GradiFrac User Manual - GE Healthcare Life Sciences
Contents
1. 5x1 ml 17 0416 01 HiTrap NHS activated 1x5 ml 17 0417 01 43 Appendix System configurations 44 Appendix system configurations Pump P 1 PSV 50 1 IN SS Eluent A Eluent B UV 1 GradiFrac GradiFrac System two valve configuration with Pump P 1 Appendix System configurations Valve IV 7 GradiFrac REC 102 GradiFrac System two valve configuration with HiLoad Pump P 50 PSV 50 IN US Eluent B EluentA Sampie GradiFrac REC 102 GradiFrac System with auxiliary valve for sample application 45 Index Index A accessories COMMOCUION sser 5 18 ordering information secere 42 adjusting the delivery arm 16 alar Issa easet rok bec POE a PEU ER ke tivated 29 Arrow KOS aliena 4 assembling tube racks T2 toli Tae cooper lalla 13 T8 IMI TACK Siac vectra inten 13 30 MAMI TACKS uio BA end donet pi Dai tede 15 automatic sample application 8 10 auxiliary valve n ctii rede etre 8 10 CONNECCION a Nodo Ha cute t tes dx 19 Menual cornitroli sociale 34 POURING eos eos ae io 17 ROAMING sss et does dat fette esi 28 B bowl mounting corrente ene 15 break DOMES o osse Pith Cete tatit ia atis 26 changing parameters 29 dini 29 MISErENO e EE Ls 29 C calibrating pulmips uu ueteri ast e eec 25 8 1 QI 01 cocaine deoa RNA FIGI es talia da Da seu 37 connecting accessories i 5 connections electric2. Conc B Flow Fractn Valve Peak Slope Comment 0 0 0 5 0 N N Start method 5 0 0 5 0 5 N Y 5 Start gradient start fractions 29 9 60 5 0 5 N Y 5 End gradient 30 0 100 5 0 5 N Y 5 Start column wash 44 9 100 5 0 5 N Y 5 End column wash 45 0 0 5 0 N N End fractions start re equilibration 50 0 5 0 N N End method Alarm at 1 min Alert for sample application 30 5 Setup and programming N ote the final breakpoint at 50 minutes which ensures that the method continues for 5 minutes after the concentration of B is reduced to 0 Example 2 shows illustrates a method with the same gradient as above but using an auxiliary valve for sample application see system configuration in Appendix In example 2 fractions are collected between 10 and 20 minutes starting and stopping within the gradient N ote how these breakpoints are entered after the gradient is programmed allowing GradiFrac to calculate the value for CONC B Table 5 2 Breakpoints for example method 2 Values actively entered for each breakpoint are shown in bold Unchanged values are grey Time Conc B Flow Fractn Valve Peak Slope Comment 0 0 0 5 0 N N Start method 1 0 0 5 0 Y N Start sample application 2 0 0 5 0 N N End sample application 5 0 0 5 0 N N Start gradient 29 9 60 5 0 N N End gradient 30 0 100 5 0 N N Start column wash 44 9 100 5 0 N N x End co
3. langle 28 threshold SIOPE co e rari 28 power conversion unit 6 power supplyu uu eneateta cet tareteastbenrt buti aa tained 6 CONNECTION v rio ii 20 POWE SWIECh o cde ope o odes ha eaae 5 program method MENU 26 programming an alarm 29 programming auxiliary valve 28 programming flow rate 27 programming fraction SIZ 28 programming gradientS i 27 47 Index programming peak collection 28 pump calibration PAGCOR PRETI 24 PEOCEdUTe ceste hu tnde e desc 24 pump calibration factor for HiLoad Pump P 50 secsec 26 PUMP CONNECEOTF ad oid Perder da Sn Da a 9 Pulp Pelo cau etes Ops e teta ni dede PUES 9 pumps CONNEC OM vi ci sepe ta vr ctr e d d Y rn 19 CODU OT ii eer oe ru eed De deae 9 l COITif Tieni ded canos freti rb rc eee 9 R PACK de 9 component placing cse yea s 18 rear panel uus ean vat henna Calica wired 5 FOCOPGEY Pre 10 chart Speed usse dateci 20 CONNECCION aus dete nor ers ioo Be Kas eevee 20 recorder SEapt SbO Dro aeui a sq pnmo hime eo EM ER RuS 10 run Method MENU ede eec eten texta erra deo 35 running a Method 35 S sample application valve 10 SetU p Tien uie cei eser Receta pent rh cea 22 setup parameters siriana 22 slope for peak collection 28 solenoid Vales leet bet here e 7 10 CONTECLHON terrorizzati altri ont 19 MOUNN saio aisi 16 17 911 0 L 03sec thu RO VR
4. of trade mark degeneration it is respectfully suggested that authors wishing to use these designations refer to their trade mark status at least once in each article Copyright 1997 Amersham Biosciences AB All rights reserved N o part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means without permission in written form from the company Contents WEP OGUCEION mL 3 Desci ORION eC PN 4 2 Idi e 4 2 Rafale 5 2 3 Power SU I idi comercio esa rers tmd bate a 6 MN ip e 6 2 5 Delivery arm and tube sensor siii 7 2032 9 0 90 cm 7 ZI DAVE viaria 8 Be AGCESI EQUIPE 9 dl System compolel Ils iii 9 LE PRA SS 9 Bide PUMPS DRM E 9 34 rl 10 2 9 SONG Valves uisi spine pb eon a oiera D EVE P pe ERE er 10 3b injection Valve asolo FE aeea Es EPA ERI EDIE 10 SE ip Licia 10 40 Mollica 11 amp Unpacking and asse i 12 al Upea 12 4 2 AssemblingtuberackS iii 12 4 3 Mounting the DOW cc 15 4 4 Inserting tubes nnnm 16 4 5 Adjusting the delivery arm mm 16 4 6 Fitting the delivery tubing 16 4 7 Mounting solenoid valves 16 4 9 System mouritillf sssesoeoa coconut px Fas tRRE apad Cena acp ai prt ea cu Fe lupi na nk ncs 17 4 9 System CODIGCHIOE asi coats cure tatto Seen epar ere oed eb tex a iaia 18 410 TUBING Connectors soar checa ERR EaE a 20 4 11 Starting tie Syste ii 21 4 12 Turning the System Off
5. parameters during method controlled operation are those which were last entered N ote that the method base and fractionation base are defined by those used in the most recently accessed method either in M ethod Run or in Program method unless they are actively reset in Setup Switch on GradiFrac After a short interval the display shows Main Menu Run Method Press the 1 key three times until the display shows Main Menu Setup 5 Setup and programming Press ENTER to enter the setup block Main menu RUN METHOD Main menu MANUAL RUN Main menu PROGRAM METHOD ENTER METHOD BASE time vol FRACTIONATE BY time vol drop CALIBRTN VALUE pls 0 1 ml ENTER PUMP CALIBRATION START COLLECTING PROCEDURE ENTER DELAY UV TO FRAC PRESS end TO ul STOP COLLECTING MIX VOLUME u1 DROP SYNC ACTIVE Y N ENTER COLLECTED VOLUME m1 Fig 16 The Setup menu block 5 2 1 Setting the method base Set the method base with M ethod base Figure 16 Choose time min or volume ml The chosen base will apply for all subsequent programming of new methods Previously stored methods however retain the method base in which they were programmed METHOD BASE VOLUME ml Y N METHOD BASE TIME min Y N Fig 17 Setting the method base 5 2 2 Setting the fractionation base Set the fractionation base with Fractionat
6. 8 Power supply 24 V 18 1032 06 Optional HiLoad Pump P 50 19 1992 01 Mixer 6 ml 110 V 18 3500 01 Mixer 6 ml 220 V 18 3501 01 Solenoid valve PSV 50 19 1994 01 Accessories refer to Figure 10 1 Position no Tube holder guide 12 mm 1 19 7242 02 Tube holder guide 18 mm 2 19 8689 02 Tube holder guide 30 mm 3 18 1124 68 Tube support 4 18 3054 02 Bowl 5 18 3051 03 Tube rack complete 12 mm 6 19 8684 03 Tube rack complete 18 mm 7 18 3050 03 Tube rack complete 30 mm 8 18 1124 67 Tube spring metal for 18 mm holder guide 9 19 6057 01 Valve PSV 50 10 19 1994 01 Valve holder kit 11 19 6069 01 Tubing holder 12 18 6464 01 Tube sensor kit 13 19 7815 02 Funnel 14 19 6044 01 Funnel tubing 12 8 250 mm 15 18 3020 01 Drive sleeve 16 19 6067 02 Spiral cable 19 19 8638 01 Sensor positioning kit 20 18 6470 01 Accessories available but not illustrated Drop Counter Kit 18 1012 05 Communication cable Pump P 1 19 6005 02 Signal cable UV 1 18 0577 01 Tube holder kit 8 11 mm 18 8522 01 10 Accessories Designation Code No Green connector 4 pole female 18 0863 01 Green connector 2 pole female 19 8629 01 254 nm Filter Kit 19 2432 01 405 nm Filter Kit 19 4724 01 Inlet tubing 19 4448 01 Assorted sample loops 25 50 100 200 500 ml 18 0404 01 SuperloopTM 10 ml together with Pump P 50 19 7585 01 SuperloopTM 50 ml together with Pump P 50 19 7850 01 Clamp holder pkg 5 18 1032 03 Gel filtration columns HiLoad 16 60 Superdex 75 prep
