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akta system - Sevier Lab

1. L9 1 2 EOS Built in air sensor Valves FV 923 SV 903 Switch valve Injection valve Loop valve Column valves Outlet valve IV 908 PV 908 PV 908H INV 907 port 1 7 port 2 3 INV 907H port 1 7 port 2 3 INV 917 V9 IA V9 IB V9 IS V9 l2 V9 IX V9H IA V9H IB V9H IS V9H I2 V9H IX Internal volume 10 ul 10 ul Internal diameter 1 2 mm 2 5mm 1 5mm 1 2 mm 1 5mm 1 5mm Internal volume 173 ul 113 ul 113 ul 17 ul 16 ul 16 ul 15 ul 26 ul 7 ul 30 ul 9 Ul 5 ul 41 ul 28 ul 15 pl 88 Ul 212 ul Back pressure from 10 ml min water at 20 C 0 2 MPa 2 bar 29 psi 0 4 MPa 4 bar 58 psi Connector to use Fingertight connector 1 16 M Tubing connector for o d 3 16 with blue ferrule for 3 16 o d tubing or Tubing connector for o d 1 8 with yellow ferrule Tubing connector for o d 1 8 with yellow ferrule Fingertight connector 1 16 M Tubing connector for o d 3 16 with blue ferrule for 3 16 o d tubing or Tubing connector for o d 1 8 with yellow ferrule Max pressure 0 2 MPa 2 bar 29 psi 0 2 MPa 2 bar 29 psi 0 2 MPa 2 bar 29 psi 3 MPa 30 bar 435 psi 3 MPa 30 bar 435 psi 3 MPa 30 bar 435 psi 3 MPa 30 bar 435 psi 2 MPa 20 bar 290 psi 25 MPa 250 bar 3625 psi 3 5 MPa 35 bar 508 psi 25 MPa 250 bar 3625 psi 3 5 MPa 35 bar 508 psi 35 MPa 350 bar 5075 psi 1 MPa 10 bar 1
2. 48 29 0108 31 AA The delay volume depends on the tubing and components included in the flow path Determine the delay volume theoretically or experimentally by including the volume from all tubing and components between the absorbance detector and the fractionation tip Appendix 3 provides a detailed description of how to determine the delay volume CP Remember to include the tubing to and from the fraction collector s accumulator if that is used Spillage free fractionation To minimize spillage a drop synch function is often included in AKTA fraction collectors A sensor at the fraction collector outlet detects the presence of droplets and synchronizes tube change The maximum flow rate for drop sync depends on the surface tension of the liquid and the i d and shape of the fractionation tubing tip When the liquid starts to flow continuously it cannot be used The maximum flow rate is also limited by how fast droplets can be detected Typically drop sync can be used for lower flow rates i e below 2 to 3 ml min Another way to avoid spillage between fractionation tubes is to include an accumulator During tube change the accumulator stores liquid which is then pushed out rapidly when a new tube is in position for collection The accumulator can be used for higher flow rates and is included in Frac 950 and the fraction collector of AKTA avant See Appendix 5 for how to troubleshoot fraction collection issues 29 0108 31 AA 49
3. 80 29 0108 31 AA HiTrap columns are convenient and reliable columns 1 or 5 ml with a bed height of 2 5 cm for fast and easy preparative purifications either alone or connected in series Fig A8 3 They are designed for use with a syringe peristaltic pump or chromatography system There are HiTrap columns for a broad range of chromatography media for AC immobilized metal affinity chromatography IMAC IEX desalting and HIC A range of Sephadex Sepharose High Performance Sepharose XL Sepharose 4B and Sepharose Fast Flow columns as well as Capto ImpRes Capto MabSelect MabSelect xtra MabSelect SuRe and MabSelect SuRe LX media The HiTrap column inlet is molded with 1 16 female threads and the outlet has 1 16 male threads for direct coupling to AKTA systems without the need for extra connectors HiTrap columns cannot be opened and repacked HiScreen columns are part of the process development platform available from GE Healthcare Fig A8 4 The columns are prepacked with a range of BioProcess chromatography media for AC IMAC IEX and HIC and designed for parameter screening and method optimization HiScreen columns have small bed volumes 4 7 ml thus requiring low sample and buffer volumes Process fluid velocities can be applied because the 10 cm bed height gives enough residence time and the results can then serve as the basis for linear process scale up If necessary two columns can easily be co
4. Not recommended or not applicable AKTAprime plus AKTApurifier ekeke fe Ho AKTAxpress one module Fig A6 1 The standard AKTA system configurations epek EN EN _ bef fee de fe _ AKTA avant pe fers foe de fe o ekeke epee o AKTAmicro 29 0108 31 AA 69 Fig A6 2 AKTAprime plus system Fig A6 4 KTApurifier system 70 29 0108 31 AA KTAprime plus is an economical and easy to learn system for the purification of proteins Fig A6 2 With push button control it offers simple one step purification of proteins Fig A6 3 This system includes preprogrammed methods for the purification of affinity tagged proteins histidine GST Strep tag Il and MBP tags and antibodies There are preprogrammed methods for the use of any HiTrap column The chromatography runs are monitored with PrimeView software In addition recovery of the recombinant protein is often better than when the same protein is purified manually With optimized purification protocols and prepacked columns yields and purity are highly consistent Microgram to gram scale quantities of tagged proteins can be purified in a single chromatography step on KTAprime plus used in conjunction with the appropriate columns Fig A6 3 Typical procedures using KTAprime plus A Prepare the buffers B Connect the column C Prepare the fraction collector D Load the sample and press start KTApurifier is designed for v
5. amp o o 2 F O With the cork on When the cork is no gas air bubbles removed gas air are visible bubbles emerge Fig 7 4 The flow restrictor can be compared to the cork on a champagne bottle A hypothetical example of how a flow restrictor affects the packed column at a flow rate of 1 ml min is shown in Figure 7 5 With no flow restrictor Fig 7 5A the flow rate generates a pump pressure reading of 0 3 MPa This pressure equals the pressure drop over both the chromatography medium and the column hardware For simplification the back pressure generated by tubing after the column is excluded in this example When a flow restrictor generating a back pressure of 0 2 MPa is added after the column Fig 7 5B the pressure over the column hardware p1 is affected and will be 0 5 MPa Hence the system pressure reading at the system pump will be 0 5 MPa However the pressure drop over the packed bed is still 0 3 MPa because Ap p1 p2 36 29 0108 31 AA Flow rate 1 ml min Flow rate 1 ml min System pressure System pressure 0 3 MPa 0 5 MPa Medium top pressure p1 0 5 MPa Medium top pressure p1 0 3 MPa eon hardware pressure 0 5 MPa 0 3 0 2 Column hardware pressure 0 3 MPa 0 3 0 0 Ap 0 3 MPa Medium bottom pressure is Medium bottom pressure is p2 0 MPa p2 0 2 MPa Medium pressure drop Ap 0 3 MPa Medium pressure drop Flow restrictor 0 2 MPa Fig 7 5 Flow restrictor effec
6. M6 F Union 1 16 F 1 16 F Union Fingertight i d 0 3 mm Union 1 16 M M6 F Union 1 16 F 1 16 F Ti Union 1 16 F M6 M Union Luer F 1 16 M Union 1 16 M 1 16 M i d 0 13 mm Union 1 16 M 1 16 M i d 0 25 mm Union 1 16 M 1 16 M i d 0 5 mm Union 5 16 F M6 M Union 5 16 F 1 16 M Union M6 F 1 16 M Union 1 16 M 1 16 M i d 0 5 mm Union M6 F M6 F SRTC 2 Ferrules Ferrules for 1 16 tubing connector Blue Ferrules for 1 8 tubing connector Yellow Ferrules for 3 16 tubing connector Blue Stop plugs Stop plug 1 16 Narrow Stop plug 5 16 M Stop plug 1 16 M Quantity pack size 2m 2m 2m 2m 2m 2m 2m 3m 3m 3m 10 10 10 10 10 10 mM mM NM COW PM MPO FPN DW RFP COO LF WN PO 10 10 10 Code number 18 1120 95 18 1121 36 18 1113 68 18 1120 96 16 1112 53 18 1119 74 18 1115 83 18 1142 38 18 1121 16 18 1112 47 18 1172 63 28 4010 81 16 1112 55 16 1127 07 18 1121 17 18 1112 49 18 1172 64 18 1027 12 11 0003 39 11 0008 52 18 1112 58 18 5855 01 16 1112 57 18 1112 51 18 1120 90 18 1120 92 18 1120 95 16 1127 76 18 1142 08 18 3858 01 28 9543 26 19 2143 01 18 1127 06 18 1121 18 18 1112 48 11 0003 55 18 1112 50 18 1112 52 29 0108 31 AA 85 Description Sample loops Assorted sample loops PTFE 25 50 100 200 500 ul Sample loops PTFE 1 and 2 ml Sample loop PTFE 10 ml Sample loop PEEK 1 ml Sample loop PEEK 10 ul
7. Tubing material and dimensions PEEK PEEK polyetheretherketone is a biocompatible material that is often used for medium to high pressure systems For a color description see Table A2 1 ETFE and PTFE In low or medium pressure parts of the system e g inlet and outlet tubing ETFE ethylene tetrafluoroethylene or PTFE polytetrafluoroethylene tubing is often used With these transparent materials for example air bubbles can easily be detected ETFE and PTFE are both biocompatible materials ETFE is the more rigid of the two Steel and titanium High pressure systems often use steel or titanium tubing Steel is prone to corrosion which often makes it unsuitable for purification of biomolecules Table A2 1 Tubing data 10 cm tubing 100 cm tubing i d Color of tubing corresponds to generates Standard tubing with 0 13 mm Red 1 3 ul 24 MPa Optional for AKTA avant to generate high pressure 0 15 mm Purple 1 8 ul 13 MPa AKTAmicro 0 25 mm Blue 4 9 ul 1 7 MPa AKTApurifier UPC 10 AKTA purifier 10 AKTA explorer 10 0 50 mm Orange 20 ul 0 11 MPa AKTArpLc AKTApurifier UPC 10 AKTA purifier 10 AKTA explorer 10 AKTA avant 25 0 75 mm Green 44 Ul 0 02 MPa AKTApurifier UPC 100 AKTA purifier 100 AKTA explorer 100 AKTA avant 25 1 0 mm Beige 78 ul 0 007 MPa AKTA avant 150 1 0 mm Transparent 78 ul 0 007 MPa AKTAxpress 1 6mm Transparent 200 ul Inlet tubing to AKTAFPLC AKTApurifier UPC 10 KTA purifier 10 AKTA exp
8. allow some time usually hours for it to adjust to ambient temperature Wipe off spillage to avoid corrosion of metal parts Turn off the UV Vis absorbance detector lamp on UPC 900 KTAxpress and KTAprime plus when not in use If system is controlled by a computer reboot the system and PC at least every 14 days Remove temporary files regularly Fig 10 1 Inlet filter A is mounted on the inlet tubing Inline filter B is connected in the flow path after or in the mixer Note An inline filter is also referred to as an online filter in some literature 29 0108 31 AA 51 Cleaning the system Minimal cleaning Because salt from buffer solutions might precipitate and clog valves and tubing wash the entire system flow path with buffer or water after every run It is also important to remove any sample from the inlet tubing as soon as possible after each run Thorough cleaning A general cleaning recommendation is to flush the system once a week when it is in use with 0 5 to 1 M NaOH Start with flushing the pumps at a moderate flow rate Prepare a system cleaning method and use it Perform cleaning without a column attached or by bypassing the column s Make sure that the entire flow path is cleaned and change valve position while washing the system flow path with cleaning solution Clean all tubing including the fraction collector tubing accumulator and the manual injection port See Table 10 2 for suggested cleaning
9. 0108 31 AA 35 Pressure alarms To protect column hardware and packed chromatography medium bed against pressure that is too high it is important to use correct settings for the pressure alarms To find out the pressure limits check the column and medium instructions and set the alarm s as described below For systems that measure the pressure only at the system pump the pressure alarm limit should be set to the lowest limit of either the column hardware or the packed medium bed For a system with three pressure sensors the pressure alarm for precolumn pressure p1 should be set to the column hardware limit This is affected by the pressure generated by the column plus the system flow path located after the column The pressure alarm for Ap should be set to the limit for the packed bed if available The relation between the pressures is Ap p1 pressure generated after and by the column itself p2 pressure generated after the column Function of a flow restrictor A flow restrictor creates a steady back pressure It therefore prevents air bubbles which may disturb detector signals from forming after the column due to the column pressure drop In addition a flow restrictor can be used to prevent siphoning If for example solutions are placed above the pump A flow restrictor can be compared to a cork on a bottle of champagne Fig 7 4 The pressure generated by the restrictor will help to keep the air dissolved in the solution
10. 7 7 shows some examples of how the viscosity varies for commonly used liquids and temperatures in chromatography 38 29 0108 31 AA A Viscosity Ammonium sulfate mPas 16 Sodium chloride Urea 1 4 1 2 1 0 0 2 4 6 8 Conc M B Viscosity mPas 3 5 30 lsopropanol 2 5 2 0 1 5 1 0 0 20 40 60 80 100 Conc Ethanol C Viscosity mPas 1 8 water An 4 C 25 C 0 68 mPas 000 5 10 15 20 25 Temp ec Fig 7 7 Effect of type of salt salt concentration A content of organic solvents in water B and temperature C on viscosity 1 mPas 1 cP The viscosity of common buffers and solutions even including 1 M NaCl will only be slightly higher than water and will therefore normally not become an issue during chromatographic runs see Fig 7 7A When mixing water with organic solvents e g 20 ethanol the viscosity will be significantly higher see Fig 7 7B and the generated back pressure substantially increased This phenomenon can often be noticed when washing ethanol from a column To keep within the pressure limit the flow rate might need to be reduced The pressure increases with decreasing temperature because viscosity is temperature dependent At cold room temperature approximately 4 C the pressure generated will be nearly twice as high compared with a room temperature approximately 25 C run see Fig 7 7C Due to the column pressure limit a decreased flow rate is needed t
11. Table 8 2 Effect of UV Vis flow cell path length Absorbance signal Low High Effect of UV Vis flow cell path length Switching to a longer cell will increase the signal If the absorbance is outside of the linear range switching to a shorter cell will decrease the signal cr The amino acid sequence of a protein can be used to calculate its theoretical absorbance coefficient Web based calculators are available to assist in determining this number See for example http www biomol net en tools proteinextinction htm for one such calculator Liquids and compounds During purification runs different solutions and compounds can cause deviations in the UV Vis absorbance curve Table 8 3 includes a list of some common examples and how to address them Table 8 3 Dealing with deviations in the UV Vis absorbance curve Effect Unexpected drift in the UV Vis absorbance curve or False negative or positive peaks High UV Vis absorbance baseline Cause Difference in refractive index e g when switching from Water to organic solvent during RPC High salt to no salt during HIC The solution is absorbing UV light e g Citrate buffers at 214 nm Impure imidazole at 280 nm Oxidized form of DTE at 280 nm What to do Switch to a solvent with a different refractive index if possible When evaluating results and performing peak integration adjust the baseline Use another buffer system instead of citrate i
12. The easiest way to determine this is to calculate the volume theoretically To do this all tubing and components from the sample vessel to the injection valve should be included See Appendix 3 for details It is also possible to determine the volume experimentally Disconnect the column from the flow path Fill the system with buffer and use buffer containing 1 acetone as sample Use the pump to apply the acetone solution Note the volume it takes until the UV Vis absorbance detector using A detects acetone NaCl can be used instead of acetone In this case measure the volume it takes for the conductivity monitor to detect the salt Note the volume obtained from experimental determination is slightly higher than when using the calculation method because the path from the injection valve to the detector is added 29 0108 31 AA 21 Applying a fixed sample volume To apply a fixed sample volume using a pump first determine the volume needed to prime the flow path with sample as described above Place the sample inlet in buffer and remove air bubbles from all pumps that will be used by purging as described in Chapter 5 Immerse the sample inlet in the sample container and start the priming After priming the system is ready for sample application Applying all of the sample using an air sensor To apply all the sample use a pump and an dir sensor Prime the sample inlet to be used with buffer and remove air bubbles from the pumps as
13. and emptying a Superloop In these images sample is colored yellow and buffer blue Considerations when using a Superloop The flow rate delivered from a Superloop is determined by the system flow rate In situations where it is more important to inject the entire sample run the pump for slightly more than the estimated sample volume to make sure that the Superloop and tubing are completely emptied see Fig 4 6C vy Superloops have a limited pressure range 4 MPa for the 10 and 50 ml loops and 2 MPa for the 150 ml loop Always make sure that the system pressure alarm limit does not exceed these values when the Superloop is connected inline If using a column with a higher pressure tolerance than that of the Superloop being used remember to lower the pressure limit during sample application Also bypass the Superloop before increasing the flow rate to normal fluorocarbon rubber that has limited chemical resistance It can be used in aqueous cr The moveable seal in the 10 ml and 50 ml Superloops has an O ring made of buffer solutions and alcohols while other solvents should be used with caution Solvent resistant O rings for Superloops 10 ml and 50 ml are available as accessories 18 29 0108 31 AA How to prepare a Superloop Before connecting a Superloop to the system remove the upper end piece as shown in Figure 4 7 Fig 4 7 Upper end piece removed from a Superloop Position the moveable seal in the bottom of the Superloop an
