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GE AxiChrom 100, Operating Instructions Manual

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4.7

Over-pressure situation

General

Introduction

WARNING

Never exceed the maximum rated pressure for the column. Be
aware that the column has a 'packing pressure' and an 'operating
pressure'. For details of packing and operating pressure, refer to
Section 3.2 Primary specifications, on page 22.

CAUTION

Use pressure safety equipment to ensure that the maximum
packing pressure of the column is not exceeded.

NOTICE

The software Intelligent Packing and UNICORN at ÄKTApilot is not
verified as a safety monitoring system for the columns.

Causing over-pressure

An over-pressure situation can occur due to:

Situation

Error type

•

Operator enters a high pressure in the software

•

Operator selects wrong column size

•

The mechanical locking equipment is attached to the
column while trying to move the adapter upwards

User error

System runs towards a closed valve

Mechanical error

A system error occurs in the monitoring system

Reliability error

50

AxiChrom 50, 70 and 100 columns Operating instructions 28-9331-08 AG

4 Getting started

4.7 Over-pressure situation

Summary of Contents for AxiChrom 100

Page 1: ...GE Healthcare Life Sciences AxiChrom 50 70 and 100 columns Operating Instructions Original instructions ...

Page 2: ......

Page 3: ...ther equipment 36 4 6 Routine procedures 50 4 7 Over pressure situation 53 4 8 Preparing the column for a run 57 4 9 Installation in basic configuration 60 4 10 Installing the UNICORN Intelligent Packing strategy on ÄKTAexplorer and ÄKTApi lot 61 4 11 Selecting the Intelligent Packing strategy and component 62 5 Operation 63 5 1 Intelligent Packing of the column with ÄKTAexplorer and ÄKTApilot 79 ...

Page 4: ...mance 149 7 2 Adapter 152 7 3 Top plate 152 7 4 Mechanical locking equipment 152 7 5 Other 153 8 Ordering information 153 8 1 Useful accessories and spare parts 155 8 2 Contact information 156 A Parts list and diagrams AxiChrom 50 column 166 B Parts list and diagrams AxiChrom 70 column 176 C Parts lists and diagrams AxiChrom 100 column 186 D Tubing kits and connections 190 Index 4 AxiChrom 50 70 a...

Page 5: ...and use materials classified according to USP Class VI and FDA CFR 177 The material is free from material of animal origin or has been produced under manufacturing conditions complying with EMEA 410 01 1 2 Scope of this Operating Instructions This Operating Instructions is designed as a general introduction and guide to using AxiChrom 50 70 and 100 It is recommended that the user reads the complet...

Page 6: ...n the Appendices In some cases bold style characters either a number or letter may be contained in brackets These correspond to parts on the 100 column only that deviate from the commonly assigned items identified in square brackets see above 1 3 Important user information Read this before using the AxiChrom system All users must read the entire Operating Instructions before installing using or ma...

Page 7: ...and NOTICES concerning the safe use of the product See definitions below Warnings WARNING WARNING indicates a hazardous situation which if not avoided could result in death or serious injury It is important not to proceed until all stated conditions are met and clearly understood Cautions CAUTION CAUTION indicates a hazardous situation which if not avoided could result in minor or moderate injury ...

Page 8: ...directives and standards that are fulfilled by AxiChrom 50 70 and 100 Manufacturing information Address of manufacturer Name of manufacturer GE Healthcare Bio Sciences AB GE Healthcare Bio Sciences AB Björkgatan 30 SE 751 84 Uppsala Sweden Other regulatory information may be found in the Declaration of Conformity supplied with the product CE Conformity This product complies with the European direc...

Page 9: ...and the corresponding Declaration of Conformity is valid for the instru ment when it is used as a stand alone unit or connected to other CE marked products recommended or described in the user documentation and used in the same state as it was delivered from GE Healthcare except for alterations described in the user documentation The Declaration of conformity is valid only for systems that are mar...

Page 10: ...uctions for installation operation and maintenance use of proper personal protective equipment and or other arrangements needed to operate the process safely For personal safety it is important that the user has a proper knowledge of the entire system of which the column is a part of and that this Operating Instructions and the rest of the System documentation have been studied Additionally the us...

Page 11: ...sing hazardous chemicals take all suitable protective measures such as wearing appropriate personal protective equipment resistant to the chemicals used Follow local regulations and instructions for safe operation and maintenance of the column General cautions CAUTION Ensure that there is no pressure in the column before disconnecting any tubing CAUTION Ensure that all tubing and hoses are placed ...

Page 12: ... For details of packing and operating pressures see specifications CAUTION Use a pressure gauge pressure relief valve or other pressure safety equipment to ensure that the maximum operating pressure of the column is not exceeded General notices NOTICE Ensure that the liquids and samples used in the process are com patible with the chemical resistance properties of the materials of construction NOT...

Page 13: ...ree down to 1 µm as particles can block and damage the bed supports NOTICE Handle stainless steel parts especially the bed supports with care Damage to surfaces may lead to corrosion NOTICE Chlorides and low pH can cause corrosion on stainless steel Rinse thoroughly with clean water after use Inspect the column regularly for signs of corrosion which may cause column damage if untreated NOTICE Keep...

Page 14: ... the columns these pressure limits shall be observed Particular attention shall be taken when connecting manual pumps to the column or deviation in normal operating procedures using ÄKTAexplorer or ÄKTApilot that can lead to an over pressure AxiChrom 70 and 100 columns are not compatible with ÄKTAexplorer GE Healthcare recommends that the pressure inside the column is continually measured to detec...

Page 15: ...ated on the outer edge of the top bayonet Figure 2 1 The AxiChrom 50 70 and 100 label for serial number code number and manufacturing year Label for operating conditions The label for operating conditions is located on the column tube near the bottom flange Figure 2 2 The AxiChrom 50 70 and 100 label for operating conditions AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 15 2 ...

Page 16: ...erating temperature Symbols used in safety labels Meaning Label Warning Read the user documentation before using the system Do not open any covers or replace parts unless specifically stated in the user documentation The system complies with applicable European directives 16 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 2 Safety instructions 2 3 Labels ...

Page 17: ...rced out of the slurry B The slurry forms a consoli dated bed C The adapter reaches the surface of the consolidated bed D The bed is compressed to a predetermined packing factor Axial compression is achieved by driving the adapter downwards in the column tube by pumping hydraulic liquid typically 20 v v ethanol or 0 01 M NaOH into the space be tween the top plate and the top of the adapter This sp...

Page 18: ...dated bed height packed bed height Packing Factor PF where the consolidated bed height is read when the adapter reaches the consolidated bed where the packed bed height is read when the bed has been mechanically compressed to its final condition for use CF gravity settled bed height packed bed height Compression Factor CF where the gravity settled bed height is measured after settling the medium b...

Page 19: ...e 70 and 100 columns with the AxiChrom pivot stand not shown here Function Part Top mobile phase inlet outlet 1 Vent valve Hydraulic chamber hidden behind adaptor rod 2 Unpacking Hydraulic chamber outlet 3 Hydraulic chamber inlet 4 Bottom mobile phase inlet outlet 5 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 19 3 Specifications and characteristics 3 1 Principles of column ...

Page 20: ...The hydraulic chamber inlet has an integrated check valve Bottom mobile phase inlet outlet on the bottom plate note that the column may be run upflow or downflow Port inner diameter mm of inlets and outlets Column diameter mm Inlet outlet connection thread 100 70 50 2 9 mm 2 9 mm 1 7 mm Bottom mobile phase inlet outlet 5 16 2 9 mm 2 9 mm 1 7 mm Top mobile phase inlet outlet 5 16 2 9 mm 2 9 mm 1 7 ...

Page 21: ...ring compression there is a rise in pressure X When compression is complete UNICORN stops the flow of hy draulic liquid for some media flow conditioning will then start If selected in the Intelligent Packing wizard in UNICORN an evaluation test of the packed bed is automatically per formed A B X C Adapter height Column pressure Figure 3 3 Principle of Intelligent Packing The adapter starts from a ...

Page 22: ...Operating temperature C 1 14 Operating pH range 10 20 Stainless steel bed support pore sizes µm 10 20 Plastic bed support pore sizes µm 16 5 19 5 10 0 11 5 6 5 7 5 Column weight empty kg 3 Short column tube Long column tube N A 7 0 7 5 1 0 7 0 7 5 Foot pivot stand weight kg AxiChrom foot Pivot stand Short column tube Long column tube 1 65 1 8 1 4 1 7 Max working height m 4 Short column tube Long c...