7. GradiFrac User Manual 56 1006 08 Amersham Edition AF e Biosciences Important user information Reading this entire manual is recommended for full understanding of the use of this product The exclamation mark within an equilateral triangle is intended to alert the user to the presence of important operating and maintenance instructions in the literature accompanying the instrument Should you have any comments on this manual we will be pleased to receive them at Amersham Biosciences AB S 751 82 Uppsala Sweden Amersham Biosciences AB reserves the right to make changes in the specifications without prior notice Warranty and Liability Amersham Biosciences AB guarantees that the product delivered has been thoroughly tested to ensure that it meets its published specifications The warranty included in the conditions of delivery is valid only if the product has been installed and used according to the instructions supplied by Amersham Biosciences AB Amersham Biosciences AB shall in no event be liable for incidental or consequential damages including without limitation lost profits loss of income loss of business opportunities loss of use and other related exposures however caused arising from the faulty and incorrect use of the product Trade marks GradiFrac HiLoad HiTrap RESOURCE and Superloop are the exclusive trade marks of Amersham Biosciences AB In view of the risk
8. ISRT N PORCI ac erit Gra GE ai Pu dn dad 7 tubing connections editi eine 20 48 specifications GAO gig oe PN M ET 40 step gradient arret 33 PFOGMAMIMINVG s aas bao rattrista 27 system components cne 9 system configuration ii 44 system SNUL GOW Ihe so restet terne rion ot Eo ass 21 system SLANE eo eni eec ere aaa 21 T three buffer systems esere 7 10 iroubleshooEInig oi cete e eben retis 38 IUDE rack durata 6 assembly nei 12 COMPONENTS noire il ati 12 tube SENSOR initial usd vehi em dee audes 6 COFITDOCEI ON sisi beo easet Tas iniezioni dinzinia 19 tube sensor switch eee 7 tubing connections 20 tubing olde o eo ra eee men pie bone 7 tubing length guide 7 16 V valves see solenoid valves W waste collector eseee Hee 7 Y CINE alia sie o doo aal A 4 Amersham e Biosciences Printed in Sweden by TK i Uppsala June 1997
9. In a domestic environment this product may cause radio interference in which case the user may be required to take adequate actions Note The declaration of conformity is valid for the instrument when it is used in laboratory locations used in the same state as it was delivered from Amersham Biosciences AB except for alteration described in the User Manual used as stand alone unit or connected to other CE labelled Amersham Biosciences products or other products as recommended This product meets the requirement of the Low Voltage Directive LVD 73 23 EEC through the harmonized standard EN 61010 1 10 Accessories 10 Accessories Please order accessories according to the designation and code numbers given below Refer to Figure 24 for position number where given Fig 24 GradiFrac accessories 41 10 Accessories 42 11 Ordering information Designation Code No GradiFrac System components Peristaltic Pump P 1 18 1110 91 Monitor UV 1 Control and Optical Unit 18 1003 66 Flow cell 10 mm 19 2504 02 280 nm Filter Kit 19 2433 01 Recorder REC 102 2 channel 18 1001 43 Mixer 0 6 ml 19 6700 01 Injection Valve IV 7 19 1996 01 Sample loops 1 and 2 ml 18 5897 01 Rack 18 1993 05 Valve Bracket Kit 19 1994 50 Tubing connector M6 male pkg 5 19 1993 50 Tubing ETFE 0 8 mm i d 1 8 mm o d 2 m 19 7435 01 Valve Holder Kit 19 1994 70 Recorder Cable 18 1107 8
10. UFALION cicci 44 MOUNN Goei ii den 17 H HiLoad Pump P 50 sis ot tertie 9 Hold Reysan lie alla 5 35 l injection valve ricci rio aan ariani 10 Interrupting a method i 35 K keyboatdo us x enrico el ila 4 L linear gradient MIDI 33 DEOOF aiti Mig oscar a merrier eaten ters 27 M man MANU gees iz DRM ai tan ales 22 frisirtenall6 cis ceasetzatsedszeerersedsteukssabesiganatzas 37 manual auxiliary valve control 34 manual flow rate Setting 34 manual fraction size setting 34 manual gradientSs 33 manual operation 33 manual peak collection 34 Manual Fun MENU oos aecenas 33 method base e rte eite 22 23 method number selecting for programming 26 methods do CU mentiri aus ii 30 amp xatmples cs e eter ve S 30 interrupting NOR DE E ence meer erste 35 manual intervention 35 programming aan 26 running methods reri ene 35 SEGUE IS cos iuc choro ntt ae uode ele d eus 36 Mix VOLUME sire Rte peces 24 MIC E DEL DEC 10 COBTYECHIOD occae iei tie ibat qe 19 TIOTIELO E dits tete D e et toit 11 CONNEectionia usalo eio mts Ets 11 20 monitor range ecd pe ict duae ae Fires acis 6 11 20 monitor to collector delay 24 mounting the DOW rina 15 MOUNTING Valves rrt rtu Ea redes 16 N numerical Keys 4 P pause Key cina 4 35 peak collection Manual cContiolsas aze aree poll 34 programming ite
11. al ce teo vo tete does 18 UDING D 20 continue key odere tb eere 5 35 custom tube TACKS air 6 46 D delay UV to TEC een etas e eode em 24 daivery SET alii 7 delivery tubing ANG vache Eee dt a cde tds 16 r comimiended i ced ae disp t betae ertet rats 8 display Method Stats delia 36 display MESSAGES iii 38 display window mm 4 documenting Methods 30 drop CoUnter a eme rot ter ape rete 7 drop synchronization ii 25 E editing SIMU Cais en eene teri ete pigs 29 electrical connections 5 teretes 18 ena Key aos e ai ecu 4 35 ENTER KOY siriani 4 GuOLC TIE E os cuti ec easi oe tede SN 11 example methods 2 e papato 30 F feed tube Key cies ecce dace o PCS stes 4 fitting delivery tubing eeeee 16 flow diversion cete t Pre recent 28 flow diversion Valve 7 10 GOMNECLION E 19 VOOUIDEITIG Sii iio aei de Seeds tere tan nd rare 16 flow rate Mandala ia 34 PLOGrAmMIMI Ori iit eduntur potet pret int doaus 27 flow rate Tanga id en ola tea 9 fraction size manual settings 34 programming araba die xia Pie tents 28 Fractionation baSe 22 23 28 front pane Mis oido erdt a 4 function Keys ucc cipere e roe car Pt eter n eir 4 Index G gradient vale cranio iaia 7 10 CONNECTION pesati nr Taste 19 MOUNTNG sn ii 17 gradients manual alteratiONS ii 34 Manual Control aree 33 programa saio ideata 27 GradiF rac System ccecce 9 CONFIG
12. base time volume or drops is determined by the current setting in Setup see Section 5 2 As long as the fraction size is set to 0 the flow diversion valve will divert effluent to waste 5 5 6 Programming the auxiliary valve Control the auxiliary valve where used with Valve active Y N Figure 19 The auxiliary valve is connected to the Aux port Enter Y to activate the valve and N to deactivate the valve The current setting is indicated by Y or N in the instruction prompt 5 5 7 Programming peak collection Set peak collection with Peak collect Y N Figure 19 Enter Y to activate peak collection The fraction size must be set to gt 0 for peak collection to be activated This may be done in the same breakpoint as that in which peak collection is activated If peak collection is activated enter the threshold slope for peak detection as Slope ml Values may be set from 1 to 99 96 ml The fraction collector will change tubes whenever the slope exceeds the set value The peak end is determined automatically by GradiFrac The set fraction size applies both within and between peaks The following procedure is recommended for determining a suitable slope value 1 Usea previous chromatogram from an identical run to determine the slope ml see Figure 19 Peak collection is not recommended with an unknown sample 2 Perform a blank run with the chosen setting to check that tube changes do not occur as a result of basel
13. collection tubes into the rack beginning at position 1 g Ensure that the tubes are pushed down as far as possible All tubes in one collection sequence must be of the same length and diameter and there should be no empty spaces 4 5 Adjusting the delivery arm 4 6 Fitting the Li delivery tubing 2 M ove the arm by lifting it a little so that the tube sensor comes to rest against a tube in the outer row Loosen the lock knob on the column Adjust the height of the arm so that the tube sensor is about 5 mm below the top of the tubes and tighten the lock knob Rotatethe rack anti clockwise by hand until the sensor comes to rest against the tube in position 1 Ensure that the red sensor switch is correctly set for the tube diameter to the smaller marker for 12 mm racks and to the larger marker for 18 and 30 mm racks Cut the end of the delivery tubing cleanly at 90 Lift out the tubing holder from the delivery arm and loosen the nut 3 Fit the delivery tubing through the tubing holder 4 di Place the tubing holder over the length guide hole Push the tubing through the holder until it reaches the bottom of the length guide then tighten the nut on the tubing holder This ensures that drops are formed in the correct position with respect to the drop counter Fit the tubing holder into the delivery arm 4 7 Mounting Screw the valve holder to the back of the delivery arm using the screws l id val provided A
