Eicom HTEC-500 Manuel utilisateur

HPLC-ECD Stand Alone System

(HTEC-500)

User’s Guide

Eicom Corporation

AC01-0319

Contents

1. Important Checklist Before Operation ............................................. 1

2. Terminology .......................................................................................... 2

3. A List of Fitting Parts and Directions for Use...................................... 3

4. Mobile Phase Preparation.................................................................. 4

5. System Installation ............................................................................... 5

5-1. Wiring.......................................................................................... 5

5-2. Tubing and Degasser............................................................... 5

5-3. Connecting to the Autoinjector and the Autosampler

(Plumbing) ......................................................................................... 5

5-4. Connecting to the Autoinjector and the Autosampler

(Electrical Signal Connection) ....................................................... 6

6. Online Elution Degassing System ...................................................... 7

6-1. If you think the degassing efficiency is reduced,

you may diagnose the problem by the

following procedure: .................................................... 7

7. HPLC Pump Operation ....................................................................... 8

7-1. Run/Stop .................................................................................... 9

7-2. Flow Rate Setting...................................................................... 9

7-3. Pulse Free Mode Setting.......................................................... 10

7-4. Function of Pulse Free Mode .................................................. 10

8. How to Fix Pump Errors and Problems .............................................. 11

8-1. Less Flow Rate, Less Pump Pressure or

Unstable Pump Pressure ............................................... 11

8-2. Types of Pump Errors ................................................................ 12

8-3. How to Fix these Errors.............................................................. 13

9. Manual Sample Injector and Autoinjector...................................... 16

9-1. Structure..................................................................................... 16

9-2. Operation of Manual Injector ................................................ 16

9-3. Autosampler and Autoinjector Setup ................................... 17

10. Precolumn ............................................................................................ 18

10-1. Types of Precolumn Housing and Line Filters........................ 18

10-2. Identification of Precolumn Quality....................................... 19

10-3. How to Repack ......................................................................... 20

10-4. Filter Exchange ......................................................................... 21

11. ECD Operation..................................................................................... 22

11-1. Basics.......................................................................................... 22

11-2. Working Electrode.................................................................... 23

11-3. Applied Potential...................................................................... 23

11-4. Time Constant ........................................................................... 25

11-5. Background Current ................................................................ 25

11-6. If Background Current is High ................................................. 25

11-7. Caution for the Platinum Working Electrode........................ 25

12. Storage of the System......................................................................... 26

12-1. Short-term (A few days to a few weeks) ................................................26

12-2. Long-term (more than a few days or a few weeks) ............................26

13. Storage of a Standard Sample ......................................................... 27

14. Regular Maintenance Schedule....................................................... 28

Daily Maintenance.............................................................................. 28

Occasional Maintenance.................................................................. 28

Yearly Maintenance ........................................................................... 28

15. Troubleshooting ................................................................................... 30

Appendix. A ................................................................................................... 33

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HTEC-500

1. Important Checklist Before Operation

• The separation column and the enzyme column must be held firmly by the column clip. The stainless

steel surfaces should make good contact with each other in order to ground static electricity. This

may be the cause of noise in the ECD.

• Place the mobile phase container and the waste container on the same level as the HTEC-500.

• Use glass bottles as mobile phase and ultrapure water containers. Do not use any plastic containers.

• Rinse behind the pump pistons with ultrapure water before and after use through the pump piston

washing port. Do not use any organic solvents in the rinse solution. If precipitation or something blocks

the line, do not push out with high pressure or force. First, disconnect the line and then remove the

source of the blocking.

• Before opening the detector cell for maintenance, remove the outlet screw and aspirate all the

remaining mobile phase in the detector cell with a syringe.

• Pay attention to prevent any tubing from becoming blocked. Salt solvents must be rinsed out by

ultrapure water before an organic solvent of a high concentration is used. Drying out of the salt

solvent should also be avoided.

• Pay attention not to allow any leakage from any connection points of the tubing. Leakage is not only

harmful for the equipment but also generates pump pulse noise.