Page 23: ... and 20 μm 3 4 Materials The materials used to manufacture AxiChrom columns have been chosen for their bio logical and chemical compatibility with the solvents used during operation and cleaning in place CIP procedures The columns have also been designed to comply with the varying hygienic requirements at the different stages of development and production Polymermaterialsin AxiChrom columnsin cont...

Page 24: ...ttom plate 50 and 70 columns Yes PEEK Bed support ring Yes Stainless steel EN1 4404 316L Bed support Yes Stainless steel EN 1 4571 316Ti EN1 4462 Duplex Adapter 50 column Yes Stainless steel EN1 4404 316L EN1 4462 Duplex Adapter 70 column No Stainless steel EN1 4404 316L EN1 4462 Duplex Adapter 100 column Yes PP End cell 100 column Plastic bed support dependent parts Yes EPDM FKM FPM and UHMWPE Se...

Page 25: ... PS PolyStyrene PVDF PolyVinylideneDiFluoride POM C PolyOxyMethylene PP PolyPropylene PE PolyEthylene PTFE PolyTetraFluoroEthylene For components made of Stainless steel EN1 4404 stainless steel EN 1 4432 316L and 1 4435 316L may also have been used All these are to standards EN 10028 7 and EN 10272 For components made of Stainless steel EN1 4401 stainless steel EN 1 4436 316L may also have been u...

Page 26: ...water between the use of salt and low pH liquid This information has been collected from several published sources not from individual tests on column components It should be used only as a guide The effects of a chemical will generally be more severe at higher temperatures Note also that the combined effects of agents have not been taken into account in this table Note NOTICE Low pH solutions con...

Page 27: ... 2 CIP 24 h RT to 30 C 1 M pH 14 Sodium hydroxide 1310 73 2 64 17 5 CIP 3 h 1 M 20 Sodium hydroxide ethanol 7757 82 6 Adsorption 3 h 1 M4 Sodium sulfate 57 13 6 Purification CIP 5 h 8 M4 Urea Equilibration adsorption elution 24 h 10 to 250 mM pH 3 to 10 Commonly used aqueous buffers for chromatographic use 1 Recommended temperature for usage is 2 C to 30 C for all chemicals listed in this table 2 ...

Page 28: ...r versus other possible rinsing solutions like ethanol solutions Both the outside and the inside of the column should be thoroughly rinsed with purified water Residual chloride ions can be corrosive to stainless steel over time Therefore it is recom mended to remove any buffer containing chloride ions that comes into contact with the top bayonet during pouring of slurry into the column by rinsing ...

Page 29: ...ml min 35 8 ml min AxiChrom 100 The flow rate needed into the hydraulic chamber to move the adapter downwards at a given linear velocity is lower than the flow rate needed to achieve a linear velocity through the bed This is due to the adapter rod volume in the hydraulic chamber AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 29 3 Specifications and characteristics 3 6 Packing ...

Page 30: ...rom column is delivered in two pieces Column tube assembly B with bottom plate assembly C mounted Adapter and top plate assembly A Additionally the following components are delivered AxiChrom foot with AxiChrom 50 column pivot stand available as an accessory Pivot stand with AxiChrom 70 or 100 Bayonet tool x2 Centering tool x2 one assembled onto the column Scraper seal x2 one assembled onto the co...

Page 31: ...s and outlets AxiChrom 50 column is delivered with AxiChrom foot shown in picture and AxiChrom 70 and 100 columns with the AxiChrom pivot stand not shown here Function Part Function Part Vent valve Hydraulic chamber hidden behind adaptor rod 5 Adapterand top plate assembly 1 Unpacking Hydraulic chamber outlet 6 Column tube assembly 2 Hydraulic chamber inlet 7 Bottom plate assembly 3 AxiChrom 50 70...

Page 32: ...ort Using a bed support with a larger porosity results in particles passing or getting caught in the bed support leading to poor bed efficiency and for a clogged bed support high pressure Separate 10 µm and 20 µm parts stainless steel bed support For 50 and 70 columns with stainless steel bed supports only All affected parts that are compatible with 10 µm bed supports are marked with 10 µm The mar...

Page 33: ... support 502 Bed support top 10 μm 20 μm 301 Bed support bottom 10 μm 20 μm 302 Screw stainless steel bed support 50 70 401 End cell 100 stainless steel bed support 402 Adapter tube id 1 7 stainless steel bed support 50 Adapter tube id 2 9 stainless steel bed support 70 100 405 O ring stainless steel bed support 50 70 410 O ring stainless steel bed support 411 1 Position number in the exploded dia...

Page 34: ...ber in the exploded diagrams in Appendices A C 4 4 Wetting instructions for plastic bed supports Follow these instructions to wet top and bottom plastic bed supports before packing the column 1 Before using new plastic bed supports wet the bed supports in 20 v v 1 propanol for at least 12 hours in an appropriate container Make sure that the bed supports are submerged below the surface of the solut...

Page 35: ...round the cir cumference 7 If necessary place the adapter with the bed support downwards into a container of 20 ethanol to prevent air from entering the flow path 8 Mount the adapter on to the column as described in this Operating Instructions 4 5 Other equipment In addition to the AxiChrom column and media other equipment that is needed is de scribed below General equipment The following equipmen...

Page 36: ...cifications on page 22 4 6 Routine procedures The following sections describe procedures that are routinely performed when operating the column and or servicing the column These sections are cross referenced from rele vant points in this Operating Instructions Identifying the hydraulic vent valve and hydraulic outlet valve The hydraulic vent valve and the hydraulic unpacking valve are both located...

Page 37: ...end of the stand arm Fitting the column onto the AxiChrom pivot stand Assemble the column onto the pivot stand as follows 1 Make sure that the pivot stand is placed on an even foundation 2 Make sure the stand arm is locked with the pin at the back of the vertical stand bar CAUTION The pivot stand locking pin is secured to prevent the column from turning over by accident 3 Lift the column verticall...

Page 38: ...own position to facilitate removal of the bottom plate WARNING Keep fingers away from the locking mechanism while rotating the column to avoid injury Tilting the column tube 1 Release the pin at the back of the vertical stand bar 2 Tilt the column for emptying or swing the column upside down 3 Secure the arm again by first pushing and then screwing the pin into the stand arm NOTICE The adapter and...

Page 39: ... information The locking equipment allows the position of the adapter to be locked without the need for hydraulic pressure CAUTION High pressure Never move the adapter in a column while mechan ical locking equipment is fitted Remove the mechanical locking equipment first Fitting the mechanical locking equipment Use of the mechanical locking equipment assumes that the bed has been packed by hydraul...

Page 40: ...d to compress the bed further than what was intended during the packing of the column 5 Check that the mechanical locking equipment is firmly fixed onto the column and that the parts sit securely 6 To avoid air entering the column after disconnection connection to the system apply upflow through the column to evacuate any air trapped in the tubing 7 Insert and hold a syringe into the top of the hy...

Page 41: ...at the top mobile phase inlet outlet 3 Remove the nuts and withdraw the mechanical locking equipment from the column Fitting the top plate and adapter The following procedure is used to fit the top plate 803 and adapter A to the column 1 Prime the adapter and bottom bed supports see Priming the column on page 73 2 Fill the column with an appropriate liquid or slurry up to the start of the glass in...

Page 42: ...bout 10 cm from the adapter Grip the adapter rod with one hand on each side of the top plate 5 Carefully insert the adapter into the mouth of the column tube at an angle so that the adapter enters into the liquid or slurry and down into the column tube without trapping air bubbles See Figure 4 4 The adapter shall rest on the tube chamfering when the lid is closed with the bayonet tools If the adap...

Page 43: ...n between the top plate and the adapter or top plate and top bayonet when the top plate slides down the adapter rod CAUTION To avoid use of excessive force and risk of trapping fingers lu bricate the adapter rod with 20 v v ethanol to make sure that the top plate can move smoothly AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 43 4 Getting started 4 6 Routine procedures ...

Page 44: ...ear head of liquid 1 2 cm above the top surface of the settling slurry before inserting the adapter into the column tube NOTICE Never force the adapter into the column manually Only use hydraulic pressure as described in this Operating Instructions to move the adapter in the column tube 6 Align the hole for the bayonet tool on the top plate with the furthest right machined mark at the top of the b...