14. cribed above GradiFrac will perform the calculation automatically M ove through the program method menu with the arrow or ENTER keys setting breakpoint parameters as required If several breakpoints have been set you can move up and down through the breakpoints changing parameters as required 5 5 3 Programming gradient shape Set the concentration of buffer B with CONC B Figure 19 Two breakpoints with different values for concentration of B will create a linear gradient from the first to the second value over the interval between the breakpoints For example the following breakpoints give a gradient from 25 to 45 B starting at 5 minutes and extending over 10 minutes TIME 5 0 min CONC B 25 TIME 15 0 min CONC B 45 Two gradient breakpoints separated by 0 1 ml or min will create a step gradient with an immediate change in buffer B concentration For example the following breakpoints cause an abrupt change from 25 to 45 B at time 5 min TIME 4 9 min CONC B 25 TIME 5 0 min CONC B 45 5 5 4 Programming flow rate Set the flow rate with Flow ml min Figure 19 A method cannot be programmed with zero flow rate Any changes in flow rate are made instantaneously at the breakpoint time or volume where the new value is requested 27 5 Setup and programming 28 5 5 5 Programming fraction size Set the fraction size with Fraction min ml drop Figure 19 The fraction
15. ctor M ix volume too low for requested CONC B at this flow rate Flow rate too high or mix volume too low Check for obstructions to rack movement and remove them Contact Amersham Biosciences service engineer Fill rack with more tubes Check for obstructions to rack movement and remove them Position the delivery arm correctly Choose a different method number Position the GradiFrac bowl so that a tube is directly under the flow outlet Set a flow rate manually or as the first breakpoint in the method Press hold or pause and stop the gradient first Set a lower flow rate or recalibrate the pump Set a higher flow rate or recalibrate the pump Set a higher mix volume The warning may be ignored at CONC 96B closeto 0 or 100 in a gradient Set a lower flow rate or higher mix volume 38 8 Troubleshooting Message Cause Action Gradient is already on Warning Pump is not operable Pump O verPresssure Pump RUN isnot pressed Too short time between feeds No fraction size N o drops check sensor Continuous flow check sensor M ethod memory is full N o alarm after end of method Bad drop sensor or not connected Endpoint missing edit your method Attempt to change CONC B during a running gradient manual mode Attempt to start pump failed The pressure limit for the pump has been reached HiLoad Pump P 50 only TheRUN button on the pump is
16. default settings for a new breakpoint are taken from the previous existing breakpoint in the method 5 5 1 Selecting a method number Choose Program M ethod in the main menu Figure 15 and press Enter Enter the number of the method to program GradiFrac shows the number of the lowest empty method in the window and suggests the most recently used number as default If the method number is occupied GradiFrac responds with the message Method occupied To choose a new method number press end to return to the main menu To clear the existing method press EN TER to acknowledge the message then answer Y to the Clear method prompt Answering N here allows you to edit the method see Section 5 6 5 Setup and programming 5 5 2 Entering breakpoints A method consists of a series of breakpoints which define changes in one or more control parameters M ethods are programmed on a time or volume base according to the current setting in Setup see Section 5 2 T he first breakpoint must be at time volume 0 0 An alarm can be set anywhere in the method e g to alert the operator for manual sample application or to signal the end of the method Note If FRACTN VALVE ACTIVE or PEAK COLLECT breakpoints are to be programmed within a gradient then these breakpoints should be added after the gradient programming is completed This avoids the need to calculate the exact value for CONC 96B within the gradient By programming as des
17. delay volume from the length of tubing The volume of 0 8 mm i d tubing is 50 ul 10 cm The delay volume will apply for all subsequent manual and method controlled operation until a new value is set 5 2 5 Setting the mixing volume Set the mixing volume with M ix volume Figure 16 The mix volume is the total volume ul of liquid buffer A buffer B which passes through the gradient valve during one cycle of operation The value for the mix volume together with the flow rate thus determines the switching frequency of the valve If the mix volume is too low the switching frequency may betoo high the minimum cycle time for valve PSV 50 is 30 ms If the mix volume is too high the gradient will be inaccurate at low flow rates The mix volume for Pump P 1 is calculated according to the table below 5 Setup and programming 5 3 Calibrating the pump Pump tubing Flow rate Mixer Mix volume i d mm range ml min ml pl 1 0 0 1 0 6 Mix volume 25 000 pump calibration factor 2 1 1 4 0 6 Mix volume 18 000 pump calibration factor 3 1 3 5 0 6 Mix volume 3 B00 pump calibration factor 3 1 5 8 6 0 Mix volume 18 000 pump calibration factor The following values are recommended for HiLoad Pump P 50 Flow rate Mixer Mix volume ml min ml Hl 0 5 0 6 60 gt 5 6 0 600 N ote that the mix volume is not the same as the volume of the mixer The mix volume will apply for all subsequen
18. e instrument memory Stored methods are retained in memory even when the power is switched off GradiFrac can also be used in manual control mode and manual adjustments can be made while a method is running GradiFrac can be programmed to collect fractions based on time volume or drop count Fractions can also be collected in a peak detection mode when a suitable monitor is connected Tube racks accommodate tubes with diameter 10 18 mm and length 50 180 mm 2 Description 2 1 Front Panel ENTER 5 dg feed tube E YIN 2 Description The front panel consists of an alphanumerical LCD display window and a keyboard with membrane keys 1 Pharmacia LKB GradiF rac Pharmacia Fig 1 GradiFrac front panel The keyboard is divided into numerical keys and function keys 2 1 1 Numerical keys are used for entering values during programming and manual control 2 1 2 Function keys are used during both programming and operation The main functions are briefly summarized below and are described in more detail as required in the appropriate sections Step through the control and programming menus You can use the arrow keys whenever the display shows an up down or double arrow Enters parameters as shown in the display during programming and manual control Acknowledges error messages Stops manual operation Interrupts method operation before the method is completed Finishes work in the variou
19. e by Figure 16 Choose time min volume ml or drops The chosen base will apply for manual operation and for all subsequent programming of new methods Previously stored methods however retain the fractionation base in which they were programmed 23 5 Setup and programming 24 FRACTIONATE BY VOLUME ml Y N FRACTIONATE BY TIME min Y N FRACTIONATE BY DROPS drp Y N Fig 18 Setting the fractionation base 5 2 3 Setting the pump calibration factor Set the pump calibration factor with Calibrtn value Figure 16 The pump calibration factor is the number of pulses required to deliver 0 1 ml from the pump Enter the value directly if known and press ENTER If the calibration factor is unknown see Section 5 3 N ote that the calibration factor for Pump P 1 depends on the tubing diameter You are advised always to perform pump calibration whenever the tubing is changed The pump calibration factor will apply for all subsequent manual and method controlled operation until a new value is set 5 2 4 Setting the monitor to collector delay Set the delay volume between the UV monitor and the fraction collector with Delay UV to Frac Figure 16 Enter the volume of liquid in ul in the delivery tubing between the UV monitor flow cell and the very end of the tubing This will ensure that event marks on the UV absorbance trace on the recorder are synchronized with tube changes Calculate the
