Valving Schemes for the BioLogic Duo-Flow Chromatography System

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Horace Hamilton
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1 Valving Schemes for the iologic Duo-Flow Chromatography System

table of contents pplication asic ion Valve Operation using,, and Purge Positions position is for filling a loop with sample or for running buffer through a column; position is for injecting contents

Multiple uffer Selection Gradient pump inlet valves may be used to select a different buffer, to add sanitation or storage buffer at the end of a run, or to load sample directly through the gradient

The protein is bound at the top of the column and nonbinding material is washed out.

2 table of contents pplication asic ion Valve Operation using,, and Purge Positions position is for filling a loop with sample or for running buffer through a column; position is for injecting contents of the sample loop onto a column; Purge position is for priming the Duo-Flow workstation 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 buffer, to add sanitation or storage buffer 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. -Column Switching Test columns side by side and compare their separation characteristics. dditional gradient pump inlet valves for multiple buffer selection may also be used with this application. 8-Column Switching Test up to 8 columns side by side and compare their separation characteristics. dditional gradient pump inlet valves for multiple buffer selection typically are used with this application. Mini utosampler with utomated Loop Fill and Rinse Use an auxiliary pump and a stream select valve as a - or 8-place mini autosampler. Combine a peristaltic pump, such as the Model EP- Econo TM pump or Econo gradient pump, with an inlet valve for automated filling and rinsing of a sample loop. Large-Volume ion onto a Low ack-pressure Column Use an auxiliary pump and a valve for direct injection 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 buffer selection may also be used with this application. 8 Large-Volume Fraction Collection Use an VR9-8 stream select valve as a large volume fraction collector. 9 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 TM or other sample loop. fter injection of the collected sample, it is eluted from the second column. Gradient pump inlet valves are used if different buffers are needed for equilibration and elution of each column. Valves Needed and x VR9-8* *Or x or SVT- x x Up to x SVT- or are optional for buffer selection and x VR9-8 Up to x SVT- or are optional for buffer selection and VR9-8* *Or or SVT- Up to x SVT-, or, or VR9-8 are optional for sample and buffer selection and VR9-8 x, VR9-8, SVT- Up to x are optional for buffer selection

pplication 0 Flow-Through Profiling sample is loaded onto one column and nonbinding components (flow-through proteins) pass through up to other columns. Each column may then be eluted individually.

$Sequential inding and sample is loaded onto one column, nonbinding proteins are washed away, and then a fraction of eluted proteins is diverted to a second column.$

3 pplication 0 Flow-Through Profiling sample is loaded onto one column and nonbinding components (flow-through proteins) pass through up to other columns. Each column may then be eluted individually. Gradient pump inlet valves are used if different buffers 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 buffers are needed for equilibration and elution of each column. Using the Econo Gradient Pump and a Splitter Valve to Collect Sets of Fractions The splitter valve is connected to and powered from the Econo gradient pump, which itself is controlled by the Duo-Flow system. The % Split is set in the Duo-Flow software. Valves Needed x Up to x are optional for buffer selection x Up to x are optional for buffer selection Splitter valve and an Econo gradient pump The flow exiting the column passes through the detectors and goes to the common port of the splitter valve. portion of the flow, defined by the user, is split and goes to a primary collector. The remaining portion of the flow goes to a secondary collector. The secondary collector runs in stand-alone mode, and is started manually. typical application for stream splitting is to automatically collect a small amount of each fraction for biological activity assays, thus eliminating post-run liquid transfer.

valve selection dvanced Chromatography pplications using Valving Introduction The iologic Duo-Flow chromatography system is a powerful and versatile biocompatible chromatography system for the

$The modular nature of the Duo-Flow system makes it very easy to upgrade the gradient pump and to add peripheral devices such as detectors, auxiliary sample loading pumps, and fraction collectors.$ For the user, this means reliable operation and reproducible results, run after run, year after year. This guide describes typical valving applications.

For the user, this means reliable operation and reproducible results, run after run, year after year. This guide describes typical valving applications.

The Duo-Flow system 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.