Page 45: ... aligned with the machined mark at the left close open Figure 4 5 Opening and closing of the bayonet WARNING Ensure that the top plate is completely closed by confirming that the tool in the top plate is aligned with the machined mark on the top bayonet AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 45 4 Getting started 4 6 Routine procedures ...

Page 46: ... outlet in the bottom plate and connect a tubing from this outlet to waste 2 Ensure that the inlets outlets in the top plate including the top mobile phase in the adapter rod are sealed 3 Connect one end of a tubing to the hydraulic chamber inlet on the side of the top bayonet Connect the other end of the tubing to a system or pump 4 Pump in hydraulic liquid typically 20 v v ethanol or 0 01 M NaOH...

Page 47: ...top the pump This is to facilitate safe removal of the liquid above the adapter 6 Ensure that the hydraulic chamber outlet and or the hydraulic chamber vent valve are opened 7 Ensure that the column is depressurized 8 Insert a bayonet tool into each of the holes on the top plate 803 and top bayonet 804 9 Hold the tool still that is in the top bayonet and move the tool in the top plate coun tercloc...

Page 48: ...n the adapter rod Limitation of adapter movement in the long column tube The length of the adapter rod in a long column tube is designed for packing bed heights from 30 cm to 50 cm The adapter rod length is insufficient to allow adapter movement much below a 30 cm packing height If the adapter moves too far into the column the pressure is automatically reduced in the hydraulic chamber thus prevent...

Page 49: ... is stable in the mouth of the column or hold the adapter in an upright position 6 Dampen the adapter rod 802 using a cloth wetted with 20 v v ethanol and carefully slide the top plate approximately 15 cm up the adapter rod while keeping the adapter in place CAUTION Beware when handling the sharp edges of the bayonet threads NOTICE Never force the adapter out from the column manually Only use hydr...

Page 50: ...Intelligent Packing and UNICORN at ÄKTApilot is not verified as a safety monitoring system for the columns Causing over pressure An over pressure situation can occur due to Situation Error type Operator enters a high pressure in the software Operator selects wrong column size The mechanical locking equipment is attached to the column while trying to move the adapter upwards User error System runs ...

Page 51: ...edure 1 Stop the pump 2 Alert people in the room that the column is over pressurized 3 Monitor the pressure 4 Take all necessary safety precautions before depressurizing the column 5 Try to release the pressure by opening the flow path through the column using the system software If this is not possible loosen the tube connected to the adapter at the pump side at least 1 5 meters from the column t...

Page 52: ...ulic chamber vent valve 107 and hold it in place 2 Open the hydraulic chamber vent valve and collect the liquid in the syringe until the excess pressure in the hydraulic chamber is normalized Do not touch the syringe plunger during this step Note 3 If the syringe fills up before the pressure has normalized close the valve discard the contents of the syringe and repeat from step 1 52 AxiChrom 50 70...

Page 53: ...ubing and plug the top and bottom inlets outlets and the hydraulic chamber inlet before the column is moved NOTICE Do not hold the column by the adapter rod as this can damage the column NOTICE Take care not to subject the adapter rod to mechanical forces as this could lead to malfunctioning of the column CAUTION Use protective footwear whenever the column is moved to avoid injury AxiChrom 50 70 a...

Page 54: ...he mechanical locking equipment when moving a packed column Note CAUTION Make sure not to touch the locking equipment for the arm of the pivot stand when moving the column to avoid injury and rotation of the column If transporting the column in the pivot stand without the top plate and adapter A fitted place a centering tool into the mouth of the column to secure the tube in place For correct posi...

Page 55: ... the X direction i e the tilt of the whole back section between the wheels The other two points are the adjustable feet b and c at the front of the stand and allow levelling in the Y direction 1 Screw in a b and c as far as they will go 2 Place a spirit level in the X direction on top of the column 3 Adjust a until X is level If the surface on which the column is situated is sloping ensure that bo...

Page 56: ...xample water and or 20 v v ethanol to remove any surface residues acquired during removal from the packaging Before use carefully pump 1 0 M NaOH into the column empty and then pump a washing liquid for example water through the column to ensure that all inner surfaces are clean With exception to plastic parts all parts of AxiChrom 50 70 and 100 may be autoclaved if required 56 AxiChrom 50 70 and ...

Page 57: ...ing kits and connections on page 186 Installation to ÄKTAexplorer For details about tubing see section Tubing used between column and system on page60 Column Valve V2 Column Valve V3 2 3 2 3 Figure 4 8 Basic configuration for connecting AxiChrom columns to ÄKTAexplorer system AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 57 4 Getting started 4 9 Installation in basic configur...

Page 58: ...KTA avant 150 Connect the column to the system as described in the illustration in the Intelligent Packing phase and according to those column valves chosen in the method see step 6 in section Creating an Intelligent Packing method using GE approved media on page84 For details about tubing see section Tubing used between column and system on page60 Use a T connection on the outlet tubing Note 58 A...

Page 59: ... 9888 89 Tubing kit AxiChrom 50 ÄKTA avant desk Pos id 1 7 L 1600 x2 id 1 7 L 1200 x2 1 id 1 7 L 1200 x1 id 1 7 L 800 x1 2 id 2 9 L 1600 x1 id 2 9 L 1600 x1 3 id 1 7 L 200 x2 id 1 7 L 200 x2 4 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 59 4 Getting started 4 9 Installation in basic configuration ...

Page 60: ...Intelligent Packing strategy on ÄKTAexplorer and ÄKTApilot Install the correct UNICORN strategy containing Intelligent Packing for the appropriate system according to the instructions on the cover of the strategy CD supplied with the column The latest Wizard can be downloaded from the AxiChrom web page see Sec tion 8 2 Contact information on page 155 60 AxiChrom 50 70 and 100 columns Operating ins...

Page 61: ... ÄKTA explorer 1 Open the UNICORN manager module 2 Under Administration select System Setup 3 Select the appropriate system and click on Edit System 4 Select the appropriate strategy and then click on Component 5 Ensure that the check box for the component IntelligentPacking is checked Figure 4 10 Component dialog in UNICORN 6 Click on OK in Component and Edit System dialogs For more details on ho...

Page 62: ...make it possible to detect leakage at an early stage with minimized consequences and the risk for potential leakage of hazardous liquids is diminished Note Before packing the column pump 1 0 M NaOH through the column empty the column and then pump for example purified water through the column to make sure that all inner surfaces are clean Note The adapter rod attachment to the adapter may loosen a...

Page 63: ...rmed These require that flow occurs into the process chamber via the mobile phase inlet outlet By contrast during packing the method is con structed so that the air trap is bypassed as hydraulic liquid is pumped into the hydraulic chamber of the column Preparing the slurry Prior to filling the column it is necessary to calculate the volume of slurry based on slurry concentration needed to achieve ...

Page 64: ...d repack the column or c Filter smaller volumes of medium using a vacuum filtration system with a glass filter funnel Slurry volume The method wizard calculates and presents the needed slurry volume in the wizard summary page according to the following formula Slurry volume Target bed height x PF x Column cross sectional area x SVF Slurry concentration where the Slurry Volume Factor SVF is a compe...

Page 65: ...lt values for variables in the method wizard may be changed to optimize an Intelligent Packing method Changes using the variables page in the Method Editor module is for advanced users only Note The description below is for ÄKTApilot Pages may differ slightly for ÄKTAexplorer 1 Locate the method wizard for the relevant ÄKTA system in UNICORN 2 On the Main page of the wizard click on the Special Me...

Page 66: ...propriate material The only available option is Glass Column material Select the appropriate bed support type and material The mate rial options are stainless steel and plastic The porosity choice 10 or 20 µm is dependent on the bead size of the medium Bed support type Select the appropriatemedia type For custom media see Creating an Intelligent Packing method based on custom media on page 71 Show...

Page 67: ...ed range Note There are different scales for stainless steel and plastic bed supports on the column Target bed height Select the appropriate inlet Inlet for hydraulic chamber liquid Select the appropriate inlet Inlet for mobile phase AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 67 5 Operation 5 1 Intelligent Packing of the column with ÄKTAexplorer and ÄKTApilot ...

Page 68: ...acking Factor Unchecking this option for example for custom media see Creating an Intelligent Packing method based on custom media on page 71 allows the user to enter a Packing Factor in the range of 1 00 1 30 NOTICE Over compression during packing can damage the medium In the Intelligent Packing tests performed by GE Healthcare on its base media selection the default Packing Factor for each mediu...