20. grade 17 1068 01 HiLoad 26 60 Superdex 75 prep grade 17 1070 01 HiLoad 16 60 Superdex 200 prep grade 17 1069 01 HiLoad 26 60 Superdex 200 prep grade 17 1071 01 HiLoad 16 60 Sephacryl S 100 HR 17 1165 01 HiLoad 26 60 Sephacryl S 100 HR 17 1194 01 HiLoad 16 60 Sephacryl S 200 HR 17 1166 01 HiLoad 26 60 Sephacryl S 200 HR 17 1195 01 HiLoad 16 60 Sephacryl S 300 HR 17 1167 01 HiLoad 26 60 Sephacryl S 300 HR 17 1196 01 lon exchange columns HiLoad 16 10 Q Sepharose HP 17 1064 01 HiLoad 26 10 Q Sepharose HP 17 1066 01 HiLoad 16 10 S Sepharose HP 17 1065 01 HiLoad 26 10 S Sepharose HP 17 1067 01 HiLoad 16 10 Q Sepharose FF 17 1060 01 HiLoad 26 10 Q Sepharose FF 17 1062 01 HiLoad 16 10 S Sepharose FF 17 1061 01 HiLoad 26 10 S Sepharose FF 17 1063 01 RESOURCE Q 1 ml 17 1177 01 RESOURCE Q 6 ml 17 1179 01 RESOURCE S 1 ml 17 1178 01 RESOURCE S 6 ml 17 1180 01 Hydrophobic interaction columns HiLoad 16 10 Phenyl Sepharose HP 17 1085 01 HiLoad 26 10 Phenyl Sepharose HP 17 1086 01 HiLoad pump P 50 is recommended for best performance with these columns Affinity chromatography columns HiTrap Protein A 5x1 ml 17 0402 01 HiTrap Protein A 1x5 ml 17 0403 01 HiTrap Protein G 5x1 ml 17 0404 01 HiTrap Protein A 1x5 ml 17 0405 01 HiTrap Heparin 5x1 ml 17 0406 01 HiTrap Heparin 1x5 ml 17 0407 01 HiTrap Chelating 5x1 ml 17 0408 01 HiTrap Chelating 1x5 ml 17 0409 01 HiTrap Blue 5x1 ml 17 0412 01 HiTrap Blue 1x5 ml 17 0413 01 HiTrap NHS activated
21. he accumulated volume Press continue to resume gradient formation end interrupts the method with the prompt End method Y N Entering Y terminates the method Entering N resumes method operation 35 6 Operation 36 6 2 3 Examining current method status During method operation the arrow keys can be used to scroll through display of the current method status The following information is displayed Method Elapsed method Method no status time or volume Current parameter Possible program value direction Parameter currently displaye 6 2 4 Manual control during method operation The parameter values in method status display may be replaced with new values to modify method operation A parameter which is changed manually continues to apply until it is reset by a breakpoint with a value different from that originally programmed in the method For example consider a method with breakpoints at 0 5 and 10 minutes with flow rate 2 2 and 4 ml min respectively 0 00 FLOW 2 0 ml min 5 00 FLOW 2 0 ml min 10 00 FLOW 4 0 ml min If the flow rate is changed manually to 3 ml min after 2 min this value will continue to apply until the breakpoint at 10 min Gradients can only be changed in H old or Pause 7 Maintenance 7 Maintenance GradiFrac should be cleaned thoroughly after use Inadvertently spilled liquid should not be allowed to dry on the instrument and the delivery arm should always be
22. how Enter collected volume 7 M easurethe collected volume enter the value ml and press ENTER GradiFrac will calculate the calibration factor and the display will show the calculated value UJ 25 5 Setup and programming 5 4 Program Method menu 5 5 Programming 26 a new method 8 Press end to return to the main menu or Uto continue with the setup menu The calibration factor for HiLoad Pump P 50 is 234 pulses 0 1 ml Enter this factor directly in the Setup menu if Pump P 50 is used Pump calibration should only be necessary for Pump P 1 and other pumps with unknown calibration factors GradiFrac can store up to 10 methods of 52 breakpoints each end Main menu d SELECT METHOD RM Y HUN METEOD 10 9 FREE n Method occupied Main menu CLEAR METHOD MANUAL RUN Y N ENTER Y new method N edit method CE EDIT BREAKP TIME VOLUME time volume CONC amp B iii ENTER t TIME VOLUME FLOW ml min REMOVE CURRENT ti 1 BREAKPOINT Y N COnUnUE TIME VOLUME CTN nd OINT z 1 TIME VOLUME VALVE ACTIVE Y N END METHOD Y N 1 TIME VOLUME mm TIME VOLUME PEAK COLLECT Y N SLOPE ml SHOW CONC B ON REC OUT Y N Fig 19 The Program method control block Each holds settings for all method parameters During programming the
23. ica 21 Di Setup and POO OF ANN NINN csr n a cruenta 22 51 GradiFrac Ed E indais teia e a e 22 MEE sepu ccm 22 5 9 Calibrating the PUD oce io inediti bara ene beau tate 25 5 4 Program M ethod MEnu sio e rec nali 26 5 5 Programming a new Tett eiiebuit cce e ecu neum 26 5 6 Editing an existing method urico iii 29 5 7 Documenting methods oa etia adi ditis doni ote eins 30 5 8 Method examples tirrenia anale 30 DO PErAofica aerea ety oie ca Bi debates ini 33 6 1 Manual Operations ou ene rta hes 33 6 2 Method controlled operation 35 7 ANGELIII CC seco seo av eG ee ten de tie ini 37 8 Troubleshooting rete rr ter etii End d 38 9 Technical specifications ted cepe bedient 40 TO JACCOSSOTLGS occisi aeree prone estet etapa rir UE DRM EM 41 11 Ordering Information 0h ai edi 42 Appendix System contigul atl OTI c coco ala 44 1 Introduction 1 Introduction GradiFrac Code No 18 1993 01 from Amersham Biosciences AB combines the functions of gradient programmer and programmable fraction collector for standard chromatography applications It forms the basic unit of GradiFrac System which includes GradiFrac Pump P 1 M onitor UV 1 Recorder REC 102 Valve IV 7 Switch Valve PSV 50 and a mounting rack Gradients are generated using a single pump and a switch valve Easy to use programming allows automatic gradient formation for pre programmed separation methods Up to ten methods can be stored in th
24. ine disturbances If unwanted tube changes occur set a higher slope value and repeat the run AU 0 5 4 Monitor FSD 0 5 AU 0 4 4 Slope at peak start 0 05 AU ml 1096 FSD 0 3 4 Peak collect slope 10 ml 0 2 4 0 1 4 0 1 ml 0 5 10 Elution vol ml Fig 20 Determining the slope value for peak collection ml 5 Setup and programming 5 6 Editing an existing method 5 5 8 Ending the method The method will end at the last breakpoint If you require a period of constant parameters at the end of the method enter a final breakpoint with the same parameters as the penultimate breakpoint see the examples in Section 5 8 When all breakpoints have been entered press end to end method programming and answer Y to the End method prompt 5 5 9 Programming an alarm Set an alarm with Alarm at Figure 19 If you want an alarm to sound during or after the method enter a non zero value here For example entering Alarm at 1 min will sound an alarm 1 minute after the method is started To edit an existing method choose Program method from the main menu Select the number of the method and press enter GradiFrac responds with the message Method occupied Press ENTER to acknowledge the message then answer N to the Clear method prompt You may now edit the breakpoints in the existing method Use the arrow keys at the Edit breakpoint prompt to scroll through the existing breakp
25. inning of the manual run menu The display indicates the current gradient direction with the symbols increasing 96 B constant isocratic B decreasing B 6 1 2 Setting flow rate Set the flow rate with Flow ml min Figure 22 The pump will start or change speed if already started when you press ENTER 6 1 3 Setting fraction size Set the fraction size with Fraction min ml drop Figure 22 The fraction base time volume or drops is determined by the current setting in Setup see Section 5 2 The fraction size will apply as soon as you press ENTER As long as the fraction size is set to 0 the flow diversion valve will divert effluent to waste 6 1 4 Operating the auxiliary valve O perate the auxiliary valve where used with Valve active Y N Figure 22 The auxiliary valve is connected to the Aux port Enter Y to activate the valve and N to deactivate the valve The current setting is indicated by Y or N in the instruction prompt 6 1 5 Setting peak collection Set peak collection with Peak collect Y N Figure 22 Enter Y to activate peak collection The peak collection function is explained on p 28 6 Operation 6 2 Method controlled operation This section describes the use of GradiFrac for method controlled runs It is assumed that GradiFrac has been installed as described in Chapter 4 and that at least one method has been programmed M ethod programming is described in Section 5 4 6 2 1 Run