14. by increases in organic solvent concentration most commonly acetonitrile Samples that are concentrated during the binding and separation process are collected in a purified concentrated form The key stages in a separation are shown in Figure A7 8 column sample gradient clean after NREN equilibration application elution gradient Fes eq ESS Opes 2 4 CV wash out unbound molecules lt i nm before elution begins z cd 2 Z E JE 9 10 15 CV 5 CV J 2 CV 0 Column volumes CV Fig A7 8 Typical RPC gradient elution Blue line absorbance red line elution buffer CP RPC is often used in the final polishing of oligonucleotides and peptides and is well suited for analytical separations such as peptide mapping eT RPC is generally not recommended for protein purification if recovery of activity and return to a correct tertiary structure are required because many proteins are denatured in the presence of organic solvents Exceptions exist Method development 1 Perform a screening and select chromatography medium from the results 2 Select optimal gradient to give acceptable resolution As a starting point a linear gradient from 0 to 100 B of 10 to 20 columns volumes is recommended 3 Select highest flow rate that maintains resolution and minimizes separation time 4 For large scale purification transfer to a step elution 5 Proteins that bind strongly to a chromatography medium are more easi
15. column and media used in laboratory scale chromatography often generate varying back pressure the pump must also function under both high and low pressure CP Always make sure to remove all air bubbles in the pump before starting a run vy For optimal separation make sure that the pump delivers the correct flow rate Conditioning the pump for accurate liquid delivery Some systems have two pumps to be able to create accurate gradients Other systems use one pump and a switch valve to form gradients Each pump normally contains two pump heads that work in opposite mode to create a homogeneous flow rate How to detect air bubbles in the pump Air bubbles present in the pump cannot be detected by visual inspection of the pump Instead the pressure curve can be analyzed When the pump runs against a back pressure above 0 2 MPa air bubbles present will be seen by disturbances in the pressure curve Fig 5 1 To generate a back pressure above 0 2 MPa a reference capillary can be used see Table A2 1 in Appendix 2 10 No air bubbles Air bubbles in one of the pump heads 0 8 0 6 O Qa 0 4 0 2 0 33 4 33 8 34 2 34 6 35 0 Volume ml Fig 5 1 System pressure curve appearance when air bubbles are present in the pump The accuracy of the volume delivered is affected by even very small air bubbles a few microliters trapped in the pump 29 0108 31AA 23 How to remove air bubbles Air bubbles are removed from the pump by using a s
16. from AKTA avant 25 8 29 0108 31 AA Buffer mixing The mixer has two important functions in the system The first is to provide a homogeneous mix during gradient formation where two or more liquids are used to create the gradient The second is to even out pulsation effects from the pump The mixer that is delivered with the system will cover most of the applications within the flow rate range of the system but in some cases it may be necessary to switch to a mixer of a different volume to obtain optimal results See Chapter 6 Gradient formation and mixers for more information on this topic See also Figure 2 4 Programmed gradient B Conductivity curve correct mixer Conductivity curve mixer too large elution buffer B Distorted gradient Mixers of different sizes Volume Fig 2 4 The actual gradient will differ from the programmed gradient column in a system with too large a mixer Pressure High performance media used to achieve high resolution require a high performance pump that can operate under high pressure Generally such pumps can generate a higher pressure than the column hardware and media can withstand It is therefore important to monitor the system pressure so that it does not exceed the limits of the column Read more about this in Chapter 7 System pressure Detectors Different detectors are used to follow the progress of the purification For protein detection multip
17. run in the same system and with the same mixer For example if the gradient delay volume is 5 ml add 5 ml when running the columns in the example above Thus for the 1 ml column the total volume added would correspond to 10 ml 5 ml and for the 10 ml column it would correspond to 100 ml 5 ml CP If the system comes with preprogrammed methods the gradient delay volume is included in the method e g in the system volume compensation block CP Appendix 3 describes how to calculate delay volumes W During scale up or scale down make sure that the optimal mixer size is used see above If the new scale requires a mixer change remember to also update the gradient delay volume in the system UNICORN software 29 0108 31 AA 31 32 29 0108 31 AA Chapter 7 System pressure A back pressure will be generated when running liquid through the system If the back pressure exceeds any of the set pressure limits an alarm will be triggered and the system will stop This may be the most common problem in chromatography It is therefore important to understand the cause of high pressure to be able to avoid it Back pressure contributions It is important to keep the back pressure as low as possible because columns and system components are often sensitive to high pressure Table 7 1 highlights contributions to high back pressure and includes suggestions on how to avoid it Table 7 1 Contributions to back pressure Source How to
18. ul load a Se ae Overfilling high reproducibility nn aad 100 ul sample loop Fig 4 2 Filling a sample loop here i d was 0 50 mm Emptying a tubing loop To avoid dilution when emptying a tubing loop empty it in the opposite direction from which it was filled The volume to achieve complete recovery will vary with the flow rate loop dimensions and the properties of the sample but usually three to five times the loop volume is sufficient Figure 4 3 shows an example of the recovery achieved at different volumes when emptying a 100 ul loop at 0 5 ml min To empty the loop completely in this example a buffer volume corresponding to three times the loop volume was needed 1x 2x 3x Loop volume 82 0 97 0 99 5 Sample recovery Fig 4 3 The elution profile and recovery when emptying the contents of a completely filled sample loop In this setup a tubing loop with i d 0 50 mm was used 16 29 0108 31 AA To achieve high sample recovery use a large volume to empty the loop For nonbinding techniques e g desalting and GF there are sample volume limitations due to the size of the column used Figure 4 4 shows an example of how resolution can be improved by decreasing the volumes used to empty the loop during sample injection This is a common way of working when analytical studies are performed A280 3 times loop volume 1 time loop volume Loop volume 50 ul Flow rate 0 3 ml min Column Superdex
19. 1 4 ml 10 MPa Sample pump to KTA avant 25 for wash up to 100 bar 1450 psi 65 ml min P9H 0 01 150 ml min 429 ul 1 8 ml 5 MPa System and sample pump to 50 bar 725 psi KTA avant 150 Mixers Internal volume Max pressure Used with M 925 magnetic stirrer 90 ul 35 MPa 350 bar 5075 psi AKTAmicro M 925 magnetic stirrer 0 2 ml 35 MPa 350 bar 5075 psi AKTAmicro M 925 magnetic stirrer 0 6 ml 25 MPa 250 bar 3625 psi AKTAreic AKTApurifier UPC 10 AKTApurifier 10 AKTAexplorer 10 M 925 magnetic stirrer 2 ml 25 MPa 250 bar 3625 psi AKTApurifier UPC AKTApurifier AKTAexplorer M 925 magnetic stirrer 5ml 25 MPa 250 bar 3625 psi AKTApurifier UPC 100 AKTApurifier 100 AKTAexplorer 100 29 0108 31 AA 55 Mixers continued Internal volume Max pressure Used with M 925 magnetic stirrer 12 ml 10 MPa 100 bar 1450 psi AKTApurifier UPC 100 AKTApurifier 100 AKTAexplorer 100 AKTAxpress mixer static 0 37 ml 3 MPa 30 bar 435 psi AKTAxpress M9 0 6 ml 20 MPa 200 bar 2900 psi KTA avant 25 M9 1 4 ml 20 MPa 200 bar 2900 psi KTA avant M9 5 ml 20 MPa 200 bar 2900 psi KTA avant M9 15 ml 5 MPa 50 bar 725 psi KTA avant 150 Inline filter Internal volume Max pressure Used with Filter holder 115 ul 35 MPa 350 bar 5075 psi AKTApurifier UPC 100 AKTApurifier 100 AKTAexplorer 100 Filter holder 20 ul 35 MPa 350 bar 5075 psi AKTApurifier UPC 10 AKTApurifier 10 AKTAexplorer 10 AKTAmicro Filt
20. 20 39 18 1115 98 18 1113 99 18 1114 02 18 1140 53 11 0005 02 28 9044 38 18 1115 81 18 1113 82 18 1023 85 18 6500 01 18 1104 97 18 1147 24 18 1147 21 18 1118 90 18 1118 91 18 1118 92 18 1119 95 28 9561 86 28 9562 25 28 9562 46 28 9803 09 18 1128 25 18 1128 24 18 1111 10 18 1147 25 18 1111 11 28 9793 86 28 9793 80 28 9563 78 18 6324 01 18 6524 02 18 6324 04 18 6324 05 18 6324 06 Description Quantity pack size pH detectors for AKTA systems excluding AKTA avant PH electrode with cell and holder round tip pH electrode round tip Dummy electrode round tip Air sensors Air 900 N control unit Air 912 N flow cell 1 2 mm i d Air 925 N flow cell 2 5 mm i d 1 each 1 1 Racks and cassettes for AKTA avant fraction collector Cassette holds 6 x 50 ml tubes Cassette holds 15 x 15 ml tubes Cassette holds 40 x 3 ml tubes Cassette holds 1 x 96 48 or 24 deep well plate Rack holds 55 x 50 ml tubes Rack holds 18 x 250 ml bottles Cassette holds 24 x 8 ml tubes Racks and options for Frac 950 Rack A 18 mm and 30 mm tubes Rack B 12 mm tubes Rack C 4 x 96 well and 30 mm tubes Rack D 30 mm tubes Prep mode Prep Mode Conversion Kit for use with Rack E and Rack F Rack E for Prep mode using 30 mm tubes Rack F for Prep mode using 250 ml bottles Funnel to Flask Kit with funnels tubing and tubing organizer for use with Rack E Microfractionation Microfraction Collection K
21. 200 5 150 GL AQS 0 0 2 0 4 0 6 0 8 1 0 Volume ml Fig 4 4 The chromatogram shows how the separation in GF is affected by the different volumes used to empty the loop during sample injection CP Before starting decide whether the most important aim is high recovery or high resolution Superloop Superloops are available in three different sizes 10 50 and 150 ml and can be used for sample volumes in the range of 100 ul to 150 ml They can be used to inject the complete sample volume onto the column or to make repeated injections of a sample without manual interactions in between Figure 4 5 depicts a 10 ml Superloop Buffer Moveable seal Sample Fig 4 5 Schematic drawing of a 10 ml Superloop 29 0108 31 AA 17 How to fill and empty a Superloop A Superloop is connected to the injection valve and is initially filled with buffer Sample is loaded from the bottom either manually using a syringe or by using a sample pump Fig 4 6A The sample is injected onto the column by pushing buffer into the top of the Superloop so that the seal moves downward pushing the sample out of the Superloop The seal hinders mixing of sample and buffer Fig 4 6B When the moveable seal reaches the bottom position the buffer will automatically bypass the seal to the column following the sample Fig 4 60 A Sample load B Sample inject C Sample flush h sm d d f T Pump or Column Column syringe Fig 4 6 Filling
22. 45 psi 2 MPa 20 bar 290 psi Used with AKTAprime plus AKTAFPLC AKTApurifier 100 AKTAexplorer 100 AKTAxpress AKTA avant AKTApurifier UPC 10 AKTApurifier 10 _ AKTAexplorer 10 AKTAmicro Used with AKTAre c AKTApurifier UPC AKTApurifier AKTAexplorer P 960 AKTAre c AKTApurifier UPC AKTApurifier AKTAexplorer P 960 AKTAretc AKTApurifier UPC AKTApurifier AKTAexplorer AKTAxpress P 960 AKTA avant AKTA avant KTA avant inlet valves V9 IA V9 IB and V9 IS Used with AKTAretc AKTApurifier UPC AkKTApurifier AKTAmicro AKTApurifier AKTAexplorer AKTAmicro AKTAxpress AKTAxpress AKTAxpress AKTAxpress AKTAxpress AKTAretc AKTApurifier UPC AKTApurifier AKTAexplorer AKTAretc AKTApurifier UPC AKTApurifier AKTAexplorer High Flow kit for AKTApurifier UPC 100 AKTApurifier 100 AKTAexplorer 100 AKTAretc AKTApurifier UPC AKTApurifier AKTAexplorer AKTAreic AKTApurifier UPC AKTApurifier AKTAexplorer AKTAmicro AKTA avant 25 AKTA avant 150 29 0108 31 AA 57 Valves continued V9 Inj V9H Inj V9 C V9 C2 V9H C V9H C2 V9 DH V9H pH V9 O V9 O2 V9 03 V9H O V9H O2 V9H O3 Fraction collectors Frac 920 Frac 950 Built in with AKTAxpress Built in with AKTAprime plus Built in with AKTA avant Autosamplers A 900 A 905 Internal volume 9 ul 23 ul 110 ul 191 ul 15 ul via bypass 36 ul via bypass 11 ul 82 ul Capacity 95 tube
23. 50 29 0108 31 AA Chapter 10 Cleaning and storage of system components System lifetime and performance will be maximized If proper cleaning and storage routines are followed This chapter describes how to maintain the system Table 10 1 provides maintenance tips to help keep the system running problem free for a long period of time Table 10 1 Tips on preventive maintenance Purpose Keep back pressure low by preventing particles entering the flow path Clean system to prevent carryover between runs and contamination of the flow path Keep system clean to prevent microbial growth in the flow path Avoid condensation in system components Protect exterior of the instrument Prolong lifetime of the UV Vis absorbance detector lamp Avoid software connection problems A B What to do Use filtered solutions A filter pore size of 0 45 um is recommended Use inlet filters on all inlet tubing Fig 10 1A Replace the inline filter regularly Fig 10 1B Clean the system flow path regularly with 0 5 to 1 M NaOH Create appropriate cleaning procedures Replace pump rinse solution 20 ethanol once a week only applicable for pumps with a rinsing system Use 20 ethanol as storage solution when system will not be used for 2 days or more Leave the power ON if the system is in cold room the UV Vis absorbance detector lamp can be turned off to save lamp run time When the system is moved to a new temperature
24. 82 20 9502 95 20 9363 19 28 9644 99 28 4007 37 18 1113 17 18 3094 60 18 1126 32 18 1149 98 18 1113 19 18 1158 31 18 1144 61 18 1175 93 16 5050 65 18 1148 62 29 0108 31AA 89 For local office contact information please visit www gelifesciences com contact www gelifesciences com akta GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden imagination at work KTA AKTAexplorer AKTAreic AKTAmicro AKTAprime AKTApurifier AKTAxpress BioProcess Capto FPLC HiLoad HiPrep HiScale HiScreen HiTrap MabSelect MabSelect SuRe MabSelect Xtra Mini Q Mini S Mono Q Mono S PrimeView RESOURCE Sephacryl Sephadex Sepharose SOURCE Superdex Superloop Superose Tricorn and UNICORN are trademarks of GE Healthcare companies GE imagination at work and GE monogram are trademarks of General Electric Company Decon is a trademark of Decon Laboratories Inc Strep tag is a trademark of IBA GmbH PEEK is a trademark of Victrex plc StrepTrap HP and StrepTactin Sepharose High Performance These products are covered by US patent number 6 103 493 and equivalent patents and patent applications in other countries The purchase of StrepTrap HP and StrepTactin Sepharose High Performance includes a license under such patents for non profit and in house research only Please contact IBA info iba go com for further information on licenses for commercial use of Strep Tactin Histidine tagged protein purifica
25. 905 P9 P9 S and P9H pumps Figure 5 4 shows the pump head design As the piston moves out of the chamber during the suction phase the inlet check valve will open and the outlet check valve will close allowing the chamber to fill up with liquid During the delivery phase the outlet check valve will open while the inlet check valve will close During this phase the piston will move into the chamber pushing the liquid out of the pump 29 0108 31 AA 25 Liquid out Outlet check valve Chamber Inlet check valve High pressure piston seal Liquid in Fig 5 4 Schematic view of one pump head from P 901 P 903 P 905 P9 P9 S and PYH Piston seal rinsing system The piston seal rinsing system has two functions 1 It protects the piston seals and pump heads by preventing a buildup of deposits consisting of components from solutions used for example salt crystals 2 It prolongs the lifetime of the seal by preventing it from drying The inlet and outlet tubing of the rinsing system are most often placed in the same container The rinsing system should always be filled with 20 ethanol which then circulates on the back side of the pump head as shown in Figures 5 5 and 10 2 In this process deposits will be flushed out and the ethanol prevents microbial growth Wash out T Inline one way valve Piston Backwash pump diaphragm Wash in Fig 5 5 Schematic view of the piston seal rinsing system of
26. A MOVE a EEEE EE EA EEE AE EN EE E NE 20 Samplelogding using O PUNI capasnczasvvscesnnsceseavnnsvins tediovsssletvsvasdvsnsidegunsnstio sondasivusioasoobsssiasivea vinstabesieabsntivistensenests 21 Chapter 5 Liquid delivery ANd PUMPS cccssscssccsssssssscssssssessesscsssssssseessscsesssssssessssssssassssessssensssceescenseass 23 Conditioning the PUMP for accurate liquid delivery 0 ceeecsesssessssessssessseesseesssecsseccsssessecssecsssecssecssecsssecsseeeses 23 Description of the pump and rinsing SYSTOM u eecesssesssssssecssecsssessseessecsssecssscsssesssscsssssssscssecessesssecssecesseessecs 25 Pil en orta prote C COUN ennan n AER O 27 Chapter 6 Gradient formation and MiIXePS cscscscssccsssssssssssssessessecsscsssssssssssssesceessessecsessacsacscsassseseeees 29 OS a E E EEE EN perering ve avereoatioscartasiemrossina ET 29 CORE GS Fh OK SES asoni E A E N 29 adent CSN VoU C argan A EET EEN EN 30 2 29 0108 31 AA Chapter 7 SYS PRESSE EN A E E E AA A A 33 hole ae FOSS USE CONDUTOR ariaeiesroiini n EE O A 33 TUBING contribution to back pressure css tccecenacneasnaidonnavdverigetavatectuotanseieteisrinaraentiraadaserieonniesanten 34 The effect of back pressure on column and packed bed ou eeeceeessesssssssssessssessecsssecssecssessesssecesssesseessecs 34 Presoure MONTO sensies EAR AAAA AEE AA EDER ED FEE Eee Er 33 PRES US MEE AEA EEE NA E AEEA AO 36 Funcion ora TOWT e C1 Ol araga a E E EE E EA A 36 Removal HOW TOS tic
27. Aexplorer or as stand alone AKTAretc KTA purifier UPC AKTApurifier AKTAexplorer AKTAmicro AKTAxpress AKTAprime AKTAprime plus AKTA avant Used with AKTApurifier AKTAexplorer AkTApurifier AKTAexplorer AKTAmicro 2 AKTAprime plus refers also to AKTAprime except regarding the pump AkTAprime has a P 950 pump AKTApurifier UPC refers to AKTApurifier UPC 10 AKTApurifier UPC 100 AKTApurifier refers to AKTApurifier 10 AKTApurifier 10 plus AkTApurifier 100 AkTApurifier 100 plus AkTAexplorer refers to AkTAexplorer 10 AkTAexplorer 10S AkTAexplorer 100 AkTAexplorer 100 Air AKTAxpress refers to AKTAxpress Single AKTAxpress Twin and AKTAxpress module AKTA avant refers to AKTA avant 25 and AKTA avant 150 AKTApurifier 10 refers also to AKTApurifier 10 plus AKTApurifier 100 refers also to AKTApurifier 100 plus AKTAexplorer 10 refers also to AKTAexplorer 10S AKTAexplorer 100 refers also to AkTAexplorer 100 Air 3 To connect to AKTAretc AKTApurifier UPC AKTApurifier and AKTAexplorer use the Air 900 N control box 58 29 0108 31 AA Appendix 2 Tubing guide Many different sizes types of tubing can be connected to a chromatography system Tubing with a smaller inner diameter i d holds less delay volume and will therefore generate less dilution of the protein peak Narrow tubing however increases the system pressure especially when running at high flow rates The tubing used should match the application needs