Page 69: ...and test liquid for the selected medium Equilibration volume Leave checked to use the suggested default liquid velocity of 30 cm h Use default liquid velocity during equilibration Leave checked to use the suggested default liquid velocity of 30 cm h Use default liquid velocity during packing test Select whether to perform packing tests upflow downflow or both Both are selected by default Packing t...

Page 70: ... flow condition ing 6 Click on Next to display the Column Packing Summary wizard page Figure 5 5 Intelligent Packing Wizard 5 of 5 ÄKTApilot This page provides a summary of the options selected in the previous wizard pages A Suggested slurry recipe and estimated buffer consumption are also presented The summary page gives a reminder that it is important to change to the recom mended packing liquid...

Page 71: ...cking methods may also be developed for custom media using the wizard The wizard facilitates this by providing an extended range of variables that may be modified to optimize the method For example it is possible to Pack according to a desired Packing Factor or bed height If selecting the latter option the user stops the compression when the final bed height has been reached Select flow conditioni...

Page 72: ...izard NOTICE Over compression during packing can damage the medium In the Intelligent Packing tests performed by GE Healthcare on its base media selection the default Packing Factor for each medium has been used and verified 72 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 5 Operation 5 1 Intelligent Packing of the column with ÄKTAexplorer and ÄKTApilot ...

Page 73: ...eral times Gently tap the column and shake the tubing while flowing packing liquid into the column to help free the air bubbles Drain the column through the bottom phase inlet until 1 cm of liquid remains at the bottom of the column Repeat the procedure once 5 Hold the adapter with the bed support facing upwards and pump packing liquid at high flow velocities through the top mobile phase inlet out...

Page 74: ...on of the media stirrer and additional liquid volume from rinsing Note 5 Add more packing liquid into the column right up to the edge of the glass column tube Medium stuck on the column wall or in the hydraulic chamber inlet should be flushed down into the gel slurry with packing liquid Adding too little liquid increases the risk for air bubbles becoming trapped whereas too much liquid will lead t...

Page 75: ...ot turn over by accident CAUTION Make sure that the top plate is completely closed by confirming that the bayonet tool in the top plate is aligned with the ma chined mark on the top bayonet 2 Make sure that the hydraulic chamber outlet has been sealed 3 If any of the tubing from the system to the column has a 5 16 straight valve or SRV 1 valve make sure that these are open NOTICE Ensure that the f...

Page 76: ...ion NOTICE Ensure that the column is free from air before the process run is started NOTICE Do not seal the hydraulic chamber vent valve too early i e wait for the instruction to close the vent valve Otherwise the adapter will be pushed down in the column without UNICORN recognizing that the actual consolidation step with timed countdown has started and will require that the bed is repacked d The ...

Page 77: ...k on Continue in the wizard when the adapter comes into contact with the consolidated bed surface to achieve the right compression Clicking continue too early or too late will deliver a too low or too high Packing Factor respectively It is also important to correctly judge the correct level of the bed surface since medium can sometimes accumulate at the edges of the consolidated bed AxiChrom 50 70...

Page 78: ...e different scales for stainless steel and plastic bed supports on the column Note 5 When the packing test is completed open the Evaluation module of UNICORN and perform an integration of the peak using the packed bed height value 6 It is recommended to replace the tubing with a stop plug 101 at the check valve 116 78 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 5 Operation ...

Page 79: ... Ensure that the air trap is filled with mobile phase liquid This is because the methods created using the Intelligent Packing wizard are constructed so that the airtrap is in line with the column when equilibration and testing operations are being performed These require that flow occurs into the process chamber via the mobile phase inlet outlet By contrast during packing the method is con struct...

Page 80: ... For more information on Column Handling refer to the documentation for your UNICORN software Open the Column Handling dialog To open the Column Handling dialog select Tools Column Handling in any of the UNICORN modules or click the Column Handling icon in the Toolbar where available 80 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 5 Operation 5 2 Intelligent packing of the c...

Page 81: ...pe Parameters tab in the Column Handling dialog click 1 Result The New Column Type dialog opens Note The Intelligent Packing method is only compatible with AxiChrom columns AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 81 5 Operation 5 2 Intelligent packing of the column with ÄKTA avant 150 5 2 1 Create a column type ...

Page 82: ...how medium types by technique drop down list above Result The following parameters are automatically filled in can be edited if appropriate 2 Enter the remaining parameter values for the new column type in the Run Parameters Details and Ordering Information tabs Fields marked with must be filled in 3 Values in the gray fields are calculated and automatically filled in based on entered values for t...

Page 83: ... database and displayed in the Column types list Note If traceability of result is requested a column individual can be created under column type for logging of the data see UNICORN 6 1 Method Manual AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 83 5 Operation 5 2 Intelligent packing of the column with ÄKTA avant 150 5 2 1 Create a column type ...

Page 84: ...ligent Packing method using GE approved media This section describes the creation of an example Intelligent Packing method based on GE approved media If custom media is selected instead the method settings allow a wider range of variables to be defined to optimize the method Follow the instruction below to create an Intelligent Packing method based on GE approved media Action Step Open the Method ...

Page 85: ...layed in the Method Outline pane to the left and the default settings for each of the phases are shown in the Phase Properties pane to the right AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 85 5 Operation 5 2 Intelligent packing of the column with ÄKTA avant 150 5 2 2 Creating an Intelligent Packing method using ÄKTA avant 150 ...

Page 86: ...llustration below shows the Method Outline pane to the left and the Phase Properties pane of the Method Settings phase to the right Make sure that all parameter values are correct Note If the check box Column Performance Test is checked automatic logging of the result will be saved in the column logbook if a column individual is chosen 86 AxiChrom 50 70 and 100 columns Operating instructions 28 93...

Page 87: ... Intelligent Packing phase 4 The illustration below shows the Method Outline pane to the left and the Phase Properties pane of the Intelligent Packing phase to the right Note If the user wants to make changes in parameter values Custom packing settings can be used See Creating an Intelligent Packing method using custom media on page 92 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 ...

Page 88: ...d packing buffert for the selected medium is also shown in the dialog 5 Result The Suggested Slurry Recipe dialog opens Note The Slurry Recipe button is not available in Custom Packing settings Click OK to close the dialog 88 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 5 Operation 5 2 Intelligent packing of the column with ÄKTA avant 150 5 2 2 Creating an Intelligent Packin...

Page 89: ...e How to connect the column to the system dialog opens Note The text will be updated dependent on the column positions chosen but the illustration will remain the same Click OK to close the dialog AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 89 5 Operation 5 2 Intelligent packing of the column with ÄKTA avant 150 5 2 2 Creating an Intelligent Packing method using ÄKTA avant ...

Page 90: ...olumn volumes 7 The illustration below shows the Method Outline pane to the left and the Phase Properties pane of the Equilibration phase to the right Note All parameters in this phase can be changed 90 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 5 Operation 5 2 Intelligent packing of the column with ÄKTA avant 150 5 2 2 Creating an Intelligent Packing method using ÄKTA ava...

Page 91: ...quality The eluate is monitored by measuring the UV absorption at 280 nm or the conductivity and the curve can be evaluated in Evaluation See Performance eveluation of the column in ManualNameComplete for more information The illustration below shows the Method Outline pane to the left and the Phase Properties pane of the Column Performance Test phase to the right Note To check if the test is run ...

Page 92: ...ay be modified to optimize the method For example it is possible to Pack according to a desired Packing Factor or bed height If selecting the latter option the user stops the compression when the final bed height has been reached Select flow conditioning with associated variables Specify desired adapter velocity during packing within defined range In the Phase Properties pane of the Intelligent Pa...

Page 93: ...Aavant Follow the instructions in this Operating Instructions to prepare the slurry prime the column pour slurry into the column and to pack the column The UNICORN pages may differ slightly for ÄKTAavant 150 but the concept is the same as for ÄKTAexplorer and ÄKTApilot AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 93 5 Operation 5 2 Intelligent packing of the column with ÄKTA...

Page 94: ...ructions see below CAUTION Ensure that the pivot stand locking pin is secured before starting the process so that the column cannot turn over by accident 1 Prepare the media slurry see Preparing the slurry on page 63 Remember to com pensate for the compression and the Slurry Volume Factor when calculating the slurry volume needed to achieve the target bed height The target bed height is cal culate...

