Protein Purification iologic DuoFlow Valving Guide Valving Schemes for the iologic DuoFlow Chromatography System
Protein Purification iologic DuoFlow Valving Guide Introduction The iologic DuoFlow Chromatography System is a powerful and versatile biocompatible chromatography system for the purification of proteins, peptides, nucleic acids, and other small molecules. The modular nature of the iologic DuoFlow System makes it very easy to upgrade the gradient pump and to add peripheral devices such as iologic Maximizer Valve System Detectors, auxiliary sample loading pumps, and fraction collectors. In addition, the unique versatility of the iologic DuoFlow System is enhanced by the availability of several different valves for automating both routine and more complex separation protocols. For the user, this means reliable operation and reproducible results, run after run, year after year. This guide describes typical valving applications. The applications range from use of a single VR- ion Valve to simply equilibrate a column, inject sample, and elute the column, to multidimensional chromatography by using a combination of VR-, VR9-8, SV-, and a iologic Maximizer Valve System to connect multiple columns for multistep purifications. iologic DuoFlow Software makes working with valves easy and intuitive. The software automatically senses each valve and displays it in a graphical format on the Manual screen. The Setup Editor makes it simple to program any valving scheme by its valve aliasing feature and its ability to save an unlimited number of user-identified valving configurations.
The Make-efore-reak dvantage To accommodate the wide range of pressure and flow rates of the iologic DuoFlow System, the VR- and VR9-8 Valves use a patented make-before-break design that prevents pressure spikes when the valves rotate from one port to another. This is beneficial when using fragile columns or flow-sensitive detectors and prevents pump shutdowns due to transient overpressure situations. Reliability Guaranteed Reliability is a vital prerequisite of any valve, especially when used with the wide range of s, salt solutions, and solvents seen in today s laboratory. Thousands of io-rad chromatography valves are in daily use worldwide, handling everything from.0 M salt solutions to 00% acetonitrile. ll valves are completely biocompatible with no metallic parts in contact with the liquid flow. iologic DuoFlow System Valve Selection Guide Valve* Type Ports SV- Low-pressure solenoid valve for stream selection Common,,,, SVT- Low-pressure solenoid valve for stream selection or flow diversion Common,, VR9-8 High-pressure make-before-break stream-select valve Common, 8 VR- High-pressure make-before-break valve (,, and Purge, positions) iologic Maximizer Valve System Enables blending, double flow rates, and twice the valving capacity of the standard iologic DuoFlow System * Up to three SV and three VR valves can be connected to the iologic DuoFlow System. Up to six SV and six VR valves can be connected to the iologic DuoFlow Maximizer and iologic DuoFlow Pathfinder Systems.
Table of Contents pplication asic VR- ion Valve Operation Using,, and Purge Positions position is for filling a loop with sample or for running through a column; position is for injecting contents of the sample loop onto a column; Purge position is for priming the iologic DuoFlow Gradient Pump and purging lines to waste without removing the column from the system. Multiple uffer Selection Gradient pump inlet valves may be used to select a different, to add sanitation or storage at the end of a run, or to load sample directly through the gradient pump. Reverse-Flow Chromatography for ffinity Purifications This is especially useful when purifying antibodies using protein or other affinity columns. The protein is bound at the top of the column and nonbinding material is washed out. Flow to the column is then reversed and the bound proteins elute from the top of the column in very concentrated form, which helps prevent denaturation. Using an VR- Valve for Two-Column Switching Test two columns side by side and compare their separation characteristics. dditional gradient pump inlet valves for multiple selection may also be used with this application. Using Two VR9-8 Valves for Eight-Column Switching Test up to eight columns side by side and compare their separation characteristics. dditional gradient pump inlet valves for multiple selection typically are used with this application. Mini utosampler for Multiple ion up to samples through the gradient pump using SV- and VR9-8 Valves. Mini utosampler With utomated Loop Fill and Rinse Use an auxiliary pump and a stream-select valve as a four- or eightplace mini autosampler. Combine a peristaltic pump, such as the Model EP- Econo Pump or Econo Gradient Pump, with an inlet valve for automated filling and rinsing of a sample loop. 8 Large-Volume ion Onto a Low ackpressure Column Use an auxiliary pump and a valve for direct of sample onto a column. This application uses a peristaltic pump such as the Model EP- Econo Pump or Econo Gradient Pump to load large amounts of crude sample onto a column for a first-step separation. dditional pump inlet valves for multiple sample selection, rinsing, and selection may also be used with this application. Valves Needed VR- VR- and x VR9-8* * Or x SV- or SVT- x VR- x VR- Up to x SVT- or SV- are optional for selection VR- and x VR9-8 Up to x SVT- or SV- are optional for selection x VR9-8 and SV- VR- and VR9-8* * Or SV- or SVT- VR- Up to x SVT-, or SV-, or VR9-8 are optional for sample and selection Page 8 9