28. Check that the pump is delivering the correct flow rate at 1 ml min If not make sure that the pump has no air bubbles within it see Chapter 5 2 Filla small sample loop i e 100 ul with a 1 to 5 acetone solution 3 Fill the system with water Run the pump at 1 ml min and inject the acetone solution as a sample The volume from point of injection to peak appearance in the chromatogram is equal to V1 4 Reconfigure the system a To replace the UV Vis flow cell disconnect the two tubing segments and connect them with a low dead volume connector Use for example a 1 16 female 1 16 female union connector b Mount the fractionation tubing tip into the top of the UV Vis flow cell and connect a waste tubing from the bottom of the UV Vis flow cell 5 Set the Frac size to a large volume for example 100 ml so that the valve is in the Frac position during the entire run Start the pump at 1 ml min and inject acetone solution The volume from point of injection to peak appearance in the chromatogram is equal to V2 6 Subtract V1 from V2 to obtain the delay volume 1 This method cannot be used with KTA avant because the fractionation tubing cannot be disconnected by the user 62 29 0108 31 AA Weighing water To determine the delay volume experimentally by weighing water a preweighed container e g a fractionation tube is needed 1 Make sure that the system flow path is set up so that the liquid is directed from the UV
29. GE Healthcare Life Sciences AKTA Laboratory scale Chromatography Systems Instrument Management Handbook Handbooks from GE Healthcare er mi ieee bighi p y a a a w z E f rr d 3 a i p Y a i wah eee ara SW gt i Aa 63 gt i oa al of we at ya Hadi Puri g E pant ripia a gt tcp AE Chalengrg Md amenat a pe i par am eee aan S N i x pot ig me Simi a Ken e 9 b e Tarain a a w P ER A 2 EF A far agnen niet DECENT icy Grian 27 b a bed a _ wo a 8 Ln o gt GST Gene Fusion System Gel Filtration Handbook Principles and Methods 18 1157 58 18 1022 18 Affinity Chromatography Recombinant Protein Purification Handbook Principles and Methods Principles and Methods 18 1022 29 18 1142 75 Antibody Purification Hydrophobic Interaction and Handbook Reversed Phase Chromatography 18 1037 46 Principles and Methods lon Exchange Chromatography and Chromatofocusing Principles and Methods 11 0004 21 Cell Separation Media Methodology and Applications 18 1115 69 Purifying Challenging Proteins Principles and Methods 28 9095 31 Isolation of mononuclear cells Methodology and Applications 18 1152 69 High throughput Process Development with PreDictor Plates Principles and Methods 28 9403 58 AKTA Laboratory scale Chromatography Systems Instrument Management Handbook 29 0108 31 11 0012 69 2 D Electrophoresis using im
30. NG secsi serii san enian O 59 Tubing material and GIMENSIONS ccccccsessssesssescsseesssssssecssecsssecssecssucsssscssscssuscssecessecssecssssssuecsseessusessecssecesseesseeeses 59 eaae Ee eE aa E A E AE E A A E EIA V T A EES T ERE SE 60 POC P E U aE E E E E EEN EEEE NE ENTE EE N ANA 60 29 0108 31 AA 3 Appendix 3 Determination of delay VOIUIMOES siccsec cecssesesesesarctvececcastesedsacsosevesaneeseatececseaucendsachesensecsctesencessaetaness 61 Theoretical determination preferred method ccesssssssssecssecsssecsssscsssecsssecsssesssuessssecssecssccsssecsssecsseeesses 61 ze sales almeo SPEER ERR SEERE RESEN EEN ERE EET even nna ee eta EDER na castors nade 62 Appendix 4 Troubleshooting column ISSUES 422 45 ore renere reder 65 Appendix 5 Troubleshooting fraction collection esesoesoesesoeseseesossesosseseesossesoeseseesossesoesesossessesossesesseesesess 67 Appendix 6 Introducing laboratory scale KTA SYStEMS cccscssescssssessescscssestssessssssessssessssesssssssssssssessessees 69 Appendix 7 Principles and standard conditions for different purification techniques 73 Vaal avis gels ge gg erre eve re aly AG mamererr treme errr enter Mrmr a yt entire tr Mnnerttnrtr Tete terry 73 lon exchange chromatography WEA asensin 74 Hydrophobic interaction chromatography HiChina 75 Gel filtration GF or Size exclusion chromatography SEC ov eeceescseessssessssessssescssesssseesssesssseecsseessseeesseeen IR Reversed ph
31. Sample loop PEEK 100 ul Sample loop PEEK 500 ul Sample loop PEEK 2 0 ml Sample loop PEEK 5 0 ml Sample loop for AKTAxpress ETFE 10 0 ml Loop extension kit for AKTAxpress ETFE 10 0 ml Superloop 10 ml 1 16 fittings Superloop 50 ml 1 16 fittings Superloop 150 ml M6 fittings Solvent resistant O rings to Superloop 10 and 50 ml O ring to movable seal 11 3 x 2 4 mm KAL O ring to movable seal 11 3 x 2 4 mm FFPM FFKM Mixers Mixer M 925 Mixing chamber 90 ul Mixer M 925 Mixing chamber 200 ul Mixer M 925 Mixing chamber 0 6 ml Mixer M 925 Mixing chamber 2 ml Mixer M 925 Mixing chamber 5 ml Mixer M 925 Mixing chamber 12 ml AKTA avant Mixer chamber 0 6 ml AKTA avant Mixer chamber 1 4 ml AKTA avant Mixer chamber 5 ml AKTA avant Mixer chamber 15 ml UV Vis flow cells Flow cell 2 mm for UPC 900 Flow cell 5 mm for UPC 900 Flow cell 2 mm for UV 900 Flow cell 3 mm for UV 900 Flow cell 10 mm for UV 900 UV flow cell 0 5 mm for U9 UV flow cell 2 mm for U9 UV flow cell 10 mm for U9 To determine the exact UV Vis flow cell lengths UV 900 cell 1 mm calibration kit UV 900 and UPC 900 cell 2 mm calibration kit UV 900 cell and UPC 900 5 mm calibration kit UV 900 cell 10 mm calibration kit UV 900 cell calibration XL file 86 29 0108 31 AA Quantity pack size 1 each 1 each ji e e e OP PP PP BP he e e e e PP PP PP PB e e e re PP PP BE Code number 18 0404 01 18 5897 01 18 1161 24 18 1114 01 18 11
32. Vis flow cell to the fraction collector 2 Replace the inlet tubing of the UV Vis flow cell with a Luer adapter 3 Filla syringe with at least 5 ml of water and inject it into the flow cell to make sure that the flow path to the fraction collector tubing tip is filled with water 4 Filla syringe with at least 20 ml of air because of compression and collect the water that is replaced while injecting the air 5 Determine the delay volume by weighing the water 6 Repeat at least two times for calculation of a mean value eT 1 mg of water is equal to 1 ul at 4 C is temperature dependent 29 0108 31 AA 63 64 29 0108 31 AA Appendix 4 Troubleshooting column issues If high system pressure is due to the column the following procedure Fig A4 1 can help to resolve the problem High column back pressure Decreased capacity Gap between adapter and medium Adjust adapter Is resolution OK Pressure still high Has resolution improved Clean column Clean column Pressure still high Capacity still decreased Column is OK New column is needed Column is OK New column is needed Fig A4 1 Decision tree for dealing with column issues CP For information about how to clean the column see the column s instruction sheet It is not unusual that the system back pressure increases for a short time at the start of the cleaning process 29 0108 31AA 65 66 29 0108 31 AA Appendix 5 Troublesh
33. after purging using buffer use 100 methanol instead Make sure the pump contains water then use a syringe to draw 100 methanol into the pump and let it run at 10 of the system s maximum flow rate until the pressure curve disturbances Fig 5 1 disappear To remove the methanol stop the pump and switch to water Make sure that no air bubbles are introduced Run the pump at a flow rate of 1 ml min for 5 min to wash away the methanol Then purge the pump again using a syringe Wi If the pump gives an inaccurate flow rate even after removal of air bubbles contact your GE Healthcare Life Sciences Service representative Air bubble origin Air bubbles may be released from the liquid when the pressure drops When the pump is running the pressure inside the pump will be higher than the atmospheric pressure during liquid delivery When the pump is in the suction phase the pressure will be below atmospheric pressure and air bubbles may be released To avoid this situation place the bottles above 24 29 0108 31 AA or at least at the same height as the pumps NEVER place the liquids below the pump unless the user manual states that it is possible some systems have a pump design that allows such placement Because solutions are always in contact with air it is recommended to degas them prior to use Er Pay special attention to liquids stored at low temperature that will be used at room temperature More air is dissolved at lower temperatur
34. an the column please see the column s instruction sheet 29 0108 31 AA 37 High back pressure Bypass or remove column Pressure still high Locate system blockage DA connectors and tubing Clean column Fig 7 6 How to troubleshoot high back pressure To find the flow path blockage start the pump at a flow rate that will keep the pressure low enough so that the alarm is not triggered Take note of the measured system pressure Then starting from the fraction collector loosen the first connector If there is no change in pressure tighten it again and move to the next connector toward the pump Loosen this connector check for any change in pressure tighten etc until the one that releases the pressure has been located This is where the flow path blockage is Very often the blockage is caused by obstructed tubing If this is the case replace as needed In less severe cases perform cleaning in place of the system flow path In some cases a system calibration is needed to reset the pressure sensors This should be performed without any column inline For details see the specific system user manual Viscous samples and solutions The system back pressure is affected by the viscosity of a liquid Some salts high salt concentrations and low temperatures increase viscosity of the liquids as well as mixtures between organic and aqueous solutions Crude samples e g cell lysates are often highly viscous Figure
35. ase chromatography IR PC sisisi aiana annakaan a raean anceis 78 Appendix 8 Columns for AKTA SyS teM excess cece tects ce aTa 79 Related itera Hg RENEE Taise rE EEEN E EE E E ER 84 Ordering information sesseseeseseesesessesseseesesoesessesessesessessesossesossessesessesessesecsessesesseseesessesesseseesesseses 85 4 29 0108 31 AA Chapter 1 Introduction This handbook AKTA Laboratory scale Chromatography Systems is focused on liquid chromatography systems used for protein purification at research laboratory scale Beginners can use the handbook to obtain an overview of how purification systems work and to learn about important considerations for achieving successful results Experienced system users will also find valuable and detailed information on different hardware modules A chromatography system should be used when reproducible results are important and when manual purification becomes too time consuming and inefficient Systems provide more control than manual purification because of the ability to automatically control the flow rate and monitor the progress of the purification as well as to make controlled gradients and automatically collect fractions Systems can perform automatic simple step gradient elution as well as high resolution separations using accurately controlled linear gradient elution This handbook addresses different aspects of AKTA chromatography systems such as the effect of system peak broadening on res
36. ations and amounts Simply enter the extinction coefficient for the protein and the path length of the UV Vis flow cell used and the software will calculate concentration and amount based on the UV absorbance data at 280 nm To obtain highly accurate results two criteria are very important 1 The UV Vis absorbance signal must be within the linear range of the UV Vis detector 2 The exact UV Vis flow cell path length should be used in the calculation For AKTA avant and AKTAxpress the exact path length has been predetermined and is included in the software calculations For other systems the exact path length of each individual system needs to be determined experimentally by measuring the absorbance of one or several solutions with known absorbance see ordering information to determine which UV Vis flow cell calibration kit to use When using a UV Vis flow cell calibration kit the exact path length is determined according to Lambert Beer s law see below The exact path length must then be manually entered into the system setting of the software Which UV Vis flow cell to use According to Lambert Beer s law the relationship between absorbance and concentration can be described as A sxbxc where A absorbance e extinction coefficient b cell path length and c concentration To get the absorbance signal within the linear absorbance range different cell lengths can be chosen described in Table 8 2 42 29 0108 31 AA
37. ble ranging from a few microliters up to 10 ml When filling the loop it is important to consider the fluid dynamics as explained in Figures 4 1 and 4 2 The flow rate of the sample entering the loop will be higher toward the middle compared with close to the walls of the tubing this creates a parabolic flow profile in the loop Fig 4 1 Thus in order to fill the loop completely a larger volume needs to be loaded which is explained in the next section and illustrated in Figure 4 2 Fluid dynamics This is a common view of a x LL SE a Maat it is not correct E i This is the way the sample Fig 4 1 Fluid dynamics through tubing 29 0108 31 AA 15 Filling a tubing loop There are two ways to fill a tubing loop partial filling and overfilling see Figure 4 2 With partial filling there is no sample loss but reproducibility is lower if the same procedure Is repeated With overfilling a better volume accuracy is obtained For a complete fill load three to five times the loop volume to obtain high accuracy The needed volume depends on the loop dimensions length and i d Generally the larger the loop volume the less overfill is needed Cr For a partially filled sample loop do not fill more than half of the total loop volume If more is applied a portion of the sample may pass through and out of the loop as shown in Figure 4 2 Partial filling minimal sample loss 50 ul load ZF 60 ul load ee 100 ul load E 200
38. broadening Narrow peak from column Final peak broadened Wide peak from column Final peak similar System contribution peak broadening Fig 3 4 Smaller peaks are more affected than larger peaks same system in both cases It is important to know that the effect of the system contribution on the peak width is nonlinear as can be seen in Figure 3 5 This graph shows the contribution from a typical laboratory scale system with 0 5 mm i d tubing In this example the system contribution has little effect on peaks that are larger broader than 3 to 4 ml On the other hand if the peaks are less than 1 ml the system contribution becomes significant 29 0108 31 AA 13 A B mAU Peak broadening 0 2 4 6 8 10 i i Peak width ml Peak width ml Fig 3 5 A The relative system contribution depends on the peak width B Peak width is in this example determined at the half height of the peak Besides tubing diameter peak broadening is also affected by tubing length and the dimensions of valves and flow cells It is therefore important to determine if the column to be used is Suitable for the system Do not run smaller columns than recommended for the system see the selection guide in Related literature If a smaller column is needed consider minimizing the system volume by modifying the system for example by changing tubing to a smaller i d and or excluding components from the flow path to minimize t
39. column This is called the gradient delay volume In the chromatogram the actual gradient will be delayed compared with the programmed gradient B curve as seen in Figure 6 2 The shape of the gradient is also affected by mixer effects Make sure that the conductivity reaches the programmed gradient value by the end of the run by continuing to run at the final elution conditions until the target value is reached The volume to add needs to be determined experimentally CP In AKTA design systems the default mixer effect has been included in the so called gradient delay volume of the system UNICORN software B Gradient delay volume in this 100 example corresponding to the volume from the mixer to the conductivity monitor 80 N 60 Added volume to compensate for 40 both the gradient delay volume and the mixer effect 20 0 5 10 15 20 25 ml Fig 6 2 Actual gradient blue compared with programmed gradient green Maintaining a constant gradient slope when changing column size Gradient length is often defined in terms of X column volumes CV Maintaining a constant gradient will ensure that the slope of the gradient will not change when scaling up or down For example if the gradient length is 10 CV for a 1 ml column this corresponds to 10 ml and for a 10 ml column this corresponds to 100 ml 30 29 0108 31 AA The gradient delay volume is independent of the column used it will be the same as long as the columns are
40. d fil it by pouring buffer into the glass cylinder as shown in Figure 4 8 Reassemble the top piece and make sure that no air bubbles are trapped inside as shown in Figure 4 9 Fig 4 8 Moveable seal should be in bottom position left Buffer is poured into the cylinder right Fig 4 9 How to mount the top piece left to reassemble the Superloop right ET To apply sample at a preferred temperature allow water of the desired temperature to circulate in the outer shield of the Superloop 29 0108 31 AA 19 How to connect a Superloop to the injection valve A Superloop is connected to the same ports as a tubing loop To find out which port should be connected to the bottom of the Superloop connect a syringe to the injection valve and turn the valve to position load Inject liquid and check where it comes out as shown in Figure 4 10 Connect the tubing from the bottom of the Superloop to this port Be careful not to introduce air bubbles in the sample compartment Fig 4 10 Check where to connect the bottom of the Superloop by injecting buffer into the injection valve The top of the Superloop should be connected to the port where liquid from the pump is directed to during injection mode If unsure start a flow rate and change to position inject Where the liquid comes out Is the port to which the Superloop top should be attached How to clean a Superloop A Superloop ca