Page 95: ...e adapter into the mouth of the column tube at an angle to ensure that no air is trapped in the column 7 Place a syringe without plunger onto the hydraulic chamber vent valve and open the vent valve 8 To pump liquid into the hydraulic chamber For ÄKTApilot ÄKTApilot in the UNICORN System Control module choose Manual in the menu and Flowpath Columns Col1_Col2_Packing AxiChrom 50 70 and 100 columns ...

Page 96: ...ving down and the media is packed NOTICE Make sure to remove the syringe otherwise it may become hooked under the adapter rod top and hinder the packing es pecially if packing a low bed height 11 Note when the adapter comes into contact with the consolidated bed There are different scales for stainless steel and plastic bed supports on the column Note 12 Continue to compress the bed until the targ...

Page 97: ...utes The instruction is for ÄKTApilot The commands differ slightly for ÄKTAex plorer Note 15 Run the column upflow for a few minutes to remove residual air possibly trapped in the adapter The column is now packed Before using it evaluation of the packing is recommended see Section 5 4 Performance evaluation of the column on page 100 16 It is recommended to replace the tubing with a stop plug 101 a...

Page 98: ...cking factor 2 Prime the column see Priming the column on page73 and close the bottom mobile phase inlet outlet 3 Pour the slurry into the column see Pouring slurry into the column on page 74 4 Fit the adapter and top plate onto the column see Fitting the top plate and adapter on page 41 NOTICE Ensure that the full flow path is unrestricted when pumping liquid into the column NOTICE Insert the ada...

Page 99: ...different scales for stainless steel and plastic bed supports on the column Note 11 Continue to compress the bed until the target bed height is reached Calculate the achieved Packing Factor PF or Continue to compress until the target Packing Factor PF is reached PF consolidated bed height packed bed height Read the bed height Calculate the achieved Packing Factor PF 12 Turn off the pump when the t...

Page 100: ...the packing is evaluated Note The sample volume should be approximately 1 to 2 of the total bed volume and 1 to 2 v v acetone can be used Alternatively 0 8 M sodium chloride or equivalent can be applied to a 0 4 M NaCl elution buffer depending on the media that is used Use a liquid velocity 30 to 40 cm h for 34 µm and 50 µm media and 20 cm h for 75 and 90 µm media Running the HETP test at a higher...

Page 101: ...y acceptable A change in the shape of the peak is usually the first indication of bed deterioration due to use The asymmetry factor As describes the deviation from an ideal Gaussian peak shape and is calculated from the peak width at 10 of peak height As b a The following test chromatogram shows the parameters used to calculate peak broad ening and peak symmetry 0 0 0 0 296 253 0 85 0 20 10 0 0 0 ...

Page 102: ...quid has accumulated under the packed bed increase the flow rate successively again monitoring the pressure carefully During this procedure the adapter will move upwards in the column and the medium bed will successively break up NOTICE Never exceed an adaptervelocity of 120 cm h to avoid damage to the scraper seal 6 As the adapter begins to reach its upper position slow the flow rate for the fina...

Page 103: ... and the expected working life cycle of the medium and equipment For more information about which chemicals to use for CIP see the table on page 27 Ensure that the media and the column withstands the chemicals used for CIP Note 5 7 Sanitization Description Sanitization is the use of chemical agents to reduce a microbial population to an accept able predetermined level A sanitization procedure is u...

Page 104: ...cm h b 2 column volumes downflow at a velocity of 60 cm h 3 For 50 and 70 columns with plastic bed support Recirculate 5 column volumes of 1 0 M NaOH upflow for 2 hours followed by downflow for 2 hours at a pressure of 1 bar For other column configurations Recirculate 5 column volumes of 1 0 M NaOH upflow for 4 hours at a velocity of 60 cm h 4 Wash the column with buffer or water of desired qualit...

Page 105: ... column on page 102 2 Remove the adapter and top plate assembly see Removing the top plate and adapter on page 49 3 Release the pin at the pivot stand if used tilt the column and pour out medium into a container 4 Pump in packing liquid upflow if needed to help in pouring the medium Figure 5 8 Emptying the column using the pivot stand AxiChrom 50 70 and 100 columns Operating instructions 28 9331 0...

Page 106: ... fit the mechanical locking equipment see Mechanical locking on page 39 onto the column Plug the mobile phase inlets outlets and the hydraulic chamber inlet Ensure that the storage conditions of the column do not cause an increased pressure in the column Storage of a packed column for longer than 24 hours For storage periods longer than 24 h it is recommended that the process chamber containing th...

Page 107: ...te 803 placed at the end of the adapter rod right below the adapter rod top 105 Ensure that the top plate rests on its planar side to keep it in place For the stainless steel bed support ensure that the bed support O ring and scraper seal on the bed support are stored in a way that prevents mechanical damage For plastic bed supports ensure that the snap ring on the bed support is stored in a way t...

Page 108: ... valve performing a drainage test performing a column leakage test Referencingcolumncomponents Many of the procedures in this Operating Instructions make reference to specific com ponents on the AxiChrom column To facilitate easy identification of a specified compo nent numbers or letters are given within square brackets These correspond to a commonly assigned item in the diagrams for each of the ...

Page 109: ...s been emptied and properly cleaned If this is not pos sible wear protective clothing CAUTION When handling the adapter and top plate assembly ensure that fingers or hands are not caught in between the top plate and adapter or top plate and top bayonet when the top plate slides down the adapter rod CAUTION Heavy object At least two persons are required to lift objects over 20 kg NOTICE If leaving ...

Page 110: ... 5 2 5 3 4 Allen key mm 13 10 Torque wrench key with socket mm 2 5 Torque driver with bit mm Yes x2 Bayonet tool1 Yes x2 Centering tool1 Yes Gloves Yes for lubrication of friction surfaces Bottle of 20 v v ethanol Yes x2 Mounting aid for O Rings1 Note Two are supplied but only one is needed Yes For maintenance of Stainless steel bed sup ports Hook wrench key1 Yes For maintenance of plastic bed sup...

Page 111: ...gs are replaced once a year The scraper seal on the adapter bed support shall be regularly checked for scratches and other damage see Servicing the stainless steel adapter bed support on page112or Servicing the plastic adapter bed support tubing and rod top on page 124 On sign of any damage the scraper seal shall be replaced Otherwise it is recommended that the scraper seal is replaced after ten p...

Page 112: ... the bed support with water and then inspect for signs of damage If necessary replace the entire bed support 6 Clean the bed support if required for example due to clogging as determined by a drainage test see Section 6 3 Performing a drainage test on page 142 or a change of medium type in the column It is recommended to use an ultrasonic bath for cleaning see below or alternatively compressed air...

Page 113: ...d water 5 Test the cleaning efficiency with a drainage test Reassembling the stainless steel adapter bed support 1 Fit the O ring 603 411 into the groove in the adapter 501 for the 50 and 70 columns or into the groove of the end cell 402 for the 100 column 2 Fit the scraper seal 605 and O ring 604 using the following procedure NOTICE Improper fitting of the scraper seal and O ring or damage caused...

Page 114: ...g out from the bed support wall and moving it downwards a fraction and then repeating on an adjacent section of the O ring and so on The scraper seal can be mounted in a plastic container filled with water to facilitate the placement of the scraper seal and O ring and to avoid trapping air Note CAUTION Avoid rolling the O ring down the bed support as this may lead to twisting of the O ring which m...

Page 115: ...h the mounting tool on top of the O ring when in place in the groove and then draw the mounting tool around the circumferance of the O ring while applying pressure downwards Draw the mounting tool in both directions around the O ring circumferance This is to release any tension in the O ring arising from the assembly of the scraper seal and the O ring onto the adapter bed support A B Figure 6 4 Co...

Page 116: ...the 100 columns Tightening of the screws 401 will cause the O ring 603 411 to be pushed onto the bed support and damage the bed sup port Approx 1 mm A B 4 For the 100 column only Check and replace the O rings 413 if necessary 5 Use an Allen key to carefully refit the screws 401 on the adapter Using a torque driver tighten each screw a little at a time crosswise to 0 5 Nm for 50 and 70 columns and ...

Page 117: ...om their holes b There are three additional threaded holes on the bottom plate assembly Use three of the removed bolts 801 and insert them into these three holes c Gently screw in each bolt a little at a time to jack the bottom plate assembly C until it slides out from the bottom flange 805 Figure 6 5 Inserting the bolts to jack out the bottom plate assembly 3 Remove and discard the O ring 606 on ...

