basicmarathon

USER’S MANUAL

Version 1.4, March 1995

Part no. 0060.103-14

DECLARATION OF CONFORMITY We Spark Holland B.V.

Pieter de Keyserstraat 8, NL- 7825 VE Emmen

declare that the product

"Basic-Marathon" autosampler (p.n. 0816.90x)

is in conformation with the following documents:

# EEC directives 89/392 incl. 91/368 and 93/44 (machine safety) and EEC directives 73/23 and 93/68 (low voltage safety), applied with the following standard:

IEC 1010-1 Safety requirements for laboratory equipment (Class I,

Installation cat. II, Pollution degree II)

Spark Holland will not accept any liability for damages direct or indirect caused by connecting this instrument to devices which do not meet relevant safety standards.

# EEC directives 89/336 and 92/31 (EMC requirements, applied with the following standards:

EN 55011 Radio frequency emission EN 50082-1 Voltage fluctuations IEC 801-2/3/4 Electro magnetic immunity

Use shielded cables and connectors for all remote connections.

Emmen, December 11, 1995 G. de Pee, QA manager

Basic-Marathon user's manual Version 1.4, March 1995

0060.103-14 i

Please read this manual carefully before starting to use the Basic-Marathon autosampler.

This manual describes the Spark Holland BASIC-MARATHON autosampler. The sections deal with appearance and usage of the BASIC-MARATHON autosampler and contains necessary information regarding operation and installation.

Using the BASIC-MARATHON not according these instructions may cause unsafe operation!

If you have any comments on this manual, we will be pleased to receive them at:

SPARK HOLLAND BV, P.O.BOX 388, 7800 AJ EMMEN, THE NETHERLANDS.


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SAFETY PRACTICES General The following safety practices are intended to insure the safe operation of the equipment. Electrical Hazards 1. Removal of some panels exposes potentially dangerous voltages. Disconnect the instrument from all

power sources before removing protective panels. 2. Replace blown fuses with size and rating stipulated on the fuse panel or holder, and in the manual where

listed. 3. Replace or repair faulty or frayed insulation on power cords. 4. Check actual line voltage to confirm it is the value for which this instrument is wired. Be sure power

cords are plugged into correct voltage sources. General Precautions 1. Perform periodic leak checks on supply lines. 2. Do not allow flammable and/or toxic solvents to accumulate. Follow a regulated, approved waste disposal

program. Never dispose of such products through the municipal sewage system.


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SPECIFICATIONS

GENERAL Sound pressure level

<70 dB

Working temperature

10 - 35 °C

Installation category

Category II (according to IEC 1010)

Pollution degree

Degree 2 (according to IEC 1010)

Injection volume

flushed loop:

5 - 500 µl (dispenser controlled), 5 - > 5000 µl (by headspace pressure).

partial loopfill: (only Basic-plus) 10 - 490 µl in steps of 10 µl. (depending on the loopvolume, maximal 50% of the loopvolume)

Injections per vial

max. 9

Analysis time

max. 99 min 59 sec

Needle wash

Selectable, 50 µl fixed volume between vials.

Headspace pressure

Built in compressor. (±0.05 MPa)

Switching time injection valve

electrically <100 msec.

ANALYTICAL PERFORMANCE Reproducibility

Flushed loop injections:

RSD <= 0.5 % Partial loopfill injections: (only Basic-plus)

RSD <= 1.0 % Memory effect

better than 0.2 %

ELECTRICAL Power requirements

115 VAC; ±10%; 50/60 Hz; 150 VA 230 VAC; ±10%; 50/60 Hz; 150 VA


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COMMUNICATIONSee section 4.2.2 OUTPUTS

Inject marker (Relays & TTL) Stop I/O (TTL) Vial number BCD output (TTL) Auxiliary 1 (open collector) Auxiliary 1 inverted (open collector) Auxiliary 2 (open collector) Auxiliary 2 inverted (open collector)

INPUTS

Next injection input (TTL) Next vial input (TTL) Freeze runtime (TTL) Stop I/O (TTL)

OPTIONS Sample tray cooling

Build-in Peltier cooling

Temperature : 4 ±2 °C (fixed) Coolcapacity : Ambient - 20°C ( upto 70% relative

humidity ) King Size tray

48 positions for 5 ml vials (specify cool or no-cool)

Micro injector

0.5, 1, 2, 5 µl loop (Rheodyne)

PC Control

SPARKLINK software for autosampler control.

PHYSICAL Dimensions ( W × D × H )

300 mm × 440 mm × 283 mm 11.8 in × 17.3 in × 11.1 in

Weight

15.5 kg (34 lbs.) 18.5 kg (41 lbs.) , with cool option.


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CHAPTER 1 INTRODUCTION The BASIC-MARATHON autosampler is dedicated to routine analysis of large series of samples in an environment where zero sample loss and injection flexibility are of little significance but where reliability, stability and reproducibility is mandatory. The BASIC-MARATHON therefore makes use of flushed loop injection, guaranteeing reproducibility of injection better than 0.5 % and the Basic-plus has additional the partial loopfill injections mode, guaranteeing reproducibility of injection better than 1.0%. Sample tray cooling (Optional) Sample tray cooling down to 4°C is provided by a build-in Peltier cooling, which is controlled by the BASIC-MARATHON. Large sample volumes (Optional) A special sample tray for 48 large sample vials (5 ml) can be obtained as an option. Loopfilling on a time base by sample headspace pressure can be selected for quick filling of large sample loops as an alternative to regular dispenser controlled loopfilling on a volume basis.


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CHAPTER 2 INJECTION PRINCIPLE A six-port high pressure switching valve can perform the injections based on the flushed loop principle. Loopfilling can also be done on a volume basis, controlled by the digital dispenser system or on a time basis by headspace pressure on the sample (ref. SYSTEM SETTINGS, 5.5.1) to shorten filling time for larger loop volumes. The switching patterns of the valve in "LOAD" and "INJECT" position are shown in Figure 2.1. An injection is performed as follows: 1.1 DISPENSER CONTROLLED INJECTION

1. Dispenser aspirates approximately 10 µl air bubble in the sample needle for more efficient removal of the previous sample.

2. With the injection valve in inject position, the coaxial needle pair is inserted into the sample. Air pressure, applied to the sample through the outer air needle, ensures that no air or vapour bubbles are formed during sample withdrawal.

