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マニュアル Gradient Programmer GP

1. Fig 10 Connection of pumps to the GP 250 Plus HiLoad Pump P 50 Code No 19 1992 01 may be used as an eluent delivery pump For gradient applications a switch valve is required supplied in Gradient Kit P 50 Code No 19 1992 50 HiLoad Pump P 50 that should be controlled from the controller as an eluent pump is connected to the PUMP A socket on the controller Use the Communication cable Code No 19 6005 02 or 18 0012 75 Do not use the PUMP B socket on the controller Connect the Gradient Kit P 50 switch valve to pins 5 and 7 on the controller Fig 11 Controller 57 PumpA PumpB m edad dadadasentdbesdaadiatatd J Solenoid valve Pump P 50 Y Optional sample valve Switch valve Fig 11 Connection a Pump P 50 as an eluent pump 6 Installation and connection of equipment 6 3 Connection of recorder Speed signal Event mark from the fraction collector Gradient 1 V signal The Recorders REC 101 and REC 102 are connected to the GP 250 plus with the Recorder cable Code No 18 1017 66 supplied in the Cable kit GP 250 plus This signal is used to control the chart drive speed from the GP 250 Plus refer to Fig 12 GP 250 Plus ee Communication Cable Code No 19 6005 02 FRAC 100 200 REC 102
2. Recorder Cable Code No 18 1017 66 Fig 12 Connection of the recorder to the GP 250 Plus 1 Connect the External control cable Code No 18 1017 66 15 pin D connector to the recorder 2 Connect the green 6 pole connector to the RECORDER input pos 12 17 on the rear panel on the GP 250 Plus To get an event mark signal at the tube change connect the long cable that exits from the 15 pin D connector to the Even mark port at the rear of the fraction collector refer to Fig 12 This signal is used to record the gradient profile Use the short cable that exits from the 15 pin D connector refer to Fig 12 1 Connect the wire marked to the red Pin Connector supplied with the recorder Connect the wire marked to the black Pin connector supplied with the recorder 2 Insert the Pin connector in the lower signal input terminals short pen on the back of the recorder Now you also have the event mark from the fraction collector on this channel If you want the event mark on the same channel as UV you have to switch the positions of the gradient Pin connectors with the UV pin connectors on the chart recorder 27 6 Installation and connection of equipment 6 4 Connection of fraction collector 6 5 Connection of mixer 6 6 Connection of 28 accessories to the program mable outports The Fraction Collectors FRAC 100 FRAC 200 and SuperFrac can be controlled from the GP 250 Plus
3. ea Fg we jaran 24 25 26 27 28 Fig 16 Connection of a P 50 or a P 1 pump to the GP 250 Plus 6 Installation and connection of equipment Note on programming By default a pump connected to port 1 and 2 will run when the port is activated using the PORT SET no 1 instruction on the controller and stop when the port is deactivated As an example when a pump is connected to port and PORT SET set to 1 1 the pump will run The pump stops when PORT SET is set to 1 0 The reverse is true for port 8 and 9 Note on port polarity If required the port polarity can be changed with P POL instruction in the controller calibration block The polarity setting determine the effect of the PORT SET instruction as below PORT SET instruction P POL 0 1 0 inactive active 1 active inactive By default the polarity is 0 for all ports except 1 and 2 which have polarity 1 Note Do not use this connection for controlling pumps which are required for gradient formation Use the sockets marked PUMP A and PUMP B instead 31 7 Operation 7 1 Start up 7 2 Programming 32 a method 7 Operation Before programming and starting any method please check the system according to the following list 1 Check that all components are correctly installed according to section 6 Installation and connection of equipment 2 Turn on the pumps monitor recorder and fraction collector 3 S
4. Connection to FRAC 100 FRAC 200 Connect the Communication Cable Code No 19 6005 02 or the fraction collector cable in the Cable Kit GP 250 Plus Code No 18 1017 68 between the socket marked FRACTION COLLECTOR on the GP 250 Plus and the socket marked REMOTE on the FRAC 100 or FRAC 200 see Fig 12 Communication Cable Code No 19 6005 02 cE fmm EXTERNAL 10 RAG FLOW METER EI ve Q O eue Rae ae Fig 13 Connection of the SuperFrac to the GP 250 Plus A 0 6 ml fluoroplastic mixer and a rod for attaching it to a laboratory stand are supplied with the GP 250 Plus The mixer can be used at working pressures up to 5 MPa When running at flow rates higher than 4 ml min the Mixer 1 6 ml Code No 18 4580 01 or the Mixer 6 ml Code No 18 3500 01 110V and 18 3501 01 220 V are recommended In the two small mixers the buffers are stirred in a mixing chamber powered from the GP 250 Plus The Mixer 6 ml does not need to be connected to the GP 250 plus Connection to the GP 250 Plus The mixer is connected to a 2 pole connector which is connected to the socket marked MIXER no 8 and 9 on the GP 250 Plus no polarity Fig 14 The GP 250 Plus has seven programmable outports Outports 1 2 8 and 9 are programmabl
5. MIXER 24 off at about 20 kHz with a 40 duty cycle i e 0 02 msec ON and 0 03 msec OFF A solenoid valve is too slow to respond to such short power offs and will therefore remain ON when activated Each outport is protected against overcurrent by a PTC resistor that is activated if current continuously exceeds 300 mA Power Switched power for the switch valve used in one pump gradients Same as above No 5 on GP 250 Plus rear panel A port 0 For control of the switch valve Mix volume is programmed in the Calibration block Active equal to 0 Can sink 300 mA When two pumps are used for gradient formation it has the same function as outports 3 and 4 and is adressed as port No 0 No 6 on GP 250 Plus rear panel B Inverted to A port 0 Floating when not used for gradient formation No 7 on GP 250 Plus rear panel Functions Positive or negative logic can be selected for outports 0 4 and 8 9 in the Calibration block Positive logic active floating voltage shown as 1 or negative logic active 0 V shown as 0 Default means that all outports have negative logic except 1 and 2 To change the polarity for outport 3 to positive logic enter 3 1 and press do store at PORT POL in the Calibration block PORT SET X Y X portnumber 0 1 2 3 4 8 or 9 Y status 1 active 0 inactive PT XXXXXXXXXX same port numbers and status as in programmable functions A 24 V power source for external devices No 10 on GP 2
6. The tenth level of CALL METH will be ignored However as a called method is executed and returns to the calling method the return address stored in the call memory will be cleared To avoid overcalling when using many sub methods link the methods with a separate control method that controls the sequence of sub methods to be called see figure below 13 4 Description of keyboard and programming LOOP TMS END OF LOOP FEED TUBE HOLD ALARM WASH A B 14 control method sub method CALL METH 1 METHOD 1 method Y loop CALL METH 2 Ex METHOD 2 wv CALL METH 3 METHOD 3 etc Y Fig 3 The principles of linking methods by using the CALL METH instruction During a call a C will appear before the method number in the display The instructions stored between LOOP TMS and END OF LOOP are repeated the number of times max 99 specified Loops within a loop are not allowed i e only one level loop nesting in a specific method However each method has its own unique set of loop variables This means that loops within a loop can be constructed with a combination of LOOP instructions and CALL instructions Example A program for 1 000 loops Method no 0 0 0 LOOP TMS 20 0 0 CALL METH 1 0 0 0 END OF LOOP Method no 1 0 0 LOOP TMS 50 Instructions to be looped XXX END OF LOOP RUN METHOD 0 will execute the instructions in Method no 1 1000 times Note If a LOOP TMS in
7. 1 Majns power supply Before operating the unit read section 6 Installation and connection of equipment carefully 4 Description of keyboard and programming 4 Description of key board and programming The keyboard Fig 1 is of touch type and has very good chemical resistance It contains function keys and numerical keys The function keys can be divided into three groups control keys for manual control e g of a method in progress special function keys for entering different blocks which contain instructions to be executed or programmed and special keys for the Review block The different blocks available are shown in Fig 2 step forward 7 step backward do store Fig 1 The keyboard method file manual Evaluation oc Calibration block Programming block Fig 2 Block scheme Keys for data entry 0 1 9 Data entry Decimal point CE Clear entry Resets entered data to 0 4 Description of keyboard and programming 4 1 Control keys end hold gradient step forward step backward store Feeds paper on the recorder Resets a programmed alarm or check code Stops a running method resets all activities and sets functions to initial inactive conditions RUN METHOD O is displayed Concentration is set to 0 Flow rates are set to 0 Chart speed is set to 0 Outports are set to the inactive state
8. 800 mA SB 100 110V 19 8687 01 oa 2 Communication Cable 19 6005 02 1 Signal Cable 19 6006 01 1 Mains cable 120 V 2m 19 2447 01 1 Mains cable 220 V 2m 19 2448 01 1 Solenoid Valve PSV 50 19 1994 01 1 Remote Control Adaptor 19 6008 01 1 Stop plug for mixer 19 5170 01 2 Mixer 24 V 19 6700 01 1 Cable Kit GP 250 Plus 18 1017 68 1 2 Pole connector female 19 8629 01 2 3 Pole connector female 18 0012 52 2 4 Pole connector female 18 0881 01 1 5 Pole connector female 18 0012 53 1 6 Pole connector female 18 0012 54 1 42 Application Part 2 Application This section describes the programming of some of the different methods used in our laboratories for practical applications Each example given is supplemented with a short description of the method schematics showing the components involved and a programming example In some cases the programming examples are limited to a particular function In such cases further instructions should be added to get a complete separation method Most of the examples of FPLC System given in this chapter are based on P 500 pumps and the manual V 7 and V 8 valves Of course these components may be exchanged for other FPLC equipment i e P 1 P 50 or P 6000 pumps may be used instead of P 500 and FRAC 100 or SuperFrac fraction collectors instead of FRAC 200 The examples given are just suggestions There are other ways to program the GP 250 Plus to obtain the same result 43 1 Elut