26. ity and may be connected either way round Connect the monitor cable with the polarity shown on GradiFrac rear panel and recorder cables as shown in Figure 12 All sockets on the rear panel must have their respective green connectors fully inserted to avoid the risk of electrostatic discharge ESD 4 Unpacking and assembly slosh Sos Fig 12 Electrical connections 4 9 1 Tube sensor Connect the tube sensor cable from the delivery arm to the Tube sensor contact on the rear panel of GradiFrac This cable may already be connected on delivery 4 9 2 Pump Connect the Remote contact on the pump to the Pump contact on the rear panel of GradiFrac 4 9 3 Valves Connect the flow diversion valve to the Frac port on the rear panel of GradiFrac Connect the gradient valve to the Switch port on the rear panel of GradiFrac Connect the auxiliary valve if used to the Aux port on the rear panel of GradiFrac 4 9 4 Mixer Connect M ixer 0 6 ml to the M ixer port on the rear panel of GradiFrac 19 4 Unpacking and assembly 4 10 Tubing connections 20 4 9 5 Recorder Connect the recorder to the three pin port marked Recorder Start stop event mark GND and the two pin port marked Recorder GRAD 1V A GND on the rear panel of GradiFrac using the special Recorder Cable packed in the GradiFrac box Code No 56 1136 67 The ordering number for the Recorder Cable is 18 1107 88 Connect the 15 pi
27. k stops for each tube is determined by the tube sensor on the delivery arm see below so that the rack will not stop in an empty tube position A safety feature stops collection automatically if rotation of the bowl is obstructed 2 Description 2 5 Delivery arm and tube sensor 2 6 Solenoid valves The delivery arm carries the delivery tubing tube sensor and drop counter The cable carrying both tube sensor and drop counter signals is connected to the Tube sensor contact on the rear panel or GradiFrac tubing holder lock knob sensor control switch tubing length guide valve tube sensor bracket Fig 4 Delivery arm and tube sensor The delivery arm has three stationary positions At rest it is located over the waste collector in the centre of the bowl It can also be drawn gently outwards to two positions beyond the radius of the bow for unhindered access to the tube rack During operation the delivery arm is tensioned inwards and rests with the tube sensor against the edge of a tube where present The height of the arm can be adjusted with the lock knob on the support rod The tube sensor detects the presence of tubes in the tube rack If a tube is missing the delivery arm will swing inwards until a tube is detected or the centre position is reached N ote that the spiral tube sequence will be interrupted in such circumstances The red sensor control switch at the end of the deliver
28. lumn wash 45 0 0 5 0 N N i Start re equilibration 50 0 0 5 0 N N End method 10 0 12 5 0 5 N Y 5 Start fractions 20 0 36 5 0 N N End fractions Alarm at 50 min Signal method end 31 5 Setup and programming GOIUMN TY Pes wcities en ene Auxiliary valve used Setup parameters Method base time Q volume Q Pump calib value Fractionate by time Q volume Q drop Delay UV to frac i MIX VOL iuit Drop sync active i Notes Breakpoints Time volume Conc B Flow Fractn Valve Peak Slope Comment 32 6 Operation 6 1 Manual operation 6 Operation To control GradiFrac manually without using a preprogrammed method choose M anual Run in the main menu and press ENTER ain me PEEL E MANUAL RUN METHOD CONC B ENTER MANUAL FLOW ml min MANUAL FRACTN Gradient is already on Main menu PROGRAM METHOD if gradient already running MANUAL GRADIENT TARGET CONC B enter MANUAL REACH TARGET VALVE ACTIVE Y N lt aE TER ml min MANUAL M 5 MANUAL PEAK COLLECT Y N SLOPE min Fig 22 The Manual Run control block Usethe arrow keys to move through the menu options setting parameters as required Settings take effec
29. mersham Biosciences REC 101 and REC 102 as well as other recorders are suitable for use with GradiFrac The recorder is connected to the port s marked Recorder on the rear panel of GradiFrac Use the Recorder Cable packed in the GradiFrac box Code No 56 1136 67 The ordering number for the Recorder Cable is 18 1107 88 The port connections have the following functions Start Stop Start and stop the recorder chart The chart drive will operate as long as the flow rate is not zero 3 Accessory equipment 3 8 Monitor Event Event marker signal activated at fraction collector start and markat each tube change T he signal is a short 50 ms pulse GND Ground for start stop and event marker signals For two channel recorders GradiFrac supplies a signal corresponding to the buffer gradient Grad 1V Signal for recording the gradient 0 1 V corresponding to 0 100 B AGND Analogue ground for the gradient signal M onitor UV 1 is recommended for use with GradiFrac The output from the monitor is connected to the M onitor ports on the rear panel of GradiFrac to supply a signal for peak detection functions Recommended cable Signal cable Part N o 19 6006 01 TheRange switches on the rear panel of GradiFrac should be set to the full scale output from the monitor 10 mV for M onitor UV 1 The output from the monitor should be connected separately to the recorder and to GradiFrac There is no output from GradiFrac for the
30. monitor signal An adaptor cable for connecting UV 1 to both GradiFrac and a recorder is supplied with GradiFrac 11 4 Unpacking and assembly 4 Unpacking and assembly 4 1 Unpacking Unpack the GradiFrac carefully and check the contents of the carton against the included packing list Inspect each item for damage that may have occurred during transit Save the packing material and carton for later use Tube racks are assembled in the GradiFrac bowl from some or all of tube 4 2 Assembling holder tube guide and tube support Figure 5 Details of assembly differ tube racks according to the length and diameter of the tubes tube support cutout tube guide single cutout tube holder Fig 5 Tube rack components Components of the tube rack are positioned in the bowl by engaging the cutouts either single or L shaped on the edge of the component with the ribs on the inside of the bowl This ensures that the different components are correctly aligned with each other 12 4 Unpacking and assembly 4 2 1 Tube racks 12 mm and 18 mm Tube lengths 50 85 mm L shaped cutout single cutout Fig 6 Assembly of 12 mm and 18 mm tube racks for 50 85 mm tubes 4 Unpacking and assembly 14 Fit the tube support using the L shaped cutouts The support should be seated in the lower position in the bowl with the circular marks on the underside Fit the tube guide in the bowl wi
31. n D SUB connector to the port on the rear panel of the recorder and the red and black pin connectors to channel 2 red on the top of the recorder Set the red switch on the recorder voltage selector for channel 2 to 1 V Note Chart speed must be set locally on the recorder The int ext button on REC 102 should bein position int The Rec on off button should be in position on 4 9 6 Monitor Set the DIP switch on the rear panel of GradiFrac see Section 2 2 to correspond to the full scale output from the monitor 10 mV for M onitor UV 1 Use the Y cable supplied with GradiFrac to connect the monitor output to both GradiFrac and recorder Connect the monitor output to the M onitor port on GradiFrac taking care to match the and poles Connect the monitor output separately to channel 1 input on the recorder Set the blue switch on the recorder voltage selector for channel 1 to correspond to the full scale output from the monitor 10 mV for M onitor UV 1 4 9 7 Power supply Connect the 24 V DC outletfrom the power conversion unit to the Power Socket on the rear panel of GradiFrac Connect the power conversion unit to the mains supply The conversion unit adjusts automatically to mains voltages in the range 100 240 V AC 50 60 Hz Connect tubing as shown in Figures 13 and 14 M ake surethat the tubing connections to the solenoid valves are made to the correct valve outlets as shown in Figure 14 Fig 13 Connecti