3. Dispenser withdraws "preflush volume" from sample vial to fill the sample line with sample and to remove previous sample. (30 - 999 µl, programmable)

4. Injection valve switches into load position; dispenser transports 2 or 3 x loopvolume through the loop to fill the loop quantitatively. 2 x loopvolume for loops equal to or larger than 100 µl, 3 x loopvolume for loops smaller than 100 µl. In case of partial loopfill injections (Basic-plus) the loop is filled with the programmed injection volume.

5. Injection valve switches into inject position. Sample loop is now part of the HPLC mobile phase flow path: sample is transported to the column. Analysis time starts.

If one injection is to be made out of each sample vial, the needle pair withdraws after analysis time and sequence 1-5 starts anew for the next vial. If more than one injection is to be made out of the same sample vial, the needles remain in the vial and after analysis time the next injection is made starting the sequence at 3 with a fixed preflush of 4 µl.


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1.2 PRESSURE CONTROLLED INJECTION

1. With the injection valve in load position, the coaxial needle pair is inserted into the sample. Air pressure, applied on the sample through the outer (air-) needle, will force the sample via the inner (sample-) needle through the sample loop. The duration of the headspace pressure is programmable; appropriate duration has to be determined experimentally.

2. Injection valve switches into inject position. Sample loop is now part of the HPLC mobile phase flow path: sample is transported to the column. Analysis time starts.

A special sample drain, that prevents siphoning, must be connected to the injection valve (replacing the dispenser connection), refer to section 4.2.3.3.

If one injection is to be made out of each sample vial, the needle pair withdraws after analysis time and sequence 1-2 starts anew for the next vial. If more than one injection is to be made out of the same sample vial, the needles remain in the vial.

1.3 DIGITAL DISPENSER SYSTEM (DDS) Working principle The DDS is a miniature "finger pump". The operating principle is shown in Figure 2.3: Fingers 1 and 3 act as inlet and outlet pinch valve respectively, finger 2 provides the fluid displacement. The stroke of finger 2 is factory adjusted to provide 2 µl per stroke. Since the volume dispensed is always a multiple of one stroke volume (digital dispensing), i.e. 2, 4, 6 µl etc., odd values for volumes will be round down to even values.


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CHAPTER 3 INSTRUMENT DESCRIPTION 1.4 GENERAL DESCRIPTION

1.Sample tray, consisting of 4 removable segments 2. Keyboard 3. Display 4. Cover with hinges 5. Needle arm with sample needle 6. Injection valve with sample loop 7. Dispenser cassete screw 8. Dispenser 9. Outlet tubing holder 10.Common leakage/condensation water drain for injection valve and sample tray.


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1.5 INJECTION SYSTEM

1. Side-port sample needle 2. Air needle 3. Air tubing to provide sample headspace pressure 4. Needle penetration depth adjustment screw 5. Injection valve 6. Digital Dispenser System 7. Pump tubing Cassette 8. Dispenser drain 9. Sample tubing needle → valve (port 2) 10. Sample tubing valve (port 3) → dispenser 11. Sample loop 12. Sample drain for loopfilling by sample headspace

pressure. Replaces sample tubing (10) 13. Vial stripper. 14. The silicon tubing connector with air inlet hole must be

approximately at the same level as the liquid in the sample vial to prevent hydrostatic sample flow during analysis time.

15. Air inlet hole in connector to prevent emptying of sample vial by hydrostatic suction.

16. Needle connection nut 17. Ferrule (PTFE) 18. Sample needle connection hub 19. Air seal 20. Vial sensor


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CHAPTER 4 INSTALLATION 1.6 UNPACKING Unpack the BASIC-MARATHON carefully. When unpacked, inspect the instrument for damage. Should the instrument not be in good order, or if any of the items listed below is missing, please notify the supplier immediately. For contents of the shipping container see packing list in container. 1.7 CONNECTIONS

1. Fuses and voltage selector 7. Connector vial number output 2. Mains input 8. Connector control I/O

3. Mains switch 9. Connector auxiliaries 4. Cooling fan, only in case of tray cooling. 10. Optional communication connector 5. CE mark 11. Type label 6. Standard RS232 connector


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1.7.1 POWER CONNECTIONS - Before plugging in the power cable, check voltage settings of the modules at the input socket on the rear

panel. - Make sure that the voltage settings of the modules are identical with the voltage of your local power

supply. - Use only a supply appliance with protective grounding. - If the indicated voltage is not correct, select the proper voltage by removing, inverting, and than re-

inserting the voltage selector. - When the voltage selection and fuses are correct for your power source, plug in the power cable. The correct fuses should be installed.

For 115 V (AC) ± 10%, use two 5.0 AT fuses (slow) For 230 V (AC) ± 10%, use two 2.5 AT fuses (slow)

RISK OF FIRE, REPLACE FUSES AS MARKED!

1.7.2 COMMUNICATION CONNECTORS 1.7.2.1 RS232 connector RS232 serial interface. BASIC-MARATHON can be controlled by a PC using the RS232 interface and the SPARKLINK application software from Spark Holland (optional). Refer to the SPARKLINK manual for operating instructions. 1.7.2.2 Auxiliaries connector

1.7.2.3 Control I/O connector

Pin No. I/O description

1 + 24 volt DC 2 Auxiliary output 2 inverted (open collector, max 250 mA) 3 Auxiliary output 2 (open collector, max 250 mA) 4 Auxiliary output 1 inverted (open collector, max 250 mA)

5 Auxiliary output 1 (open collector, max 250 mA) 6-8 Ground


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The duration of input 5, 7, and 8 must be at least 20 msec!

1.7.2.4 Vial number output connector 8-bit BCD-coded VIAL NUMBER OUTPUT (True logic), representing vial numbers 1-96; active when corresponding vial is processed.

Pin No. I/O description

1 Inject marker output (TTL, active low)

2Inject marker common (for N.O./N.C. contacts) 3 Inject marker output (N.C. contact) 4 Inject marker output (N.O. contact)

Note:Inject marker contacts are closed/open during the programmed marker pulse length. Starting at the moment of injection.

5Stop I/O (TTL, pulled up); output is low when BASIC-MARATHON ends processing (READY); pull low input will stop processing (BASIC-MARATHON goes to READY).

6Freeze (HOLD) input (active low, pulled up, TTL) This input has the same function as the HOLD/CONT key (ref. 5.6.1).