9. Active if the fraction collector is operable When attempting 9 to control a fraction collector that is not operable a check code is displayed Not collecting 0 Active when fraction collector is not collecting If the 11 GP 250 Plus is controlling the fraction collector with an active RUN signal the GP 250 Plus will be paused if this signal is active Outports all open collector Pause 0 Sets the fraction collector in pause mode when active 1 Automatically active if GP 250 Plus is paused otherwise controlled as PORT 5 Run 0 Starts fractionation if active stops if inactive Controlled as 2 PORT 6 Feed A 0 1 sec pulse will feed a new tube when the 3 programmable function FEED TUBE is executed Recycle 0 Enables the fraction collector to find the first tube when 5 active Should be inactive to start fractionation Controlled as PORT 7 Operable 0 Active signal tells the fraction collector that the GP 250 10 out Plus is operable Connected to signal ground i e indicates operable as soon as the GP 250 Plus is connected Flow rate Frequency proportional to the flow rate 13 Ground Voltage reference for the signals above Same as logic 15 ground Recorders such as the REC 101 and REC 102 as well as other recorders driven by stepper motors can be controlled from the GP 250 Plus Chart speed and pen down can be controlled by programmable functions or controlled in the Manual block Pen down signal is automatically given
10. and an inport in order to define an electric level when the inport is not connected Programmable Read Only Memory This type of memory contains information on how the instrument should work Random Access Memory or read and write memory This type of memory contains variables e g methods and peak data An electric circuit is given e g from 24 V to solenoid valve to outport The outport can then sink max 300 mA current to activate the valve This indicates the lowest permitted resistance of the solenoid of the valve Defines how digital information is electrically represented ON logic 1 represented by high voltage OFF logic 0 represented by low voltage 0 V To avoid overheating solenoid valves 24 V is only supplied for about 30 msec Power is then reduced by switching the 24 V on and off at about 20 kHz with 40 duty cycle ie 0 02 msec ON and 0 03 msec OFF A solenoid valve is too slow to respond to such short power offs and will therefore remain on when activated Transistor Transistor Logic A family of integrated digital semiconductor components Input and output voltage levels are standardized which makes it easy to interface instruments which use TTL components 41 12 Spare parts and accessories 12 Spare parts and accessories Please order spare parts and accessories using the designations and code numbers given below Designation Code No No per pack Fuse 400mA SB 220 240V 19 8686 01 Fuse
11. in called methods 49 3 Fraction collection 50 3 Fraction collection Fraction collection is achieved by controlling a FRAC 100 or FRAC 200 Controlling FRAC 100 and FRAC 200 The fraction collectors are controlled by programming the instructions below at appropriate time volume Time min Function Value 0 00 PORT SET 6 1 start 2 00 PORT SET 6 0 stop PORT SET 5 1 pause PORT SET 5 0 continue PORT SET 71 recycle will move thefraction collector back to where collection started only FRAC 200 and SuperFrac PORT SET 7 0 reset 4 Looping a method for manual repeated injection 4 Looping a method for manual repeated injection A separation may be repeated manually a number of times The most convenient way is to loop the method with the instructions LOOP TMSEC and END OF LOOP e 100 en 2 S 80 tz D o S O 50 40 5 5 2 10 18 20 21 2 Time min LOOP TMS END OF LOOP Fig 26 Looping a method Method Gradient segmented with different slopes from 5 5 to 80 B in 18 ml Flow 1 0 ml min Chart speed 0 5 cm min Sample loop 200 ul Fraction collector FRAC 200 Programming base time min 51 4 Looping a method for manual repeated injection 52 Method no 1 Time min Function Value 0 00 ML MIN 1 00 initial conditions 0 00 CM MIN 0 50 0 00 LOOP TMS 7 00 0 00 HOLD sample injection 0 00 ALARM 0 1 2 00 CONC B 5 50 2 00 FEED TUBE 2 00 PORT SET 61 0 10 00 C
12. of the risk 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 1995 Amersham Biosciences AB All rights reserved No 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 Part 1 Technical description 1 2 3 4 5 Introduction 5o irrito titt ha PO EEKE Po gott Poet pope bugs 3 Specifications vocis opas aticep buteri ens eut edu aa e Quran er beno t noa 4 Unpacking i iius ceno a ee tested Gai eve Lodi ein EE a D 6 Description of keyboard and programming ssssssssssseeeee 7 41 Control keys eee eee eee eee 8 42 Cahbration block itt b i rhopebee ip orte SER P eheu 9 43 Evaluation block o c bee c E PO EC CC EA Gas 10 4 4 Manualblock ERU C EN ER TEE si lena abbr MER Odd 11 45 Method block oC oe oo er SAG rds 12 4 6 Programming block sssssese eee 12 4 7 Listofinstructions sssssssse eme eee een 13 4 8 Review block uoo eeu TR e qe res rena eds 15 4 0 Display c E 16 410 EED indicators zresa ana EE od cay erg bh EAST 17 4 11 Short instruction esee eee 17 Description of rear panel and connections sees 18 51 Mainspower supply ooo te e entered ees 18 5 2 Pumps sene nnne enne eene enn 18 5 3 Fraction collect
13. or as an with an instruction in rate the method at volume 0 before running the method 35 8 Check codes Check Explanation Action code number 14 Attempt to drive pumps A and B with a frequency greater than Set a lower flow rate and check pump calibration value 15 kHz Pump frequency must be less than 15 kHz 20 The concentration of solvent B has been set greater than 100 Set the concentration of solvent B less than or equal to manually or a method has been started with a CONC B instruction 100 greater than 100 21 Attempt to set a port manually to an illegal value or method has been The port set format is X Y where X is port number 0 9 started with a PORT SET instruction having an illegal value and Y is active 21 or inactive 0 22 Attempt to copy a method to a method that is not empty This check code will also be given if inport 1 goes active external control of the GP 250 Plus and method 1 is empty and cannot be starfed 23 Attempt to run or copy a method to an empty method Delete destination method and try again or find an empty method 24 Insufficient memory for copy There is not enough memory left to Delete method s to get a sufficient number of available copy the whole method No copy has been made instructions for copying Reduce peak memory space specified in the Calibration block 25 Cannot enter a collected volume equal to zero after the calibration Enter a volume not equal to zero proce
14. ul Change mix time to a value within the set range divided by flow rate ml min times 0 06 Mix time must be more than 100 msec and less than 500 sec 109 Mix time too long gt 500 sec See 108 110 Too deeply nested methods Only 9 levels are allowed Check that method does not call itself directly or indirectly 111 Invalid bank number Allowed numbers are 1 5 112 Sync not allowed when MIX VOLUME gt 0 i e if you are forming gradients with one pump and a switch valve 190 Incorrect power down Press alarm reset If persistant contact service technician 191 Master reset Memory has been cleared because of use of RESET DIAGNOSIS button on rear panel of GP 250 Plus 192 Error in stored stack value Press alarm reset If persistant contact service technician PROM RAM and TIME failures PROM and RAM and timer failures are indicated in another way The GP 250 Plus tests PROM RAM and timer when power is switched on An error is indicated by a blank display and an alarm The alarm can either be a continuous signal or an on off signal The type of error is indicated by the LEDs to the left of the display They light up in different combinations depending on the fault LED Run OFF OFF ON OFF Hold OFF ON ON ON Pause OFF OFF OFF ON Alarm ON OFF etc ON OFF etc ON OFF etc ON OFF etc Error PROM RAM RAM TIMER 37 9 Maintenance and servicing 9 Maintenance and servicing Instrument housing Wipe the instrument r
15. when chart speed is set to a non zero value The GP 250 Plus automatically gives a voltage proportional to the current concentration B which can be plotted e g along with the chromatogram from GRADIENT 1 V and A GND analog ground Signal cable Code No 19 6006 01 or Cable Kit GP 250 Plus Code No 18 1017 68 Electrical interface Chart speed and pen down can be used with any logic ground as reference Gradient 1 V should be used with analog ground A GND as reference TTL compatible open collector with a protecting resistor 100 Q in series with the outport Max 15 V 21 5 Description of rear panel and connections Pen down Gradient 1 V Calibration value Chart speed Logic ground Gradient Analog ground Pen down Programmable functions Manual functions 5 5 Method control inports METHOD CONTROL Start 1 Hold 22 TTL compatible open collector can sink 300 mA Max 30 V 0 1 V corresponding to 0 100 B Resolution 0 001 V 0 1 B Inaccuracy max 0 2 FSD See calibration below Use analog ground A GND as voltage reference Calibration The chart speed of a stepper motor driven recorder is controlled by sending out a frequency proportional to the chart speed When connecting a recorder the GP 250 Plus has to be calibrated i e told how many pulses it has to send to the recorder in order to get 1 cm of paper fed The calibration value for the Recorders REC 101 and REC 10
16. 2 is 200 pulses per cm For other recorders please consult the relevant instruction manual Note Control frequency has to be within the range 0 15 kHz otherwise the chart speed range of the recorder is reduced Functions The outport SPEED controls the chart speed for a recorder The sockets GND allows ground connection of TTL compatible signals Offset is adjusted by pressing the key end whereby the GP 250 Plus will give an output corresponding to 0 B The recorder is then adjusted to 0 by using the zero knob on the recorder The spans of the GP 250 Plus and the recorder are then matched by entering CONC B 100 in the Manual block and setting the recorder to 100 by adjusting the span knob on the recorder also marked variable on some recorders The socket A GND allows ground connection of analog signals Pen down is obtained by setting a chart speed other than 0 The pen is lifted as soon as the chart speed is 0 CM MIN ML DL Functions controlled in the Manual block CM MIN ML DL The 4 inports for method control are external start hold pause and end of methods Starts Method no 1 in currently selected bank if activated 0 V and no method is running otherwise ignored No 24 on GP 250 Plus rear panel Only valid during run Will hold a running method when activated 0 V Holds on leading edge continues on trailing edge Trailing edge will continue even if hold was initiated manually from the k