Page 118: ... 3 Refit the bed support 302 onto the bottom plate 502 being careful to align the holes around the circumference on both parts NOTICE The bed support must be mounted in the position according to figure A below before the bolts 403 are tightened Damage to the bed support can occur if the bed support slides on only partway as in figure B below This implies that the O ring 607 411b has snapped off th...

Page 119: ... removed from the column see Removing the top plate and adapter on page49 It is also required that the adapter bed support is removed for this operation see Servicing the stainless steel adapter bed support on page 112 Disassembling the adapter rod adapter tubing and top plate 1 Undo the five stop screws 106 on the adapter rod top 105 using an Allen key Re move the adapter rod top It is recommende...

Page 120: ...age to unscrew the adapter from the adapter rod 3 Gently pull on the adapter to withdraw the tubing from inside the adapter rod Figure 6 7 Withdrawing the tubing from inside the adapter rod 120 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 6 Maintenance and service 6 2 Performing maintenance and service ...

Page 121: ...and if nec essary replace the O rings 127 and 411a Figure 6 9 Unscrewing the top end cell on AxiChrom 100 column Allowing the O ring 411a to remain on the end cell facilitates removal Note 6 Slide the top plate 803 down the length of the adapter rod 802 and remove Apply a small amount of ethanol 20 v v on the adapter rod for easier movement of the top plate along the adapter rod AxiChrom 50 70 and...

Page 122: ...w O ring and roll it onto the adapter d Remove the Parafilm Reassembling the adapter rod and top plate 1 For the 100 column only Screw the top end cell 402 back by hand Ensure that it is screwed tight against the adapter plate 2 Screw the adapter tubing with O ring into the adapter finger tight only For column 100 always ensure that the top end cell is in place before screwing the adapter tubing i...

Page 123: ... 6 If necessary remove and replace the O ring 104 in the adapter rod top This O ring does not normally suffer from wear and its replacement is a difficult procedure Note 7 Fit the adapter rod top onto the adapter rod and i tighten the three lower screws so that the rod top is centered on the adapter rod and then ii tighten the two upper stop screws 106 with an Allen key to fix the adapter tubing i...

Page 124: ... the support net 422a as well B A Figure 6 11 Remove the snap ring and the bed support from the distributor by hand A or using the removal tool B To service or change distributor scraper seal and adapter tubing 1 Undo the five stop screws 106 on the adapter rod top 105 using an Allen key Re move the adapter rod top It is recommended that the stop screws are not fully removed from the adapter rod t...

Page 125: ...ry 6 Remove the scraper seal 605 and O ring 604 by gently moving them up the dis tributor a bit at a time around the circumference The O ring is secured within the scraper seal so they will most likely be removed together Note 7 Check the scraper seal for scratching and wear and the O ring for twisting and wear If damaged or if there is any doubt about their integrity discard them 8 Rinse the dist...

Page 126: ...the flat surface face downwards until it sits on the distributor flange B c Fit the O ring 604 by gently moving it down the distributor a bit at a time around the circumference Use 20 v v ethanol for lubrication This may be achieved by gently pulling a section of the O ring out from the distributor wall and moving it downwards a fraction and then repeating on an adjacent section of the O ring and ...

Page 127: ...ove created by the scraper seal and the wall of the distributor Press down one part of the O ring and then at an opposite point on the O ring circumference Continue until the O ring is located within the groove Figure 6 16 Location of the O ring into the groove of the scraper seal AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 127 6 Maintenance and service 6 2 Performing maint...

Page 128: ...nd that has the connector with a smaller diameter shall be connected to the system end see exploded diagrams in Appendix A C according to the column type Note 4 Feed the free end of the adapter tube through the top of the adapter rod until it be comes visible and points out through the middle hole of the adapter Ensure that the O ring 127 is in place 5 Refit the adapter tube onto the distributor 6...

Page 129: ...esulting from the adapter tubing coming loose Figure 6 18 Tightening the stop screws 10 a For 50 and 70 columns Flush the adapter downflow with 20 ethanol to ensure that the adapter and tubing are filled with liquid and free from air Place a new bed support in the snap ring Place a new support net on the distrib utor Fit the bed support and snap ring assembly onto the distributor Ensure that the e...

Page 130: ...Fit the bed support and snap ring assembly onto the distributor Ensure that the bed support is in contact with the distributor all around the circumference Check that the snap ring is correctly and evenly mounted around the whole circumference of the distributor Note Figure 6 20 Fitting the bed support in the snap ring and the snap ring onto the distributor 100 column 130 AxiChrom 50 70 and 100 co...

Page 131: ...oles on the bottom plate assembly Use three of the removed bolts 801 and insert them into these three holes c Gently screw in each bolt a little at a time to jack the bottom plate assembly C until it slides out from the bottom flange 805 Figure 6 21 Inserting the bolts to jack out the bottom plate assembly 3 Remove the snap ring 421b and the bed support 302 from the distributor 404 see Figure 6 11...

Page 132: ...the distributor all around the circumference Check that the snap ring is correctly and evenly mounted around the whole circumference of the distributor Note Figure 6 22 Fitting the bed support in the snap ring and the snap ring onto the support net and distributor 50 and 70 columns b For 100 column Place a new bed support in the snap ring Fit the bed support and snap ring as sembly onto the distri...

Page 133: ...e at a time crosswise Do not overtighten 5 Lubricate the inside of the tube end 201 and the O ring 606 of the bottom plate assembly C and refit it onto the column Tighten the screws crosswise and a little at a time NOTICE Do not physically push the bottom plate assembly into the column Carefully place it so that the holes on the circumference are aligned and tighten the bolts 801 crosswise AxiChro...

Page 134: ...leverage to unscrew the adapter from the adapter rod 2 Slide the top plate 803 down the length of the adapter rod 802 and remove Apply a small amount of ethanol 20 v v on the adapter rod for easier movement of the top plate along the adapter rod NOTICE The adapter rod seal 103 may be damaged if the top plate is removed over the key handle on the adapter rod 3 Check the O ring 108 on the outer rim ...

Page 135: ...nol 20 v v on the adapter rod for easier movement of the top plate along the adapter rod NOTICE Always wear gloves when handling the adapter rod to avoid fat from unprotected fingers and scratching the sealing surface 2 Screw the adapter rod onto the adapter Tighten by holding the adapter rod with a wrench and then tighten using another wrench The parts can loosen after using the column several ti...

Page 136: ...remove the nuts 806 and washers 119 connected to the tie bars underneath the bottom flange 805 Carefully remove the bottom flange 4 Unscrew 1 2 tie bars see Replacing tie bars on page 139 5 Carefully remove the column tube 201 6 Check the O ring 114a and 114b in the bottom flange and top bayonet for visible signs of damage Replace if necessary a Use a plastic implement or the removal tool to remov...

Page 137: ...lanar surface on the outer rim of the bottom flange is aligned with the planar surface of the top bayonet and that the holes for the pivot stand are aligned 4 Place the washers 119 onto the tie bars at the bottom flange and then loosely screw the nuts 806 onto the threads Do not tighten 5 Insert a centering tool in one end of the column tube 6 Carefully lift the column tube assembly B and lay it o...

Page 138: ...ool into the top A and bottom B of the column It is important that the centering tools are placed into the column tube to the correct marking point at the respective ends otherwise the column will not be correctly centered Note 9 Rotate the column tube so that the scale is set in the desired position There are different scales for stainless steel and plastic bed supports Ensure that the correct sc...

Page 139: ...loosen and unscrew the damaged tie bar s 901 connected to the top bayonet 804 2 Screw in a new tie bar to one of the tie bar holes by hand and tighten using a wrench at the key handles One of the ends of each tie bar has a short flattened length before the thread This flattened end should be used to screw into the top bayonet Note 3 Repeat for other tie bars as appropriate 4 Continue reassembly of...

Page 140: ...lve represents the flow di rection and shall be fitted so that it points inwards to the column Figure 6 27 Positioning of the check valve on AxiChrom 50 or 70 column CAUTION To avoid risks caused by over pressure the check valve shall be fitted into the valve housing such that the direction of the printed arrow on the valve points inwards to the column 5 Fit in a new O ring 117b on top of the chec...