7 Next vial input (active low, pulled up, TTL) This input triggers a next vial to be processed when BASIC-M ARATHON is in REMOTE CONTROL mode.

8Next injection input (active low, pulled up, TTL) This input triggers a next injection when BASIC-M ARATHON is in REMOTE CONTROL mode. Can be used as START input (also when BASIC-MARATHON is not in REMOTE CONTROL mode).

9-15 Ground

Pin no Output Pin no Output 1 Bit 0 units 1 9 Bit 1 units 2 2 Bit 2 units 4 10 Bit 3 units 8 3 Bit 4 units x 10 11 Bit 5 units x 20 4 Bit 6 units x 40 12 Bit 7 units x 80 8 Ground 15 Inject marker (TTL, active low)


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BCD: Binary Coded Decimal The 2-digit vial number on the BCD output is represented as two four bits nibbles, where D3-D0 represent the ones (0-9) and D7-D4 represents the tenths. The highest possible vial number is therefore 99 (see Table 4).

Output

Value

l = ON

D0 (1)

D1 (2)

D2 (4)

D3 (8)

0

1

l

2

l

3

l

l

4

l

5

l

l

6

l

l

7

l

l

l

8

l

9

l

l

Tenth

Ones

BCD

D7 (8)

D6 (4)

D5 (2)

D4 (1)

D3 (8)

D2 (4)

D1 (2)

D0 (1)

Vial number

1

0

0

1

0

1

1

0

Output

9 × 10 (101)

6 × 1 (100)

96

90 + 6 0

1

0

1

1

0

0

1

Output

5 × 10 (101)

9 × 1 (100)

59

50 + 9 0

0

0

1

0

0

0

0

Output

1 × 10 (101)

0 × 1 (100)

10

10 + 0


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1.7.3 FLUID CONNECTIONS The instrument has been tested with isopropanol: make sure that the mobile phase of your HPLC system is miscible with isopropanol. If not, start up with an intermediate solvent as mobile phase (disconnect column). Connect the HPLC pump to port 5 and the column to port 6 of the injection valve. To get access to the fluidics, it is necessary to open the front cover of the BASIC-MARATHON, therefore remove the screw on the lefthand side of the cover. 1.7.3.1 Sample loop The BASIC-MARATHON is factory-equipped with a 20 µl sample loop. If a different volume is to be injected, the sample loop must be replaced. Please note that 3 x the loop volume is withdrawn from the sample vial for loops smaller than 100 µl and 2 x the loop volume, to fill the loop quantitatively. For partial loopfill injections the maximum injection volume for a loop is 50% of the loopvolume. Loops ranging from 5 µl to 5 ml are available, either from SPARK HOLLAND or directly from Rheodyne. 1.7.3.2 Digital Dispenser System (DDS) The DDS pumptubing cassette is factory-installed and connected to the injection valve. Fasten the pumptubing cassette before use with the screw on top of the pumptubing cassette. If the pumptubing cassette needs replacement, refer to Appendix A. 1.7.3.3 Sample drain for loopfilling by headspace pressure TIME based loopfilling by sample headspace pressure can be selected (SYSTEM SETTINGS, ref. 5.5.1) to shorten loopfill time for larger loop volumes (Ref. Chapter 2, INJECTION PRINCIPLE). To allow time based loopfilling and to prevent hydrostatic sample loss, the special sample drain for headspace pressure loopfill must be connected at the injection valve, replacing the dispenser connection at the valve (Ref. 3.2 for description).


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1.8 PREPARATION OF VIALS 1.8.1 VIAL DIMENSIONS To avoid malfunctioning of the instrument, purchase standard vials from Spark Holland (ask your local dealer):

Chromacol ref. : 2-CV (1.5 ml vial) 11-AC4 (crimpcap)

Spark p/no's: 3760.401 (vial, 1.5 ml)

3760.402 (crimpcap); For small sample volumes or for low sample loss (low rest volume, see 4.3.2) use Chromacol 1,1 ml conical vials with support sleeves (ask your local SPARK dealer):

Chromacol ref. : 1.1-CTV (conical 1.1 ml vial) TTS-312 (support sleeve) 11-AC4 (crimpcap)

Spark p/no's : 3760.407 (conical 1,1 ml vial)

3760.408 (support sleeve) 3760.402 (crimpcap)

For large sample volumes (max 5 ml), use

Spark p/no's : 3760.409 (5 ml vial) 3760.410 (crimpcap)

If other vials are to be used, make sure that the following specifications are met:

minimum height: 33 mm maximum height: 40 mm (including cap) maximum diameter: 11.8 mm (standard tray) minimum cap diameter: 10 mm minimum space for septum penetration: 4 mm diameter (hole in cap)

The use of caps with a diameter smaller than 10 mm may cause damage to the BASIC-MARATHON.

The use of Eppendorf microcentrifuge tubes may cause malfunctioning of the vial sensor of the BASIC-MARATHON.


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1.8.2 SAMPLE CONSUMPTION Rest volume: Since the sample needle is always at some (very short) distance above the bottom of the sample vial, a certain volume of sample always remains in the vial, out of reach of the sample needle. This volume is called the rest volume. For standard 1.5 ml vials, the rest volume is approximately 200 µl. For a conical vial, the rest volume is only a few µl. Refer to Appendix A for adjustment of needle penetration depth. Pre flush volume: Prior to the FIRST injection, the sample needle and tubing (needle → injection valve) are flushed with sample (for dispenser controlled loopfill, ref. chapter 2, INJECTION PRINCIPLE). Depending on the concentration differences between samples and the cross-contamination allowed, this so-called pre flush volume may range between 30-999 µl (programmable). (note that only dispenser controlled loopfilling uses a pre flush). If sample loss > 30 µl for pre flush is not acceptable, select NEEDLE WASH between sample vials in SYSTEM SETTINGS (ref.5.5.1) to avoid cross contamination. Injection volume: Due to the reduced solvent velocity near the wall of the tubing (loop), it takes more than the nominal loopvolume to fill the loop reproducibly: BASIC-MARATHON takes for flushed loop injections twice the loop volume from the sample vial for a sample loop equal to or larger than 100 µl and three times the loopvolume for a sample loop smaller than 100 µl. In case of partial loopfill injections (Basic-plus only) the programmed sample volume is aspirated into the sample loop, but because of the same phenomenon only the half loopvolume can be injected. Sample consumption formula (dispenser controlled): The minimum amount of sample needed for N injections per vial: Flushed loop injections for 100 µl loop or larger:

rest volume + pre flush volume + 2N x loopvolume + (N-1) x 4 (µl) Flushed loop injections for loop smaller than 100 µl:

rest volume + pre flush volume + 3N x loopvolume + (N-1) x 4 (µl) Partial loopfill injection (Basic-plus only):

rest volume + (1+ (N-1)/2) x pre flush volume + N x injectionvolume (µl) Example: 3 flushed loop injections out of a standard sample vial using a 20 µl loop and 30 µl pre flush

volume, minimum sample volume needed is:

200µl+30µl+3x3x20µl+ 2x4µl=418µl


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Sample consumption for headspace pressure controlled loopfill If the sample loop is filled on a TIME base by sample headspace pressure (ref. 5.5.1, SYSTEM SETTINGS and chapter 2, INJECTION PRINCIPLE), sample consumption has to be determined experimentally since the flow is dependent on the viscosity (for water the flow rate is approximately 1 ml/min). Make sure that programmed filltime is sufficient to flush the loop with at least twice its volume. If more injections are to be taken from the same vial, please note that a volume equal to max. 20% of the headspace in the vial after loopfill may flow to waste during analysis time due to expansion of the headspace back to atmospheric pressure. 1.8.3 FILLING AND SEALING THE VIALS The 1.5 ml vials, as well as the conical vials, can best be filled by means of a narrow-end pipette to allow air to escape when filling the vial. Do not fill vials to the edge! In that case sample will be forced into the air needle, risking extra cross-contamination of samples. It is important that the seal be airtight, to maintain a constant pressure on the vial for air bubble prevention and reproducible injections. For loopfill by headspace pressure, obviously leaking vial septa will impair reproducibility.

DO NOT RE-USE A VIAL WITHOUT REPLACING ITS CAP OR SEPTUM.

Check seal after crimping: if cap can be turned easily, the seal is not airtight (readjust handcrimper). 1.8.4 LOADING THE SAMPLE TRAY To allow remote loading of the sample tray, the sample tray consists of four removable segments that fit on one UNIQUE position of the tray holder only. Use the STEP key (ref. 5.3, KEY FUNCTIONS) to rotate the tray (functioning only when the BASIC-MARATHON is in READY status).

DO NOT ROTATE THE TRAY MANUALLY; YOU MAY DAMAGE THE TRAY DRIVE MECHANISM!

Positioning of vials Sample vials should be placed in sequential positions on the tray. Empty spaces between vials of a sample series are allowed, since the BASIC-MARATHON can detect empty positions. After detecting an empty place the BASIC-MARATHON will continue with the next position. Additional samples can be placed during RUN since LAST VIAL value can be changed during RUN (Ref. 5.6, RUN). Needle wash vials If needle wash is programmed (ref. 5.5.1, SYSTEM SETTINGS), place 4 vials with washing solvent (1.5 ml each, usually mobile phase) on positions 93-96 (place all 4 vials; they will be used alternately).


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CHAPTER 5 OPERATION Operating the BASIC-MARATHON is easy; after programming the system SETTINGS, only a few parameters need to be programmed for regular automatic processing of a series of samples. All parameter values are saved in memory after power switch OFF, except tray temperature setting, if tray cool option is installed: tray cooling is always OFF after power UP. 1.9 PRINCIPLE DIFFERENT PROGRAMMING MODES To simplify regular operation, common injection parameters like "analysis time" and "first/last vial" are programmed in the RUN PARAMETER mode. Less frequently altered parameters like "auxiliaries YES/NO" are programmed in the SYSTEM SETTINGS mode. An additional COOLING CONTROL mode takes care of the sample tray temperature, when the option is installed. OTHER MODES:

RUN mode: automatic processing of samples. REMOTE CONTROL mode: autosampler control via I/O contacts. SERIAL RS232 mode: autosampler control via serial interface.

READY FIRST READY status is the "ground state" of the BASIC-MARATHON which is initiated automatically after power UP. Every mode can be entered from READY status, except PRIORITY mode, which can be entered only when BASIC-MARATHON is in RUN mode. In addition, RUN PARAMETER and COOLING CONTROL mode can be entered when BASIC-MARATHON is in the RUN mode. Function codes are used to enter a certain mode except for the RUN PARAMETER mode which can be entered directly via the PROG/END key and RUN mode (entered by the START command). Every mode uses the same function code (F,0) to exit, except for RUN PARAMETER mode and RUN mode which are left via PROG/END and STOP respectively. 1.10 FUNCTION CODES The following table shows all the possible modes of the BASIC-MARATHON Autosampler. See also the programming chart.

Function code

Selected mode

F,9

Select SYSTEM SETTINGS mode.

F,7

Select COOLING mode.

F,2

Select PRIORITY mode.

F,3

Select REMOTE CONTROL mode.

F,4

Select SERIAL: RS232 mode.

F,0

To exit all modes, selected by a function code.


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1.11 KEY FUNCTIONS

Key:

Function:

STEP key; used to step through the parameter list. The STEP key is also used to rotate the sample tray (in READY status only!).

Start/Stop automatic processing of samples.

Hold/Continue countdown of analysis time. Analysis time is extended with the time that hold is active.

Enables/Ends programming of parameters in the selected mode. Note that the PROG/END key selects the RUN PARAMETER mode and enables the programming of its parameters at the same time.

Clears the value of the parameter on display.

Function key; used in combination with a numerical key to select or exit the various modes.

Numerical keys; used to enter values for programmable parameters. Values are stored when going to the next parameter (STEP) or when ending programming (PROG/END).


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1.12 PROGRAMMING CHART

READY

RUN PARAMETER LIST

SYSTEM SETTINGS

COOLING CONTROL

Use to step.

1. FIRST VIAL (1→ 48/92/96) 2. LAST VIAL (1→ 48/92/96) 3. NO OF INJ'S/VIAL (1→ 9) 4. PREFLUSH VOLUME (30→ 999µl) FLUSH TIME (1→ 999SEC) 5. INJECTION VOLUME (10→ 490µl)‡ 6. ANALYSIS TIME (0:00→ 99:59) 7. TIME AUX 1 ON (0:00→ 99:59) 8. TIME AUX 1 OFF (0:00→ 99:59) 9. TIME AUX 2 ON (0:00→ 99:59) 10. TIME AUX 2 OFF (0:00→ 99:59)

(4,5 and 7-10 depend on setting)

Use to step.