pplication 9 Large-Volume Fraction Collection Use an VR9-8 Stream-Select Valve as a large-volume fraction collector. 0 Tandem Chromatography Tandem chromatography is used to automatically collect selected fractions from one column and inject them onto a second column. Eluted fractions from the first column are collected into a DynaLoop or other sample loop, which are then eluted through the second column. Gradient pump inlet valves are used if different s are needed for equilibration and elution of each column. Flowthrough Profiling Using Three VR- Valves sample is loaded onto one column and nonbinding components (flow-through proteins) pass through up to two other columns. Each column may then be eluted individually. Gradient pump inlet valves are used if different s are needed for equilibration and elution of each column. Sequential inding and sample is loaded onto one column, nonbinding proteins are washed away, and then a fraction of eluted proteins is diverted to a second column. This column is washed, eluted, and fractions diverted to a third column, which in turn is washed and eluted. Gradient pump inlet valves are used if different s are needed for equilibration and elution of each column. Valves Needed VR- and VR9-8 x VR-, VR9-8, SVT- Up to x SV- are optional for selection x VR- Up to x SV- are optional for selection x VR- Up to x SV- are optional for selection Page 8
Column pplication asic VR- ion Valve Operation Using,, and Purge Positions position is for filling a loop with sample or for running through a column; position is for injecting contents of the sample loop onto a column; Purge position is for priming the iologic DuoFlow Gradient Pump and purging lines to waste without removing the column from the system. VR- in Position (Figure ) The column may be equilibrated or eluted. is loaded into the loop. If the loop is overfilled, the sample runs to waste. Never remove the syringe before the sample is injected onto the column. VR- in Position (Figure ) The sample is injected onto the column. VR- in Purge Position (Figure C) Flow from the gradient pump goes directly to waste, bypassing the column. Column Column Column Column Column Fig. Fig. Purge Purge Column Column Fig. C Purge
pplication Multiple uffer Selection Gradient pump inlet valves may be used to select a different, to add sanitation or storage at the end of a run, or to load sample directly through the gradient pump. ny valve may be attached to gradient pump inlets or. Two examples are shown. Use the Manual screen valve control to switch between valve ports and to prime each valve position using a syringe before running a method. C* 8 Example C 8 VR9-8 VR9-8 Example SV- Water C SVT- NaOH (sanitizing solution) * Common port. VR- VR- Column
iofrac pplication Reverse-Flow Chromatography for ffinity Purifications This is especially useful when purifying antibodies using protein or Valve other affinity columns. VR- The protein is bound at the top of the column and nonbinding material is washed out. Flow to the column is then reversed and the bound proteins elute from the top of the column in very concentrated form, which helps prevent denaturation. Step (Figure ) has first been loaded into the loop of valve in position. Valve moves to position and the sample passes to valve. With valve in position, sample is pumped onto the top of the column and nonbinding material is washed out of the column (top to bottom). Step (Figure ) Note that ports and of valve have been connected using a short piece of tubing. Valve moves to position for column washing and elution. The loop is now out of the flow path. With valve in position, the column is eluted by reverse flow (bottom to top). Valve VR- Column iofrac Valve VR- Valve VR- Column Valve VR- Valve VR- Valve VR- Column iofrac iofrac Valve VR- Valve VR- Valve VR- Fig. Fig. 8
pplication Valve VR- Using an VR- Valve for Two-Column Switching Test two columns side by side and compare their separation characteristics. Flow-through Column (Figure ) Two columns are connected to valve as shown. With valve in position, flow from the gradient pump goes to column. Flow-through Column (Figure ) Column With valve in position, flow from the gradient pump goes to column. Valve VR- Column iofrac Valve VR- Valve VR- Column Valve VR- Column Valve VR- Column Column iofrac iofrac Valve VR- Column Fig. Valve VR- Fig. Column 9
pplication (continued) Two-Column Switching With uffer Selection (Figure C) dditional gradient pump inlet valves for multiple selection may also be used with this application. SV- SV- NaOH (sanitizing solution) Water Valve VR- Column Valve VR- Column iofrac Fig. C 0
pplication Using Two VR9-8 Valves for Eight-Column Switching Test up to eight columns side by side and compare their separation characteristics. dditional gradient pump inlet valves for multiple selection typically are used with this application. Two VR9-8 Valves act as column inlet and outlet valves to determine which column is in line. SV- Valves act as gradient pump inlet valves for selection. When VR- Valve is in position, sample is injected onto the selected column. SV- SV- NaOH (sanitizing solution) valve VR- For sample application Up to 8 different columns C* 8 C* 8 Column inlet valve VR9-8 (cts as column switching valve to receive sample.) Column outlet valve VR9-8 (cts as column switching valve to direct eluent to detectors and fraction collector.) iofrac * Common port.