41. described in Chapter 5 Then immerse the sample inlet in the sample container and use the pump to apply the sample Fig 4 12A Apply the sample to the column until the air sensor detects air bubbles Fig 4 12B After air has been detected the sample valve switches to a buffer inlet allowing the remaining sample from the sample valve to the injection valve to be applied onto the column Fig 4 12C A Sample application B Switch from sample application to buffer application after detection of air Sample pump Injection valve elle OBO i Air sensor Sample Air sensor Sample Inlet valve inlet valve Injection valve Waste Buffer ig Buffer Sample C Continued buffer application to deliver sample to column Injection valve Air sensor Sample inlet valve Sample ia Buffer Fig 4 12 Sample application example where a sample pump and air sensor are used to apply sample In the figure sample is colored green and buffer blue Refer to text in images for descriptions of A B and C panels CP In some systems preprogrammed methods are available that can be used to prime the sample pump and air sensor with sample 22 29 0108 31 AA Chapter 5 Liquid delivery and pumps This chapter describes the high performance pumps used in laboratory scale AKTA systems Accurate flow rate reproducible liquid delivery and low pulsation are essential for an optimal purification result Because the
42. e molecules Unwanted 2 4 CV sample injection i RR so volume N J 4CV elute equilibration re equilibration 5 CV Column volumes CV Fig A7 4 Step elution Blue line absorbance red line conductivity salt concentration Further information Handbooks Strategies for Protein Purification 28 9833 31 Purifying Challenging Proteins Principles and Methods 28 9095 31 lon Exchange Chromatography and Chromatofocusing Principles and Methods 11 0004 21 Hydrophobic interaction chromatography HIC HIC media separate proteins with differences in hydrophobicity The technique is well suited for the capture or intermediate steps in a purification protocol Separation is based on the reversible interaction between a protein and the hydrophobic surface of a chromatography medium This interaction is enhanced by high ionic strength buffer which makes HIC an excellent next step after precipitation with ammonium sulfate or elution in high salt during IEX Samples in high ionic strength solution e g 1 5 M ammonium sulfate bind as they are loaded onto a column Conditions are then altered so that the bound substances are eluted differentially Elution is usually performed by decreases in salt concentration Fig A7 5 Changes are made stepwise or with a continuous decreasing salt gradient Most commonly samples are eluted with a decreasing gradient of ammonium sulfate Target proteins are concentrated during binding and collect
43. e therefore allow time for the liquids to adjust to room temperature before use Air bubbles might also be generated when switching between aqueous and organic solvents in the pump Due to different capacities to dissolve air bubbles may be released when two liquids are mixed To avoid this situation when switching between different liquids direct the flow path to the waste position of the injection valve and pump at a fairly high flow rate gt 50 of the system s maximum flow rate for some time Description of the pump and rinsing system Functionality of the pump Due to the design of the KTA pumps they are virtually pulse free and do not introduce sheer forces that may disrupt or break down proteins mechanically The pumps can also operate at both high and low pressure which makes them convenient for various conditions encountered in protein purification To generate the set flow rate during operation the pumps use an algorithm to control how the pistons move As long as there are no air bubbles in the pump the flow rate accuracy will be high with an error rate typically lt 2 Most KTA pumps consist of two pump heads Fig 5 3 The individual heads are identical but operate in opposite phase to each other using individual stepper motors The two pistons and pump heads work alternately to provide continuous low pulsation liquid delivery Piston Pump head Stepper motor Fig 5 3 Schematic view of the P 901 P 903 P
44. e drop sensor If the light path is blocked an error message will be received during fractionation CP For fraction collectors that have an accumulator for spillage free fractionation remember to also add an accumulator wash in the system cleaning method 54 29 0108 31 AA Appendices Appendix 1 System components in laboratory scale AKTA systems Table A1 1 highlights some system components and the AKTA system they relate to For each component information such as pressure limit flow rate range internal volume etc is noted Table A1 1 Parameters for AKTA system components see page 58 for footnotes Stroke volume total internal Pumps Flow rate range volume Max pressure Used with AKTAprime 0 1 50 ml min 200 ul 1 ml 1 MPa AKTAprime plus plus pump 10 bar 145 psi P 901 0 01 100 ml min 286 ul 1 4 ml 10 MPa AKTApurifier UPC 100 100 bar 1450 psi AKTApurifier 100 AKTAexplorer 100 P 903 0 001 10 ml min 36 ul 0 6 ml 25 MPa AKTApurifier UPC 10 AKTApurifier 250 bar 3625 psi 10 AKTAexplorer 10 P 905 0 001 2 ml min 36 ul 0 6 ml 35 MPa AKTAmicro 350 bar 5075 psi P 920 0 01 20 ml min 10 ml 10 ml 5 MPa AKTAFPLCTM 50 bar 725 psi P 960 0 1 50 ml min 200 ul 1 ml 2 MPa Sample pump to AKTApurifier UPC 20 bar 290 psi AKTApurifier AKTAexplorer AKTAxpress 0 1 65 ml min 286ul 14ml 3 MPa KTAxpress pump 30 bar 435 psi P9 0 001 25 ml min 54ul 0 55ml 20 MPa KTA avant 25 200 bar 2900 psi P9 S 0 01 25 ml min 286 ul
45. e is applied under conditions that favor specific binding to the ligand Unbound material is washed away and bound target protein is recovered by changing conditions to those favoring elution Elution is performed specifically using a competitive ligand or nonspecifically by changing the pH ionic strength or polarity Samples are concentrated during binding and the target protein is collected in purified and concentrated form The key stages in an AC separation are shown in Figure A7 1 AC is also used to remove specific contaminants for example Benzamidine Sepharose 4 Fast Flow removes serine proteases adsorption of wash alte equilibration Sample and__ away bound gt regeneration of medium wash of unbound protein s unbound material material begin sample change to Absorbance application elution buffer gt 1 lt x CV 2 5CV CV 2 3 CV Column volumes CV Fig A7 1 Typical affinity purification Further information Handbooks Strategies for Protein Purification 28 9833 31 Purifying Challenging Proteins Principles and Methods 28 9095 31 Affinity Chromatography Principles and Methods 18 1022 29 Antibody Purification Principles and Methods 18 1037 46 29 0108 31AA 73 lon exchange chromatography IEX IEX media separate proteins based on differences in surface charge generating high resolution separations with high sample loading capacity The separation is based on the reversible interactio
46. ed in a purified and concentrated form Other elution procedures include reducing eluent polarity ethylene glycol gradient up to 50 adding chaotropic species urea guanidine hydrochloride or detergents changing pH or temperature 29 0108 31 AA 75 sample _ gradient equilibration gt salt free wash gt re equilibration application elution 1M g D tightly bound molecules E unbound molecules elute elute under salt free conditions before gradient begins O E E 10 15 CV 4 CV PR 5 CV O Column volumes CV Fig A7 5 Typical HIC gradient elution Blue line absorbance red line conductivity salt concentration Method development in priority order 1 The hydrophobic behavior of a protein is difficult to predict and binding conditions must be studied carefully Use HiTrap HIC Selection Kit or RESOURCE HIC Test Kit to select the chromatography medium that gives optimal binding and elution over the required range of salt concentration For proteins with unknown hydrophobic properties begin with a starting buffer containing for example 1 M to 1 5 M ammonium sulfate Knowledge of the solubility of protein in the binding buffer is important because high concentrations of for example ammonium sulfate may precipitate proteins 2 Select a gradient that gives acceptable resolution As a starting point a linear gradient from O to 100 B of 10 to 20 columns volumes is recommended 3 Select the highest f
47. either warm Decon 90 solution approximately 60 C or leave the flow cell with the detergent overnight Table 10 3 lists other solutions recommended for cleaning the UV Vis flow cell Table 10 3 Solutions for cleaning absorbance detector flow cell To remove Use Buffers salts and detergents Water Proteins 0 5 to 1 M NaOH for 15 min then flush with water Lipids and other hydrophobic components 30 to 100 isopropanol then flush with water 29 0108 31 AA 53 pH electrode The pH electrode is one of the most sensitive components in the system Recommended solutions for cleaning the pH electrode are listed in Table 10 4 Table 10 4 Solutions for cleaning pH electrode To remove Use Salt deposits Alternating 0 1 M HCl and 0 1 M NaOH Lipid deposits Detergent or organic solvent Protein deposits 1 pepsin in 0 1 M HCI Remove thoroughly afterwards After the electrode has been cleaned it needs to be restored and calibrated See the user manual for guidance Fraction collector It is important to keep both the exterior of the fraction collector and the drop synch photo cell clean Wipe off spillage immediately and use a cloth and water or mild cleaning agent to clean the exterior The drop sync photo cell should be wiped carefully with a damp cloth Usually the tube rack can be disassembled for cleaning Wi After cleaning check that the fractionation tubing is positioned correctly and that it does not block the light path of th
48. em flow path AKTA chromatography systems measure this pressure at the system pump Fig 7 3 Some systems have additional pressure sensors before and after the column Fig 7 3 p1 and p2 that allow calculation of the pressure drop Ap over the column This gives useful information about the condition of the packed bed A Ap that is too high for a newly packed column indicates that packing can be improved With time as the column collects impurities due to nonspecific adsorption the Ap will increase To regain optimal column conditions perform cleaning in place CIP and or change the column top filter Pressure generated before the column 22ers System pressure is measured here X Tubing P umps af Ap TA Column Buffers solutions UV Vis absorbance D ma Fraction collector pH Conductivity Cd Pressure generated after the column Fig 7 3 System pressure generated by the complete system flow path is measured in AKTA chromatography systems at the system pump Ap p1 pressure generated after and by the column itself p2 pressure generated after the column The maximum pressure over the packed bed Ap is an approximate limit It is dependent on the characteristics of the chromatography medium and on sample liquid viscosity The measured value also includes the pressure generated by the column tubing For many columns the recommended flow rate is a better guideline for protecting the packed bed 29
49. en as disturbances in conductivity curve A current is applied across the conductivity cell and the electrical resistance between the electrodes is measured and used to calculate the conductivity in the eluent The conductivity is linear only to a salt concentration of approximately 0 3 M It is therefore important to measure the conductivity of the solution that is used rather than calculating it In a salt gradient a decrease in linearity will be seen with increasing salt concentration Figure 8 2 Conductivity a linear relation actual relation Salt concentration Fig 8 2 Illustrated relationship between conductivity and salt concentration Conductivity measurements are temperature dependent The conductivity signal will increase with temperature according to C C leo t cal where C the measured conductivity C conductivity at reference temperature and At the difference between reference temperature and actual temperature The constant a is concentration and salt dependent but 0 02 is a good mean value for many salts In all AKTA systems except for AKTAxpress a temperature sensor is mounted within the conductivity cell to allow temperature compensation The compensated conductivity value is displayed which means that conductivity curves generated at different temperatures can be compared 44 29 0108 31 AA Monitoring pH For most KTA systems a pH electrode can be connected at the low pressure s
50. epharose Sephacryl Superdex prep grade Superose lon exchange Capto Capto ImpRes Sepharose Fast Flow Sepharose High Performance Sepharose XL SOURCE Affinity Capto Capto Blue Sepharose 6B 4B CL 4B Sepharose Fast Flow Sepharose High Performance MabSelect MabSelect Xtra MabSelect SuRe MabSelect SuRe LX Reversed phase SOURCE Hydrophobic interaction Capto Capto Phenyl Capto Butyl Sepharose Fast Flow Sepharose High Performance SOURCE Systems AKTAmicro AKTAprime plus AKTApurifier 10 AKTApurifier 100 AKTA avant 25 AKTA avant 150 AKTAxpress e Recommended combination o Can technically be used but not an optimal combination Not recommended or not applicable 1 Not recommended for XK 50 Tricorn 02 Recommended column XK el ol 02 2 For optimal performance use prepacked columns where purification parameters are predefined HiScale For more information visit www gelifesciences com protein purification www gelifesciences com bioprocess or www gelifesciences com purification_techsupport 29 0108 31 AA 83 Related literature Handbooks GST Gene Fusion System Affinity Chromatography Principles and Methods Antibody Purification Principles and Methods Purifying Challenging Proteins Protein Sample Preparation Strategies for Protein Purification Recombinant Protein Purification Principles and Methods Gel Filtration Principles and Methods Hydrophobic Interaction and Reve
51. er holder included in 50 ul 20 MPa 200 bar 2900 psi KTA avant mixer M9 Absorbance detector Cell volume flow cells Total volume Max pressure Used with 2 mm for UPC 900 2 ul 30 ul 4 MPa 40 bar 580 psi AKTAprime plus AKTAretc AKTApurifier UPC AKTAxpress 5 mm for UPC 900 6 ul 20 ul 4 MPa 40 bar 580 psi AKTAprime plus AKTAFPLC AKTApurifier UPC 2 mm for UV 900 2 ul 7 ul 2 MPa 20 bar 290 psi AKTApurifier AKTAexplorer 3 mm for UV 900 0 7 ul 3 ul 2 MPa 20 bar 290 psi AKTAmicro 10 mm for UV 900 8 ul 13 ul 2 MPa 20 bar 290 psi AKTApurifier AKTAexplorer 0 5mm for KTA avant 1 pl 10 ul 2 MPa 20 bar 290 psi AKTA avant 2 mm for AKTA avant 2 ul 11 ul 2 MPa 20 bar 290 psi AKTA avant 10mm for KTA avant 8 ul 12 ul 2 MPa 20 bar 290 psi AKTA avant Conductivity flow cells Internal volume Max pressure Used with Flow cell 24 ul 5 MPa 50 bar 725 psi AKTAprime plus AKTAFPLC AKTApurifier UPC AKTApurifier AKTAexplorer AKTAxpress Flow cell 22 ul 5 MPa 50 bar 725 psi AKTA avant Flow cell 2 ul 35 MPa 350 bar 5075 psi AKTAmMicro pH flow cells Internal volume Max pressure Used with Standard cell 88 ul 0 5 MPa 5 bar 73 psi AKTAprime plus AKTArpic AKTA purifier UPC AKTApurifier AKTAexplorer V9 pH 76 ul 0 5 MPa 5 bar 73 psi AKTA avant 25 V9H pH 76 ul 0 5 MPa 5 bar 73 psi AKTA avant 150 56 29 0108 31 AA Flow restrictors FR 902 FR 904 Air sensors Air 912 N5 Air 925 N Air 915 N
52. ermining the delay volume of a system The easiest and recommended method is to perform a theoretical determination Theoretical determination preferred method A theoretical determination is performed in three steps 1 Identify all components in the system flow path that contribute to the delay volume of interest 2 Determine the internal volumes of all parts See Appendix 1 with respect to hardware components and Appendix 2 with respect to tubing 3 To obtain the total delay volume sum up all the volumes Example Determination of fractionation delay volume that is components between the UV Vis absorbance detector and the fraction collector In this example an AKTApurifier UPC 10 is used See Figure A3 1 for system parts UV Vis absorbance Il SEER 7 1 fis nil Fraction collector Delay volume The peak is detected by the The peak reaches the absorbance detector at time To fraction collector at time T Fig A3 1 Identification of parts of the system used in example 1 Identify all system parts In this example the following parts were identified UV Vis absorbance detector with 2mm cell tubing conductivity cell tubing pH cell tubing outlet valve tubing fraction collector Frac 950 2 Create a table and fill in all the internal volumes of each system component Measure tubing lengths with a ruler and use Appendices 1 and 2 or the system manual to find out all the components internal volum
53. ersatile FPLC purification of proteins and peptides Fig A6 4 There are four core KTApurifier systems that can be combined with automation kits into an advanced setup to reduce time consuming steps increase productivity or meet new purification challenges Besides the core AKTApurifier two additional systems KTApurifier 10 plus and KTApurifier 100 plus give further automation possibilities They are both preassembled with convenient automation kits and geared for media screening and optimization For purification of proteins at microgram and milligram scale choose KTApurifier 10 10 plus or UPC 10 systems Purification of larger gram scale quantities of protein is achieved with AKTApurifier 100 100 plus or UPC 100 systems Fig A6 6 AKTA avant system AKTAxpress is designed for unattended multistep purification of tagged proteins and antibodies Fig A6 5 Up to 12 AKTAxpress systems can be controlled from one computer allowing parallel purification of up to 48 different samples Due to its small footprint two systems can fit in a cold cabinet The purification protocols consist of up to four purification Steps A typical four step protocol begins with AC followed by desalting IEX and GF In addition automatic on column or off column tag removal steps can be integrated in the purification protocols Extended and automated washing procedures enable processing of a larger number of samples with minimal risk of cros