4 valve selection dvanced Chromatography pplications using Valving Introduction The iologic Duo-Flow 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 Duo-Flow system makes it very easy to upgrade the gradient pump and to add peripheral devices such as detectors, auxiliary sample loading pumps, and fraction collectors. In addition, the unique versatility of the Duo-Flow 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 sample injection valve to simply equilibrate a column, inject sample, and elute the column, to flow-through profiling using columns and valves. The Duo-Flow system 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 rate of the Duo-Flow, the 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 over-pressure situations. Reliability Guaranteed Reliability is a vital prerequisite of any valve, especially when used with the wide range of buffers, 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. Up to SV and VR Valves Can e Connected to the Duo-Flow System SVT- Valve Type Ports VR9-8 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 High-pressure make-before-break injection valve (,, and Purge positions)

5 uffer uffer Column ion Purge C Column Column uffer uffer uffer uffer application 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. in Position (Figure ) The sample is injected onto the column. in Purge Position (Figure C) Flow from the gradient pump goes directly to waste, bypassing the column. asic ion Valve Operation using,, and Purge Positions position is for filling a loop with sample or for running buffer through a column; position is for injecting contents of the sample loop onto a column; Purge position is for priming the Duo-Flow workstation gradient pump and purging lines to waste without removing the column from the system.

6 C* 8 Example C 8 application Multiple uffer Selection Gradient pump inlet valves may be used to select a different buffer, to add sanitation or storage buffer 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. uffer * common port uffer VR9-8 Water (Sanitizing Solution) Example ion VR9-8 C SVT- Column

7 Valve uffer uffer Column ion Valve uffer Valve uffer Valve Model 8 Model 8 Column application 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). 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.

8 Valve uffer uffer application Flow Through Column columns are connected to valve as shown. With valve in position, flow from the gradient pump goes to column (Figure ). Flow Through Column With valve in position, flow from the gradient pump goes to column (Figure ). Using an for -Column Switching Test columns side by side and compare their separation characteristics. ion Valve ion Column Column Model 8 uffer Model 8 Valve uffer Valve Column Column

9 iologic uffer uffer uffer uffer Valve ion uffer (Sanitizing Solution) Column Model 8 uffer Valve Water Column application -Column Switching with uffer Selection dditional gradient pump inlet valves for multiple buffer selection may also be used with this application.

10 uffer uffer uffer uffer application Using VR9-8 Valves for 8-Column Switching Test up to 8 columns side by side and compare their separation characteristics. dditional gradient pump inlet valves for multiple buffer selection typically are used with this application. VR9-8 valves act as column inlet and outlet valves to determine which column is in line. valves act as gradient pump inlet valves for buffer selection. When valve is in position, sample is injected onto the selected column. uffer Valve For sample application Up to 8 different columns uffer ion C* C Model 8 (Sanitizing Solution) 8 8 uffer Column Inlet Valve VR9-8 s column switching valve to receive sample Column Outlet Valve VR9-8 s column switching valve to direct eluent to detectors and fraction collector * common port

11 Step Valve Loop Overfill to uffer uffer Valve VR9-8 C* * common port Econo Gradient Pump 8 Rinse Step Valve is in position for sample loading into the loop. Valve is an VR9-8 valve for sample and rinse selection. The Duo-Flow 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 injection 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 injection. Model 8 Step Valve VR9-8 Step C Valve Econo Gradient Pump 8 Rinse Model 8 application uffer uffer Mini utosampler with utomated Loop Fill and Rinse Use an auxiliary pump and a stream select valve as a - or 8-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.

12 Step 0 psi Valve uxiliary Pump Purge Low Pressure Column uffer uffer application Step Valve is in position and the column is equilibrated. The auxiliary pump is primed with sample. Step Valve moves to position and the sample is pumped directly onto the column by the auxiliary pump. The iologic Duo-Flow workstation gradient pump stops while the auxiliary pump is injecting sample. Step (not shown) When sample injection is complete, valve is automatically returned to position and the workstation gradient pump is used to elute sample from the column. Large-Volume ion onto a Low ack-pressure Column Use an auxiliary pump and a valve such as the SVT- for direct injection of sample onto a column. This application uses a peristaltic pump such as the Model EP- Econo pump or the Econo gradient pump to load large amounts of crude sample onto a column for a first-step separation. 0 psi back-pressure device may be placed after the workstation gradient pump to improve pump performance. dditional pump inlet valves for multiple sample selection, rinsing, and buffer selection may also be used with this application. SVT- C* C Valve Econo Gradient Pump Rinse SVT- Rinse Econo Gradient Pump Model 8 Step 0 psi Low Pressure Column Model 8 * common port uffer uffer

13 Valve uffer uffer Example Example Fraction Fraction Fraction * common port Valve VR9-8 s stream select valve Fraction Valve VR9-8 C Fraction ion Fraction 8 Fraction Fraction Fraction Fraction C* Fraction 8 Fraction Fraction Fraction Fraction Fraction 8 Fraction 0 Fraction Valve VR9-8 C Fraction 9 8 Fraction Fraction 8 Fraction Fraction application 8 Large-Volume Fraction Collection Use an VR9-8 stream select valve as a large-volume fraction collector. n VR9-8 valve can function as an 8-place collector in place of a regular fraction collector (Example ). n additional VR9-8 can be plumbed to the first one to provide -place capacity (Example ). Individual change valve steps are programmed in the method Protocol to collect the desired fractions.