32. ng tubing to PSV 50 4 Unpacking and assembly 4 11 Starting the system 4 12 Turning the system off Valve iV 7 GradiFrac Fig 14 System tubing connections Turn on the power to GradiFrac first then to the other components in the System Turn off the power to the pump and recorder first If GradiFrac is turned off while power is still supplied to pump or recorder these instruments will start to run according to the settings on the instruments themselves This can be particularly important for the pump since liquid or air may otherwise inadvertently be pumped through the system 21 5 Setup and programming 5 1 GradiFrac Main menu 5 2 Setup menu 22 5 Setup and programming The GradiFrac main menu is divided into four blocks Main menu RUN METHOD Main menu MANUAL RUN Main menu PROGRAM METHOD Main menu SETUP Fig 15 GradiFrac main menu When GradiF rac is first switched on the Run method option is displayed Press the arrow keys to move between menu options The Setup menu block sets general operation parameters Setup should be performed when GradiFrac is first installed and whenever parameters need to be changed Current setup parameters apply during manual operation and method programming During method controlled operation the method base time volume and fractionation base time volume drop which were current at the time of programming apply All other Setup
33. ning a method end Main menu RUN METHOD RUN METHOD Main menu MANUAL RUN Main menu PROGRAM METHOD Main menu SETUP arrows to view or change TIME VOLUME CONC 8B TIME VOLUME FLOW ml min TIME VOLUME FRACTN TIME VOLUME VALVE ACTIVE Y N TIME VOLUME PEAK COLLECT Y N Method ended TIME VOLUME SLOPE amp min Method completed Fig 23 The Run method control block 1 Fill the tube rack with tubes starting at position 1 and adjust the delivery arm so that the tube sensor rests against the outer side of tube no 1 2 Choose Run method from the main menu and press EN TER 3 Enter the number of the method to run 0 9 the numbers of programmed methods are displayed and press ENTER to start the method If you enter the number of a method which has not been programmed GradiFrac will respond with the message M ethodisempty Press ENTER to acknowledge and select another method number 4 When the method is finished GradiFrac will display the message M ethod is completed Press end to return to the main menu Press ENTER twice to re run the method 6 2 2Interrupting a method There are three ways to interrupt a method pause stops method operation but does not end the method Press continue to resume method operation hold holds method time volume and stops gradient changes but continues to operate the pump and fraction collector Press hold again to display t
34. not pressed HiLoad Pump P 50 only Flow rate too high or fraction size too small Attempt to use peak collection with no fraction size N o drops detected drop sync mode or drop fraction base only N o drops detected due to too high flow rate or dirty drop sensor Attempt to program more than 52 breakpoints in a method Attempt to set an alarm later than method completion Drop sensor cable not connected or dirty drop sensor setup mode only The method consists of only one breakpoint Wait until the gradient is complete or press pause or hold followed by ENTER to stop the gradient Check that the pump is switched ON and that the communication cable is properly connected Press ENTER then continue to ignore the message pump must be controlled separately Find and correct the cause of the overpressure Tubing or column may be blocked or the flow rate may be too high for the column Press the RUN button on the pump Reduce the flow rate or set a larger fraction size Set a fraction size before activating peak collection Check that buffer drops from the outlet Clean the drop sensor If the error persists contact Amersham Biosciences service or run without drop sync and drop counting Reduce the flow rate or clean the drop sensor If the error persists contact Amersham Biosciences service or run without drop sync and drop counting Reduce the number of breakpoints in the method Se
35. o a power supply recommended by Amersham Biosciences Fraction tubes are held in racks which fit into the bowl of GradiFrac Three standard racks are available 1 12 mm rack for 175 tubes diameter 12 mm 2 18 mm rack for 95 tubes diameter 10 18 mm 3 30 mm rack for 40 tubes diameter 30 mm GradiFrac is delivered with the 18 mm rack The other racks are available as optional accessories The 12 mm and 18 mm racks can be assembled to accommodate tube lengths 50 85 mm or 85 180 mm The 30 mm rack can be assembled to accommodate tube lengths 30 50 mm 50 85 mm or 85 180 mm For correct operation all tubes in a collection sequence must be of the same size See Section 4 2 for details of tube rack assembly Note In the 18 mm rack tubes longer than 100 mm should have diameters at least one tenth of the length Otherwise pressure from the delivery arm can force the tube toward the centre of the rack Tubes are arranged in the rack in a counter clockwise spiral from the outside edge in to the centre Tube positions are numbered on the tube holders Custom tube racks can be made from block styrofoam or by drilling tube holders from plastic plates of the appropriate size It is important that the angle and distance between tubes are not too great so that tube changes can be made smoothly During operation the bowl and tube rack are turned by the rubber covered drive sleeve at the side of the GradiFrac base The position at which the rac
36. oints or enter the time or volume for an existing breakpoint to move directly to that breakpoint 5 6 1 Inserting a breakpoint To insert a breakpoint enter the breakpoint time or volume at Edit break point Figure 19 and press ENTER Set the parameter values as required A newly entered breakpoint inherits default parameter settings from the closest previous breakpoint in the method 5 6 2 Changing breakpoint parameters To change the time or volume for a breakpoint insert a new breakpoint at the required time or volume as described in Section 5 6 1 then delete the unwanted breakpoint as described in Section 5 6 3 You may need to edit parameter settings for the new breakpoint To change other parameters for an existing breakpoint move to the required breakpoint press ENTER then change the parameters as required Remember that all parameters are set at every breakpoint A new parameter value must be entered for all breakpoints where it is to apply This is particularly important for flow rate and fraction size which are often set early in a method and apply throughout the method If for example the flow rate is changed only at the first breakpoint the previous flow rate will be restored at the second breakpoint 5 6 3 Deleting a breakpoint To remove an existing breakpoint move to the required breakpoint and set the time volume to 0 use the CE key to clear the existing value GradiFrac will ask you to confirm that the break
37. point should be deleted 29 5 Setup and programming 5 7 Documenting The programmed gradient shape for any stored method may be documented methods on the recorder when method programming is completed Set the recorder chart speed to 10 or 20 mm s and answer Y to the prompt SHOW CONC B ON REC OUT at the end of the Program M ethod block Figure 19 The complete method is recorded in 10 s It is important that you make notes of the purpose and structure of methods stored in GradiFrac Without these it is easy to forget the purpose of a method which is identified by number alone and it may be difficult to reconstruct the method if it is accidentally deleted The form on page 32 suggests a standard for recording method notes and breakpoints 5 8 Method Example 1 illustrates a simple GradiFrac method for a gradient elution with a linear gradient from 0 to 60 96 B then an instantaneous change to 100 96 B examples Peak detection is set to on The sample is applied manually after 1 minute and fraction collection starts at the beginning of the gradient allowing 25 ml flow through to run to waste 100 B 80 60 40 201 0 10 20 30 40 50 Fraction collection Fig 21 Buffer gradient in the example method Table 5 1 Breakpoints for example method 1 Values actively entered for each breakpoint are shown in bold Unchanged values are grey Time