pplication Mini utosampler for Multiple ion SV- and VR9-8 Valves can be used as a mini autosampler to direct load samples through the gradient pump when connected to a iologic DuoFlow System. In this configuration, up to samples can be loaded. One port must be plumbed to a rinse solution in order to rinse the valve and tubing between sample s. The example below shows a plumbing configuration that can be used to load samples with 8 different systems on different columns. These runs could be used for scouting experiments that scout on sample preparation, or alternatively for purification of a variety of samples as part of a queue in conjunction with a column switching valve. VR- valve C 8 Column inlet valve VR9-8 VR9-8 gradient SV- gradient pump inlet valve pump inlet valve C 8 S S S S uffer uffer C 8 Column outlet valve VR9-8 To detector
pplication Mini utosampler With utomated Loop Fill and Rinse Use an auxiliary pump and a stream-select valve as a four- or eight-place mini autosampler. Combine a peristaltic pump, such as the Model EP- Econo Pump or Econo Gradient Pump, with an inlet valve for automated filling and rinsing of a sample loop. Step Valve is in position for sample loading into the loop. Valve is an VR9-8 Valve for sample and rinse selection. The iologic DuoFlow Software automatically loads the selected sample into the loop. Step Valve moves to position and the sample is injected onto the column. Step When sample is complete, valve is automatically returned to position. The software can then automatically select a defined rinse position and rinse the loop prior to another sample. Loop overfill to waste Valve VR- Step Valve VR9-8 C* Econo Gradient Pump 8 iofrac Step C* Econo Gradient Pump 8 iofrac s Rinse s Rinse * Common port. Step
pplication 8 Large-Volume ion Onto a Low ackpressure Column Valve VR- Use an auxiliary pump and a valve such as the SVT- for direct of sample onto a column. Low This application uses a peristaltic pump such as the Model EP- Econo uxiliary Pump pump purge or the Econo Gradient Pump to load large amounts of crude sample onto a column for a first-step separation. 0 psi backpressure device may be placed after the workstation gradient pump to improve pump performance. dditional pump inlet valves for multiple sample selection, rinsing, and selection may also be used with this application. Step Econo Gradient Pump Valve is in position and the column is equilibrated. The auxiliary pump is primed with sample. C* Step SVT- Valve moves to position and the sample is pumped directly onto the column by the iofrac auxiliary pump. The iologic DuoFlow Gradient Pump stops while the auxiliary pump is injecting sample. Rinse Step (Not Shown) When sample is complete, valve is automatically returned to position and the workstation gradient pump is used to elute sample from the column. 0 psi pressure column 0 psi 0 psi Valve VR- uxiliary pump purge Low pressure column Valve VR- Lowpressure column Econo Gradient Pump Econo Gradient Pump C* SVT- iofrac C* SVT- iofrac Rinse Rinse * Common port. 0 psi Valve VR- Lowpressure column Econo Gradient Pump Step Step iofrac
pplication 9 Large-Volume Fraction Collection The iologic DuoFlow System provides three mechanisms for doing large-volume fraction collection. Example Use one VR9-8 Valve as a fraction collector to collect 8 fractions. Individual change valve steps are programmed in the method protocol to collect the desired fractions. Example Use two VR9-8 Valves as a fraction collector to collect fractions. Example Use the preparative fraction collector rack option for the iofrac. The iofrac Prep-0 Preparative Rack can be used to collect fractions in up to 0 collection vessels of any size, with milliliter to liter collection volumes. It can be used at flow rates up to 80 ml/min. Tygon Tubing /8 OD, / ID is typically used to plumb the Prep-0 funnels to the collection vessels. Valve VR- Valve VR9-8 Valve VR9-8 Valve VR- s stream-select valve Fraction Fraction Fraction C* Fraction 8 Fraction Fraction Fraction 8 Fraction Fraction Fraction Fraction Fraction C* 8 C* 8 Fraction Fraction Fraction Fraction Fraction 0 Fraction 8 Fraction Fraction 9 * Common port. Example Fraction Fraction Fraction * Common port. Example Example
pplication 0 Tandem Chromatography Tandem chromatography is used to automatically collect selected fractions from one column and inject them onto a second column. Eluted fractions from the first column are collected into a DynaLoop or other sample loop, which are then eluted through the second column. Gradient pump inlet valves are used if different s are needed for equilibration and elution of each column. For example, monoclonal antibodies can be purified with column as a cation exchanger and column as ceramic hydroxyapatite, at a netural ph. This could be an alternative to affinity chromatography, which requires low-ph s to elute antibodies. Step (Not Shown) The sample is loaded into the loop of valve and injected onto column. With the appropriate selected from the gradient pump inlet valves, column is eluted. Valve directs the flow to the fraction collector for collection or diversion to waste. Step t a predetermined time or volume, valve moves to position and a portion of the column eluate is captured in the loop attached to valve. Valve VR- Column SV- uffer Valve VR- SV- DynaLoop Loop Column inject NaOH (sanitizing solution) Valve VR9-8 C* 8 C* Valve SVT- iofrac * Common port. Step