Page 141: ...umn Figure 6 28 Positioning of the check valve on AxiChrom 100 column CAUTION To avoid risks caused by over pressure the check valve shall be fitted into the valve housing such that the direction of the printed arrow on the valve points inwards to the column 4 Screw the check valve adapter back into the top bayonet 5 Screw the check valve housing back into the check valve adapter Be careful not to...

Page 142: ... harm the bed support Use a new bed support as reference and the same weight as used with the used bed support Note 5 Open the bottom outlet and record the time it takes for the liquid level to move every 5 cm 6 Repeat steps 3 5 using clean bed supports 7 Compare the drainage times If the drainage times are at least 10 longer for the used bed support compared with the new then For stainless steel ...

Page 143: ...for the hydraulic chamber Other wise the correct pressure is not registered during measurement by the check valve at the hydraulic chamber inlet Note 7 Close the hydraulic chamber vent valve when only liquid escapes the valve 8 The adapter moves down Stop the pump when the adapter is at a suitable height Water should be seen emerging from the open mobile phase inlet outlet 9 Close the open mobile ...

Page 144: ...able height Water should emerge from the open mobile phase inlet outlet 7 Assemble the mechanical locking equipment see Mechanical locking on page 39 8 Pump liquid into the process chamber at a low liquid velocity while monitoring the pressure Stop the pump when pressure is equal to maximum packing pressure see Section 3 2 Primary specifications on page 22 In order for the pressure to continue to ...

Page 145: ...ouble peaks The extra peak is before the main peak i e leading Try to compress the bed slightly and retest Otherwise repack the column with a higher Packing Factor Cracks in the bed or a gap between the chromatogra phy medium and adapter The column may have been packed too loosely Double peaks The extra peak is after the main peak i e tailing To remove protein Clean in placeaccordingto instruction...

Page 146: ...aluation Decreased leading Repack the column with a higher or lower Packing Factor Bed packed too densely or too loosely Poor stability when repeating packing evaluation Decreasing number of plates Ensure the equipment is the same or identical to your test routine or Standard operating procedure SOP Extra volume in the system e g new tubing longer shorter or different diameter affects peak positio...

Page 147: ...ge and replace if necessary Leaking bottom bed support Liquid velocity should be in the range 15 60 cm h depending on particle size The smaller the particle the higher the velocity Liquid velocity too high or too low High HETP value Use a sample volume that is maximum 2 of the column volume Too large sample volume gives a broad peak Shorten these distances minimizes dilu tion Too great a distance ...

Page 148: ...ase buffer before starting the Intelligent Packing method A dip in the baseline during performance evaluation Ensure that no connections are leaking Change gaskets if necessary Leakage in the tubing between system and column or in the connectionbetween the end cell and adapter rod tubing The retention volume observed during evaluation is higher than expected Make sure that the correct sample with ...

Page 149: ... instructions underServicing the adapter rod adapter tubing and top plate stainless steel bed support on page 119 Adapter does not move upwards in the column despite applied pressure upflow in the process chamber In the beginning of the packing the adapter might be just above the glass incline Give the adapter a very gentle push downwards approximately 0 5 to 1 mm If this is not done immediately t...

Page 150: ... the Intelligent Packing wizard and be careful to add the right volume of slurry Wrong amount of medium added to the column Check that defaults values were used during Intelligent Packing Performance of media available in Intelligent Packing wizards has been verified by GE in the range default Packing Factor 0 02 Therefore it is known that a small devi ation in Packing Factor does not result in ba...

Page 151: ...t on page 119 The parts can loosen after using the column several times Check that the adapter rod is tight before performing each packing procedure The adapter is not tight enough The adapter is partially unscrewed from the adapter rod Prime the adapter at a higher flow ve locity Spray 20 v v ethanol on the bed support to lower the surface ten sion and thereby facilitate air removal The adapter w...

Page 152: ... adapter down by 1 2 mm Loosen the nuts Make sure not to over pressurize the column or the bed Nuts at the mechanical locking are too tight to loosen by hand 7 5 Other Action Comment Problem Replace damaged parts or add missing O rings Damaged missing O ring Damaged thread on tube connection Leakage from top mobile phase inlet outlet bottom mobile phase inlet outlet or hy draulic chamber inlet Ens...

Page 153: ... 4018 41 EN 1 4401 1 Mechanical locking 100 Yes 28 9231 80 PP 1 Media stirrer 40 Yes 28 9191 03 PP 1 Media stirrer USP 6 80 mm Yes 28 9055 41 PVDF 1 Tubing kit AxiChrom 50 ÄKTAexplorer desk Yes 28 9056 03 PVDF 1 Tubing kit AxiChrom 50 ÄKTAexplorer floor Yes 28 9056 76 PVDF 1 Tubing kit AxiChrom 50 ÄKTApilot desk Yes 28 9888 89 PVDF 1 Tubing kit AxiChrom 50 ÄKTAavant desk Yes 28 9888 92 PVDF 1 Tubi...

Page 154: ... Stop plug 5 16 No 18 1127 76 PEEK 3 Union 5 16 female M6 male Yes 28 9608 75 PEEK 1 Vent valve M8 No 19 5170 01 PP PTFE 2 Stop plug M6 male No 18 1142 08 8 Union 5 16 female 1 16 male No 19 2145 01 ETFE Titanium 1 SRV 1 M6 female Yes 28 9076 74 PEEK 1 Valve 5 16 straight Yes 18 1169 22 PEEK 2 Connector TC25 5 16 female Yes 18 1169 23 PEEK PFR 2 Connector TC25 5 16 male Yes 28 4034 13 EPDM 10 TC g...

Page 155: ...y must be ex changed The bed support kits are numbered 28 3664 46 through 28 3664 49 Note Spare parts recommended to keep on site It is recommended to keep the following spare parts on site Then keep the following spare parts on site If bed support type is A complete set of O rings bed supports and scraper seals Stainless steel A complete set of snap rings bed supports O rings and scraper seals Pl...

Page 156: ...108 Tube connection 5 16 id 1 7 Fem fem 109 O ring 11 1x1 6 111 O ring 54x2 114 a b Tube support 50 115 a b Check Valve holder 116 O ring 3 6x1 6 117 a b Check Valve 118 Washer 6 4x12 119 AxiChrom foot 50 123 1 Hex socket round head screw M6x30 123 2 O ring 3x1 126 Stop plug 5 16 USPVI 131 a b Glass tube 50 300 Short 201 Glass tube 50 500 Long 201 156 AxiChrom 50 70 and 100 columns Operating instr...

Page 157: ...28 3x1 78 410 O ring 34 646x1 78 411 Washer 3 2x9x0 8 412 O ring 2 5x1 3 413 O ring 2 5x1 3 416 Snap ring 50 421 a b Support net 422 a b Adapter 50 501 Bottom plate 50 502 O ring 3x1 602 a b O ring 44 12x2 62 604 Scraper 50 605 O ring 44 2x3 FPM 606 Screw M4x16 SS 801 Adapter rod 50 802 Top plate 50 803 Bottom flange 50 805 Hex nut M6 806 AxiChrom 50 70 and 100 columns Operating instructions 28 93...

Page 158: ... D Adapter assembly E Bayonet tool Hook wrench key Mounting aid for o rings Centering tool 50 Diagrams Meaning Symbol Number corresponds to Pos no in Material conformity and Spare part list No Illustrating parts in contact with process liquids and or that are pressure retaining 158 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG A Parts list and diagrams AxiChrom 50 column ...

Page 159: ...Figure A 1 Column and foot AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 159 A Parts list and diagrams AxiChrom 50 column ...

Page 160: ...Figure A 2 Column tube adapter and bottom plate 160 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG A Parts list and diagrams AxiChrom 50 column ...

Page 161: ...Figure A 3 Adapter assembly stainless steel bed support AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 161 A Parts list and diagrams AxiChrom 50 column ...

Page 162: ...Figure A 4 Adapter assembly plastic bed support 162 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG A Parts list and diagrams AxiChrom 50 column ...

Page 163: ...igure A 5 Adapter plate assembly and bottom plate assembly stainless steel bed support AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 163 A Parts list and diagrams AxiChrom 50 column ...

Page 164: ...Figure A 6 Adapter plate assembly and bottom plate assembly plastic bed support 164 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG A Parts list and diagrams AxiChrom 50 column ...

Page 165: ...Figure A 7 Column tube assembly AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 165 A Parts list and diagrams AxiChrom 50 column ...