54. es See Table A3 1 for this example 29 0108 31 AA 61 Table A3 1 Data for determining delay volume in example System part Details Internal volume Comment UPC 900 UV cell 2mm cell s total volume 30 ul 2 15 ul From Appendix 1 Blue tubing UV Vis absorbance i d 0 25 mm 8 cm 3 9 ul Formula in Appendix 2 detector Cond cell Cond cell Standard 14 ul 14 ul From Appendix 1 Blue tubing Cond cell pH cell i d 0 25 mm 10 cm 4 9 ul Formula in Appendix 2 pH cell 88 ul From Appendix 1 Blue tubing pH cell Outlet i d 0 25 mm 12 cm 5 9 ul Formula in Appendix 2 valve Outlet valve PV 908 7 ul From Appendix 1 Orange tubing Outlet valve gt i d 0 5 mm 30 cm 58 9 ul Formula in Appendix 2 Fraction collector Accumulator was bypassed Total volume 198 ul Use half of the total internal volume of the UV Vis absorbance cell 3 The total volume is then used to update the delay volume in UNICORN System Control CP If a fraction collector accumulator is used remember to also include the volume of the tubing to and from the accumulator Experimental methods Experimental determination is also possible Two methods are described below Measuring delay volume using the UV Vis absorbance detector To determine the delay volume experimentally two volumes must be measured These are V1 and V2 V1 volume between injection valve and UV Vis absorbance detector V2 volume between injection valve and fractionation tubing tip 1
55. f possible Use imidazole of high purity DTE a reducing agent oxidizes over time Use only freshly made solutions Noisy and insensitive UV Vis absorbance measurements The most common cause of noisy and insensitive UV Vis absorbance curves is a dirty flow cell Clean the flow cell as described in Chapter 10 The problem can also be due to air bubbles within the flow cell See the discussion on flow restrictors in Chapter 7 for more details An aging UV lamp With time the light intensity of the UV Vis absorbance detector lamp will decrease When a low intensity warning Is given it is time to replace the lamp Note The displayed UV Vis absorbance signal will be the correct value as long as no intensity warning is issued This is possible because the detector uses a reference signal against which the measured UV Vis absorbance is normalized 29 0108 31 AA 43 Monitoring conductivity The conductivity monitor is used to detect changes in salt concentration and other charged molecules during a chromatographic run It can be used to gather a variety of information as described in Table 8 4 Table 8 4 Examples of information gathered from conductivity monitoring Used during Used for Equilibration Stable signal indicates that column is equilibrated Sample application and wash Detection of salt peaks Gradient elution Monitoring gradient formation Desalting Detection of salt peaks System troubleshooting Erroneous flow rate se
56. for micropreparative liquid chromatography applications and for rapid purity analysis in method development and protein characterization Fig A6 7 Microscale purifications can be performed starting with samples containing extremely small amounts of target protein using microbore to analytical scale columns The highest possible sample recovery and stability are obtained when the complete flow path is manufactured from inert and biocompatible materials and assembled to give minimal peak broadening The pump design gives a flexible flow rate range with low pulsation and a broad pressure range enabling high as well as low pressure separations Appendix 7 Principles and standard conditions for different purification techniques Affinity chromatography AC AC media separate proteins on the basis of a reversible interaction between a protein or a group of proteins and a specific ligand attached to a chromatographic matrix The technique is well suited for a capture or as an intermediate purification step and can be used whenever a suitable ligand is available for the protein s of interest AC offers high selectivity and usually high capacity It is frequently used as the first step capture step of a two step purification protocol followed by a second chromatographic step polishing step to remove remaining impurities The target protein s is are specifically and reversibly bound by a complementary binding substance ligand The sampl
57. ging Tricorn columns are available with an i d of 5 mm with lengths of 20 50 100 150 and 200 mm and with an i d of 10 mm and in lengths of 20 50 100 150 200 300 and 600 mm The maximum pressure is 100 bar for the 5 mm i d column and 50 bar for the 10 mm i d column 29 0108 31AA 81 T F he a4 Fig A8 9 XK columns as Fig A8 10 HiScale columns 82 29 0108 31 AA XK columns are specified to run most chromatography media including Superdex prep grade and Sepharose High Performance Fig A8 9 They are jacketed and available as 16 26 and 50 mm i d columns XK16 XK26 and XK50 with lengths from 20 to 100 cm The maximum pressure is 5 bar for XK 16 and XK 26 columns and 3 bar for XK 50 columns Prepacked XK columns go under the name HiLoad HiScale columns are designed for preparative laboratory scale purification and for process development using standard chromatography media Fig A8 10 HiScale columns are available with i d of 16 26 and 50 mm and lengths of up to 20 or 40 cm The maximum pressure is 20 bar The QuickLock mechanism of the adapter shaft facilitates rapid and easy movement of the adapter simplifying adjustments as well as disassembly and cleaning Turning the column end caps enables controlled axial compression of the medium bed which is suitable during packing of rigid media Table A8 1 Empty column and chromatography media guide Loose media Gel filtration Sephadex S
58. he collected protein peaks peak fractionation can be used The UV Vis detector is then used to determine when to start and stop peak fractionation as shown in Figure 9 1B Straight fractionation and peak fractionation can also be combined during a run 29 0108 31 AA 47 A Straight fixed fractionation Absorbance UDE B Peak fractionation Absorbance ml oUo UU Ud Fig 9 1 Straight fractionation A Peak fractionation B Fractionation delay volume The fractionation delay volume is the volume between the UV Vis detector s flow cell and the fraction collector It is important that the correct delay volume is entered in the software The defined delay volume will be used by the system to calculate the time T which is when the peak reaches the fraction collector T is used to synchronize the fractionation marks in the chromatogram with the tube switch of the fraction collector see Fig 9 2 At the start of the fraction collection the delay volume is directed to waste or the first fractionation tube depending on which system is used Valve UV Vis lt p gt A Conductivity pH Fraction collector Delay volume The time when a peak is detected The peak reaches the fraction by the UV Vis detector is called Ty collector at time T Fig 9 2 T is the time when the fraction collector moves in order to collect the fractions to match what was detected in the UV Vis detector T T Delay volume flow rate
59. he fraction collector in the user manual If problem cannot be solved contact Service To avoid waste blockage make sure that the waste tubing is not bent curved or in touch with the bottom of the waste container The scanning in the AKTA avant fraction collector reads only number and type of racks If no tubes plates are present the system will still run the method resulting in spoiled samples 68 29 0108 31 AA Appendix 6 Introducing laboratory scale AKTA systems AKTA systems are designed for protein purification ranges from micrograms to kilograms of target protein All systems are controlled by UNICORN software with the exception of AKTAprime plus which is monitored by PrimeView software UNICORN has the benefits of one common control platform and user interface for all scales of operation in chromatography and filtration Research scale AKTA systems are briefly described on the following pages in Figures A6 1 to A6 7 Table A6 1 lists the standard AKTA system configurations Table A6 1 Ways of working with standard AKTA systems Way of working Scale Laboratory scale Process development Regulatory demands System control and data handling for regulatory requirements Type of work Method development Generic methods Micropreparative and analysis Automation Buffer preparation function pH scouting Media or column scouting Multistep purification Software UNICORN PrimeView e Recommended e Optional
60. he system volume CP If making hardware changes that will affect the system volume remember to update relevant delay volumes in the software For more information refer to Chapter 9 and Appendix 3 Effect of sample volume on resolution Sample volume does not affect resolution in chromatography techniques involving adsorption of the target protein onto the column Examples of binding techniques are affinity chromatography AC ion exchange chromatography IEX and hydrophobic interaction chromatography HIC Gel filtration GF however is a nonbinding chromatography technique and a sample zone is therefore broadened during passage through the GF column As a result the sample gets diluted and the resolution will decrease with increasing sample volume Figure 3 6 shows a GF example in which different volumes of a sample were applied to a Superdex 200 10 300 GL column In the first case 250 ul of sample was applied which correspond to 1 of the column volume In the second case 1000 ul of sample was applied which corresponds to 4 of the column volume As can be seen the resolution was higher when a smaller sample volume was used 250 ul 1 0 ml min 76 cm h 1000 ul 1 0 ml min 76 cm h A280 A280 0 15 0 15 0 10 0 10 0 05 0 05 0 0 0 5 10 15 20 25 Time min 0 5 10 15 20 25 Time min Fig 3 6 Effect of sample volume on resolution in GF Sample volume as of media volume Column Superdex 200 10 300 GL Aa The loaded samp
61. ide i e after the UV Vis absorbance detector To receive accurate measurements it is important to calibrate the pH electrode pH electrodes are sensitive to for example 20 ethanol and it is therefore important to store them in appropriate storage solutions see the pH detector s user manual If a FR 904 flow restrictor is used make sure that the pH sensor is placed after the flow restrictor because it cannot withstand the back pressure generated Other detectors With some systems it is possible to incorporate signals from an external detector that is from a non KTA detector This can be useful for applications where for example highly sensitive detectors or more qualitative information is needed Common detectors used in combination with AKTA systems includes fluorescence light scattering and refractive index 29 0108 31 AA 45 46 29 0108 31 AA Chapter 9 Fraction collection Preparative chromatography requires that material eluted from the column is collected Two common methods employing either a fraction collector or a multiport outlet valve are used to direct the eluent to different containers tubes or bottles Table 9 1 compares these methods Table 9 1 Two methods for collecting purified sample Fraction collector Outlet fractionation Fraction size 100 ul to 250 ml Fraction size gt 5 ml Possible to collect many fractions typically 20 200 Number of fractions limited to number of outlet valve ports t
62. it Racks for Frac 920 Tube rack 95 x 10 18 mm Complete Tube rack 175 x 12 mm Complete Tube rack 40 x 30 mm Complete Filter assemblies Inline filter 10 and 20 ml min systems Inline filter kit 10 ml min systems Inline filter holder 20 50 and 100 ml min systems Inline filter kit 20 50 and 100 ml min systems 1 Inline filter is sometimes also referred to as online filter 2 PMO FR e N N N e e he e e e he e 10 Code number 18 1134 84 16 1111 26 16 1111 05 16 1121 22 18 1174 15 18 1174 16 28 9564 02 28 9564 04 28 9564 27 28 9542 12 20 9005 19 28 9818 73 28 9564 25 18 6083 11 18 6083 12 18 6083 13 18 6083 14 18 6083 18 18 6083 15 18 6083 16 18 6083 17 28 9487 80 18 3050 03 19 8684 03 18 1124 67 16 1118 91 18 1120 94 18 1112 44 16 1027 11 29 0108 31 AA 87 Description Column holders AKTA avant Column block for 5 columns Column holder Flexible column holder Column clip Column holder HiScale 50 AKTAxpress Large column holder Other AKTA Column holder short plastic Column holder XK 50 Column holder extra long metal Column clamp small column Clamp for lab rods AKTA extension equipment holder Autosamplers Autosampler A 900 cooled Autosampler A 905 Autosampler A 905 for AKTAmicro For external detection AD 900 Analog Digital Converter 88 29 0108 31 AA Quantity pack size Pm e e e e e e e rpe he Code number 28 9562 70 28 9562
63. ith empty tubes prior to start Lower the flow rate Make sure to position the fractionation tubing tip so that it is not blocking the light path for the drop sync Clean the drop sync photo cell see Chapter 10 Preventive corrective action Make sure that the same tubes and racks used are selected in the method Make sure that there is enough free space for the fraction collector movement If the alignment is incorrect even after a restart contact Service so the Frac 950 can be recalibrated Make sure that the UniNet 1 cable is placed in the correct socket Consult the user manual for a detailed description Preventive corrective action Change drive sleeve Change tube sensor Switch to the correct tube option on the fractionation arm allowing the droplets to fall in the center of the tube Make sure that the arm is positioned toward the tube as described in the manual 29 0108 31 AA 67 General problems continuing AKTA avant fraction collector problems Issue Cause Failed scanning There can be a number of reasons for a failed scanning Liquid appears when Liquid has entered the frac frac door is opened compartment instead of the waste container Preventive corrective action Open and close fraction collector to allow system to repeat the scanning Inspect cassettes and replace if for example identification bars are damaged or blocked If problem remains check the troubleshooting section for t
64. l nE E R E AA 37 Troubleshooting high Dack pressure opisnicinicustisiesisinntesantenrensttonnistsiensihainineiedimnnianaawelien 37 viscous campes and SON IONS arrene aa TNA 38 Pressure controlled sample application eeccescssessssesssecssesssecssecsssscsseessecsssecsssssssssssecssecssuscssecsssecssecssesssseessecs 39 Chapter 8 Sample monitoring and detectors wscsssezssncsanecezesceancccpeonasasvascaresesederessaunecensecaansiaactntriaevencoagrseenseces 41 Manitoriha UVV absarhah Er sairia A E ele nee ne 41 Monitoring ah EEN BES aa ar EDER EEN entre rrr 44 KOORD else Faan n E E EE E EE reer ERE REE ERE EDER DEERE ENDE Y airy te 45 BD MS OU omme obese an pares Era Ai 45 Chapter 9 Fraction collectio srs pcage cae occcevesi nes op venaysia sever vei distriv seri sr skio betast iot re E NESEY EEEE cdorsssearee ences 47 Straight Fractionation and peak Traction ao na osse den kinderne iii 47 PROACTONGUON CSI GV OMIT SEERE EET Er 48 SPIE ree TCC UOT ICOM sisean a AS 49 Chapter 10 Cleaning and storage of system componentS eseseeseseeseesesesseseeseeseseesesoeseeseseeseseesesseseesesees 51 Ge TE a e a a T EET EET treet terme ety eater REE 32 Shs alg EET 8 6 samen O A AEA A E EE EA E A ES ee renee nee ERE EDEN Ce eee 52 deaning recommend one occur rr ee A eaae i E eea pTi Ee idee 53 Appendices Appendix 1 System components in laboratory scale KTA SYStEMS c ccccscsscscssssessestsssssstssesssessssssesseees 55 Appendix 2 MUM FENG U
65. le volume should be kept small when using a nonbinding chromatography technique To achieve the highest resolution in GF a sample volume of less than 2 of the total column volume is recommended 14 29 0108 31 AA Chapter 4 How to choose sample injection technique There are three common ways of applying the sample to the column 1 From a prefilled sample loop 2 Direct injection via the sample pump 3 Direct injection via the system pump Table 4 1 Sample application techniques Technique Sample volume Important Benefit Tubing loop Small Filling and emptying the loop Handles small volumes 10 ul 10 ml in a correct manner High reproducibility Minimizes sample loss if partially filled Can be used at high pressure Superloop Intermediate Filling and cleaning the loop Minimizes sample loss 100 ul 150 ml Allows repeated injections without manual interactions in between Sample or Large Removing air bubbles from Is convenient for large volumes system pump 5 ml severalliters pump Priming the tubing with buffer sample before start Cleaning the pump afterward Sample volume in the lower range requires tubing with small i d to minimize sample loss Tubing loop Tubing loops are used for smaller sample volumes The loop must be filled and emptied in a correct manner Reproducibility when using a loop will be high because the sample application is independent of any variation in flow rate Sample loops of different volumes are availa
66. le wavelength absorbance detectors are often used The majority of proteins can be detected by measuring UV absorbance at 280 nm but other wavelengths can also be used to gather additional information see example in Fig 2 5 The conductivity monitor is used to follow column equilibration and salt gradient formation For some applications it is important to also monitor pH For more information see Chapter 8 Sample monitoring and detectors Absorbance MAU 2000 A280 Asso 1500 Conductivity 1000 500 0 JC V 0 5 10 15 20 25 Volume ml Fig 2 5 Specific detection of green fluorescent protein GFP at 490 nm 29 0108 31 AA 9 Fraction collection Preparative purification requires that the purified protein can be collected and fractionated The eluted materials are collected in fractions using a fraction collector or an outlet valve See Chapter 9 Fraction collection for information about different ways of controlling protein peak fractionation and what the important parameters are for successful protein collection See also Figure 2 6 Absorbance ml oUo UU Ud Fig 2 6 During this run peak fractionation was used to collect the eluted proteins System cleaning To ensure the long term performance of the system regular maintenance is important When not using the system for some time it is important to store it properly Chapter 10 Cleaning and storage of system components describes how to properly c