14 application 9 Valve Column Tandem Chromatography uffer uffer 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. fter injection of the collected sample, it is eluted from the second column. Gradient pump inlet valves are used if different buffers are needed for equilibration and elution of each column. n example of this application is the purification of monoclonal antibodies, where column is a cation exchanger and column is ceramic hydroxyapatite. This automated procedure can be performed at neutral ph, as an alternative to affinity chromatography, which requires low-ph buffers to elute the antibodies. Step (not shown) The sample is loaded into the loop of valve and injected onto column. With the appropriate buffer 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/dynaloop attached to valve. uffer uffer Step Model 8 Valve Valve VR9-8 C* 8 uffer (Sanitizing Solution) C Valve SVT- * common port DynaLoop Column step

15 Valve Purge Column uffer uffer uffer Valve uffer DynaLoop Column uffer (Sanitizing Solution) Valve VR9-8 C 8 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 buffer selected from the gradient pump inlet valves, the sample stored in the loop/dynaloop is injected onto column when valve moves to position (shown). Step (not shown) Valve reverts to position and column is eluted. Model 8 C Valve SVT- steps &

16 Purge Valve application 0 uffer uffer uffer Flow-Through Profiling using Valves uffer sample is loaded onto one column and nonbinding components (flow-through proteins) pass through up to other columns. Each column may then be eluted individually. Gradient pump inlet valves are used if different buffers 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 buffers from the gradient pump inlet valves. 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 uffer (Sanitizing Solution) uffer T-union uffer T-union ion Column Column UV Detector Column Model 8 Valve Valve step

17 Valve uffer uffer uffer uffer Column uffer uffer (Sanitizing Solution) uffer Valve Column 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. Column UV Detector Model 8 Valve step

18 Step To elute column, the gradient pump inlet valves both change to buffer. Valve changes to Purge position and valves and remain in position. The flow elutes column, bypassing columns and, and goes to the fraction collector. uffer uffer uffer uffer Purge step Column Valve uffer uffer (Sanitizing Solution) uffer Valve Column Purge Valve Valve Column uffer uffer uffer uffer Column uffer uffer Step To elute column, the gradient pump inlet valves change to buffer. (sanitizing solution) uffer Column Valve UV Detector Model 8 Valve remains in Purge position, valve is in position and valve remains in position. The flow elutes column and goes to the fraction collector. Column Valve step UV Detector Model 8

19 Valve uffer uffer uffer uffer Step ion Column uffer uffer (Sanitizing Solution) uffer Valve Column 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. Column UV Detector Model 8 Valve step

$Purge Valve application uffer uffer uffer uffer uffer Sequential inding and using Valves sample is loaded onto one column, nonbinding proteins are washed away, and then a fraction of eluted proteins$

20 Purge Valve application uffer uffer uffer uffer uffer Sequential inding and using 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 buffers 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 buffers from the gradient pump inlet valves. 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 (Sanitizing Solution) uffer T-union uffer T-union ion Column Column UV Detector Column Model 8 Valve Valve step

21 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 Valves changes to position and sample is loaded onto column. uffer uffer uffer uffer ion Column step Valve uffer uffer (Sanitizing Solution) uffer Step Valve Column Valve Valve Column uffer uffer uffer uffer Column uffer uffer (Sanitizing Solution) Step Valve changes to position to wash column. The flow goes to the fraction collector or to waste via the fraction collector diverter valve. uffer Column Column Valve Valve UV Detector Model 8 step UV Detector Model 8

22 application Step Valve changes to position. The flow from column passes through column and then to the fraction collector. The flow path is shown. uffer uffer uffer uffer uffer (Sanitizing Solution) 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). uffer uffer uffer Purge Column uffer Column UV Detector uffer Column Model 8 uffer Valve Valve Valve uffer (Sanitizing Solution) uffer uffer Column Column UV Detector Column Model 8 step Valve Valve Valve step

23 Step Valve changes to position. The flow passes through column and column. The flow path is shown. uffer uffer uffer uffer Purge Column step Valve uffer uffer (Sanitizing Solution) uffer Valve Column Purge Valve Valve Column uffer uffer uffer uffer Column uffer uffer (Sanitizing Solution) Step Valve changes to position. Column is now bypassed. uffer Column Valve UV Detector Model 8 Valve changes to position and column is now washed and eluted (shown). Column Valve step UV Detector Model 8