1. LOOPVOLUME (0→999µl) 2. SAMPLE VOLUME (STD/LSV) 3. INJ. MODE (FLUSHED/PARTIAL)‡ 4. FLUSH (VOL/TIME) 5. NEEDLE WASH (YES/NO) 6. AUXILIARIES (YES/NO) 7. MARKER PULSE LENGTH (0.1→2.0)

TRAY COOLING (ON / OFF)

PRESS TO ADVANCE SAMPLE TRAY

REMOTE CONTROL MODE

SERIAL RS232 MODE

DEVICE IDENTIFIER (10 → 19)

RUN MODE

ANALYSIS TIME

Accessible: Run parameters, Cooling control.

PRIORITY MODE

default vial no 00!

NOTE

‡: Only available in the Basic-plus autosampler

Enter number to activate

FREEZE ANALYSIS TIME

Accessible: Run parameters, Cooling control.


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1.13 PROGRAMMING Only four modes contain programmable parameters:

- SYSTEM SETTINGS mode to "set" system parameters. - RUN PARAMETER mode for regular programming of sample processing. - COOLING CONTROL mode to program the tray cooling, if installed. - SERIAL RS232 mode for autosampler control via serial interface. The device identifier is also

programmable in this mode. 1.13.1 SYSTEM SETTINGS With BASIC-MARATHON in READY status, key in F,9 to select SYSTEM SETTINGS mode. Press PROG/END to enable programming of the following parameters (use STEP key to go to the next parameter): - LOOP VOLUME; Enter volume of the installed loop; 0-999 µl - SAMPLE VOLUME (STD/LSV); Enter 0 to select STANDARD sample tray. enter 1 if the

optional LARGE SAMPLE VOLUME tray has been installed 48 vials of 5 ml).

- INJECTION MODE (FLUSHED/PARTIAL); (Basic-plus only)

Enter 0 to select FLUSHED loop injection mode. Enter 1 to select the PARTIAL loopfill injection mode.

- FLUSH (VOL/TIME); Enter 0 to select dispenser controlled loopfilling on VOLUME base.

Enter 1 to select loop filling by sample headspace pressure on TIME base. Filling on time base may be used for large injection volumes (> 500 µl) to shorten loopfill time.

FOR LOOPFILLING ON A TIME BASE, MOUNT THE SPECIAL DRAIN TO ALLOW FOR PRESSURE CONTROLLED SAMPLE TRANSPORT AND TO PREVENT HYDROSTATIC SAMPLE FLOW WHICH MAY EMPTY YOUR SAMPLE VIAL DURING ANALYSIS TIME (Ref. 3.2)!

- NEEDLE WASH (YES/NO); Enter 1 to select needle wash.

Position 93-96 are now used as containers for washing solvent. After every sample vial the needle and connecting tubing is flushed with solvent from one of the "wash vials" (alternating between 93 and 96). Enter 0 if no needle wash is wanted (position 93-96 can now be used for sample vials).

- AUXILIARIES (YES/NO); Enter 1 if time programmable auxiliary output(s) are to be

used. (ref. 4.2.2) Parameters will appear in the RUN PARAMETER mode.

Enter 0 if no auxiliaries are to be used. - MARKER PULSE LENGTH; Enter the pulse length of the inject marker; 0.1 - 2.0 sec.


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1.13.2 RUN PARAMETERS With BASIC-MARATHON in READY status, press PROG/END to select RUN PARAMETER mode and to enable programming at the same time. The following parameters can be programmed (use STEP key to go to next parameter): - FIRST VIAL; enter position number of the first vial of the sample series in the sample tray.

1-96 without needle wash, 1-92 with needle wash, 1-48 in case of LSV. (ref. 5.5.1 SYSTEM SETTINGS)

- LAST VIAL; enter position number of the last vial of the sample series in the tray.

(see FIRST VIAL for valid numbers) - INJ/VIAL; up to 9 injections can be taken from the same vial.

Enter number 0-9. - PRE FLUSH VOLUME; (parameter shows up for flush on volume basis only; ref. 5.5.1 SYSTEM

SETTINGS); enter volume (of sample) which is to be used to flush needle + connecting tubing prior to filling the loop.

30-999 µl. - FLUSH TIME; (parameter shows up for flush on time base only; ref. 5.5.1 SYSTEM SETTINGS);

enter duration of headspace pressure for loopfilling. 1-999 sec.

- INJECTION VOLUME; (Basic-plus only)

Enter the injection volume for the partial loopfill injections. 10 - 490 µl in steps of 10 µl. (maximal 50 % of the loopvolume)

- ANALYSIS TIME; enter the time interval needed before the next injection takes place.

0-99'59". Time countdown starts at moment of injection. - TIME AUX 1 ON; (parameter shows up only when auxiliaries are enabled in System settings (ref.

5.5.1). Enter time at which auxiliary 1 must switch ON. 0-99'59".

- TIME AUX 1 OFF; (parameter shows up only when auxiliaries are enabled in System settings (ref.

5.5.1). Enter time at which auxiliary 1 must switch OFF. 0-99'59".

- TIME AUX 2 ON; see AUX 1. - TIME AUX 2 OFF; see AUX 1.


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1.13.3 COOLING CONTROL (optional) Sample tray temperature control is switched ON/OFF in the COOLING CONTROL mode. With BASIC-MARATHON in READY status, key in F,7 to select COOLING CONTROL mode. Press PROG/END to switch ON or OFF the cooling control. The sample tray cooling has a fixed temperature of 4°C - TRAY COOLING (ON/OFF); Enter 0 to switch OFF the sample tray cooling.

Enter 1 to switch ON the sample tray cooling.

AFTER POWER UP THE SAMPLE TRAY COOLING IS ALWAYS OFF!


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1.14 RUN When programming is ready (Ref. 5.5), system is ready (Ref. CHAPTER 4, INSTALLATION) and BASIC-MARATHON is in READY status, press START/STOP key to start automatic processing of the samples. During RUN, BASIC-MARATHON will show information relevant to the actual process like: SEARCH VIAL 45, LOOPFILL, WASHING etc. During analysis time the display will show the following information continuously (example):

The first line shows that the second injection from vial 15 is being processed, vial 82 is the last vial to be processed. The temperature of the cooling tray shown at the end of the first line will only be displayed if the option is present. The second line shows the time since the injection. The status of the auxiliaries, the number will be displayed if the auxiliary is ON. (Only shown when enabled in the settings) The first character of the second line will be a blinking r if the BASIC-MARATHON is under remote control or a blinking p if a priority sample is programmed. It is possible to reprogram RUN PARAMETERS during RUN; simply press PROG/END to enter the RUN PARAMETER mode (ref. 5.5 for programming). Press PROG/END to exit RUN PARAMETER mode and to continue the RUN program.