67. lean the different components of the system To minimize the risk of salt precipitation which may damage the seals avoid long term exposure of system components to high salt concentrations 10 29 0108 31 AA Chapter 3 System volume effects on resolution and fraction collection This chapter describes how the internal volume of the system affects liquid transportation and protein purification results The main applications for protein chromatography are either to analyze a protein sample or to prepare pure protein sometimes referred to as preparative purification For a successful result in both of these applications high resolution is often important High resolution is obtained by the use of chromatography media with combined high selectivity and efficiency High selectivity ensures that the protein is bound to the media High efficiency means that the protein peaks obtained are narrow and that good separation can be achieved between them Analytical chromatography systems generally handle small sample volumes To minimize sample dilution and loss components in an analytical system should have small internal volumes and allow usage of high resolution media In preparative chromatography it is important to use a chromatography medium and column that generate an appropriate resolution It is important to keep the distance between the column and the fraction collector short to avoid dilution of the separated proteins This is also important
68. lorer 10 AKTAxpress AKTA avant 25 2 9mm Transparent 660 ul Inlet tubing to AKTApurifier UPC 100 AKTA purifier 100 AKTA explorer 100 AKTA avant 150 1 For water at 10 ml min and room temperature 2 Negligible pressure 29 0108 31 AA 59 Internal volume To calculate the internal volume V of specific tubing use the formula V Lx Tx d 4 L length in mm d i d in mm CP If stating the dimensions in millimeters the volume will be presented in microliters Back pressure To calculate the back pressure in MPa generated over specific tubing use the following formula which is based on Hagen Poiseuille s work P cx Lx Q x v d c 0 000000679 L length in mm Q flow rate in ml min v viscosity in mPas d i d in mm eT This formula also applies to the back pressure generated over a column However the constant c differs and is dependent on the chromatography medium cr Keep in mind that the viscosity increases with lower temperatures See Figure 7 7 CP 1MPa 10 bar 145 psi Table A2 2 Viscosity values for common solutions at room temperature Solution Viscosity in mPas at 25 C Water 0 89 1M NaCl 0 97 1 M NaOH 1 11 8 M Urea 1 66 6 M Guanidine hydrochloride 1 61 20 ethanol 1 87 50 ethanol 2 41 100 ethanol 1 07 50 methanol 1 62 100 methanol 0 54 50 isopropanol 2 65 100 isopropanol 2 04 60 29 0108 31 AA Appendix 3 Determination of delay volumes A number of methods exist for det
69. low rate that maintains resolution and minimizes separation time Check recommended flow rates for the specific medium and column 4 If samples bind strongly to a medium separation conditions such as pH temperature chaotropic ions or organic solvents may have caused conformational changes and should be altered Conformational changes are specific to each protein Use screening procedures to investigate the effects of these agents Alternatively change to a less hydrophobic chromatography medium CP To reduce separation times and buffer consumption transfer to a step elution after method optimization as shown in Figure A7 6 equilibration gt salt free wash gt re equilibration elution of unwanted elution unbound material of target aad molecules molecule T elute 2 4 CV 2 5 tightly bound T molecules J Se a j T injection sa volume E 32 5 CV Column volumes CV Fig A7 6 Step elution Blue line absorbance red line conductivity salt concentration 76 29 0108 31 AA Further information Handbooks Strategies for Protein Purification 28 9833 31 Purifying Challenging Proteins Principles and Methods 28 9095 31 Hydrophobic Interaction and Reversed Phase Chromatography Principles and Methods 11 0012 69 Gel filtration GF or Size exclusion chromatography SEC GF SEC media separate proteins with differences in molecular size and shape The technique is well s
70. lues to separate several proteins that have distinctly different charge properties as shown in Figure A7 3 Selectivity at different pH of mobile phase Abs Abs Abs Abs V V V V Cation exchanger pH Surface net charge Anion exchanger Abs Abs Abs Abs V V V V Fig A7 3 Effect of pH on protein elution patterns V volume 74 29 0108 31 AA Method development in priority order 1 Select optimal ion exchanger using small 1 ml columns as in the HiTrap IEX Selection Kit or HiTrap Capto IEX Selection Kit to save time and sample If a longer packed bed is required use prepacked HiScreen IEX columns HiTrap columns have a 2 5 cm bed height and HiScreen columns have a 10 cm bed height 2 Scout for optimal pH to maximize capacity and resolution Begin 0 5 to 1 pH unit away from the isoelectric point of the target protein if Known This optimization step can be combined with optimizing the ionic strength of the sample and binding buffer 3 Select the steepest gradient to give acceptable resolution at the selected pH Usually start with a 10 to 20 column volume linear gradient 4 Select the highest flow rate that maintains resolution and minimizes separation time Check recommended flow rates for the specific medium and column ER To reduce separation times and buffer consumption transfer to a step elution after method optimization as shown in Figure A7 4 high salt wash elution 4 CV of target unbound elution of molecul
71. ly eluted by changing to a less hydrophobic chromatography medium Further information Handbooks Strategies for Protein Purification Handbook 28 9833 31 Purifying Challenging Proteins Principles and Methods 28 9095 31 Hydrophobic Interaction and Reversed Phase Chromatography Principles and Methods 11 0012 69 78 29 0108 31 AA Appendix 8 Columns for AKTA systems High quality column packing is essential for a good separation A poorly packed column gives rise to uneven flow dispersion peak broadening and loss of resolution A wide variety of available columns are described below covering different principles matrices and sizes For packing a column a range of empty columns is available See Table A8 1 for guidelines on how to combine media and columns Prepacked columns Prepacked columns from GE Healthcare will ensure reproducible results and excellent performance Er For more information refer to the guide Prepacked chromatography columns for AKTA systems Code No 28 9317 78 Fig A8 1 RESOURCE columns a Ag d P Fig A8 2 Precision columns Examples Mini Q PC 3 2 3 at left and Superdex Peptide 3 2 30 at right RESOURCE columns are prepacked with SOURCETM 15 media for IEX HIC and RPC RESOURCE columns are made of PEEK polyetheretherketone which has high pressure tolerance and high chemical resistance Fig A8 1 The RPC media are packed into steel columns SOURCE media are based o
72. minimize the contribution Tubing Keep the tubing as short as possible and optimize the i d Inline filter Change the filter regularly Buffer solution Decrease the flow rate when running high viscosity buffers solutions Temperature Decrease the flow rate when running at low temperature Sample Dilute viscous samples or decrease the flow rate during sample application Remove the inline filter if the system pump is used to apply the sample Column Clean the column Do not use smaller beads or column diameter than the application requires Flow restrictor When using chromatography media that generate low pressure at high flow rate consider removing the flow restrictor Note however that there is a risk of air bubbles entering the UV Vis absorbance cell Note A larger i d will decrease the back pressure but will have a negative effect on resolution see Chapter 3 The inline filter will prevent particles in the solutions from entering the flow path and column With time the filter will start to clog and the pressure will increase Mixing different liquids e g in a gradient can increase the viscosity and result in higher back pressure Viscosity increases at lower temperature To avoid over pressure some systems have pressure controlled sample application where the flow rate is decreased as the pressure increases See column instructions for cleaning procedures Smaller beads will give higher re
73. mobilized pH gradients Principles and Methods 80 6429 60 Microcarrier Cell Culture Principles and Methods 18 1140 62 Nucleic Acid Sample Preparation for Downstream Analyses Principles and Methods 28 9624 00 Western Blotting Principles and Methods 28 9998 97 Strategies for Protein Purification Handbook 28 9833 31 AKTA Laboratory scale Chromatography Systems Instrument Management Handbook Contents Chapter 1 Taha 2 8 o U o aa 9 panna me tare NET E eR Sn re eee ERE STE ee ee ee 5 COMMON ACrONVIMS dnd obrea S sxcseiniiariininsimsncrimanennnmaanauandnnbenddayranidumsyslenaeiies 5 Chromatography TErmniNOlO sarecenonentennvahatnd EET EET ES DEDE DENE STE 6 EE E EAEN AT AIEA EAE IA OAEIAE I AOE AAA earn EE 6 Chapter 2 Liquid chromatography systems and important considerations esessesessessesessesessessesessese 7 S EY PAC REE EET ENE A EaR 8 Chapter 3 System volume effects on resolution and fraction Collection ssoesesoeseseesessesossesessosseses 11 TUBING dimensions affect PESOIUTION essssiauniin a 11 Pedk broadening ater the UV VS Cele OP cunusconanian i 12 System volume consideration S siiani iini oii 13 Effect of sample volume on feso Ut ON sussurri aeiio 14 Chapter 4 How to choose sample injection technique cssccsscsscsscssscssssssssscssssssessssssecssssscenssnsceeseass 15 TOV SE e E EANA TEEPE TT E EEA NIA AATE AT 15 SS g E E e E A EEE A E E A ENET AE PE E A NO O EA een 17
74. n a hydrophilic matrix made from monodispersed rigid polystyrene divinyl benzene The media demonstrate very high chemical and physical stability The small particle size allows fast binding and dissociation to facilitate high resolution and the uniformity and stability of the particles ensure high flow rates at low back pressure RESOURCE columns cannot be opened and repacked Precision columns are designed for micropurification and analysis of proteins and peptides Fig A8 2 The columns are used extensively in peptide sequencing and protein structure function studies They are excellent for the polishing step of small scale protein purification procedures and for purity check analysis The small volume of the columns decreases the total area of the prepacked medium which minimizes nonspecific binding and dilution effects The column volumes have been scaled down 10 fold compared with Tricorn columns Precision columns are available for GF and IEX GF media are Superdex Peptide Superdex 75 and 200 and Superose 6 and 12 IEX media are Mono Q Mono S Mini Q and Mini S Precision columns require a special Precision column holder for use on AKTA systems and the columns cannot be opened and repacked 29 0108 31 AA 79 Fig A8 3 HiTrap prepacked columns pe a e Te 0 e mrs sa _ p eee ss e mer v ow Fig A8 4 HiScreen columns nt er FigA8 5 HiPrep prepacked columns
75. n be cleaned while connected to the system This is achieved by pumping a cleaning or sanitizing agent through the Superloop The standard recommendation is to pump 0 5 M NaOH for 30 min Make sure to rinse the loop properly after using NaOH for example wash with water followed by buffer until a neutral pH is achieved To avoid carryover when changing sample it is recommended to disassemble the Superloop and clean all parts separately vy Wear gloves and safety glasses when using hazardous corrosive chemicals Autosampler By using an autosampler several small sample volumes can be injected automatically which is convenient in for example protein analysis or micro purification work The autosampler makes sure that one sample at a time is used to fill the sample loop Table 4 2 lists two autosamplers and their capabilities These autosamplers can be used with AKTAmicro AKTAexplorer or AKTApurifier systems but AKTAmicro can only use A 905 Table 4 2 Autosampler options Autosampler Capacity Cooling A 900 96 1 5 ml vials or 160 0 5 ml vials Yes A 905 a 96 well plate a 384 well plate or 48 1 5 ml vials Yes 20 29 0108 31 AA Sample loading using a pump A sample or system pump can be used to apply sample directly onto the column Figure 4 11 shows an example of a flow path including a sample pump When using a pump a desired predetermined volume can be chosen or an air sensor can be used to allow loading of the entire
76. n between a charged protein and an oppositely charged chromatography medium Proteins bind as they are loaded onto a column Conditions are then altered so that bound substances are eluted differentially Elution is usually performed by increasing salt concentration or changing pH Changes are made stepwise or with a continuous linear gradient Most commonly samples are eluted with salt NaCl using a gradient elution Fig A7 2 Target proteins are concentrated during binding and collected in a purified concentrated form sample _ gradient equilibration gt _ _ gt wash re equilibration application elution high salt wash 1M 4CV tightly bound molecules unbound molecules elute Sne sk before gradient begins G N 8 J ea Column volumes CV Fig A7 2 Typical IEX gradient elution Blue line absorbance red line conductivity salt concentration 0 The net surface charge of proteins varies according to the surrounding pH Typically when above its isoelectric point pl a protein will bind to an anion exchanger e g Q Sepharose when below its pl a protein will bind to a cation exchanger e g SP Sepharose However it should be noted that binding depends on charge and that surface charges may thus be sufficient for binding even on the other side of the pl Typically IEX is used to bind the target molecule but it can also be used to bind impurities if required IEX can be repeated at different OH va
77. n purification system Such a system e Ensures more controlled conditions and reproducible results e Purifies proteins automatically without the need for user interactions during the run e Allows sensitive samples to be purified more efficiently e Allows use of high resolution media e Provides inline detection that helps in making decisions for example when the column has become equilibrated when to collect fractions etc e Allows automated collection of purified protein in small or large volumes e Uses software that makes it easy to create methods monitor runs and evaluate results Protein separation takes place in a column Buffers and other liquids are delivered via the system pump and sample can be applied in different ways e g using a syringe to filla sample loop or by using a sample pump Detectors e g UV Vis absorbance conductivity pH are placed after the column to monitor the separation process The purified proteins are collected in the fraction collector Figure 2 1 shows a typical system s flow path Sample Tubing Pumps AD D Column Buffers J solutions Ls RA p Fraction collector s J EEE pH Conductivity Fig 2 1 Typical flow path for a chromatography system 29 0108 31AA 7 Overview of chapters This section provides a short description of Chapters 3 to 10 Peak broadening and resolution To obtain a pure product it is important to optimize the system s flow path a
78. ne detectors are used in protein purification To monitor the purification process it is common to use a UV Vis absorbance detector because a majority of proteins will absorb light at 280 nm The area under the absorbance curve corresponds to the protein concentration and gives an indication of the amount of protein Other types of detectors can be used to gather more information about the purification process for example conductivity and pH monitors Monitoring UV Vis absorbance Wavelength to use Measuring UV absorbance at 280 nm will provide information about eluted proteins and the total protein content The ability of proteins to absorb UV light is predominantly due to the presence of tryptophan tyrosine and phenylalanine which strongly absorb at 280 nm However some proteins have only a few or non exposed aromatic amino acid residues and therefore show weak absorbance at 280 nm Apart from proteins other biomolecules also have the ability to absorb light For a purification scheme it is sometimes useful to check these Table 8 1 shows some examples of wavelengths that can be used to detect different biomolecules Table 8 1 Wavelength to detect different biomolecules Wavelength nm Absorption 214 Peptide bonds part of peptides and proteins 230 Organic compounds or chaotropic salts 260 DNA RNA 280 Aromatic amino acids residues tryptophan tyrosine and phenylalanine 390 420 Coenzymes e g in hemoproteins 490 Green fl
79. nnected in series to give a bed height of 20 cm The small volume makes HiScreen columns suitable also for laboratory scale purification HiScreen columns cannot be opened and repacked HiPrep prepacked columns are designed for convenient scale up purification Fig A8 5 HiPrep columns are available for GF desalting AC IEX and HIC in four different sizes 20 ml 53 ml 120 ml and 320 ml HiPrep columns for GF are prepacked with Sephacryl High Resolution media in 120 ml and 320 ml sizes The HiPrep Desalting column has a column volume of 53 ml for convenient desalting buffer exchange of sample volumes up to 15 ml IEX and HIC chromatography media are available in 20 ml HiPrep columns The column inlet and outlet are molded with 1 16 female threads for direct connection to AKTA systems HiPrep columns cannot be opened and repacked Fig A8 6 HiLoad columns 4 d I os 2 Fig A8 7 Tricorn columns Empty columns HiLoad columns are prepacked with high performance Superdex media for convenient and reliable GF SEC Fig A8 6 HiLoad columns are available in 120 ml and 320 ml formats prepacked with Superdex 30 prep grade Superdex 75 prep grade and Superdex 200 prep grade to cover a wide range of high resolution separation of proteins of different molecular weights The columns have an outer plastic tube that protects the column and provides personal safety in the event of breakage Tricorn high performance c