• When air is thought to be present in the tubing or the pump, please purge the air (see chapter 8,

page 11). Then you can connect a column.

• Pulse free mode must be switched on during the analysis.

Column Clip

Separation Column (PP-ODS)

Door

A separation column must be held firmly by the column clip.

When an enzyme column is used, the enzyme column must

also be held firmly by the column clip. The respective stainless

steel surfaces should make good contact in order to ground

static electricity. This can be the cause of noise in the ECD.

Fig. 1 Separation Column Installation

HTEC-500

2. Terminology

Display Manual Injector

HPLC Pump

(See Fig. 7)

Online Elution

Degasser

Column Clips

ECD Cell

Door Cabinet

Thermo Controller

Control Panel

ECD Terminal Ch1

Tubing Guide

Terminal Ch2

(for PEC-510)

Tubing Guide

Cell Mount

Fig. 2 HTEC-500 Front View (Door open)

Status Indicator

Cabinet Drain

Main Power Switch

(Back Side)

Fig. 3 HTEC-500 Front View (Door closed)

HTEC-500

3. A List of Fitting Parts and Directions for Use

Tough Connector

Low pressure part. For column connections.

Flat Seal for Degasser

The wide side is for the degasser and

narrow side is for the nut.

Hex Nut

High pressure part (pumps)

Rheodyne PEEK nut

Pheodyne PEEK Ferrule

(For Injector)

Nut for Inlet of Cell

Ferrule for Inlet of Cell

The narrow side is for the cell and

the wide side is for the nut.

Flat Seal Nut

Two-way Joint

Tubing connection

Connect the tubing and keep its tip sticking out of the ferrule

(as seen above), except for the connection with the degasser

which uses a flat seal. First, put the tubing through the ferrule

and put its tip into the holder. Then connect it by tightening

the screw. The connecting part should be sealed by a cross

section of the tubing and the holder of the tubing. For the

injector connection, use the nuts and ferrules for its connection

only. The slit side is for the nut (see figure).

Fig. 4 Tubing Connection Parts and Directions for Use

HTEC-500

4. Mobile Phase Preparation

Prepare the mobile phase according to the Eicom technical information. The mobile phase preparation

depends on what you are measuring with the HTEC-500. Please read below to find a general method

for mobile phase preparation. Also, the following points should be noted because the ECD has a higher

sensitivity compared to other detectors:

• Use ultrapure water (the resistance of the water needs to be at least 18.2 mega-ohm/cm). Any

solvent, such as mobile phase, or water applied to this system should be stored in a glass bottle.

• Use glass instruments for the preparation of mobile phase. Before use, these instruments should be

rinsed out by water and then by ultrapure water.

• Use reagents of super grade or HPLC grade quality. The following are our recommendations: for

sodium 1-Octanesulfonate, use Nacalai (code 3179-62, available at Eicom); for sodium

1-Decanesulfonate, use TCI (code I-0348 available at VWR); and for EDTA-2Na use Dojindo

(code 372.24). Separation patterns or ECD can be influenced by the use of other reagents.

• Methanol and buffer solution/water must be measured separately and then mixed. Otherwise the

methanol concentration will be higher than the designated level due to the solubility of methanol in

water.

• Do not use a sonicator for dissolving salts in the mobile phase. This may raise the background current

of the ECD.

• When all reagents are added, plug the bottle and shake and mix it thoroughly. Check if all reagents

are dissolved.

• Filtration is good for removing unwanted particles but can be a source of chemical contamination.

We do NOT recommend you to filter the mobile phase but rather encourage you to avoid any

contamination during the preparation of the mobile phase. Using less glassware and always putting a

cap or cover on the top of the containers helps to reduce chances of contamination. If you must filter

the mobile phase, use a chemically clean apparatus and do not store the mobile phase in plastic

bottles after the filtration.

• A filter at the end of the inlet tubing placed in the mobile phase can also be a cause of chemical

contamination or bacterial growth. Eicom does not recommend you to use these type of filters. If you

need to use one, please replace the filter frequently and keep it clean. Instead of these filters, we

recommend you to set a precolumn before the separation column. (If using the PP-ODS column,

please use in-line filter #PP-LF, before the precolumn and put a precolumn before the injectors.) The

precolumn filter after the HPLC pump does not prevent contamination of the HPLC pump.