If you do not return to RUN mode, RUN will stop after finishing the current injection!

Changes in the RUN parameters will be executed immediately after returning to RUN mode (although an injection will not be interrupted). COOLING CONTROL can also be reprogrammed during RUN; press F,7 to enter COOLING CONTROL mode (ref. 5.5 for programming). Return to RUN (F,0) to continue RUN! 1.14.1 HOLD HOLD (press HOLD/CONT) can be used to freeze the time base. During HOLD, the RUN PARAMETER mode, COOLING CONTROL mode and PRIORITY mode are accessible to reprogram/check parameters (ref. 5 .5). Exit any of these modes (if entered) and press HOLD/CONT key to continue RUN. Analysis time of the current injection has then been extended with the time that HOLD was active.

VIAL:15 →82 2'INJ 4°C

pTIME 03:45 AUX 1 2


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1.14.2 PRIORITY SAMPLE A sample run can be interrupted to process an emergency sample prior to the next sample of the programmed samples series: press F,2 to enter the priority mode, press PROG/END key and enter the number of the tray position where the priority vial has been placed. Press PROG/END to end programming. Return to RUN (F,0). Priority vial will be processed immediately after the current vial. A blinking "p" (first position of lower display line) indicates that a priority vial is to be/being processed. After processing the priority vial, RUN will continue where it was interrupted. 1.15 REMOTE CONTROL When BASIC-MARATHON is in REMOTE CONTROL mode it can only be controlled using the "next injection" and "next vial" inputs (ref. 4.2.2). RUN is executed as programmed, but analysis time is now infinite. Only a "next injection" input signal will initiate the next injection from the same vial (or from the next vial when the programmed number of injections per vial have been executed). A "next vial" input signal will initiate the first injection from the next vial. With BASIC-MARATHON in READY status, REMOTE CONTROL mode is entered with F,3; display will show:

A "next injection" input signal will start the RUN. A blinking r (first position of the lower display line) indicates that BASIC-MARATHON is under remote control. All other relevant RUN information will be displayed as usual (ref. 5.6, RUN). When all programmed vials have been processed, display will show:

Next injection or next vial inputs will no longer have any effect. Exit REMOTE CONTROL mode (F,0) to return to READY status.

REMOTE CONTROL

READY

REMOTE CONTROL

F,0 TO EXIT


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1.16 SERIAL INTERFACE BASIC-MARATHON can be controlled by a PC using the RS232 interface and the "SPARKLINK" application software from Spark Holland (optional). Press PROG/END to enable programming of the device identifier, programmable from 10 to 19 (Default: 11). Refer to the SPARKLINK user's manual for operating instructions. This manual is available on request as a separate document. When BASIC-MARATHON is used as the autosampler for Spark Holland's Programmable On-line Solid Phase Extraction system (PROSPEKT), it is controlled by the PROSPEKT using the RS232 interface. In this case the device identifier must be set to 10. Refer to the PROSPEKT user's manual for operating instructions. 1.17 SPECIAL DISPLAY MESSAGES During operation several messages may appear on display to make you aware of errors, system malfunctioning or other uncommon events. These special display messages are listed below together with the appropriate actions to take. MEMORY ERROR ALL VALUES DEFAULT

Press any key; if BASIC-MARATHON goes to READY status, all parameters are set to default values. Reprogram RUN and TEMP.CONTROL parameters and check SYSTEM SETTINGS. If message remains: call service engineer!

TRAY ROTATION FAILURE

Tray movement is obstructed; check/remove obstruction. Press any key to continue; if message remains: call service engineer!

NEEDLE MOVEMENT FAILURE

Needle arm movement is obstructed; check/remove obstruction. Press any key to continue; if message remains: call service engineer!

DISPENSER FAILURE

Call service engineer!

PROGRAM VIALS

1. SYSTEM SETTING STD/LSV was changed: FIRST VIAL and

LAST VIAL are reset to 0; reprogram (RUN PARAMETER mode). Press any key to continue.

2. NEEDLE WASH was switched ON (SYSTEM SETTINGS) with FIRST or LAST vial programmed on WASH positions. FIRST VIAL and LAST VIAL are reset to 0; reprogram (RUN PARAMETER mode). Press any key to continue.

ERROR!(.............)

An erratic value has been entered; limits for this parameter are shown in parenthesis on display. Enter correct value.

1.18 AFTER USE If BASIC-MARATHON is not to be used for a while, flush needle and valve with water and isopropanol (can be placed as last vials of a sample series)and loosen pumptubing cassette one turn. Never leave BASIC-MARATHON out of use with a buffer or salt solution in the tubings; crystals may block the flowpath and may damage the rotor seal of the injection valve. Power can be switched OFF since all parameter values are saved in memory (long-life non-chargeable battery back-up). Only sample tray cooling is changed to OFF after power UP.


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APPENDIX 1 MAINTENANCE

1. Side-port sample needle 2. Air needle 3. Air tubing to provide sample headspace pressure 4. Needle penetration depth adjustment screw 5. Injection valve 6. Digital Dispenser System 7. Pump tubing Cassette 8. Dispenser drain 9. Sample tubing needle → valve (port 2) 10. Sample tubing valve (port 3) → dispenser 11. Sample loop 12. Sample drain for loopfilling by sample headspace pressure. Replaces sample

tubing (10) 13. Vial stripper 14. The silicon tubing connector with air inlet hole must be approximately at the

same level as the liquid in the sample vial to prevent hydrostatic sample flow during analysis time.

15. Air inlet hole in connector to prevent emptying of sample vial by hydrostatic suction.

16. Needle connection nut 17. Ferrule (PTFE) 18. Sample needle connection hub 19. Air seal 20. Vial sensor


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1.1 NEEDLE PENETRATION DEPTH To keep rest volume small, the sample needle should stop close to the bottom of the sample vial. If penetration depth needs adjustment, proceed as follows (see Figure A.1):

- Open keyboard cover. - Place an uncapped vial in the tray. - Program a loopvolume of 0 µl. Press START and disconnect the power supply as soon as the needle is

in the vial. - Loosen adjustment screw one turn (counter-clockwise). - Using the needle connection nut as a grip, adjust needle penetration depth: Needle should stop not less

than 1 mm above the bottom of a standard vial and not less than 2 mm above the bottom of a conical vial.