38. r with GradiFrac are listed below Some typical system arrangements are illustrated in the appendix Component Recommended models Rack GradiFrac System Rack Pumps Peristaltic Pump P 1 HiLoad Pump P 50 Solenoid valves PSV 50 for gradient formation PSV 50 for flow diversion PSV 50 for sample application or three buffer systems optional Manual valves IV 7 Mixer Mixer 0 6 ml Monitor UV 1 Recorder REC 102 Columns Prepacked HiLoad and HiTrap columns see Chapter 10 A rack Code No 18 1993 05 is available for convenient mounting of system components Recommended pumps for use with GradiFrac are Pump P 1 for flow rates 0 8 3 ml min and HiLoad Pump P 50 for flow rates up to 50 ml min Other pumps may also be used provided that the pump speed is frequency controlled GradiFrac controls pump speed by a pulsed output signal with frequency range 0 4 kH z The programmable flow rate range is 0 99 9 ml min The pump calibration constant can be entered directly if known or the pump can be calibrated by collecting a measured volume See Section 5 3 for details of pump calibration Connection to GradiFrac 15 pin D SUB Recommended cable Communication cable Part N o 19 6005 02 3 Accessory equipment 3 4 Mixer 3 5 Solenoid valves 3 6 Injection valve 3 7 Recorder 10 Pump connector pin assignments Pin no Function 1 6 Not used in GradiFrac 7 0 V ground 8 Not used in GradiF
39. rac 9 Pump busy 10 Pump operable 11 Not used in GradiFrac 12 Pump control 13 Frequency from pump for calibration 14 Frequency to pump pump speed 15 0 V ground As long as GradiFrac is switched on the pump is controlled entirely from GradiFrac To usethe controls on the pump itself switch off the power to GradiFrac or disconnect the pump communication cable Amersham Biosciences M ixer 0 6 ml is suitable for most applications using GradiFrac For high flow rates above 8 ml min M ixer 6 0 ml is recommended M ixer 0 6 ml is connected to the M ixer port on the rear panel of GradiFrac which supplies power to the mixer 24 V AC 50 Hz The mixer operates as long as GradiFrac is turned on M ixer 6 0 ml cannot be supplied with power from GradiFrac and requires a separate power source Solenoid valve PSV 50 should be used with GradiFrac PSV 50 may be connected to the ports marked Frac Switch and Aux on the rear panel of GradiFrac with the following functions Frac Diverts flow to waste when fractions are not being collected fraction size is set to 0 Switch Switch valve for gradient formation Aux O ptional third switch valve e g for three buffer systems and for automatic application of large sample volumes Injection Valve IV 7 is recommended for sample application If desired a system may be configured with the auxiliary switch valve as a sample application valve see Appendix Recorders such as A
40. returned to its central position over the bowl when not in use N o regular maintenance is required The surfaces of GradiFrac may be wiped with mild aqueous cleaning solutions or with ethanol M aintenance of the power supply should be carried out by authorised Amersham Biosciences personnel only M aintenance of other components in GradiFrac System is described in the respective manuals 37 8 Troubleshooting 8 Troubleshooting The table below lists messages which may appear on the GradiFrac display with explanations and suggested actions If other error messages appear for which the corrective action is not obvious contact technical service Message Cause Action Tube pos error No more tube available M ethod is empty Warn Check that tubepos is OK No Flow Stop gradient in HOLD or PAUSE Recalibrate pump or flow too high Recalibrate pump or flow too low Warning Switch valve too fast Switch cycle time too fast Tube sensor active due to jammed rack Tube sensor broken No more tubes Rack movement jammed Delivery arm in stand by position Attempt to run method without instructions Tube sensor between tubes only at start of run Attempt to start a manual gradient or program a breakpoint with no flow Attempt to change CONC B during a running gradient Too high flow rate or incorrect calibration factor Too low flow rate or incorrect calibration fa
41. s menu blocks and returns to the main menu Advances the fraction collector one position except when GradiFrac is paused The tube feed is delayed according to the Setup value for Delay UV to Frac if the flow rate is not zero Switches between display of Yes and No answers to prompts in manual control and method programming Pressing EN T ER accepts the displayed answer Pauses all manual or method controlled operation without ending the method Pressing continue restarts operation 2 Description 2 2 Rear panel H olds method time or volume and holds gradient at the current concentration All other functions continue uninterrupted Pressing continue restarts the gradient Resumes normal operation from the paused or held state The power supply switch and electrical connections for accessories are mounted on the rear panel GRADIFRAG nn ART NO 18 1993 01 RECORDER MONITOR 14 voc iw OW orr VALVES TUBE SENSOR PUMP POWER MIXER FRAC AUX SWITCH 4 i e QOO c Pegga elo Fig 2 GradiFrac rear panel Therear panel switches and connectors are described briefly below See Section 4 9 for more details of connecting accessories to GradiFrac ON OFF When the red circle is visible the instrument is off Tube Connector for tube sensor and drop counter mounted on the sensor delivery arm Power Connector for 24 V DC power supply A power conversion uni
42. t for use with 100 240 VAC mains is supplied with GradiFrac Mixer Connector for supplying power to Amersham Biosciences M ixer 0 6 ml Frac Connector for flow diversion valve PSV 50 which diverts flow between fraction tubes and waste Aux Connector for auxiliary valve PSV 50 Switch Connector for gradient switch valve PSV 50 Pump 15 pin D SUB connector for control of pump e g Pump P1 or HiLoad Pump P 50 Recorder Start Stop Signal for starting and stopping the recorder chart Event mark Event marker signal activated at fraction collector start and at each tube change GND Ground for start stop and event marker signals Grad 1V Signal for recording the gradient 0 1 V corresponding to 0 100 B A GND Analogue ground for the gradient signal 2 Description 2 3 Power supply 2 4 Tube racks Monitor Range DIP switches to set the monitor signal range The switches should be set to correspond to the full scale output from the monitor Figure 3 Input Input connector from the monitor The monitor signal is used for peak detection Fig 3 DIP switch settings for monitor output voltages GradiFrac uses a 24 V DC power supply eliminating the need for fuses or circuit breakers in the instrument A power conversion unit for use with 100 240 VAC mains is supplied with GradiFrac The power conversion unit must be placed in a dry place Note GradiFrac should only be connected t
43. t as soon as the instruction is confirmed with ENTER You can change parameters at any time during manual control with the exception of the gradient Gradient parameters can only be changed 1 if there is no gradient running or 2 if the system is paused or held To end manual control press end 6 1 1 Setting a gradient Set a constant concentration of buffer B with CONC 96B Figure 22 The concentration will apply as soon as you press ENTER To changethe concentration instantaneously step gradient simply enter a new value To set a gradient move to GRADIENT Figure 22 press ENTER then enter the target concentration of buffer B and thetime or volume after which the target is to be reached The base is determined by the current method base setting in Setup see Section 5 2 For example the following entries start a gradient from the current concentration to 7096 B over a period of 10 minutes TARGET CONC B 70 33 6 Operation 34 REACH TARGET AFTER min 10 0 If a gradient is already running GradiFrac will respond with the message Gradient is already on The display changes to CONC B after 3 s Press pause or hold followed by ENTER to stop the gradient and enter new parameters A gradient can only be started if the pump is running If a gradient is requested when no flow has been set GradiFrac will respond with the message No Flow and return to the beg