17. 50 Plus rear panel Voltage reference for inports and outports Same as signal ground in the connectors for the pumps and fraction collector No 11 13 15 20 23 and 28 on GP 250 Plus rear panel These sockets should be used to power a mixer 24 VAC 50 60 Hz is supplied There are four different mixers available The one supplied has a 0 6 ml mixing chamber and is the one normally used with FPLC System For larger scale use the 6 ml mixer 6 Installation and connection of equipment 6 1 Mains installation 6 Installation and connection of equipment Before connecting the GP 250 Plus to the mains supply please read the following instructions carefully 1 There are two Mains Kits supplied one for 100 120 V and one for 220 240 V Please choose the voltage relevant to the local supply and discard the irrelevant kit immediately irreparable damage to the pump Always connect the instrument to a f Installing the wrong mains kit can endanger personal safety and cause properly grounded mains supply 2 Remove the warning label covering the fuse voltage selector unit on the rear panel 3 Open the fuse voltage selector cover with a thin screwdriver or the key provided Fig 6 and 7 4 Place the fuse into the fuseholder and install it into the instrument in the right hand position Fig 8 Note Use the 220 V setting for 230 V mains outlet 5 Remove the voltage selector switch select correct voltage and
18. AM ER SH AM BIOSCIENCES Gradient Programmer GP 250 Plus User Manual 56 1153 48 Edition AB GE imagination at work 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 751 82 Uppsala Sweden Amersham Biosciences AB reserves the right to make changes in the specifications without prior notice Warranty and Liability Amersham Biosciences 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 Amershasm 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 SuperFrac Bd FPLC and Mono Qe are the exclusive trade marks of Amersham Biosciences AB In view
19. F or to control instruments such as the P 1 pump The polarity can be changed for outports 0 4 and 8 9 in the Calibration block Use any logic ground as reference Signal cable Code No 19 6006 01 Electrical interface Programmable open collector TTL outports identified by the numbers 1 and 2 respectively No 18 and 19 on GP 250 Plus rear panel Active is equal to floating outport inactive is equal to 0 V Each can sink 300 mA Max 30 V Note Active voltage is inverted compared to outports 0 3 4 8 and 9 i e outports 1 and 2 are for straight logic and 0 3 4 8 and 9 for reverse logic control Programmable open collector TTL outports identified by the numbers 8 and 9 respectively Active is equal to 0 V inactive is floating They can sink 300 mA Max 30 V No 21 and 22 on GP 250 Plus rear panel Outports 3 and 4 are programmable for the control of solenoid valves No 2 and 4 on GP 250 Plus rear panel when connected to outports power No 1 and 3 on GP 250 Plus rear panel If connected with logic ground they have the same function as outports 8 and 9 To avoid overheating the valves the outports power will supply 24 V for only about 30 msec after activating ports 3 or 4 Power is then reduced by switching the 24 V on and 23 5 Description of rear panel and connections Switch valve Functions in Calibration block Programmable functions Manual functions POWER 24 V 5 7 Logic ground GND 5 8 Mixer
20. ONC B 40 0 18 00 CONC B 50 0 gradient 20 00 CONC B 80 0 21 00 CONC B 80 0 21 00 PORT SET 6 00 21 00 CONC B 5 50 23 00 CONC B 5 50 re equilibration 23 00 END OF LOOP 9661 YoreW gy uexoA L Soy ESRA gv UBlseq 8 sius Aq uepewg ul peiud GE imagination at work
21. ated This block is left by exit The list below shows the instructions that can be changed They are reached by using step forward and step backward For more information see section 4 7 List of instructions Changes the concentration of buffer B Changes the flow rates of pumps A and B Changes the recorder chart speed shown as CM MIN if time is used as base and CM ML DL if volume is the base Shows the status of attached outports i e active or inactive e g PT 0 0 1 0 0 0 0 0 0 0 indicates that port 2 is active To set port 5 active enter 5 1 Port 0 is switch valve or user definable outport Ports 1 4 are user definable outports Port 5 is fraction collector pause 1 continue 0 Port 6 is fraction collector run 1 end 0 Port 7 is fraction collector recycle 1 reset 0 Ports 8 9 are user definable outports Note Default values for active electrical signals are different for ports 1 and 2 than the others See section 5 6 Programmable outports Activates the wash program in pumps A and B if they have the function e g P 500 and P 6000 To activate the wash program in pump A enter 1 0 to activate the wash program in pump B enter 0 1 to activate the wash program in both pumps A and B enter 1 1 When the wash program is activated the pump s will set the GP 250 Plus in pause until the program is completed The wash program can be terminated with end Note When running the wash program the inj
22. concave shape is used to optimize the separation with respect to speed and resolution The resolution is increased for components eluting early in the gradient and at the same time the retention times for components eluting at higher concentrations are reduced A concave gradient can be regarded as a variant of a segmented gradient Concentration B 5 75 83 10 10 511 Volume ml Fig 24 Concave gradient Method Gradient concave 0 to 100 B in a gradient volume of 10 ml Flow 2 0 ml min Chart speed 2 0 cm min Programming base volume ml Volume ml Function Value 0 00 CONC B 0 00 0 00 ML MIN 2 00 0 00 CM MIN 2 00 5 00 CONC B 20 0 7 50 CONC B 40 0 8 30 CONC B 60 0 10 00 CONC B 100 10 50 CONC B 100 10 50 CONC B 0 00 11 00 CONC B 0 00 2 Sample injection 2 oample injection Manual sample injection Manual control of sample injection may be of interest when running occasional samples or samples in extremely small volumes e g partly filled sample loops Column Pump system Sample loop 9 U 9 A wast Fig 25 The components involved in manual sample injection Method Flow rate 2 0 ml min Volume ml Function Value 0 00 HOLD 0 00 ALARM 0 1 Note When volume is used as the method base the method which you run must contain an instruction which defines the flow rate at 0 00 ml or the method cannot run However it is not necessary to define the flow rate
23. ct a service technician Check mains voltage selector for setting replace mains fuse If fuse blows again contact a service technician Consult section 9 Check codes Press the RESET DIAGNOSIS button on the rear panel at the same time as you switch the power off and on The memory will be cleared and check code 191 is displayed If the display still does not light up contact a service technician Set a flow rate on the pump greater than 0 The GP 250 Plus is controlled from a computer program such as FPLCmanager Press the RESET DIAGNOSIS button at the same time as you switch the power off and on Contact a service technician Press the RESET DIAGNOSIS button at the same time as you switch the power off and on Contact a service technician 39 11 Technical appendix Address Analog Analog ground Bit CC Differential inports Digital 40 11 Technical appendix jt od me MT 1 everett pee r AA s _ 5 e as Fig 18 The construction of a circuit board The digital representation of an address to e g a valve position or information in memories A signal which can be of any level within a specified range e g 0 1 V Compare with digital Reference voltage for analog signals Often separated from digital ground as analog signals can be low e g 0 10 mV and therefore very sensitive to disturbances Binary digit Two states 1 and 0 C
24. d 5 LED indicators for status information three on the left and two on the right side of the display with corresponding text METHOD TIME VOL FLOW RATE hold gradient R u N M E r H p I volume battery i pause METTRA FUNCTION Fig 4 Alphanumeric display The text above and below the display refers to the programming and run modes in the following way Running When a method is running the text above the display applies The display gives information about 1 method number C before the number indicates that the method running is called from another method Note Bank number is not shown If you want to check that press the key method file or go to PATH in the Evaluation block 2 elution time volume since the start of the displayed method flow rate 4 concentration B W Programming During method programming the lower text applies This gives information about 1 time volume 2 function 3 value 4 Description of keyboard and Programming 4 10 LED indicators RUN HOLD GRADIENT PAUSE VOLUME BATTERY 4 11 Short instruction There are 5 LED indicators which give status information about running conditions as follows Lights up when a method is running during the calibration procedure and when a value for the flow rate or the chart speed is entered manually Lights up when a programmed hold is reached and when the key hold gradient is pressed or activated by a rem
25. de No 19 6005 02 1 5 m or 18 0072 75 0 6 m Electrical interface TTL compatible with a 10 kQ pull up resistor Max 5 5 V TTL compatible open collectors with a protecting resistor 100 W in series with the outports Max 15 V 5 Description of rear panel and connections Calibration value Programmable functions Manual functions Calibration The flow rate of stepper motor driven pumps is controlled by sending out a frequency proportional to the desired flow rate When connecting a pump the GP 250 Plus has to be calibrated i e the number of pulses it has to send to the pump to deliver 0 1 ml liquid has to be defined The calibration value for the Pump P 50 is 234 pulses 0 1 ml for the P 500 is 110 pulses 0 1 ml and for the Pump P 6000 it is 45 pulses 0 1 ml Note P 50 and P 500 cannot be run in dl mode The calibration value for P 6000 in dl mode is 4 500 0 1 dl An unknown calibration value can be determined by a built in calibration procedure The calibration is performed on the pump connected to socket PUMP A only Flow rate range 0 999 ml min if the program is time based 0 02 75 0 ml min dl min if the program is volume based The lower limit is caused by the internal round off to 0 in the GP 250 Plus The upper limit is determined by the time required for the GP 250 Plus to execute the programmed instructions at exact programmed volumes Note The control frequency has to be within the range 0 15 kHz ot