pplication 0 (continued) Step Valve moves to Purge position to put valve and column into the flow path. Valves and change position to connect column to the detectors and fraction collector. With the appropriate selected from the gradient pump inlet valves, the sample stored in the loop is injected onto column when valve moves to position. Step (Not Shown) Valve reverts to position and column is eluted. Valve VR- Purge Column SV- Valve VR- SV- DynaLoop Loop Column NaOH (sanitizing solution) Valve VR9-8 C* 8 C* Valve SVT- iofrac * Common port. Step
pplication Flowthrough Profiling Using Three VR- Valves Flowthrough profiling is a technique used to capture multiple components from a mixture in a single run. Here, a mixture of proteins is loaded onto the first column. The nonbinding components of the mixture are then allowed to flow through a second and third column. Each column may then be eluted individually. The columns used in this technique are selected based on the properties of the components to be captured. SV- or VR9-8 Gradient Pump Inlet Valves may be used for selection of multiple s. Gradient pump inlet valves are used if different s are needed for equilibration and elution of each column. Step With valve in Purge position and valves and in position, the gradient pump may be primed with the s from the gradient pump inlet valves. Two T-unions* are used to connect tubing. Port of valves and should be end-capped to prevent misdirected flow. To equilibrate columns individually: Column Valve Valve Valve or Purge Purge Purge Valve VR- SV- SV- Column NaOH (sanitizing solution) Valve VR- Column T-union* Valve VR- Column T-union* UV detector iofrac Step * P- TEE, with P-00/P-, PEEK, available from Upchurch Scientific. 8
pplication (continued) Step (Not Shown) Valve is placed initially in position. Valves and remain in position. ll three columns are equilibrated and the flow goes to waste via the fraction collector diverter valve. Step is loaded into the loop. Valve is placed in position and the sample is injected onto column. With valves and remaining in position, the unbound components of the sample pass through all three columns and then to the fraction collector (or to waste via the fraction collector diverter valve). The flow path is shown in blue. Valve VR- SV- SV- Column NaOH (sanitizing solution) Valve VR- Column Valve VR- Column UV detector iofrac Step 9
pplication (continued) Step To elute column, valve is in position. Valves and are both in position and are out of the flow path. The flow from column goes to the fraction collector. SV- SV- Column Valve VR- NaOH (sanitizing solution) Valve VR- Column Valve VR- Column UV detector iofrac Step 0
pplication (continued) Step To elute column, the gradient pump inlet valves both change to Valve changes to Purge position and valves and remain in position The flow elutes column, bypassing columns and, and goes to the fraction collector Step To elute column, the gradient pump inlet valves change to Valve remains in Purge position, valve is in position, and valve remains in position The flow elutes column and goes to the fraction collector Purge Valve VR- Purge Valve VR- SV- SV- Column SV- SV- Column NaOH (sanitizing solution) Valve VR- NaOH (sanitizing solution) Valve VR- Column Column Valve VR- Valve VR- Column Column UV detector UV detector iofrac iofrac Step Step
pplication Sequential inding and Using Three VR- Valves sample is loaded onto one column, nonbinding proteins are washed away, and then a fraction of eluted proteins is diverted to a second column. This column is washed, eluted, and fractions diverted to a third column, which in turn is washed and eluted. Gradient pump inlet valves are used if different s are needed for equilibration and elution of each column. Step With valve in Purge position and valves and in position, the gradient pump may be primed with the s from the gradient pump inlet valves. Two T-unions* are used to connect tubing. Port of valves and should be end-capped to prevent misdirected flow. To equilibrate columns individually: Column Valve Valve Valve or Purge Purge Purge Valve VR- SV- SV- Column NaOH (sanitizing solution) Valve VR- Column T-union* Valve VR- Column T-union* UV detector iofrac Step * P- TEE, with P-00/P-, PEEK, available from Upchurch Scientific.
