i n valn v le e. t is not connected to a multi- Different zone configurations Open-/Closed-Loop position valve. In standard configuration every zone consists of Every AZURA® SMB system can be switched be-two columns (2:2:2:2). To optimize the process, it tween a Open- and a Closed-Loop mode. The might be useful to change this distribution. In Closed-Loop is the common SMB mode. In Open-case of a very effective regeneration of the solid Loop mode the SMB cycle is opened between and liquid phase in zone 1 and 4, the number of the last column of zone 4 and the zone 1 pump. Feed Raffinate Feed Raffi nate columns in these zones can be reduced. With a When to choose this mode? 1:3:3:1 configuration, a much higher productivity • One impurity with very low retention time is in Zone 3 Zone 3 can be achieved. This system configuration can the sample (and eluent costs are low enough) easily be adjusted via our software PurityChrom® • To start/clean the system or change the eluent MCC. The hardware does not have to be modified. Birch xylose converted to xylitol as sweetener Column Zone 2 swi Ctch o in lumgn Zone 4 Zone 2 dir s e wicttion ching Zone 4 direction Zone 1 Zone 1 Extract Eluent Extract Schematic AZURA® SMB design Eluent Schematic AZURA® SMB design 16 17 14 KNAUER_Brosch_SMB_12do.indd 14 28.08.17 10:51 AZURA® SMB systems Evaluation of the SMB parameter The adsorption isotherms (linear or Langmuir) can be easily in- serted into the parameter wiz- Purification of xylitol by HPLC methods from fermented biomass ard of our SMB software PurityChrom® MCC. The starting pa- rameter will be calculated and transferred into the method automatically (Fig. 4). Due to a contamination in the mixtures with nearly no interac- To optimize SMB parameters can be a very time is needed. In this application, xylitol was purified tion with the material, the SMB consuming task, especially if a purity and a yield from fermentation mash of a fed-batch process. separation was done in an of more then 99 % with a maximum productivity Open-Loop mode, so without Fig.4: Parameter wizard in PurityChrom® MCC solvent recycling. Optimization of the analytical method Transfer into preparative scale The analytical HPLC method has to be trans-The next step is an overloading study based on ferred to an isocratic method (Fig. 2). The sta-the analytical method by using a column with the Separation verification μRIU tionary phase must be adapted to the later SMB same dimensions of the prospective SMB process. Figure 5 shows an overlay of process requirements too. In this case, the ma-Volume and mass overload are evaluated and raffinate / extract and waste terial was changed from 10 µm to 25 – 56 µm. adsorption isotherms are determined based on fraction of the 6th SMB cycle re-Additionally the column dimensions were the retention times (Fig. 3). The received param-vealing a successful separation changed from 300 × 8 mm to 150 × 20 mm (Fig. 3) eters are transferred into the appropriate SMB of the fractions, with 100 % pu- scale. Figure 4 shows where the chromatogram rity and recovery of xylitol. is split based on the calculated SMB parameter. RAFFINATE EXTRACT time μRIU XYLOSE XYLITOL μRIU "Acknowledgement: This project has received funding from the European Union‘s Fig.5: Overlayed analytical chromatograms of raffinate (blue); extract / Seventh Framework Program for research, technological development and de monstra-XYLITOL xylitol (light blue); waste (blue); 20 µL 1:2 dilution; Eurokat Ca 300 × 8 mm, tion under grant agreement no FP7-KB-BE-2013-7-613802." 10 µm particle, 0.5 ml/min, 75°C, H O isocratic 2 dd ARABINOSE GLYCEROL MANNITOL MANNITOL 100 % 98 % recovery recovery The comparable batch separation (same conditions as seen in Fig. 3, dark blue chromatogram) offers nearly the same purity and 1.800 252 SMB Batch mg/h mg/h time time recovery rate, but a significantly lower productivity of 252 mg/h. The throughput of Fig.2: Analytical chromatogram of fermentation mash Fig.3: Semi-preparative chromatogram of fermenta-the SMB process is with 1,8 g/h greater by 100 % 99 % with identified sugar/sugar alcohols; 20 µL 1:2 dilution; tion mash; injection: light blue – 0.5 ml, blue – 1.0 ml, the factor of seven than that of the batch purity purity Eurokat Ca 300 × 8 mm, 10 µm particle, 0.5 ml/min, dark blue 2.0 ml; Eurokat Ca 150 × 20 mm, 25–56 µm process. 