80. o avoid high pressure Pressure controlled sample application When applying sample the buildup of material on the column can be significant leading to the pressure limit being reached The buildup consists of contaminants such as denatured proteins nucleic acids and lipids This buildup of material can occur even if the sample has been clarified before the run In some AKTA systems pressure controlled sample application can be used During the run the system will then monitor the pressure and if it approaches the set pressure limit the flow rate will gradually decrease to avoid triggering the alarm 29 0108 31 AA 39 Figure 7 8 shows an example in which 150 ml of a sample was applied onto a column After approximately 110 ml i e 44 min the pressure became too high and the flow rate was automatically down regulated so that the pressure stayed at an acceptable level When the pressure decreased during the wash phase the flow rate was automatically up regulated Sample Elution pool from MabSelect SuRe pH 6 75 conditions adjusted to 15 mSiemen cm with NaCl System AKTA avant 25 Column HiScreen Capto Adhere Load Flowthrough mode 150 ml of 200 mg Mab ml Flow rate 2 5 ml min Azgo 25 Pressure T Flow rate E 2 0 15 g 2 10 aw US 0 O 10 20 30 40 50 60 70 80 90 Time min Fig 7 8 Pressure controlled sample application 40 29 0108 31 AA Chapter 8 Sample monitoring and detectors Inli
81. olumns are designed for high resolution protein purification at laboratory scale making them an excellent choice for the polishing step in multi step purification protocols Fig A8 7 Tricorn columns are available with a range of chromatography media for GF Superose Superdex IEX Mono Q Mono S SOURCE 150 and SOURCE 15S chromatofocusing Mono P and HIC SOURCE 15PHE The columns are simple to use with specially designed fittings for easy connection to AKTA systems and other high performance LC systems The columns are coated with a protective plastic film that protects the column and provides personal safety in the event of breakage Tricorn columns are also available empty for packing with a chromatography medium of choice see below To obtain a column with high quality packing and that can resist the pressure caused by the pressure drop across the selected chromatography bed select the appropriate empty column based on the guidelines given in Table A8 1 During packing follow the instructions supplied with the chromatography medium and empty column 9 P Fig A8 8 Tricorn columns 5 5 E Tricorn columns are designed for high performance chromatography media such as MonoBeads Sepharose High Performance Superdex and SOURCE Fig A8 8 When working with capture media such as Capto MabSelect or Sepharose Fast Flow a Tricorn Coarse Filter Kit is recommended to use for reducing the risk of clog
82. olution choosing sample injection technique and selecting an appropriate mixer It also gives straightforward advice on how to avoid problems such as air bubbles in the pump how to troubleshoot problems such as high back pressure and how to perform cleaning of system components The appendices include a general introduction to the different AKTA laboratory scale systems and columns as well as information on how to determine exact delay volumes for a specific system Common acronyms and abbreviations Psa AC Cr CIP CV DS FPEC GF HIC i d IMAC IEX mAU MPa mPas o d PM RPC SEC UV Vis Absorbance of light at specified wavelength in this example 280 nanometers Affinity chromatography Chromatofocusing Cleaning in place Column volume Desalting group separation by gel filtration buffer exchange Fast protein liquid chromatography Gel filtration sometimes referred to as SEC size exclusion chromatography Hydrophobic interaction chromatography Inner diameter Immobilized metal affinity chromatography lon exchange chromatography also seen as IEC in the literature Milli absorbance unit MegaPascal unit of pressure Unit for viscosity 1 mPas 1 cP i e 1 centiPoise Outer diameter Preventive maintenance Reversed phase chromatography Resolution the degree of separation between peaks Seconds Size exclusion chromatography same as gel filtration Ultraviolet visible light 29 0108 31AA 5 Chromatog
83. ooting fraction collection Some fraction collector problems and preventive corrective actions are listed in Table A5 1 Table A5 1 Potential problems and solutions with fraction collectors General problems Issue Cause Fractionation marks and Incorrect delay volume entered actual tube change do in the software not match Spillage between tubes No synchronization of collection defined in the software No tubes or filled tubes in fraction collector Too high flow rate is used Error message Incorrect positioning of Sensor dirty the tubing Dirty photo cell Frac 950 problems Issue Cause Spillage between tubes Incorrect selection of tube type and or rack in the method Incorrect alignment during Frac 950 initialization Error message Controller Board Error connection 2012 Frac not Found Frac 900 920 problems Issue Cause Tubes are not fed Drive sleeve worn out Tube change is not Tube sensor worn out performed properly e g more than one tube is fed per movement Spillage between tubes Wrong tube center position is used Fractionation arm not positioned correctly Incorrect Frac 950 UniNet 1 Preventive corrective action Make sure that the correct volume is entered See Appendix 3 for a description of how to determine the delay volume In the software select drop sync or accumulator as appropriate or collect in serpentine mode Make sure to have the fraction collector filled w
84. raphy terminology Back pressure Chromatogram Chromatography Chromatography medium media CIP cleaning in place Column Column hardware Column hardware pressure Degassing Delay volume Efficiency Flow rate Flow velocity Inline Medium media Peak broadening Pressure over the packed bed Resolution Selectivity System volume Symbols The pressure caused by column or system components in the system flow path A graphical presentation of detector response s From Greek chroma color and graphein to write The stationary phase also called resin The chromatography medium is often composed of a porous matrix base matrix The matrix is usually functionalized by coupling it with ligands that can bind molecules to be separated Common term for cleaning chromatography columns and or systems with the purpose of removing unwanted nonspecifically bound material Usually column hardware packed with chromatography medium The column tube and adapters All pieces of the column except the chromatography medium the packed bed The pressure inside the column during chromatography Column hardware pressure that is too high can break the column Removal of dissolved air from buffers solutions The volume corresponding to a part of the system Fractionation delay volume is the volume of tubing and system components between a monitor and the fraction collector Gradient delay volume also called dwell volume rela
85. rd to mix e g high salt concentration or mixing aqueous with organic solvents A larger mixer may be needed when creating gradients with aqueous and organic solvents Improper mixing of aqueous and organic solvent can be seen as disturbances in the absorbance baseline Change to a larger mixer and perform a test run without a column to make sure that the absorbance baseline is stable Conductivity disturbances Perturbations to the shape of the conductivity curve may indicate improper mixing If the internal volume of the mixer is too large the shape and slope of the gradient will be affected which can be observed on the conductivity curve as disturbances to the slope This effect is most pronounced at low and high conductivity as shown in Figure 6 1 It is especially important to be aware of this effect when scaling up to larger columns 29 0108 31 AA 29 When changing to a different mixer size the slope of the actual gradient can be compared with the programmed gradient by performing a test run without a column Programmed gradient B Conductivity curve correct mixer Conductivity curve mixer too large elution buffer B Distorted gradient Mixers of different sizes Volume Fig 6 1 The actual gradient will differ from the programmed gradient in a system with too large a mixer Gradient delay volume When planning a gradient run it is important to consider the system s delay volume prior to the
86. rsed Phase Chromatography Principles and Methods lon Exchange Chromatography and Chromatofocusing Principles and Methods 2 D Electrophoresis Selection guide Prepacked chromatography columns for AKTA systems User manuals for AKTA system Code number 18 1157 5 18 1022 29 18 103 7 46 26 9095 351 28 9887 41 28 9833 31 18 1142 75 18 1022 18 11 0012 69 11 0004 21 80 6429 60 20 951 76 Refer to www gelifesciences com and search for specific system s user manual within the Literature Documents and Downloads section CDs Column Packing CD The Movie Data files interactive selection guides apps and application notes Refer to www gelifesciences com protein purification 84 29 0108 31 AA 18 1165 33 Ordering information Description Tubing PEEK tubing i d 0 25 mm o d 1 16 ETFE tubing i d 0 25 mm o d 1 16 PEEK tubing i d 0 50 mm o d 1 16 ETFE tubing i d 0 50 mm o d 1 16 PEEK tubing i d 0 75 mm o d 1 16 ETFE tubing i d 0 75 mm o d 1 16 PEEK tubing i d 1 0 mm o d 1 16 ETFE tubing i d 1 0 mm o d 1 16 FEP tubing i d 1 16 o d 1 8 FEP tubing i d 1 8 0 d 3 16 Fingertights and tubing connectors Fingertight connector 1 16 M Narrow Black Fingertight connector 1 16 M Narrow Red Fingertight connector 1 16 M Tubing connector for o d 1 16 Tubing connector for o d 1 8 Tubing connector for o d 3 16 M6 connector Unions Union Luer F
87. s o d 10 18 mm or 175 tubes o d 12 mm or 40 tubes o d 30 mm 4 micro plates 96 wells and 8 tubes o d 30 mm or 120 tubes o d 18 mm and 8 tubes o d 30 mm or 240 tubes o d 12 mm or 45 tubes o d 30 mm Prep mode using 80 tubes o d 30 mm or 20 bottles 250 ml 1 deep well plate 96 or 24 wells 95 tubes o d 10 18 mm or 175 tubes o d 12 mm or 40 tubes o d 30 mm 6 cassettes or 55 bottles 50 ml or 18 bottles 250 ml Cassette options 6 tubes 50 ml 15 tubes 15 ml 24 tubes 8 ml 40 tubes 3 ml 1 deep well plate 24 48 or 96 wells Capacity 96 standard vials 1 5 ml or 160 microvials 0 5 ml 1 deep or micro plate 96 or 348 wells or 48 vials 0 5 ml Max pressure 20 MPa 200 bar 2900 psi 5 MPa 50 bar 725 psi 20 MPa 200 bar 2900 psi 5 MPa 50 bar 725 psi 10 MPa 100 bar 1450 psi 2 MPa 20 bar 290 psi 10 MPa 100 bar 1450 psi 2 MPa 20 bar 290 psi Other function Drop sync Accumulator Drop sync Optional prep mode Optional micro mode Drop sync Cooling Accumulator Cassette reader Mix up to 6 cassettes Other function Cooling Cooling 1 Stroke volume is the amount of liquid that is pushed out from the pump per piston Used with AKTA avant 25 AKTA avant 150 AKTA avant 25 AKTA avant 150 AKTA avant 25 AKTA avant 150 AKTA avant 25 AKTA avant 150 Used with AKTAretc KTA purifier UPC AKTApurifier AKT
88. s contamination AKTA avant represents the new generation of AKTA systems Fig A6 6 It incorporates functionality for achieving fast and secure protein purification AKTA avant is available in two versions with 25 and 150 ml min pumps AKTA avant 25 is designed for screening of media and method optimization in laboratory scale purification AKTA avant 150 is designed for scale up and robustness testing AKTA avant together with UNICORN 6 contains several features to facilitate and automate protein purification A Design of Experiments DoE software module is integrated in UNICORN 6 for AKTA avant It allows automation of the run scheme for the experimental design and maximizes the amount of information obtained while keeping the number of experiments at a minimum during method development BufferPro is an advanced inline buffer preparation function that enables buffer mixing without manual interaction The built in fraction collector provides security by cooling the purified samples and preventing dust from being introduced AKTA avant has a versatile valve configuration to facilitate the purification and increase reproducibility up to eight samples can be automatically purified the delta pressure over the column is monitored five columns can be connected in parallel and built in air sensors prevent air bubbles from being introduced 29 0108 31 AA 71 Fig A6 7 AKTAmicro system 72 29 0108 31 AA AKTAmicro is designed
89. s the actual separation obtained in the fraction collector absorbance detector It is also what is displayed on the chromatogram Fig 3 3 Consequence of the hidden system contribution if using tubing that is too long or that has a large i d between the UV Vis absorbance detector and the fraction collector 12 29 0108 31 AA In a system designed for high performance separations the recommendation is to use narrow and short tubing to keep the peak broadening low The drawback is that narrow tubing will increase the back pressure Read more about this in Chapter 7 System pressure An optimal combination of tubing length and i d is required to achieve the resolution needed and at the same time keep the back pressure within the pressure limit of the column used System volume considerations For a given chromatography system the relative system contribution to peak broadening will depend on both the bead size of the chromatography medium and column dimensions Small beads and narrow columns result in narrow peaks a high performance column whereas large beads and wide columns result in wide peaks The system volume can contribute significantly to the peak broadening of a narrow peak but will contribute almost nothing to a wide peak As illustrated in Figure 3 4 the system effect on resolution will be much larger for smaller peaks Peak volume Watch out for the system volume m contribution a System contributio t peak
90. sample undefined volume onto the column When the sample container is emptied air will trigger the air sensor and the sample valve will turn to another port This also prevents air being injected into the column For serial purification runs the pump can be used together with an inlet valve to serially load different samples Before applying sample onto the column the following preparation is important 1 To ensure correct volume delivery air bubbles must be removed from the pumpi s as described in Chapter 5 2 The flow path from the sample bottle to the injection valve must be filled with sample primed before starting the sample application Preparing sample inlet When the pump is started the volume from the sample container to the injection valve will be directed to the column If the flow path has not been prefilled i e primed with sample the actual sample volume applied to the column will be smaller than anticipated Injection Injection valve valve Fig 4 11 From the start the flow path including the sample inlet is filled with buffer A Primed tubing that is the tubing between inlet and injection valve is filled with sample B ensuring that the correct volume is injected onto the column during sample application In the figure sample is colored green and buffer blue Sample inlet preparation volume The volume needed to fill the sample inlet depends on the tubing and components included in the flow path
91. se the run and stop the flow 29 0108 31 AA 27 28 29 0108 31 AA Chapter 6 Gradient formation and mixers Gradients are used during elution of absorbed proteins from the column High accuracy in flow rate delivery is key in generating an optimal gradient How to ensure an accurate flow rate is described in Chapter 5 For proper gradient formation it is important to minimize the effect of pump pulsation and to make sure that the liquids used to form the gradient are mixed to a homogeneous solution before entering the column A mixer will accomplish these functions Different approaches may be taken and both dynamic and static mixers are used in chromatography systems Some systems have two pumps to be able to create accurate gradients Other systems use one pump and a switch valve to form gradients Choosing mixer size The delivered volume and type of solutions will determine the mixer size needed see Table 6 1 Usually the mixer supplied with the system will cover a broad range but there are occasions when changing to a different mixer size should be considered Check the system user manual to find out which mixer to use Table 6 1 Recommendations for mixer size When running What to do Small columns at a flow rate in the lower range and Changing to a smaller mixer will reduce the effects of with small gradient volume the system volume High flow rates and or using solutions that are Change to a larger mixer for proper mixing ha
92. solution but also higher back pressure The reason the flow restrictor is present is to prevent air bubbles in the UV Vis absorbance cell This is important when running columns that generate high back pressure 29 0108 31 AA 33 Tubing contribution to back pressure To keep peak broadening low the tubing should have a small i d and be short see Chapter 3 The drawback is that narrow tubing increases the back pressure in the system If the system is equipped with tubing that is too narrow the pressure generated can be too high for the column being used Pressure MPa Ls 1 0 05 es ee ee ee oe ee Pressure limit 0 25 0 35 0 50 0 75 1 00 Tubing i d mm Fig 7 1 Effect of tubing i d on back pressure Length of tubing 200 cm Flow rate 10 ml min Solution used water at room temperature Figure 7 1 shows the pressure generated by tubing of different i d s In the example a tubing i d of at least 0 35 mm is required to run a column with a pressure limit of 0 5 MPa In practice the recommendation is to not run close to the column pressure limit because the pressure alarm will stop the system In the example above the recommendation would be to use 0 5 mm tubing The effect of back pressure on column and packed bed The column and the packed bed have different pressure tolerance as described in Figure 7 2 A pl The precolumn pressure p1 affects the S P column hardware The column will break or
93. solutions CP By generating a back pressure for example by placing flow restrictors on used waste tubing the cleaning solution will during the cleaning method more easily reach dead spaces for example within valves vy Wear gloves and safety glasses when using hazardous corrosive chemicals W Make sure that valve ports without tubing connected are plugged during cleaning and that all waste tubing is inserted in waste containers Table 10 2 Cleaning solution suggestions to use for system components excluding the pH electrode To remove Use Buffers and salts Water Proteins lipids cell debris 0 5 to 1 M NaOH Proteins lipids and cell debris not removed by NaOH 1 to 10 M acetic acid Lipids and other hydrophobic components not Organic solvent for example 100 isopropanol removed by NaOH or acetic acid System storage Store the system in 20 ethanol to prevent microbial growth when not in use for more than 2 days When preparing the system for storage prevent precipitation of buffer components upon mixing with ethanol by rinsing the system with water Then fill the system with a 20 ethanol solution Make sure that the entire flow path is filled including all inlet and outlet tubing EF For some KTA systems premade methods for preparing the system for storage are included in the software W Prior to using the system after storage remove the ethanol using water 52 29 0108 31 AA Cleaning recommendations Sys