pplication (continued) Step (Not Shown) Valve is placed in position to load sample into the loop. Valve is in position and valve is in position. Columns and are now bypassed. Step Valve changes to position and sample is loaded onto column. Step Valve changes to position to wash column. The flow goes to the fraction collector or to waste via the fraction collector diverter valve. Valve VR- Valve VR- SV- SV- Column SV- SV- Column NaOH (sanitizing solution) Valve VR- NaOH (sanitizing solution) Valve VR- Column Column Valve VR- Valve VR- Column Column UV detector UV detector iofrac iofrac Step Step
pplication (continued) Step Valve changes to position. The flow from column passes through column and then to the fraction collector. The flow path is shown. Step Valve changes to Purge position to bypass column With valves and in position, the flow passes only through column and then to the fraction collector (or waste via the fraction collector, as shown) Valve VR- Loop Purge Valve VR- SV- SV- SV- SV- Column uffer uffer uffer uffer Column NaOH (sanitizing solution) Valve VR- uffer uffer NaOH (Sanitizing Solution) uffer Valve VR- Column Column Valve VR- Valve VR- Column Column UV detector UV detector iofrac iofrac Step Step
pplication (continued) Step Valve changes to position The flow passes through column and column. The flow path is shown Step Valve changes to position. Column is now bypassed Valve changes to position and column is now washed and eluted (shown) SV- SV- Purge Column Valve VR- SV- SV- Purge Column Valve VR- NaOH (sanitizing solution) Valve VR- NaOH (sanitizing solution) Valve VR- Column Column Valve VR- Valve VR- Column Column UV detector UV detector iofrac iofrac Step Step
Ordering Information Catalog # Description iologic DuoFlow Valves 0-00 VR- utomated Valve, -port, -position high-pressure valve,,00 psi ( bar) limit 0-00 VR- Valve Rebuild Kit 0-008 VR9-8 Stream-Select Valve, 9-port, 8-position highpressure valve,,00 psi ( bar) limit 0-00 VR9-8 Valve Rebuild Kit 0-00 SVT- Diverter Valve, -port, -position solenoid valve, 0 psi ( bar) limit 0-0 SVT- Valve Rebuild Kit 0-0 SV- Select Valve, -port, -position solenoid valve, 0 psi ( bar) limit iologic Dynamic (Sliding Piston) Loops 0-0 DynaLoop Kit, includes ml DynaLoop Sliding Piston Loop, DynaLoop Parts Kit, instructions 0-0 DynaLoop 90 Kit, includes 90 ml DynaLoop Sliding Piston Loop, DynaLoop Parts Kit, instructions 0-00 DynaLoop Parts Kit, includes end cap O-rings, sliding seal O-ring, filter, nut fittings, four /8" ferrules, five / 8 nuts and ferrules, 0' of /8" PTFE tubing 0-0 DynaLoop Loop, ml, replacement 0-0 DynaLoop 90 Loop, 90 ml, replacement 0-0 DynaLoop Sliding Seal Replacement Model EP- Econo Pump -8 System Cable, 8-pin mini-din to bare wires Catalog # Description Econo Gradient Pump -900 Econo Gradient Pump, 00/0 V, includes tubing and fittings kits -900 Econo Gradient Pump, 0/0 V 0-0 System Cable 8, bus communication cable,. m (') iologic Maximizer Valve System (for Use With a iologic DuoFlow System) 0-00 iologic Maximizer Kit, 00/0 V, for US, Japan, Korea; includes iologic Maximizer ase Unit, ph, iologic Maximizer, starter kit, tubing kit, system cable 0, U.S. power cord PEEK is a trademark of Victrex PLC. Tygon is a trademark of Norton Company.
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