26. different blocks Step forward to get the next function instruction and backward to get the previous function instruction Executes methods instructions or stores data entered in memory 4 Description of keyboard and programming 4 2 Calibration block MIN 0 ML 1 DL 2 PUMP CAL AB REC CAL CALIBRATE PUMP A CLEAR MEMORY MIX VOLUME Not accessible during run By pressing this key the Calibration block is entered This block contains data needed by the GP 250 Plus to control the pumps and recorder It also contains data defining outports etc This block is left by pressing exit When pressing step forward or step backward the following calibration choices are given Selects the base for advancing the method Enter 0 MIN 1 ML or 2 DL 1 deciliter DL 100 ML A LED is switched on to indicate if ML or DL was chosen Enters the calibration value for pumps A and B i e the number of pulses the GP 250 Plus has to send to the pumps to deliver 0 1 ml dl Calibration values in ml mode are 234 for P 50 110 for P 500 and 45 for P 6000 In dl base the calibration value for the P 6000 is 4 500 Note Pump P 50 and P 500 cannot be run in dl mode Enters the calibration value for the recorder i e the number of pulses the GP 250 Plus has to send to the recorder to feed 1 cm of paper 200 for REC 101 102 Allows calibration of a pump connected to the connector PUMP A For calibrating a peristaltic pump co
27. dure for the pump has been performed 27 Memory full Delete method s to get sufficient number of available instructions Reduce peak memory space specified in the Calibration block 28 Illegal loop instruction sequence The method has been started with one or more of the following errors a nested loops loops within a loop b looping without programming instruction END OF LOOP c looping without programming instruction LOOP TMS 30 Continue not allowed during wash Wait until wash program is finished or press end to abort wash 31 Cannot calibrate the pump during solvent changeover WASH Wait until wash program is finished or press end to abort wash 32 Attempt to delete a running method Press end to stop the method and try again if you want to delete it 42 Illegal parameter to WASH instruction Enter 1 0 for pump A 0 1 for pump B 1 1 for both pumps 70 External end The current running method has been terminated by a 74 signal on Method control Stop External pause The current running method has been paused by a signal on Method control Pause 80 Illegal instruction in method This check code occurs when illegal Delete method and reprogram If encountered again instructions are found in the memory Such instructions can be stored contact service technician if there are very severe disturbances to the mains voltage 81 Illegal time volume or parameter in method see 80 Delete method and reprogram If encounter
28. e for the control of equipment with TIL compatible inports Outports 3 and 4 are programmable for the control of solenoid valves such as the Solenoid Valve PSV 50 Code No 19 1994 01 Outport 0 controls the switch valve for gradient formation Note Do not connect or disconnect a solenoid valve with the GP 250 Plus turned on 6 Installation and connection of equipment GP 250 Plus 1013 121314151617 Fig 14 Connection of the mixer to the GP 250 Plus Connection of solenoid valves to outports 3 and 4 Connect the two wires from the valve to a 2 pole connector and connect it to outport 3 no 1 and 2 or outport 4 no 3 and 4 No polarity Fig 15 Outports 3 and 4 can be used as programmable TTL outports see section 5 6 Programmable outports Voltage Frequency Power 110 120 220 230 240 V 50 60 Hz 60 VA prc EE zz FUSE Sheu 300 120 V 220 230 240 V or continued protection AU against fire hazard replace 1800 m 1400 mAL 220 Y a only with the same type Code No 56 1153 48 and eating of fuse RESET DIAGNOSIS SWITCH VALVE MIXER POWER MAX300mA 24VAC 24V GP 250 Plus 100 mA SOLENOID VALVES OFF 0 MAX 300 mA FLA 2318 8 o go o O 3 C 8383 2 al O pakal ON 1 MAINS 567 89 1011 Dissconnect before service SSS PSV 50 Sive PSV 5 MP 9 24 VDC i 1 Ust Fig 15 Connection of the Sol
29. e method The GP 250 Plus controls the chromatographic run according to programmed methods Each method is built up of a number of instructions forming a sequence of events Each instruction is programmed at the time volume when it should be executed in the method It will be valid until a new instruction in the method is reached defining a new value for the function 7 Operation A typical method includes an initial equilibration step followed by a sample injection followed by a short equilibration under starting conditions Components bound to the column are eluted with a gradient The column is then washed in limit buffer and finally re equilibrated in start buffer Typical conditions for a separation on a Mono Q HR 5 5 column are as follows Column Mono Q HR 5 5 BufferA 20 mM Tris HCl pH 8 0 Buffer B 20 mM Tris 0 35 M NaCl pH 8 0 Flow rate 1 0 ml min Chart speed 0 5 cm ml Sample volume 100pl 1 ml Monitor UV M II 280 nm 1 0 AUFS The method should contain the following steps Fill sample loop 500 pl Equilibrate column with 2 ml start buffer Buffer A Ahold that will give time to fill the sample loop The sample is applied to the column with 2 ml start buffer An alarm that will remind you to return injector valve to LOAD position Run linear gradient from Buffer A to 100 Buffer B in 20 ml Wash column with 2 ml Buffer B Re equilibrate column with 2 ml Buffer A AN Oo tn FP WN L
30. ected to the switch valve socket no 5 7 on the rear panel of the GP 250 Plus 4 Description of keyboard and programming P POL 4 3 Evaluation block evaluate RETENTION LOOP NO PATH TUBE 4 4 Manual block 10 NO Recommended mix volume for the 0 6 ml mixer is 60 and for 6 ml mixer is 600 If another pump is used please contact your Amersham Biosciences representative for further information Note When running in dl mode the mix volume is 1 999 ml As the display shows 10 9 999 the entered value has to be divided by 10 to get the correct value i e 15 on the display means 1 5 ml mix volume Sets polarity for the programmable outports Positive or negative logic can be selected for outports 0 4 and 8 9 Ports 5 7 connected to the fraction collector cannot be changed 3 1 means port 3 has positive logic active floating voltage 3 0 means port 3 has negative logic active 0 V Negative logic is normally used Default means that all ports have negative logic except 1 and 2 Port 0 is designed for a switch valve when making gradients with one pump When not used for a switch valve it can be used for an extra solenoid valve If you want to connect an extra pump P 1 and both outports 1 and 2 are occupied connect the pump to outport 8 and change its polarity by entering 8 1 and pressing do store By pressing this key the Evaluation block is entered The data below will be shown by pressing step for
31. ection valve must be in the wash position in order to protect the column from the exposure of high flow rates During wash there will be no check code if the over pressure alarm is activated Changes tube on the fraction collector when do store is pressed Drives the pump pistons to an end position if the pumps are provided with the function e g P 500 and P 6000 Useful for gradients of very small volumes Enter 1 0 for driving the pump A piston to an end position 0 1 for pump B and 1 1 for both pumps When the sync program is activated the pumps will set the GP 250 Plus in pause until the program is completed The sync program can be aborted with end Note When running the sync program the injection valve must be in the wash position in order to protect the column from the high flow rates Only for use with pumps that are provided with the function e g P 500 and P 6000 Otherwise the high flow rate could seriously damage the pump 11 4 Description of keyboard and programming 4 5 Method block method file MEMORY LEFT PROGRAM METHOD DELETE METHOD COPY FROM TO BANKCOPY FROM BANKCOPY TO 4 6 Programming block The GP 250 Plus has fifty methods divided into five banks which can be programmed By pressing the method file key three blocks are entered the Method block the Programming block and the Review block The Method block contains instructions to handle methods and banks e g changing bank re
32. ed again contact service technician 82 Internal division by zero Press alarm reset If persistant contact a service technician 83 Illegal order of instructions Correct or delete the method 84 Internal stack error Could occur due to severe disturbances to the Press alarm reset If persistant press the mains voltage RESET DIAGNOSIS button and turn the GP 250 Pius off and on again 90 Internal control circuitry has detected an error Press alarm reset If persistant contact a service technician 101 Attempt to drive recorder with a frequency greater than 15 kHz Set a lower value in cm min ml dl and check calibration Recorder frequency must be less than 15 kHz value 102 Illegal parameter to SYNC A B Enter 1 0 for pump A 0 1 for pump B 1 1 for both pumps 103 Wash continue and calibrate are not allowed during SYNC A B Wait until sync program is finished or press end to abort sync 36 8 Check codes Check Explanation Action code number 104 SYNC A B not allowed during WASH Wait until wash program is finished or press end to abort wash 106 Synchronizing pumps SYNC A B lasts more than 6 min Check if pump is provided with the function Check power cable and pressure limit If error persists there is a circuit failure on the pump or the GP 250 Plus Contact a Service technician 107 Pumps are washing more than 6 min See 106 108 Mix time too short 100 msec Mix time is equal to mix volume