- Fasten adjustment screw (clockwise). - VIAL SENSOR MUST BE AROUND THE NEEDLE! - CLOSE KEYBOARD COVER! - Connect power.

1.2 PRESSURE RANGE OF INJECTION VALVE Refer to Appendix D.4 for adjustment of pressure range of RH 7010 injection valve. 1.3 FUSES If fuses need to be replaced, refer to section 4.1 of the manual.

DISCONNECT POWER BEFORE TAKING OUT THE FUSES!


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1.4 NEEDLE PAIR REPLACEMENT 1.4.1 SAMPLE NEEDLE To replace sample needle proceed as follows:

- Open keyboard cover. - Disconnect the power supply. - Loosen needle connection nut (Figure A.1) that connects PTFE tubing to sample needle. - Remove sample needle by pulling it out of its fitting with the PTFE tubing that usually sticks to the

needle. - Put in new needle (watch the air seal around the needle). - When needle connection tubing needs replacement, loosen the nut that connects the tubing to the

injection valve. - Cut the appropriate length (185 mm) of a 0.25 mm I.D. tubing taking care that cuts are made

perfectly rectangular to avoid dead volumes. - Connect tubing to needle. - Connect loose end of needle connection tubing to port 2 of injection valve (use a Rheodyne ferrule!).

Do not tighten excessively; it may close the tubing! - Check needle penetration depth (ref. Appendix A.1) and adjust if necessary. - Connect mains power supply. - VIAL SENSOR MUST BE AROUND THE NEEDLE! - CLOSE KEYBOARD COVER!

1.4.2 AIR NEEDLE To replace air needle (Ref. Figure A.1), proceed as follows:

- Remove sample needle as described in A.4.1 - Unscrew air needle and install new air needle. - Reinstall sample needle as described in A.4.1

1.5 DISPENSER PUMP TUBING Refer to Figure A.1.

- Loosen fitting that connects PTFE inlet tubing of dispenser to injection valve. - Remove pump tubing cassette including PTFE connection tubing, by loosening the screw that attaches

the cassette to the dispenser. - Install new pump tubing cassette. - Check length of PTFE tubing (160 mm). - Connect PTFE tubing to injection valve.

1.6 INJECTION VALVE For maintenance and replacement procedures, see Appendix D


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APPENDIX 2 LARGE VOLUME SAMPLES (Optional) 1.7 DESCRIPTION The large volume sample tray is an option of the BASIC-MARATHON Autosampler. This option provides the possibility to inject large volumes of sample with the DDS or by headspace pressure. The option consists of: - 4 LSV sample tray segments

- 500 µl-loop - a serum needle with serum air-needle - special sample tubing (0.5 mm ID) - sample drain

1.8 INSTALLATION Install the LSV-option according to the following procedure:

- Replace the standard installed tubing and needles by the tubing and needles of the LSV option. Follow the procedure described in appendix A.

- Replace the standard tray by the LSV tray.

- Install the 500 µl-loop to port 1 and 4.

- Check the needle penetration depth and if necessary adjust it, see appendix A.1.

- Connect the drain to port 3, if the loop will be filled on time base by headspace pressure. It is also

possible to fill the loop with the DDS, but this will take less sample, but more time.

- Change the SYSTEM SETTINGS; LOOPVOLUME : 500 µl SAMPLE VOLUME : LSV FLUSH : TIME

The last SYSTEM SETTINGS parameter change is necessary for pressure controlled injections.

If the loop is filled with the DDS, be aware that a larger preflush volume (minimal 50 µl) has to be programmed to avoid cross contamination, because of the larger internal diameters of the tubing and needle.


0060.103-14 27

APPENDIX 3 BASIC-MARATHON TEST PROCEDURE The BASIC-MARATHON is factory tested for reproducibility and carry over according to the following test procedure. The BASIC-MARATHON is tested in an analytical system with the following conditions (see Figure C.1)

Seven vials are placed in the BASIC-MARATHON tray, vial 1 and vials 4 till 7 are the vials with sample (50 ppm Uracil), vials 2 and 3 are filled with eluent (blanks).

SYSTEM SETTINGS RUN PARAMETERS

LOOPVOLUME : 20 µl FIRST VIAL : 1 SAMPLE VOLUME : STD LAST VIAL : 7 INJECTION MODE 1: FLUSHED NO. OF INJ/VIAL : 3 FLUSH : VOLUME PREFLUSH : 30 µl NEEDLE WASH : NO ANALYSETIME : 1:00 min AUXILIARIES : NO INJ. MARKER PULSE : 0.1 sec. Note 1: Only for Basic-plus

PUMP FLOW : 1.5 ml/min

ELUENT : distilled water DETECTOR WAVELENGTH : 254 nm SAMPLE : Uracil in distilled water (50 ppm)


0060.103-14 28

An example chromatogram of the test is printed above. From the integration results the Relative Standard Deviation (RSD%) can be calculated. The necessary formula's are printed below:

Calculating the RSD% from the example integration results will give a RSD% of 0.36%.

Peak Retention Peak Number Time Area ====== ========= ========== 1 0.877 14.57611 2 2.181 14.65836 3 3.480 14.71600 4 13.363 14.59072 5 14.665 14.61937 6 15.964 14.62571 7 17.513 14.59504 8 18.815 14.65542 9 20.114 14.67614 10 21.680 14.58954 11 22.983 14.64998 12 24.281 14.49563 13 25.831 14.57137 14 27.129 14.58835 15 28.430 14.62109


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APPENDIX 4 RHEODYNE INJECTION VALVE Description and replacement/adjustment instructions of the Rheodyne model 7010 injection valve. 1.9 INTRODUCTION The model RH 7010 Sample Injection valve is a six-port rotary valve with removable external sample loop, designed for high performance liquid chromatography. The valve should give years of dependable performance with easy maintenance. The RH 7010 is supplied with tube fittings to permit connection of 1/16" tubing to the valve ports. Two wrenches for servicing are also supplied. The following standard sample loops are available; each loop is supplied with fittings for direct connection to the valve:

Sample loop size Tubing bore Catalog number microlitres mm inch

RH 7022

20

0.51

0.020

RH 7023

50

0.51

0.020

RH 7024

100

0.51

0.020

RH 7025

200

0.76

0.030 1.10 DESCRIPTION OF MODEL RH 7010 The model RH 7010 operates by rotation of a flat rotor against a flat stator. Figure D.1 shows the port configuration and switching pattern. Rotation of the shaft through 60 degrees switches the valve from LOAD to INJECT. Figure D.2 is an exploded view of model RH 7010. The stator is type 316 stainless steel. The rotor is faced with a Vespel rotor seal. Slots in the rotor seal make flow paths between adjacent stator ports, and turning the rotor 60 degrees switches the flow paths as shown in Figure D.3. The diameter of the slots is 0.46 mm (0.018 in.). Holes in the stator are 0.61 mm (0.024 in.) in diameter and connect to the threaded tubing ports on the outside of the stator.