44. t manual and method controlled operation until a new value is set 5 2 6 Setting drop synchronization Set drop synchronization with Drop sync active Figure 16 If drop synchronization is active tube changes will only occur directly after a drop is registered by the drop counter to minimize spillage between tubes Drop synchronization operates in all fraction collection modes time volume and drop Drop synchronization is only possible at flow rates below 3 ml min The drop synchronization setting will apply for all subsequent manual and method controlled operation until a new value is set To calibrate the pump move to Pump calibration procedure in the Setup menu Figure 16 and press ENTER Follow the steps below 1 Set the speed on the pump to a value close to the flow rate which will be used subsequently For Pump P 1 use the flow rate guide supplied with the pump 2 M ake sure there are no air bubbles in the pump or delivery tubing Enter the setup block in the GradiFrac main menu 4 Press 4 untii the display shows Pump calibration procedure then press ENTER to enter the calibration process T he display will show Start collecting 5 Place a suitable measuring vessel under the outlet tubing and press ENTER The pump will start to operate and the display will show Stop collecting 6 Collect a volume large enough for accurate measurement then press end to stop collecting The pump will stop and the display will s
45. t the alarm within the method Use extra breakpoints with zero flow rateto extend the method time Check the drop sensor connection or clean the drop sensor Add breakpoints to the method 39 9 Technical specifications 9 Technical specifications GradiFrac Programming base Programming capacity Memory protection Pump control Gradient formation Fraction collection Time base Volume base Drop base Tube change time Rack capacity Event mark Flow diverter Monitor input range Recorder control Valve control Mixer Power supply Power consumption Dimensions w x d x h Weight Environment Safety standards EMC Safety ml or min 10 separate methods 52 breakpoints each EEPROM Remote for Pump P 1 and HiLoad Pump P 50 Single pump and switch valve 0 1 99 9 min 0 1 99 9 ml 1 999 drops 0 3 0 6 s tube rack 18 mm 95 tubes rack 18 mm 50 ms contact closure Solenoid valve PSV 50 0 10 mV 0 100 mV 0 1000 mV Controls REC 101 and REC 102 3 outports for Solenoid Valve PSV 50 switch valve flow diverter auxiliary 0 6 ml 24 V DC Power Supply for 100 240 VAC 50 60 Hz 15 VA maximum 290 x 300 x 390 mm 3 9 kg 4 to 40 C 20 95 relative humidity 84 106 kPa 840 1060 mbar atmospheric pressure This product meets the requirement of the EMC Directive 89 336 EEC through the harmonized standards EN 50081 2 emission and EN 50082 1 immunity Note This is a class A product
46. th the tube numbers facing up using the single cutouts The guide should come to rest approximately 1 cm abovethe tube support Fit the tube holder in the bowl with the tube numbers facing up using the single cutouts ensuring that tube position 1 is directly above tube position 1 in the tube guide The tube holder is held in position by the indentations in the ribs of the bowl Check that the tube holder is level and firmly seated before filling it with tubes Tube lengths 85 180 mm L shaped cutout Fig 7 Assembly of 12 mm and 18 mm tube racks for 85 180 mm tubes Do not usethe tube support Fit the tube guide in the bowl with the tube numbers facing up using the L shaped cutouts The guide should be seated in the lower position in the bowl Fit thetube holder in the bowl with the tube numbers facing up using the single cutouts ensuring that tube position 1 is directly above tube position 1 in the tube guide The tube holder is held in position by the indentations in the ribs of the bowl Check that the tube holder is level and firmly seated before filling it with tubes 4 Unpacking and assembly 4 3 Mounting the bowl 4 2 2 Tube rack 30 mm Tube lengths 30 50 mm T Tube holder 30 mm ube guide 30 mm Tube guide 18 mm S E lt gt sg SS Fig 8 Assembly of 30 mm tube racks for 30 50 mm tubes Fit the tube guide from the 12 or 18 mm rack using the single cutouts Fit the t
47. ttach the flow diversion valve PSV 50 to the valve holder solenoid valves Figure9 Fig 9 Mounting the flow diversion valve 16 4 Unpacking and assembly 4 8 System mounting Sections 4 8 4 10 describe assembly of GradiFrac System System components are listed in Section 3 1 A separate System Installation Guide is available 1 Prepare GradiFrac as described in Sections 4 2 4 7 2 Attach the solenoid valve for gradient formation to the valve bracket If an auxiliary valve is used attach this to the same valve bracket alongside the gradient valve Figure 10 Fig 10 Fitting the gradient valve to the valve bracket The auxiliary valve can be fitted to the same bracket 3 Assemble the rack as described in the instructions provided 4 Preparethe other components for use as described in the respective User M anuals 5 Placethe system components in the rack as shown in Figure 11 17 4 Unpacking and assembly 4 9 System 18 connection A IV 7 PSV 50 UV 1 i control unit gradient valve GradiFrac UV 1 optical unit REC 102 Fig 11 Placing the system components in the rack Connect components electrically to GradiFrac as described below Figure 12 Use a screwdriver to fit the green connectors supplied with GradiFrac to the valve recorder and monitor cables that are to be connected to GradiFrac rear panel Valve cables have no polar
48. ube support on top of the tube guide to give the extra height necessary when using short tubes Fit the 30 mm tube guide using the single cutouts Fit the tube holder in the top of the bowl with tube position 1 directly above tube position 1 in the tube guide Tube lengths 50 85 mm Fit the tube support using the L shaped cutouts The support should be seated in the lower position Fit the tube guide using the single cutouts Fit the tube holder in the top of the bowl with tube position 1 directly above position 1 in the tube guide Tube lengths 85 180 mm Do not use the tube support Fit the tube guide in the bowl with the tube numbers facing up using the L shaped cutouts The guide should be seated in the lower position in the bowl Fit the tube holder in the bowl with the tube numbers facing up ensuring that tube position 1 is directly above tube position 1 in the tube guide The tube holder is held in position by the indentations in the ribs of the bowl Check that the tube holder is level and firmly seated before filling it with tubes M ove the delivery arm out to the second stop Place the bow containing the rack over the central spindle Pull the spring loaded drive sleeve away from the bowl so that the bowl drops into position 15 4 Unpacking and assembly The bow should come to rest with a gap of approximately 1 cm between the bottom of the bowl and the GradiFrac base 4 4 Inserting tubes nsert sufficient
49. y arm adjusts the tube sensor for large or small diameter tubes Turn the switch to the smaller marking for tubes in the 12 mm rack and to the larger marking for tubes in the 18 and 30 mm racks The delivery tubing is inserted into the tubing holder A tubing length guide is provided midway along the upper side of the delivery arm to help in positioning the end of the delivery tubing correctly with respect to the drop counter This guide is a small hole with a white ring See Section 4 6 for a description of how to fit the delivery tubing correctly The supplied solenoid valves PSV 50 may be connected to the ports marked Frac and Switch on the rear panel of GradiFrac with the following functions Frac Diverts flow to waste when fractions are not being collected fraction size is set to 0 Switch Switch valve for gradient formation 2 Description 2 7 Delivery tubing The port marked Aux is for an optional third solenoid valve e g for three buffer systems and for automatic application of large sample volumes Solenoid valve PSV 50 has three outlets IN NO Normally open NC Normally closed See Figures 13 and 14 for tubing connections to the valves 0 8 mm i d tubing is recommended and is supplied with GradiFrac Tubing connector M 6 Code N o 19 1993 50 is used for connection 3 Accessory equipment 3 1 System components 3 2 Rack 3 3 Pumps 3 Accessory equipment Recommended components for use togethe
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