33. egularly with a damp cloth Let the instrument dry completely before use Change of battery The memory in the GP 250 Plus has battery back up so that the unit can be turned off without losing stored data or methods The lithium battery has a life time of about 5 years When the LED BATTERY lights up in the display the battery should be changed Contact a local service technician to replace the battery Warning Due to the risk of explosion always contact a Amersham Biosciences service technician to replace the battery 38 10 Troubleshooting 10 Troubleshooting Problem Possible cause Suggested action The display does not light up when the mains switch is turned on The display does not light up but the LEDs do The display does not light up when power is switched on and no LED indication is given The pump does not start when the calibration procedure is started by executing the instruction CALIBRATE PUMP No response to entire keyboard or keys in vertical or horizontal row Display faulty incomplete digits or display Power cord not plugged in No voltage at mains switch Fuse blown RAM PROM or timer failure RAM PROM or timer failure Flow rate on the pump is set to 0 Remote control Memory lock up Electronic failure Memory lock up Electronic failure Check that the power cord is plugged in Check by plugging in desk lamp If fault persists conta
34. enoid Valve PSV 50 to the GP 250 Plus 29 6 Installation and connection of equipment 30 Outport 5 and 7 are used to connect a switch valve such as PSV 50 Code No 19 1994 01 for single pump gradient formation see fig 15 Connection of an accessory e g a pump to outports 1 2 8 and 9 If an accessory such as the Peristaltic Pump P 1 or HiLoad Pump P 50 is connected to one of the sockets marked PROGRAMMABLE OUTPORTS 1 2 8 and 9 activities such as ON OFF can be controlled from the GP 250 Plus Before making any connection check which active voltage is required Outports 1 and 2 use straight logic i e floating when activated outports 8 and 9 use reversed logic i e 0 V when activated This is important if automatic OFF is required at end of method The polarity of the outports can be changed see P POL section 4 2 Calibration block To facilitate the connection a Remote Control Adaptor Code No 19 6008 01 can be used The adaptor consists of a D type connector with outlet terminal pins which correspond to different controllable functions The adaptor is plugged into the socket marked REMOTE on the P 1 pump or the P 50 pump and a Signal Cable Code No 19 6006 01 is used to connect pins 12 and 15 on the adaptor to programmable outport GND and the required port on the GP 250 Plus see fig 16 Remote Control Adaptor Code No 19 6008 01 Signal Cable Code No 19 6006 02
35. et the flow rate on the pumps to zero and press the RUN button 10 11 12 13 P 50 P 500 P 6000 Set the input ranges to correspond to monitor and controller outputs see table below Set the recorder controls as shown in the table Item Setting range gradient pen 1V monitor pen 10 mV for UV 1 typically 100 mV for UV M II typically 1 V for pH monitor and Conductivity Monitor zero suppression 0 cal var span released calibrated span both pens pen up down normally down rec off on normally down chart speed 20 mm s mm min released mm s int ext depressed ext Set the appropriate sensitivity with the sensitivity range selector on the UV monitor and adjust the baseline on the recorder with the baseline knob or press AUTO ZERO on the UV M Turn on the GP 250 Plus Enter the Calibration block and check the value for method base and calibration values for the pumps recorder etc For calibration values see section 5 2 Pumps and section 5 4 Recorders Program the fraction collector and set it in the start position Check that the pumps are filled with the correct buffers by activating the wash program in the Manual block If the method is to be programmed before starting the run it is convenient to equilibrate the column during this period by setting the initial conditions through the Manual block Program the method Check the method in the Review block Run th
36. ethod file Copies methods within the selected bank e g enter 4 5 to copy method number 4 to method number 5 in the currently selected bank The destination method must be empty After copying is completed empty methods and bank number are displayed as when pressing method file Copies methods between banks e g to copy method number 3 in bank number 1 to method number 4 in bank number 5 enter 3 1 and press do store The display will show BANKCOPY TO Enter 4 5 and press do store The destination method must be empty Methods can be programmed in the Programming block A method is built up of a number of instructions forming a sequence of events Each instruction is programmed at the time volume when it should be executed in the method It is valid until a new instruction is reached defining a new value for the function The functions that can be controlled by the GP 250 Plus are listed in section 4 7 List of instructions For more information about programming see section 7 2 Programming a method 4 Description of keyboard and programming 4 7 List of instructions CONC B ML DL MIN CM MIN ML DL PORT SET CALL METH Entered concentration in percent B will be reached at the programmed time volume The concentration will be changed linearly between two such instructions 0 100 B in steps of 0 1 The flow rate can be programmed in ml dl min in steps of 0 01 The chart speed can be programmed in cm mi
37. eyboard before method control hold was activated Remote hold will be terminated by 5 Description of rear panel and connections Pause Stop Recommended cable Inports 5 6 Programmable outports Programmable outports Recommended cable Outports 1 and 2 Outports 8 and 9 Solenoid valve pressing the key cont even if method control hold is still active No 25 on GP 250 Plus rear panel Note Continue by a trailing edge will not affect pause Only valid during run Will pause a running method when activated 0 V A check code is displayed when paused Pauses on leading edge continues on trailing edge Trailing edge will give continue even if pause was initiated manually from the keyboard before the inport was activated Remote pause will be terminated by pressing the key cont but not if the inport is still active No 26 on GP 250 Plus rear panel Note Continue by a trailing edge will affect hold Stops a running method and displays a check code when activated 0 V No 27 on GP 250 Plus rear panel Signal cable Code No 19 6006 01 Electrical interface TTL compatible inports with 10 kQ pull up resistors Max 5 5 V The signals are filtered to guarantee proper operation at contact bounces Signals must therefore have a duration of minimum 50 msec There are 7 programmable outports for ON OFF control of accessories such as solenoid valves and relays to switch mains power ON OF
38. herwise the flow rate range of the pump is reduced Inaccuracy of control frequency is max 0 2 for 0 1 5 KHz and max 1 1 for 1 5 15 kHz Pump functions ML DL MIN CONC B WASH A B SYNC A B ML DL MIN CONC B WASH A B SYNC A B Remote connection pumps A B Name Active Function Pin voltage Inports all with 10 kQ pull up Busy 0 Active when the pump is busy and unable to accept 9 remote control If the signal lasts more than 1 s the GP 250 Plus is automatically paused and a check code is displayed Direction Indicates pump piston direction Right 0 left 1 4 Operable in 0 Indicates that the unit connected is operable 10 Pump frequency Stepper motor frequency from the pump Used by the 13 GP 250 Plus to read flow rate during calibration only pump A 5 Description of rear panel and connections 5 3 Fraction collectors Connector Recommended cable Inports Outports Programmable functions Manual functions 20 Name Active Function Pin voltage Outports all open collector Wash 0 Starts a wash program if the pump is provided with that 1 function The pump will respond with busy until the wash program is completed Pump stop 0 Used by the GP 250 Plus internally to stop the pump 11 Not programmable Operable out 0 Used by the GP 250 Plus internally when flow rate is 12 controlled Flow rate Output frequency to control the flow rate 14 Ground Signal gro
39. ion schemes 44 1 Elution schemes The GP 250 Plus formsec gradients by controlling the relative flow rates of two pumps which are designated A and B in this section Pump A delivers solvent for the starting conditions and pump B delivers the eluting solvent To program a gradient profile two or more breakpoints have to be defined A breakpoint is defined by entering a retention time volume and the concentration of solvent B In this way any slope shape or segmentation of gradient can be programmed GP 250 Detector Fig 19 The components involved in gradient production Isocratic elution Concentration B 10 20 Time min Fig 20 Isocratic gradient 1 Elution schemes Method Gradient isocratic 55 B 20 min Flow 1 ml min Chart speed 1 cm min Programming base time min Time min Function Value 0 00 CONC B 55 0 0 00 MI MIN 1 00 0 00 CM MIN 1 00 20 00 CONC B 55 0 Linear gradients When using liquid chromatography techniques that require gradient elution e g ion exchange hydrophobic interaction or reversed phase it is often wise to run the sample with a linear gradient before progressing to more complex elution schemes am l 45 c Q w t 0 o c o O 5 15 Volume ml Fig 21 Linear gradient Method Gradient linear 5 to 45 B in 15 ml Flow 1 0 ml min Chart speed 0 5 cm ml Programming base volume ml Volume ml Functio
40. n The gradient will have the following profile CONC B 50 5 10 15 20 25 Volume ml Fig 17 Gradient profile This graphic representation of a linear gradient is specified by six breakpoints A breakpoint is defined as a specific concentration B at a specific time volume The segment between the breakpoints has a linear slope which represents the change in concentration B per time volume unit 33 7 Operation An example of how a method can be composed Instruction Volume ml Function Value Step 0 0 CONC B 0 0 1 2 2 0 ML MIN 1 3 0 CM ML 0 5 4 2 HOLD 3 5 4 CONC B 0 0 6 4 ALARM 0 1 4 7 24 CONC B 100 5 8 26 CONC B 100 6 9 26 CONC B 0 10 28 CONC B 0 7 When programming the parameters on the display correspond to those written beneath them Respond only to flashing parameters Whenever the display is flashing it is asking you to confirm a function or enter a value for the function which is displayed e If the number flashing is the value you want press do store If not enter the value you want and press do store e Never press do store unless you want to enter the values or function that is flashing e Every time you enter a new time volume the instruction CONC B will appear to facilitate the programming The next function in the block automatically appears on the display after you press do store f you accidently enter the wrong number and recog