75°C, H O isocratic particle, 4 ml/min 60°C, H O isocratic 2 dd 2 dd 18 www.knauer.net/smb 19 AZURA® SMB systems Whenever separation tasks are changing frequently, a classical batch LC system can be the better choice. KNAUER AZURA® Prep and AZURA® Bio purification systems are as flexible and versatile as possible. AZURA® Prep was designed for flexibility and to your products such as synthesis stages or active Complete solution for FPLC on a minimum foot-switching, buffer and sample selection as well as comfortably handle large sample volumes. Easy ingredients. The systems can be optimally adapt-print: AZURA® Bio systems combine flexibility fraction collection enable the user to automate to operate and maintain the AZURA® Prep sys-ed to the scale you need. and reliability. The biocompatible AZURA® Bio is their separation. tems are perfectly suited for the purification of the perfect choice for your protein purification From simple to complex, from lab to pilot scale: task. Design you AZURA® Bio system to your Desgin your AZURA® FPLC system according to needs. Multiple functionalities such as column your purification task. • Configure your system from injection to collection • Flexible and modular design • Choose between high or low pressure gradient systems from 50 to 1 000 ml/min • Easy upscaling – up to 1 000 ml/min • User-friendly and powerful software PurityChrom® • Powerful FPLC software PurityChrom® • Variety of great detectors to make your sample visible 20 www.knauer.net/smb 21 AZURA® SMB systems Special multi-column chromatography solutions APPLICATION SERVICES „KNAUER is the only company that could build a system SMB Method development, HPLC method according to our specifications” transfer & optmization, Rent-an-SMB-expert Our focus is on the development of optimization and control strategies for multi-column processes. We needed a flexible system that is able to perform various multi-column processes COMPLIANCE on the highest level of technology. The hardware and the soft-Qualification documents, Material certifica-ware must be flexible such that model-based optimization and tion, FAT/SAT, Capillary labeling control schemes can be tested on example processes. KNAUER KNAUER offered the complete package for us: planning, de-Prof. Dr. Ing. Sebastian Engell, Services signing, developing and manufacturing. The installation, in-Head of Process Dynamics and struction and support were very good. It was a pleasure work-Operations ing with KNAUER. Technical University Dortmund TECHNICAL SERVICE & SUPPORT Installation & Instruction, Maintenance, Repair, Support System components • 6 AZURA® Pumps P 6.1L HPG • 2 AZURA® Pumps P 2.1S ACADEMY • 2 AZURA® UV Detector UVD 2.1S HPLC trainings, Specialized courses and work- • 2 AZURA® Valve Drive V 2.1S shops for beginners and experienced users • 1 AZURA® CM 2.1S • 24 AZURA® Valve Drive V 2.1S with multi-position valves • Controlled via PCS by HiTec Zang All standard user instructions, helpful video tutorials, and a structured section of frequently asked questions is freely accessible on our web page www.knauer.net. If you need further support, our friendly Support team is happy to help you via e-mail, phone or KNAUER builds up customized Team Viewer. They will work with you personally until all issues are resolved. multi-column systems to your needs. We will support you by Phone: +49 30 809727-111 (workdays 9–17h CET) choosing the right devices, ma-Email: support@knauer.net terials and control options. Contact us: sales@knauer.net 22 www.knauer.net/smb 23 Technical data or prices are subject to change without All trademarks are the property of their respective owners. notice. Prices may vary by country and do not include Our general terms and conditions apply: taxes, customs duties or delivery. www.knauer.net/terms Distributed by: S/0.5/06.19 7741U/V 019 ER 2UAN K© KNAUER +49 30 809727-0 Wissenschaftliche Geräte GmbH +49 30 8015010 (Fax) Hegauer Weg 38 info@knauer.net 14163 Berlin, Germany www.knauer.net