Page 166: ... ring 65x3 108 Tube connection 5 16 id 2 9 Fem fem 109 O ring 14X1 5 111 O ring 72x2 114 a b Tube support 70 115 a b Check Valve holder 116 O ring 3 6x1 6 117 a b Check Valve 118 Washer 6 4x12 119 O ring 3x1 126 Stop plug 5 16 USPVI 131 a b Glass tube 70 300 201 Glass tube 70 500 201 Bed support 70 top 301 Bed support 70 bottom 302 166 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 ...

Page 167: ...2 5x1 3 413 Snap ring 70 421 a b Support net 422 a b Adapter 70 501 Bottom plate 70 502 O ring 3x1 602 a b O ring 63 2x2 8 EPDM 604 Scraper 70 605 O ring 63 09x3 53 606 Pivot stand 50 70 100 300 701 1 Pivot stand 50 70 100 500 701 1 Screw M6x12 701 2 Screw M4x16 SS 801 Adapter rod 70 802 Top plate 70 803 Top bayonet 70 804 Bottom flange 70 805 AxiChrom 50 70 and 100 columns Operating instructions ...

Page 168: ...er assembly E Bayonet tool Hook wrench key Mounting aid for o rings Centering tool 70 Diagrams Meaning Symbol Number corresponds to Pos no in Material conformity and Spare part list No Illustrating parts in contact with process liquids and or that are pressure retaining 168 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG B Parts list and diagrams AxiChrom 70 column ...

Page 169: ...Figure B 1 Column and pivot stand AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 169 B Parts list and diagrams AxiChrom 70 column ...

Page 170: ...Figure B 2 Column tube adapter and bottom plate 170 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG B Parts list and diagrams AxiChrom 70 column ...

Page 171: ...Figure B 3 Adapter assembly stainless steel bed support AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 171 B Parts list and diagrams AxiChrom 70 column ...

Page 172: ...Figure B 4 Adapter assembly plastic bed support 172 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG B Parts list and diagrams AxiChrom 70 column ...

Page 173: ...igure B 5 Adapter plate assembly and bottom plate assembly stainless steel bed support AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 173 B Parts list and diagrams AxiChrom 70 column ...

Page 174: ...Figure B 6 Adapter plate assembly and bottom plate assembly plastic bed support 174 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG B Parts list and diagrams AxiChrom 70 column ...

Page 175: ...Figure B 7 Column tube assembly AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 175 B Parts list and diagrams AxiChrom 70 column ...

Page 176: ...rge valve polished 107 O ring 98x3 108 Tube connection 5 16 id 2 9 Fem fem 109 O ring 23 52x1 78 111 O ring 104 5x3 114 a b Tube support 100 115 a b Check valve holder 116 Check valve single cpl 118 Washer 8 4x16x1 5 119 Stop plug 5 16 USPVI 131 a b Check valve adapter 124 O ring 70x2 5 127 Glass tube 100 300 201 Glass tube 100 500 201 176 AxiChrom 50 70 and 100 columns Operating instructions 28 9...

Page 177: ...ube id 2 9 plastic bed support 405 1 2 3 O ring 70x2 5 411 a b Washer 4 3x12 1 SS 412 O ring 3 69x1 78 413 Snap ring 100 plastic bed support 421 a b Adapter plate 100 SS bed support 501 Adapter plate 100 plastic bed support 501 Bottom plate 100 SS bed support 502 Bottom plate 100 plastic bed support 502 O ring 3x1 601 O ring 3x1 602 a b O ring 91 67x3 53 604 Scraper 100 605 O ring 88x4 606 Pivot s...

Page 178: ...ottom plate assembly C Adapter assembly with tube D Adapter assembly E Bayonet tool Centering tool 100 Mounting aid for o rings Hook wrench key Diagrams Meaning Symbol Number corresponds to Pos no in Material conformity and Spare part list No Illustrating parts in contact with process liquids and or that are pressure retaining 178 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG...

Page 179: ...701 4 701 3 701 1 701 2 131b 131a Figure C 1 Column and pivot stand AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 179 C Parts lists and diagrams AxiChrom 100 column ...

Page 180: ...A 801 B C Figure C 2 Column tube adapter and bottom plate 180 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG C Parts lists and diagrams AxiChrom 100 column ...

Page 181: ...5 106 107 103 101a 602a 602b 102 405 1 405 3 405 2 5x D E Figure C 3 Adapter assembly stainless steel bed support AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 181 C Parts lists and diagrams AxiChrom 100 column ...

Page 182: ...4 105 106 107 103 101a 602a 602b 102 405 3 405 2 5x 405 1 D E Figure C 4 Adapter assembly plastic bed support 182 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG C Parts lists and diagrams AxiChrom 100 column ...

Page 183: ... 412 413 127 402 411a 411b 404 Figure C 5 Adapter plate assembly and bottom plate assembly stainless steel bed support AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 183 C Parts lists and diagrams AxiChrom 100 column ...

Page 184: ...03 412 413 402 404 606 421a 421b 127 Figure C 6 Adapter plate assembly and bottom plate assembly plastic bed support 184 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG C Parts lists and diagrams AxiChrom 100 column ...

Page 185: ...01 101b 116 118 124 115a 114a 804 115b 114b 805 119 806 Figure C 7 Column tube assembly AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 185 C Parts lists and diagrams AxiChrom 100 column ...

Page 186: ...L 800 id1 2 L 800 2 Top valve P2 ÄKTAexplorer Column hydraulic chamber inlet id1 2 L 1100 id1 2 L 1100 id1 2 L 500 3 Column hydraulic chamber outlet waste id2 9 L 1600 id2 9 L 1600 id2 9 L 1600 id2 9 L 1600 4 Top valve P3 ÄKTAexplorer Column top mobile phase inlet outlet id1 2 L 1300 5 Top valve P2 ÄKTAexplorer Column hydraulic chamber inlet id1 2 L 800 6 For use with Valve SRV 1 id1 2 L 70 id1 2 ...

Page 187: ... 3 6 7 1 5 4 3x 8 7 2 9 8 9 9 7 1 3 7 9 2 4 9 7 9 10 10 Figure D 1 Kits and connections for ÄKTAexplorer AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 187 D Tubing kits and connections ...

Page 188: ...2 9 L 1600 id1 7 L 1600 id1 7 L 1600 3 For use with Valve 5 16 straight id2 9 L 200 id1 7 L 200 id1 7 L 200 4 5 16 female both valve ends 28 9076 74 Valve 5 16 straight 53 To be mounted at Pos 4 tubing end 03 0072 91 O ring 3 1 6 1 For 70 columns it is recommended to use the 50 70 tubing for media with an approximate bead size 50µm 2 For 70 columns it is recommended to use the 70 100 tubing for me...

Page 189: ...4 5 1 2 1 2 1 3 1 2 3 1 6 Figure D 2 Kits and connections for ÄKTApilot AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG 189 D Tubing kits and connections ...

Page 190: ...ning 117 C Check valve servicing 140 141 CIP 103 Cleaning 56 Adapter bed support 112 Bottom bed support 117 CIP 103 Column Accessories 153 ÄKTAexplorer connec tion 57 ÄKTApilot connection 58 AxiChrom pivot stand 37 Bed heights 22 Bed support sizes 22 Cleaning before a run 56 Cross section area 22 Drainage test 142 Emptying 105 Footprint 22 Intended use 7 Leveling 55 Moving 55 Operating pressure 22...

Page 191: ...g pressure 22 O ring Adapter servicing 113 126 O ring replacement 112 Over pressure 51 P Packing Factor Wizard setting 68 Packing list 30 Packing pressure 22 Packing the column Intelligent Packing 75 Manual packing 94 Performance evaluation of col umn 100 Pivot stand Fitting onto column 37 Tilting the column 38 Pressure Max operating 22 Max packing 22 Pressure too high in col umn 51 Releasing from...

Page 192: ... let 31 Top plate Fitting 41 Removing 49 Top plate servicing 119 124 Tubing kits 186 U UNICORN Strategy installation 60 Unpacking a packed bed 102 W Wizard Slurry concentration 69 Wizard for Intelligent Pack ing 65 Custom media 71 192 AxiChrom 50 70 and 100 columns Operating instructions 28 9331 08 AG Index ...

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Page 194: ...information UNICORN Any use of this software is subject to GE Healthcare Standard Software End User License Agreement for Life Sciences Software Products A copy of this Standard Software End User License Agreement is available on request GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healt...

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