41. n cm ml or cm dl in steps of 0 01 An automatic pen down is activated when the chart speed is set to a value other than 0 with the recorders REC 101 and REC 102 and solenoid pen lift installed The pen will lift automatically when the chart speed is set to 0 Sets outports active or inactive e g 3 1 sets port number 3 active 1 active 0 inactive Note Default means active is electrically 0 V on ports 0 and 3 9 and floating voltage on ports 1 and 2 See section 5 6 Programmable outports Port 0 when not used for switch valve rear panel no 6 1 rear panel no 18 2 rear panel no 19 3 rear panel no 2 4 rear panel no 4 5 fraction collector pause 1 continue 0 6 fraction collector run 1 end 0 7 fraction collector recycle 1 reset 0 8 rear panel no 21 9 rear panel no 22 Runs the entered method as a sub method maximum 9 levels nesting Automatic return to the calling method after the called method is ended Method number is specified as methodnumber banknumber where banknumber 0 indicates the currently selected bank When the instruction CALL METH is executed the return address method no bank no and time volume will be stored in a call memory if a second instruction CALL METH is executed in the called method a second return address will be stored upon the first in the call memory the third upon the second etc The GP 250 Plus can store up to 9 return addresses in the call memory
42. n Value 0 00 CONC B 5 0 0 00 ML Min 1 00 0 00 CM ML 0 50 15 00 CONC B 45 0 45 1 Elution schemes 46 Step gradients Affinity chromatography and chromatofocusing are two techniques where elution is done with step gradients i e instantaneous change from one buffer to another 100 Concentration B 20 40 Time min Fig 22 Step gradient Method Gradient step 100 B after 20 min Flow 1 0 ml min Chart speed 0 5 cm min Programming base time min Time min Function Value 0 00 CONC B 0 0 0 00 ML MIN 1 00 0 00 CM MIN 0 50 20 00 CONC B 0 0 20 00 CONC B 100 40 00 CONC B 100 1 Elution schemes Segmented gradients Starting with a linear gradient often leads to the use of a gradient with a more complicated shape to improve the resolution or to speed up the separation A HOLD is programmed to facilitate sample application Concentration B 2 10 18 202122 Volume ml Fig 23 Segmented gradient Method Gradient segmented with different slopes from 5 5 to 80 B in a total volume of 20 ml Flow 2 0 ml min Chart speed 0 5 cm ml Programming base volume ml Volume ml Function Value 0 00 CONC B 5 50 0 00 ML MIN 2 00 0 00 CM ML 0 50 0 00 HOLD 2 00 CONC B 5 50 10 00 CONC B 40 0 18 00 CONC B 50 0 20 00 CONC B 80 0 21 00 CONC B 80 0 21 00 CONC B 5 50 22 00 CONC B 5 50 47 1 Elution schemes 48 Concave gradients A gradient with
43. nize the mistake before pressing do store you can change your entry by first pressing CE clear entry All programmed functions are automatically reset to zero at the end of a program You do not have to tell the pump recorder or fraction collector to stop When a method is started the GP 250 Plus instantly checks all the instructions and that all instruments to be controlled e g recorders fraction collectors and pumps are connected If an illegal instruction is encountered it will immediately give a check code Called methods are automatically checked before the start If during a run the GP 250 Plus discovers that pumps or the fraction collector are not connected it will be paused and a check code is displayed identifying the fault For more information and examples about programming the GP 250 Plus please consult Part 2 Applications 34 8 Check codes 8 Check codes The GP 250 Plus is equipped with a powerful status checking facility which continually checks the status of the GP 250 Plus as well as the equipment connected to it In case of deviation from proper operation a check code will flash in the display and an alarm will sound for 30 sec If a check code is displayed look to see what it means in either the list below or the Short Instruction and take the appropriate action Check codes are cleared by pressing the key alarm reset Check Explanation Action code number 1 Pump A inoperable P
44. nnect it to the PUMP A socket and follow the procedure below Do not forget to change the connections and re enter PUMP A calibration value after you have finished the procedure The procedure is according to the following scheme 1 Fill the pump with suitable liquid e g water before connecting it to GP 250 plus 2 Connect the pump to the PUMP A connection on rear panel Turn on the pump 3 Set a representative flow rate on the control panel of the pump approx 1 ml min Place the outlet from the pump into a graduated cylinder 4 Press do store The run LED is switched on and pump A control from the GP 250 Plus is released so pump A can start with the flow rate set on the pump The GP 250 Plus is now counting pulses from the stepper motor in the pump 5 Press end when a volume large enough to be measured with good accuracy has been delivered e g 10 ml 6 VOLUME ML is displayed or VOLUME DL if dl mode is chosen Enter the collected volume and press do store 7 The calibration mode is terminated and the display will show PUMP CAL AB as above but with a new calibration value Clears all memory of the GP 250 Plus Enter 441 and press do store The display will then show RUN METHOD For two pump systems the mixvolume should be set to zero 0 If set to a non zero value 10 9 999 ml or 1 999 ml the GP 250 Plus assumes that gradients will be formed with a single pump A and a switch valve The valve must be conn
45. ole method can be looped 1 99 times The gradient is formed by the control of either two pumps or one pump and a switch valve Eluents are then mixed in a dynamically stirred mixing chamber The supplied mixer has a volume of 0 6 ml with magnetic stirring 0 100 B in 0 1 increments Any gradient profile can be programmed in linear segments of different slopes Linear gradients are programmed by entering the time volume and concentration of eluent B at the breakpoints The concentration entered will be reached at the programmed time volume The concentration will be changed linearly between the breakpoints Seven programmable ON OFF outports e g for control of accessory equipment TTL compatible Three are designed for control of a fraction collector or automatic injector The d for each outport can be programmed in the Calibration block 2 Specifications 24 V Switched power outports Power24 V Memory protection Environment Dimensions Weight Power requirement Voltage Frequency EMC standards Safety standard Power failure disturbances Flow rate Three 24 V max 100 MA switched power outports are available of which two are designed to power solenoid valves etc and one for making one pump gradients The third is also useable as an outport for solenoid valves when using two pumps for gradient formation The power outports are powered down 30 ms after any of the outports are activated to avoid ove
46. ontact closure outputs Some instruments have outports where 2 contact pins are short circuited by a contact closure If this kind of outport is connected to the GP 250 Plus inports one pin should be connected to logic ground and the other to desired inport Analog inports which are floating i e not connected to any voltage or reference The circuit will measure the voltage difference between the two inports instead of measuring the voltage with reference to an analog or digital ground which can be very noisy A signal with 2 states high and low or logical 1 or 0 For TTL outports this means 2 4 5 5 V and 0 0 4 V respectively When straight logic is used ac tive signal is 1 floating voltage when reverse logic active signal is 0 0 4 V 11 Technical appendix Digital ground Duty cycle Floating Integration time LED Open collector Pull up resistor PROM RAM Sink 300 mA Straight logic Switched power TTL Reference voltage for digital signals 0 V See Switched power below An in or outport which is not connected to any voltage The driven inport must therefore have a pull up resistor which defines the inport high when the outport is floating The time it takes for an A D converter to convert an analog voltage to digital information Light Emitting Diode A kind of outport that is either connected to ground or floating A resistor connected between a voltage positive in reference to logic ground
47. ors see eee 20 541 Recorders 2e ete t ai 21 5 5 Method control inports sse eene 22 5 6 Programmable outports sse 23 5 7 Logic ground eee 24 5 895 UMIXQE GG terre nee RR epe CUL vis dt Feet E e DH LU UR n e ea Oi b e ane 24 Installation and connection of equipment ssssssseeee ee 25 6 1 Mains installation o Re ved eek ur HM DD DONE 25 6 2 Connection of pumps eee eee eee 26 6 3 Connection of recorder sss 27 6 4 Connection of fraction collector ssssse 28 6 5 Connection of mixer see 28 6 6 Connection of accessories to the programmable outports 28 Operation oca oie etie esee ric tay in Te REA Nn d dad e ee vin cd 32 7 d Start UP uii OR GATA PUN C COR A p REPE TI EROR RI OCA 32 7 2 Programming a method sse 32 Gheck codes erts e rre NO UNDER HU EU e Nea e Hus 35 Maintenance and servicing seen 38 Troubleshooting i o e ero eere o eerie or a a i eed 39 Technical append aaae etcetera ve RO e ree reproduit 40 Spare parts and accessories sss 42 Part 2 Applications 1 2 3 4 El tiomschemes 325 Nee MEM MOS bob ER e b ea goose 44 Sample injection srice eri a E EE RE eee nennen 49 Fraction collections iere te Agee EE PER a aA TEE enn 50 Looping a method for manual repeated injection ssssssesese 51 1 Introduction Part 1 1 Introduction The Gradient Programmer GP 250 Plus Code No 20 0002 02 is a powe
48. ote hold signal on method control hold rear panel no 25 Lights up when the key pause is pressed or activated by a remote pause signal on method control pause rear panel no 26 or if connected instruments like pumps fraction collectors and valves are inoperable Lights up when ml or dl is chosen as the programming base in the Calibration block To see which base has been programmed ml or dl press cal when the method is not running Lights up when the battery has insufficient power to protect the memory if the GP 250 Plus is turned off or in case of mains power failure This indicates that the battery must be replaced The battery must be changed by a service technician Warning Due to the risk of explosion always contact a Amersham Biosciences service technician to replace the battery Included with the GP 250 Plus is a Short Instruction guide for the use of the Gradient Programmer It is located under the GP 250 Plus and is easily pulled out Fig 5 Fig 5 Short Instruction 5 Description of rear panel and connections 5 1 Mains power supply MAINS MAINS SWITCH FUSE BATTERY RESET DIAGNOSIS 5 2 Pumps PUMP A PUMP B Connector Recommended cable Inports Outports 5 Description of rear panel and connections The socket for mains inlet contains a safety fuse and a rotary switch for selection of the operational voltage The voltage range is 100 120 220 230 and 240