24 application ion Model 8 Using an Econo Gradient Pump and a Splitter Valve to Collect Sets of Fractions. The splitter valve is connected to and powered from the Econo gradient pump, which itself is controlled by the Duo-Flow system. The % Split is set in the Duo-Flow software. The flow exiting the column passes through the detectors and goes to the common port of the splitter valve. portion of the flow, defined by the user, is split and goes to a primary collector. The remaining portion of the flow goes to a secondary collector. The secondary collector runs in stand-alone mode, and is started manually. typical application for stream splitting is to automatically collect a small amount of each fraction for biological activity assays, thus eliminating post-run liquid transfer. Splitter Valve Column e.g., 9% % Econo Gradient Pump uffer uffer Model 8

For Users of the iologic HR System comparison ll the valving applications described for the iologic

However, the user must consult the following valve port comparison guide as the HR system uses V- and V9-8

The Duo-Flow system uses SVT- valves whereas the HR system uses SV- valves.

for the iologic HR System /overflow from sample loop Purge inject port to fill loop From gradient pump

25 For Users of the iologic HR System comparison ll the valving applications described for the iologic Duo-Flow system are also applicable to the iologic HR system. However, the user must consult the following valve port comparison guide as the HR system uses V- and V9-8 valves, not VR valves. These valves are not interchangeable between systems. oth systems can use valves. The Duo-Flow system uses SVT- valves whereas the HR system uses SV- valves. Comparison of Port Functions on and V- ion Valves Port Valve for the iologic Duo-Flow System Port V- Valve for the iologic HR System /overflow from sample loop Purge inject port to fill loop From gradient pump loop loop To column /overflow from sample loop From gradient pump inject port to fill loop loop loop Purge To column iologic Duo-Flow Valve Position Corresponding iologic HR V- Valve Position

26 Ordering Information Catalog # Description iologic Duo-Flow Valves 0-00 utomated ion Valve 0-00 Valve Rebuild Kit VR9-8 utomated Stream Select Valve 0-00 VR9-8 Valve Rebuild Kit 0-00 SVT- Diverter Valve 0-0 SVT- Valve Rebuild Kit 0-0 Select Valve, -port, -position solenoid random access valve, 0 psi ( bar) limit iologic Dynamic (Sliding Piston) Loops 0-0 DynaLoop Kit, includes a ml DynaLoop sliding piston loop, DynaLoop parts kit, instructions 0-0 DynaLoop 90 Kit, includes a 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, /8" ferrules, /-8 nuts and ferrules, /8" Teflon tubing, 0 ft 0-0 DynaLoop Sliding Piston Loop, ml sample loop replacement 0-0 DynaLoop 90 Sliding Piston Loop, 90 ml sample loop replacement 0-0 DynaLoop Sliding Seal Replacement iologic System ccessories 0-00 V- Manual ion Valve, -port, -position sample load valve,,000 psi (0 bar) limit 0-00 V- utomated ion Valve, -port, -position automatic sample load valve,,000 psi (0 bar) limit 0-00 V9-8 Stream Select Valve, 9-port, 8-position valve,,000 psi (0 bar) limit V9-8 utomated Stream Select Valve, 9-port, 8-position automatic valve,,000 psi (0 bar) limit 0-00 SV- Diverter Valve, -port, -position solenoid diversion valve, 0 psi ( bar) limit Model EP- Econo Pump -80 Model EP- Econo Pump, 0 V -8 Model EP- Econo Pump, 0 V -8 System Cable, connects Econo pump to iologic HR and Duo-Flow systems Econo Gradient Pump -900 Econo Gradient Pump, 00/0 V, includes tubing and fittings kit -900 Econo Gradient Pump, 0/0 V, includes tubing and fittings kit -90 Splitter Valve Kit, includes valve, tubing and adaptors Teflon is a trademark of E. I. dupont de Nemours and Company. io-rad Laboratories Life Science Group Web Site U.S. (800) IORD ustralia ustria (0) elgium 09-8 Canada (90) - China / Denmark 9 99 Finland 8 (0) France Germany Hong Kong India (9-) -00 Israel 0 9 Italy Japan Korea Latin merica Mexico -0 The Netherlands 08-0 New Zealand Norway Russia Singapore -98 Spain Sweden (0)8-00 Switzerland United Kingdom ulletin 8 US/EG Rev Sig 099

BioLogic DuoFlow Valving Guide

BioLogic DuoFlow Valving Guide 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