94. ssuring that it matches the column performance A poor match where system volume is too large may result in diluted peaks with decreased resolution and less pure protein see Fig 2 2 Tubing that is too narrow may result in back pressure that is too high for the column hardware Learn more about how to avoid these problems in Chapter 3 System volume effects on resolution and fraction collection Fig 2 2 Peak broadening in tubing Liquid flows faster in the middle of a tube as compared with closer to the walls The farther a protein peak passes through a tube the broader it becomes as depicted in the chromatograms shown on the right Sample loading The sample is typically applied to the column using either a prefilled sample loop or a pump Learn more about the different techniques and when to use them in Chapter 4 How to choose sample injection technique Liquid delivery The performance of the pump is important for ensuring reliable and reproducible results One common cause of unsuccessful chromatography is air bubbles in the pump This can cause pulsations in the flow delivery resulting in an inaccurate flow rate This effect can be observed as disturbances in the pressure curve Learn how to condition the pump in a proper manner as described in Chapter 5 Liquid delivery and pumps See also Figure 2 3 Fig 2 3 Remove air bubbles by using a syringe to draw liquid from the purge valves The figure shows a pump head
95. start leaking when the column hardware em pressure limit is reached KT For illustration only This is not how an actual column will look B p1 Delta column pressure Ap is the pressure Compressed medium due that affects the chromatography medium for example to running Ap pl p2 within the column conditions in which the J maximum Ap was exceeded p2 Fig 7 2 Be aware of the different pressure limits A precolumn pressure B delta column pressure Ap 34 29 0108 31 AA The pressure affecting the column hardware depends on the back pressure generated by the column itself and the back pressure generated by the system after the column If the pressure limit for the column hardware is exceeded the column might start leaking The pressure affecting the packed bed depends only on the flow rate and viscosity of the solution and not on the system When the flow rate is too high and or a high viscosity solution is used the pressure limit for the packed bed might be exceeded The packed bed pressure limit is the maximum allowed pressure drop over the packed bed When the pressure limit is exceeded the particles of the chromatography medium become distorted and or are forced to the bottom of the column and cause the back pressure to increase This leads to gap formation or a collapse of the packed bed resulting in poor chromatographic performance see Figure 7 2 B Pressure monitoring System pressure is generated by the complete syst
96. t also be taken into consideration Tubing i d 0 75 mm Tubing i d 0 50 mm Tubing i d 0 25 mm Tubing i d 0 15 mm mAU mAU mAU mAU 100 100 100 80 80 80 80 60 60 60 59 40 40 40 40 20 20 20 20 0 0 0 0 0 1 0 2 0 3 0 ml 0 1 0 2 0 3 0 ml 0 1 0 2 0 3 0 ml 0 1 0 20 3 0 ml Fig 3 2 The resolution increases as the tubing diameter decreases Column Superdex 200 5 150 GL CV 3 ml Flow rate 0 3 ml min Peak broadening after the UV Vis detector In a given chromatogram the UV Vis absorbance curve shows the purification result as it was while the proteins passed the absorbance detector What happens between the UV Vis absorbance detector and the fraction collector is not visible in the chromatogram This hidden effect can sometimes be dramatic especially for high resolution columns Figure 3 3 shows the effect of using larger i d and or longer tubing and thereby increasing the system volume The consequence of increasing the system volume is that the high resolution obtained in the column may be spoiled as the protein peaks progress to the fraction collector CP Use tubing that is as short as possible between the absorbance detector and the fraction collector Outlet valve V Vis absorbance U a aa Conductivity pH Fraction collector ONS Optimal system setup Increased tubing i d and or length between the UV Vis absorbance detector and the fraction collector This is the peak separation at the UV Vis This i
97. t on different pressure readings A without flow restrictor B with flow restrictor CP The flow restrictor only affects the column hardware pressure whereas the pressure on the packed bed is unaffected Removal of flow restrictor Our recommendation is to keep the flow restrictor inline because there is a risk of getting detector disturbances from air bubbles that are formed in the solution CP When using HiTrap and HiPrep columns with a system that monitors the pressure only at the pump consider the following modification Instead of removing the flow restrictor to avoid triggering the high pressure alarm increase the pressure limit to include the pressure contribution from the flow restrictor e g 0 2 MPa Do not set the pressure limit to more than 0 5 MPa however because this is the column hardware pressure limit for HiTrap and HiPrep columns Note This has already been implemented in the UNICORN column list for AKTAxpress supported columns Troubleshooting high back pressure A number of reasons could explain high back pressure see Table 7 1 A logic based approach to identify the problem is presented in Figure 7 6 First bypass or disconnect the column If the high pressure is released in bypass mode the column needs to be checked and cleaned See Appendix 4 for a workflow suggestion If the high pressure is not related to the column locate the system blockage as described below eT For information about how to cle
98. tem pump Because precipitated salts may clog valves and shorten seal lifetime it is important to rinse the pump with buffer or water as soon as possible after a run Most KTA pumps have a rinsing system with a circulating 20 ethanol solution Figure 10 2 shows an overview of the rinsing system The rinsing solution is in contact with the back side of the pump heads at all times and prevents microbial growth Change the rinsing solution once a week Note that the solution evaporates over time If the optional path without recirculation is used the rinsing solution needs to be filled more frequently Check valve Optional path without Rinsing solution recirculation 20 ethanol Fig 10 2 Schematic view of the pump rinsing system Sample pump Rinse the sample pump after each run with water buffers or cleaning agents that remove any sample traces For a sample pump with a rinsing system for example KTA avant follow the same procedure as described above UV Vis flow cell The cleaning requirement of the UV Vis flow cell will vary For general cleaning use 10 Decon 90 as described below If use of a detergent is not desired or if the cell is not sufficiently clean after use of 10 Decon 90 test one of the solutions listed in Table 10 3 CP Use a syringe to inject a small amount of 10 Decon 90 detergent directly into the flow cell and leave it for at least 20 min before rinsing with water For a rigorous cleaning use
99. tes to the volume between the point where two solutions are mixed and the column Measured as number of theoretical plates High efficiency means that sharp peaks will be obtained Flow through a column and or chromatography system Expressed in ml min Flow rate divided by the cross sectional area of a column Expressed in cm h A component that is part of the flow path Same as chromatography medium media The widening of a zone of solute e g a protein when passing through a column or a chromatography system Gives rise to dilution of the solute and reduces resolution Also termed band broadening or zone broadening The pressure drop across the packed bed upon passage of solution through the column Caused by flow resistance in the packed bed Measurement of the ability of a packed column to separate two solutes peaks Measure of the relative retention of two solutes in a column Related to the distance between two peaks The total volume of all tubing and system components outside the packed chromatography bed Sometimes referred to as system dead volume ETF This symbol indicates general advice to improve procedures or recommend action under specific situations Wi This symbol denotes mandatory advice and gives a warning when special care should be taken 6 29 0108 31 AA Chapter 2 Liquid chromatography systems and important considerations A number of benefits can be derived from using an automated protei
100. the P 901 P 903 P 905 P9 P9 S and P9H pump vy Check the 20 ethanol solution frequently Change it once a week or if the solution appears opaque or the ethanol level in the container has decreased cr To be reminded about the change place the rinsing solution where it is visible for example on top of the system or mounted on the instrument wet side 26 29 0108 31 AA Air sensor to protect column An air sensor used at the buffer inlet prevents introduction of air into the column and system Once the air sensor alarm is triggered the system will stop CP To avoid air bubbles becoming trapped within the air sensor and subsequently triggering the alarm always mount the air sensor s inlet and outlet vertically and in an upflow direction i e opposite to gravity flow Different levels of sensitivity of air detection can be set see Table 5 1 for general advice Table 5 1 Setting the level of sensitivity of air detection Level Detects Usage Low Large volume of air Provides protection against running out of buffer solution Medium Normal Air bubbles of medium size Aborts sample application when using the e g 30 to 100 ul pump to apply complete sample High Small air bubbles When the air sensor is placed between the e g tenths of microliters injection valve and column 1 Use high sensitivity with care because it may catch stray air bubbles that are not detrimental to the process and may unnecessarily activate the alarm pau
101. tion Purification and preparation of fusion proteins and affinity peptides comprising at least two adjacent histidine residues may require a license under US patent numbers 5 284 933 and 5 310 663 and equivalent patents and patent applications in other countries assignee Hoffman La Roche Inc UNICORN software Any use of UNICORN 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 2012 General Electric Company All rights reserved First published March 2012 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthcare representative for the most current information GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Japan Corporation Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan 29 0108 31 AA 03 2012
102. uited for the final polishing steps in purification when sample volumes have been reduced sample volume significantly influences speed and resolution in GF Samples are eluted isocratically single buffer no gradient Fig A7 7 Buffer conditions can be varied to suit the sample type or the requirements for further purification analysis or storage because buffer composition usually does not have major effects on resolution Proteins are collected in purified form in the chosen buffer high molecular weight low sample molecular injection weight volume intermediate molecular weight UV absorbance equilibration Column volumes CV Fig A7 7 Typical GF elution Further information Handbooks Strategies for Protein Purification 28 9833 31 Purifying Challenging Proteins Principles and Methods 28 9095 31 Gel Filtration Principles and Methods 18 1022 18 29 0108 31 AA 77 Reversed phase chromatography RPC RPC media separate proteins and peptides with differing hydrophobicity based on their reversible interaction with the hydrophobic surface of a chromatographic medium Proteins bind as they are loaded onto a column Conditions are then altered so that the bound substances are eluted differentially Due to the nature of the reversed phase matrices binding is usually very strong Binding may be modulated by the use of organic solvents and other additives ion pairing agents Elution is usually performed
103. uorescent protein GFP 600 Protein aggregates CP If obtaining a low absorption reading at 280 nm try detection at 214 nm where peptide bonds absorb light vy AKTA UV Vis absorbance detectors are linear up to 2000 mAU Signals higher than this are not proportional to the protein concentration Some chromatography systems have multi wavelength detectors that view target protein and critical impurities simultaneously Some proteins absorb at multiple wavelengths for example GFP which also has an absorbance maximum at 490 nm Measuring at both 280 and 490 nm will in this case help to identify which peak contains the target protein See Figure 8 1 29 0108 31 AA 41 Absorbance MAU 2000 A280 Asoo 1500 Conductivity 1000 500 0 JC V 0 5 10 15 20 25 Volume ml Fig 8 1 Specific detection of GFP by absorbance measurements at 490 nm The possibility of gaining information about contaminants in the sample can be useful during purification Strong absorbance around 230 nm indicates that organic compounds or chaotropic salts are present A high reading at 260 nm indicates the presence of nucleic acids P The ratio A A is a measure of DNA and or RNA purity and is thus a useful analysis method when purifying DNA or RNA If the ratio is close to 2 it indicates highly pure DNA RNA How to calculate protein concentration and amount The software for AKTA chromatography systems includes functionality for calculating concentr
104. when purifying small amounts of protein to avoid protein losses due to dilution effects Tubing dimensions affect resolution All components in the system flow cells valves etc must in some way be connected to each other with tubing Excess tubing will give unnecessary peak broadening that is the separated proteins will be diluted and resolution purity obtained will be decreased Peak broadening is due to the flow rate in the tubing being higher toward the middle compared with close to the walls of the tubing The result is that a protein peak passing through the system will become broader as it moves through the tubing as illustrated in Figure 3 1 Fig 3 1 Schematic description of protein peak broadening in tubing Liquid flows faster in the middle of a tube as compared with closer to the walls The farther a protein peak passes through a tube the broader it becomes as depicted in the chromatograms shown on the right 29 0108 31 AA 11 To achieve the best purification result it is important to find the optimal tubing parameters for the purification setup Figure 3 2 shows an example in which a sample was analyzed using tubing of different inner diameters i d Here the resolution is most affected when going from 0 75 mm to 0 25 mm i d tubing Decreasing the tubing diameter further will not have a large effect on the resolution At the same time the back pressure in the system will increase as tubing diameter is decreased This mus
105. ypically 8 10 valve Used for complex samples where several Used when a few defined peaks are expected peaks are expected The volume of the collected fractions is often different during different steps in a chromatographic run During sample application larger fraction volumes are collected as a safety measure in case the target protein were to pass straight through the column The flowthrough is collected in one or a few fractions corresponding to the volume of the sample applied and the subsequent wash During elution smaller fraction volumes are usually collected and an eluting peak is normally divided into a number of fractions in order to obtain pure protein from overlapping peaks Different fractionation modes can often be chosen for fraction collectors that have tubes or wells positioned in rows Collection can then often be performed from left to right for each row or in serpentine mode where every other row goes in the opposite direction When serpentine mode is chosen the risk of spillage is minimized Straight fractionation and peak fractionation To be able to analyze different parts of the peak the fraction size during elution is usually set to a value smaller than the expected peak volume When straight sometimes called fixed fractionation is used the fraction collector will continuously switch tubes according to the set volume throughout the entire fractionation as shown in Figure 9 1A To further increase the purity of t
106. yringe to draw liquid via the purge valve of the pump as described below This procedure is referred to as purging er To avoid air entering the pump make sure that all inlets are prefilled with liquid Also check that all tubing connections at the pump and inlets are tight To purge the pump connect a syringe to the purge valve Fig 5 2 Open the purge valve and draw liquid slowly into the syringe It is very important to draw the liquid slowly no more than 1 ml s otherwise an under pressure will be generated and more air bubbles will be released in the pump The purging will be more efficient if the pump is run at a flow rate around 10 of the system s maximum flow rate Such a flow rate will help to mechanically release any air bubbles adhering to the walls inside the pump head Normally pumps designed for higher flow rates are more easily purged because of the larger volume of the pump head EF For best results purge all pump heads of the pump A Fig 5 2 Remove air bubbles by using a syringe to draw liquid from the purge valves A Two pump heads from AKTA avant 25 and B AKTAprime plus system pump After purging check that all air bubbles have been removed by analyzing the pressure curve Fig 5 1 Start a flow and run the pump at a pressure above 0 2 MPa If the pressure curve indicates that there are still air bubbles present repeat the purging process and check the pressure curve again If air bubbles remain

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