49. place the switch into the instrument with the correct voltage showing Fig 9 6 Close the cover The correct voltage should be visible in the window 7 Connect the GP 250 Plus to a grounded mains outlet using the cable supplied in the Mains Kit Fig 6 Opening the fuse voltage selector cover When using the screwdriver to open the cover insert it at the top centre and apply pressure Fig 7 Opening the fuse Fig 8 Installing the fuseholder Fig 9 Choose the correct voltage selector cover Always use the hole at the right voltage by turning the voltage using lateral pressure with hand side when you install the selector the key provided fuseholder 25 6 Installation and connection of equipment 6 2 Connection Of pumps 26 Pumps are connected to the GP 250 Plus with the Communication Cable Code No 19 6005 02 or the pump cables in the Cable Kit GP 250 Plus Code No 18 1017 68 See the instructions for the cable kit for details The pumps are connected from the sockets marked REMOTE on the pumps to the two sockets marked PUMP A and PUMP B Fig 10 Communication Cable Code No 19 6005 02 or 18 0012 75
50. rable or is busy a check code is given in the display All signals are TTL compatible 0 999 ml min in min mode 0 02 75 0 ml min in ml mode 0 02 75 0 dl min in dl mode Increment 0 01 in the region 0 00 9 99 0 1 in the region 10 0 99 9 1 in the region 100 999 The Pumps P 1 HiLoad Pump P 50 High Precision Pump P 500 and P 6000 can be controlled within their whole flow rate range 2 Specifications 3 Unpacking Chart speed Pen down Gradient 1 V Recorder control 0 999 cm min ml dl Increment 0 01 in the region 0 00 9 99 0 1 in the region 10 0 99 9 in the region 100 999 Output to control a solenoid pen down function on a recorder Automatic pen down when chart speed is set to a non zero value Analog signal 0 1 V reflecting the current concentration in of eluent B during a run 1 V corresponds to 10096 B Resolution 0 001 V 0 196 B Fraction collector control The GP 250 Plus can control a fraction collector to RUN STOP PAUSE CONTINUE FEED TUBE and RECYCLE recycle is possible only with FRAC 200 and SuperFrac M 3 Unpacking Unpack the equipment carefully and check the contents of the carton against the packing list Save the packing material and the carton if future transport can be foreseen Check the GP 250 Plus for any visible signs of damage that may have occurred during transit Place the GP 250 Plus on the bench and check that it has the appropriate voltage setting see section 5
51. rd are pressed the delete mode is terminated and the instruction is not deleted insert This key allows you to insert new instructions in a programmed method After pressing insert the time volume of the instruction just displayed will be given as a flashing default value A default instruction and value are also displayed 15 4 Description of keyboard and programming 4 9 Display 16 A time volume has to be entered in order to change the instruction or the value If a new time volume has been stored by pressing do store the first instruction in the instruction list will be given as first choice However if the time volume was not changed the default instruction will be the one in the instruction list that follows the last instruction displayed When a time volume has been entered both the instruction and its value will now be flashing step forward and step backward are used to select other instructions The instructions will have the most recently programmed value as their default value If the time volume is not changed the new instruction will be stored immediately after the instruction just displayed If the time volume is changed the new instruction will be stored as the last instruction at that time or volume The insert mode is terminated by pressing exit The last inserted instruction will then be displayed The alphanumeric display see Fig 4 consists of 16 character positions an
52. rful multi functional control unit for chromatography systems Its programmable memory allows you to control chromatographic procedures modify separation parameters and control auxiliary equipment such as fraction collectors and chart recorders at the same time Fifty independent methods can be programmed and stored in the battery backed up memory of the GP 250 Plus Each method maybe accessed individually linked to other methods or called upon as a submethod In addition methods or parts of methods may be looped up to 99 times Programmed settings may be overridden manually at any stage Together with prepacked columns pumps valves monitors fraction collectors and recorders the GP 250 Plus forms a systems for fast high resolution liquid chromatography such as FPLC System This instruction manual is divided into two parts In the first part Technical description you will find all the technical details about the GP 250 Plus There is also a trouble shooting section and a check code list The second part Applications provides a set of useful examples which will get you started easily In the FPLC System handbook you will find chapter 3 Programming which is an easy start up course in how to program the GP 250 plus 2 Specification Method number Memory capacity Memory protection A Display Keyboard Alarm Method base Sub method call Looping Gradient formation Concentration Gradient profile
53. rheating the valves A 24 V power source for external devices Four inports for remote method control and indications e g Level sensors TTL compatible 4 to 40 C 20 95 relative humidity 84 106 kPa 840 1060 mbar atmospheric pressure 408x308x122 mm 5 5 kg 50 VA 100 120 220 230 240 V Always connect the instrument to a properly grounded mains outlet 50 60 2 Hz 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 In a domestic environment this product may cause radio interference in which cases the user may be required to take adequate actions Note The declaration of conformity is valid for the instrument when it is e used in laboratory locations e used in the same state as it was delivered from Amersham Biosciences AB except for alteration described in the User Manual e 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 In the event of power failure the system will shut down When the disturbances power returns the run will continue exactly where interrupted Pump control The GP 250 Plus can control pump functions such as RUN PAUSE flow rate and wash program If the pump is inope
54. s Programmable outports 2 Specification This section provides general specifications For full details of functions instructions and interface signals see section 4 Description of keyboard and programming and section 5 Description of rear panel and connections 50 methods can be programmed individually The methods are divided into 5 different method banks with 10 methods in each bank Maximum memory capacity is 750 instructions The memory is backed up with a lithium battery that will last for a minimum of 5 years A LED indicates when the battery should be replaced Warning Due to the risk of explosion always contact a Amersham Biosciences service technician to replace the battery 16 digit alphanumeric display with 5 LEDs for status information 29 click type keys with audible tone acknowledgement The tone will become louder when incorrect key entries are made An audible alarm can be programmed to sound at any time volume with a duration of 0 99 min in increments of 0 1 min There will also be a 30 s alarm when a check code is indicated in the display Programmed instructions are executed in either time or volume base depending on whether the GP 250 Plus is calibrated in min ml or dl 0 9 999 min ml dl Increment 0 01 in the region 0 99 99 0 1 in the region 0 999 9 1 in the region 1 000 9 999 Programmed methods can be called upon as sub methods Maximum is 9 levels of nesting A part or a wh
55. s Tube number is set to 1 Note end will have a special function during pump calibration Only valid during run When pressed current values are maintained until cont is pressed or until the trailing edge of a remote hold signal on method control hold rear panel no 25 The method is not advanced but retention time volume will be counted Values can be changed manually during hold as during run and pause A LED is switched on to indicate hold gradient Continues the run after hold gradient pause a programmed hold remote pause on method control pause rear panel no 26 or remote hold on method control hold rear panel no 25 The pumps the fraction collector and the recorder are stopped until cont is pressed or until the trailing edge of a remote pause signal on method control pause rear panel no 26 The method is not advanced and time volume will not be counted until cont is pressed Parameters can be changed manually during pause but flow rate and chart speed will not be executed until cont is pressed or until the trailing edge of a remote pause signal on method control pause rear panel no 26 A LED is switched on to indicate pause Exits Method Calibration Evaluation Review and Manual blocks and returns to initial mode i e RUN METHOD or run parameters In the Programming block exit must be pressed once more to return to initial mode Selects functions or instructions in
56. struction is changed during a run when the method is within the loop there will be no change for the current run as the number of loop times has already been stored See LOOP TMS above A pulse is given to the fraction collector to change the tube Same functions as the key hold gradient Gives an audible alarm for the entered time in minutes 0 99 min in steps of 0 1 The alarm can be reset by the key alarm reset Activates wash program in the pumps provided with that function P 500 and P 6000 For pump A enter 1 0 4 Description of keyboard and programming SYNC A B 4 8 Review block For pump B enter 0 1 For both pumps enter 1 1 When the wash program is activated the pump will set the GP 250 Plus in pause until the wash program is completed The wash program can be aborted with end Note Do not forget to set the injection valve to wash position in order to protect the column from exposure of the high flow rates Drives the pump piston to an end position for pumps that have the function e g P 500 and P 6000 Enter 1 0 for pump A Enter 0 1 for pump B Enter 1 1 for both pumps When the sync program is activated the GP 250 Plus will be paused until the program is completed The sync program can be aborted with end Remember to set the injection valve to wash position in order to protect the column from exposure of the high flow rates Note Only for use with pumps that are provided with the func