• Mobile phase can only be used for one week from the date of opening/preparation. Bottles should

be well sealed while it is being used for HPLC. Do not add new mobile phase to the bottle of old

mobile phase. This is to avoid the breeding of various germs in the system.

HTEC-500

5. System Installation

5-1. Wiring

Connect the terminal adapter (orange color) to the terminal at the right side of the system. Then

connect the AC cable to the main power supply and turn on the system. “Initializing” appears on the

display, followed by the ECD controller screen. Now you can turn off the system for the next steps

described below.

5-2. Tubing and Degasser

Pull the tubing’s for the pump rinse, the mobile phase suction and the cell drain out of the HTEC-500

system. Put all the equipment on a level place. Mobile phase and waste bottles should be placed on

the same level as the HTEC-500 (See Fig. 5).

5-3. Connecting to the Autoinjector and the Autosampler (Plumbing)

Injectors of the autoinjector (online autoinjector, EAS-20) and/or the autosampler should be set up

downstream to the manual injector. For the autoinjector, connect no. 3 port of the manual injector with

no. 2 port of the autoinjector using 0.125 mm PEEK tubing (red). Then connect no. 3 port of the

autoinjector with the separation column using the same kind of tubing. For the autosampler (model

Alcott 719D or 719AL), connect no. 3 port of the manual injector with no. 2 port of the autoinjector and

no. 3 port of the autoinjector with the separation column.

Please place the HTEC-500 on a horizontal and stable table. The mobile

phase and waste containers should be placed on the same level as the

HTEC-500.

Fig. 5 HTEC-500 Installation

HTEC-500

Mobile Phase

(Always use a glass bottle)

Pump Piston Washing

Syringe for Aspiration

Pump Piston Washing

Ultrapure Water

Mobile Phase Waste

Injector Waste

HTEC-500

5-4. Connecting to the Autoinjector and the Autosampler

(Electrical Signal Connection)

A trigger signal needs to be sent from the online autoinjector or the autosampler to the data acquisition

unit to inform it of the injection timing. It also needs to be sent to the HTEC-500 in order to give the auto

zero signal. The auto zero works to shift the baseline to the zero level. Please connect the trigger signal

output on the autoinjector/autosampler with “signal in” of the terminal adapter located on the left side

of the HTEC-500. Please also refer to Fig. 6. There is no polarity on “signal in”. To find where the output of

the trigger signal is, please look at the user’s manual of your autoinjector/autosampler. If your

autosampler has an “emergency stop input”, please connect this terminal of the autosampler to the

pump error out on the HTEC-500 signal terminal. A non-polar contact closure signal will occur when the

pump on the HTEC-500 gives error readings (see chapter 8, page 12 for the errors). This way you can

prevent loosing samples when the HPLC pump HTEC-500 accidentally stops.

Please follow this diagram to figure out the electrical connections. The ports not showing +/- have no

polarity. AUTO ZERO and SIGNAL OUT 0sec are connected before installation. With this connection,

M.INJECTOR SIGNAL IN receives a signal when the manual injector is set to the inject position from the load

position and then SIGNAL OUT 0 sec sends a signal to AUTO ZERO IN. Through this signal, AUTO ZERO is

complete. SIGNAL OUT 3 sec sends a signal to the trigger port of the EPC-300 (not included in this system).

Through this signal, a chromatograph starts to be recorded. PUMP ERROR OUT is a signal port to stop the

auto sample injection from the auto sampler when the pump flow is not good. For more details, please

refer to the instruction manual for the auto sampler. PUMP PRESSURE and TEMPERATURE MONITOR are ports

to record operation condition of this system.

Connect to

EPC-300 (option) CH terminal

Autosampler emergency stop

EAS-20/Autosampler (option)

Manual injector

EPC-300 CTL terminal

EPC-300 TRG

Fig. 6 Electrical Connections

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