0060.103-14 30

Two stop pins and the rotor pin limit the rotation to 60 degrees. Four conical spring washers and a thrust bearing are used to push the rotor seal against the stator face with sufficient force to hold 7000 psi liquid pressure. Three set screws in the stator are factory adjusted so that when the three stator screws are fully tightened, the force between stator and rotor is sufficient to hold 5000 psi. To increase maximum operating pressure to 7000 psi, follow the directions in section D.3. The isolation seal serves to prevent valve leakage from entering the spring and bearing region of the valve. It does not serve any high-pressure sealing function. The maximum operating temperature of the Model RH 7010 is 150 °C To avoid clogging of the valve passages, it is recommended that filtered solvents be used and that sample liquid be free of solids that might clog the loading tubes. 1.11 ADJUSTING FOR LEAKAGE OR HIGHER PRESSURE OPERATION The three small set screws in the stator (see Figure D.2) are factory-calibrated so that when the three stator screws are fully tightened, the spring force between the valve rotor and stator is sufficient to hold 5000 psi. If leakage is to be corrected, or if operation up to 7000 psi is to be carried out, proceed as follows: Loosen the three set screws about 1/20 turn each (rotate 18 degrees) and then tighten the three stator screws an equal amount each. If this new setting fails to accomplish leak-free operation at the pressure required, repeat the procedure with an additional 1/20 turn. Avoid excessive tightening, which accomplishes nothing but increases wear of the rotor seal. Should it be necessary to loosen the spring tension, either to lower operating pressure or to adjust for a new rotor seal which may be thicker than the one being replaced, reverse the above procedures, i.e. first loosen the stator screws and then tighten the set screws. If leakage cannot be stopped by tightening the valve, or if, a s a result of tightening to stop the leakage, the valve switch is significantly slower with the same air pressure, the rotor seal needs replacing. 1.12 DISASSEMBLY AND ASSEMBLY 1.12.1 DISASSEMBLY Disassembly of the valve for servicing of the liquid end is accomplished as follows:

a. Remove the three stator screws. Do not change the setting of the three set screws in the stator.

b. Refer to Figure D.2, and remove stator and stator ring from valve body by pulling axially to disengage the various pins.

c. Isolation seal and bearing ring are usually left in place since they rarely need changing.


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1.12.2 ASSEMBLY Reassembly of the valve requires putting all parts back together again as shown in Figure D.2, while observing the following:

a. The rotor pin can be inserted in the rotor from one side only (the side without the peened closure marks around the pin hole). Do not try to force the rotor pin in from the wrong side, and ensure that the flat on the valve shaft faces away from the protruding end of the rotor pin.

b. Ensure that the rotor seal is correctly oriented as shown in Figure D.3, with rotor seal slots facing the

stator, and with the notch in the metal rim of the rotor seal in line with the rotor pin.

c. When replacing stator ring, be sure that the two stop pins are replaced in their holes in the stator ring; then push the stator ring squarely onto the rotor assembly so that the stop pins enter the mating holes in the body (with rotor pin between the two stop pins), and so that the isolation seal slips inside the stator ring without hanging up.

d. Replace the stator by first pushing it onto the two pins on the stator ring and then adding the three

stator screws. Tighten each screw, a little at a time, to keep the stator surface parallel to the stator ring surface. If the three set screws in the stator where not changed, then tighten the three stator screws until all parts are firmly in their place. The three set screws will insure that the gap between stator and stator ring is uniform and exactly as it was before disassembly.

e. If the set screws need adjusting because a new rotor seal was installed, or because leakage needed

to be stopped, ensure that each set screw is turned an equal amount so that after re-tightening the stator screws, the gap between stator and stator ring is uniform all around. Refer also to Section D.3.

1.13 OPERATING SUGGESTIONS 1.13.1 AQUEOUS BUFFERS OR SALT SOLUTIONS To prevent formation of salt crystals in the valve, flush out the flow passages and needle port with water after using salt solutions. 1.13.2 USE OF HIGH pH SOLUTIONS The standard rotor seal is Vespel, a DuPont polyimide, which is exceptionally wear-resistant. It is, however, susceptible to alkaline attack and deteriorates rapidly when used with solutions of pH 10 or higher. An alternative material is available for alkaline applications (consult Rheodyne or local Spark dealer). 1.13.3 CLOGGED VALVE PASSAGES If valve passages are clogged, open them by removing the stator and cleaning the passages with a small wire (maximum diameter 0.015 in.).


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1.13.4 CALIBRATING SAMPLE LOOPS Sample loop sizes are designated by nominal values which can vary from the actual value by as much as 20%. This is due to the 0.001 inch tolerance of the tubing I.D. (see table below). Since standards as well as unknowns are usually analyzed with the same loop, knowledge of absolute loop volume is rarely needed. If the actual loop volume must be known, it is best to calibrate it in place on the valve, so that the flow passages in the valve (one in the rotor, two in the stator) are also taken into account.

Tubing bore

(inches)

Volume tolerance resulting from

0.001 inch bore tolerance

0.012

17 %

0.020

10 %

0.030

7 %


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APPENDIX 5 BASIC-MARATHON LOGBOOK

Customer information: Name of user

Company

Department

Address

Telephone

Fax

BASIC-MARATHON information: Serial number

Firmware version

Purchase date

Installed options

Local Dealer

Service Engineer

Address

Telephone

Fax

Comments:


0060.103-14 34

basicmarathon

Documents

following safety practices

safety requirements

low voltage safety

safety practices general

machine safety

following standards

relevant safety standards

eec directives