57. tion e g P 500 and P 6000 If used with wrong pumps it can cause serious damage to the pump In the Review block programmed methods can be checked and edited The method will be displayed according to the following format TIME VOLUME FUNCTION VALUE step forward and step backward are used to step through the method END OF METHOD will be displayed after the last instruction The method can be changed by using the three special keys change delete and insert which are described below change When programming or editing a method this key allows you to change the time volume or value of a stored instruction When the time volume is flashing enter the new value and press do store All succeeding instructions will be changed by the same time volume as the instruction changed If the entered value is less than the value of the preceding instruction then the time volume of the preceding instruction is used If the time volume is not to be changed just press do store After the time volume has been changed the value of the instruction will be flashing and can be changed The change mode is terminated when the new value if any is stored delete When programming a method this key allows you to delete the displayed instruction When delete is pressed the instruction with time volume and value is displayed flashing but will not be deleted until do store is pressed If step forward or step backwa
58. ump A is not connected to GP 250 Plus or Connect the pump and switch on power If using a P 50 pump power is off The GP 250 Plus is paused if encountered during P 500 or P 6000 pump set flow rate to 0 Check that run the power cable and communication cable are connected and that the RUN button on the pump is in the correct position If so then there is either something wrong with one of the cables or a circuit failure in the GP 250 Plus or the pump Change cable and if failure persists contact a service technician 2 Pump B inoperable The GP 250 Plus is paused if encountered Same as check code 1 during run 3 Fraction collector inoperable The fraction collector is not connected Connect the fraction collector and switch on the power If to the GP 250 not Plus or power is off The GP 250 Plus is paused if that does help change cables If failure persists there is encountered during run a circuit failure in the GP 250 Plus or the fraction collector Contact a service technician 4 Memory has been cleared The GP 250 Plus has been switched off Contact a service technician with too weak a battery 5 Power failure during running or calibration of the pump Power has Press the key alarm reset been switched off or a mains failure has occured while a method was running or during a pump calibration procedure 6 Pump A busy more than 1 sec The GP 250 Plus has received abusy Check if the pressure has exceeded the pre set pressure signal from P
59. ump A with a duration greater than 1 sec Indicates limit If so press the SET button P 500 P 6000 and pump overpressure some other real function of the pump or RUN ALARM RESET at the same time button not pressed Also given if the button SOLVENT CHANGE is pressed on the P 500 or P 6000 Activates wash program from the GP 250 Plus instead The GP 250 Plus is paused if encountered during run 7 Pump B busy more than 1 sec The GP 250 Plus is paused if Same as check code 6 encountered during run 8 Fraction collector is not collecting The Plus is paused if encountered Check the program in the fraction GP 250 collector during run Check if there are any tubes left 10 Uncalibrated pumps A and B Calibration value for pump in Enter calibration value or calibrate the pump Calibration PULSES 0 1 ML DL is equal to zero calibra tion value is gt 9 999 after value for Pump 50 is 234 pulses 0 1 ml P 500 is a calibration of pump A 110 pulses 0 1 ml and for P 6000 it is 45 pulses 0 1 ml Calibration value for P 6000 in dl mode is 4 500 0 1 dl 11 Uncalibrated recorder Calibration value for recorder in PULSES CM Enter calibration value 200 pulses cm for REC 101 and is equal to zero REC 102 12 The flow rate has been set to a value less than ml dl min or greater Set the flow rate to a value in the 0 02 range given than 75 0 ml dl min in volume mode 13 Attempt to start a method in volume mode incorrect or undefined flow Set the flow rate manually
60. und same as logic ground Voltage reference 15 for the signals above Fraction collectors such as the FRAC 100 FRAC 200 and SuperFrac can be controlled from the GP 250 Plus 15 pin female D connector Communication cable Code No 19 6005 02 Electrical interface TTL compatible with 10 KO pull up resistor Max 5 5 V TTL compatible open collectors with a protecting resistor 100 Q in series with the outport Max 15 V Calibration A frequency proportional to the flow rate is available for the fraction connector at pin 13 pump A and B frequency added If the fractionation is done with ml as base the fraction collector has to be calibrated with the same value number of pulses 0 1 ml for pump A and B as the GP 250 plus Functions FEED TUBE PORT SET X X Y port number Y new status 1 active FRAC 100 200 SuperFrac Port 5 pause 1 continue 0 Port 6 run I end 0 Port 7 recycle 1 reset 0 only FRAC 200 Functions controlled in Manual block FEED TUBE PT XXXXXXXXXX the same port numbers and status as for the programmable function TUBE NUMBER 5 Description of rear panel and connections 5 4 Recorders Recommended cable Outports Chart speed Remote Connection Name Active Function Pin voltage Inports all with 10 k Q pull up Hold 0 Will hold a running method while activated Same signalas 4 Method control hold rear panel no 25 Operable in 0
61. viewing programming copying and deleting In the Programming block methods in the currently selected bank can be programmed In the Review block programmed methods can be checked and edited All the instructions in the Method block except BANKCOPY FROM refer to methods in the selected bank Pressing step forward or step backward will give the following choices to edit methods This block is left by pressing exit The first instruction in the Method block shows the currently selected bank and empty methods in that bank This is also the place to change bank e g the display shows 0 2 3 4 5 6 7 8 9 B 1 Status of bank number 1 is shown Method number 1 is programmed and the rest are empty To change bank enter the new bank number 1 5 and press do store A method is defined by a method number and a bank number e g 2 1 method no 2 in bank no 1 Bank no 0 indicates the currently selected bank The display shows the number of instructions remaining which can be programmed in the method the maximum number of instructions are 750 To program a method enter the number and press do store If the selected method is empty the Programming block is entered automatically and programming can be performed If the method is not empty the first instruction in the Review block will be displayed The entered method is deleted if not running After deletion empty methods and bank number are displayed as when pressing m
62. volts Always connect the instrument to a properly grounded mains outlet Note Use the 220 V setting for 230 V mains outlet When turned on supplies mains power 400 mA fuse should be used for 220 240 V instruments and 800 mA for 100 120 V instruments The fuse protects the electronics from damage in the event of short circuiting The lithium battery is mounted on a PC board to replace it contact a local service technician The battery life is approximately 5 years If the power is switched off or a mains failure occurs during run or calibration of a pump the run will continue after the power returns The calibration will however be aborted A check code is given on the display If the GP 250 Plus is locked up by some severe disturbance and pressing end or switching the power off and on does not unlock it then the RESET DIAGNOSIS button will reset all values to initial conditions Press the RESET DIAGNOSIS button on the GP 250 Plus rear panel while turning the power off and on and then release the button after 2 seconds The memory will be cleared all stored methods erased and check code 191 displayed The GP 250 Plus can both receive and give information to two pumps so that pump functions such as flow rate run pause and start of wash or sync program can be controlled Pumps connected to the sockets PUMP A and PUMP B must be of the same type for gradient formation 15 pin female D connector Communication cable Co
63. ward or step backward The data reflects the current value i e the displayed values will be continuously updated This block is left by pressing exit Shows accumulated time volume Shows the loop counter value for the running method When both a calling method and a method to be called contain instructions for looping the counter value will refer to the method currently running When not in a loop no value will be shown at all Shows the current call path e g method 1 in bank 1 is started It calls method 2 in bank 3 The display will show 1 1 c2 3 Call method CALL MET is shown as c Up to four levels of calls can be shown If the nesting is deeper than four levels the four lowest levels will be shown A c directly to the right of PATH will indicate that all levels are not displayed Shows which tube number the fraction collector is presently collecting into If no fraction collector is connected or the fraction collector is not started 1 will be shown By pressing this key the Manual block is entered It allows initial conditions to be set before starting a method or parameters to be changed in a running method without changing the program If either or both the pumps and the recorder are started the run LED is switched on 4 Description of keyboard and programming CONC B ML DL MIN CM MIN PT WASH A B FEED TUBE SYNC A B The displayed value reflects the current value and is continuously upd

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