basic operation of accutof

1

Basic Operation of the AccuTOF

And Getting Specification Data 3/9/2006

Basic Procedure (This is after you will get good evacuation value 3x10-5Pa or less)

1. Cerate New Project file(Folder) (with Analysis list) (page-9 b. create new project)

2. MCP conditioning (in MS tune manager) (page-13 a-2 MCP conditioning & a-3)

3. Do “Noise Coefficient of Avarager” (page-13 a-4 Optimum of base line noise)

4. Base Line Noise Check (page-17 b Check base line noise level)

5. Resolution test (tuning) (page-18 c Resolution test)

6. Make calibration file (page-34 e Calibration)

7. Sensitivity Test (pare-43 f Sensitivity test)

8. Exact Mass Test (page-56 Exact Mass test)

9. Negative tune and making tune file (page-62)

10. (other Attachment test) (page-62)

**You have to do a-2 & a-3 every re-starting vacuum system.

2

1. Start AccuTOF

Drying Gas(to Flange) Nebulizing Gas(to Probe)

Needle 1 (ESI/APCI/etc.) Error LED

EVAC SW

Needle 2(for dual ESI or Corona ESI)

VENT SW

Heater (ESI/APCI/etc)

Power ON SW

Power OFF SW

Gas Flow Control valve

a. Turn on “Power ON SW” (VENT SW LED will be lightning)

b. Turn on “EVAC SW” (Must need N2 gas presser more than 600Mpa) > Start pumping down

EVAC SW LED will be stop the LED-blinking until evacuation presser 1.4x10¯4Pa or less

Error LED will be light if revolution of turbo 1 & 2 reach 80% after next in 8 minutes.

Or pump-system will be something wrong.

c. Start the computer, printer and etc.

d. After light on the EVAC LED must wait 4095 minutes.

It is EVAC-cont-pb has timer for protect the MCP from spark of damage.

(Finally Presser will be reach to 1or3 x10 –5 Pa or less) (at least 5x10-5pa)

(Reason: MCP is very sensitive spark of damage, easy to break by spark.)

(But! if Evacuation pressure condition is good that pressure reach to normal value

before 4095 minutes you can invalid timer on EVAC-cont-pb)

For example: After shot time vent for maintenance of orifice 1, 2 , ring lens and RP, TMP

or any short time electricity maintenance.

But either way to need check presser level (1to3 x 10-5pa)

“How to Check presser” is “e. Check evacuation condition and MS monitor” following

3

e. Check evacuation condition

You can check evacuation presser on the MS-tune or MS-monitor.

(MS-tune is in the MassCenter main software , MS-monitor on desitop)

Open from or

f. After start pump down recommend to check on MS Monitor

(Check Vacuum Gage level and MCP Elapsed Time on Exhaust tab)

>(see “a Detail of MS Monitor” as below)

2. Shutdown AccuTOF (pump stop) operation.

a. Confirm LC or any solvent is not running.

b. Close Neblizing Gas and Drying Gas valves. (Operation on PC)

c. Close MS Tune Manager and MassCenter software (On PC)

d. Confirm N2 gas presser more than 600kPa.

(If N2 Gas presser dose not reach more than 600kPa, You can not vent evacuation.)

e. Push “VENT” switch on front panel.

“VENT” switch will be blinking next 10 minutes.

f. After 10 minutes VENT switch will be light.

g. You can see N2 gas that blow out from the Orifice 1.

h. Close N2 Gas line (If necessary)

i. Shutdown Computers and LC-system. (If necessary)

j. Push “OFF” switch on front panel. (If necessary)

k. Turn off “Main Breaker” (If necessary)

Now the AccuTOF Stop completely.

A. Explanation of MassCenter

MassCenter Main program Every AccuTOF software run from her

(Expect Data-manager, MS-monitor)

Data Manager Program Maintenance MassCenter files

Periodic Table Program

Isotopic Simulator Program

MS Monitor Program Monitor every unit of AccuTOF Condition

Force System to Quit If MassCenter freeze, use this Icon for quit

MassCenter software.

4

a. Detail of MS-Monitor

[General tab]

Unit select tab

Around panel’s door SW

Actual condition

Condition LED (You have red LED, when some function is wrong)

Also if you have red-LED, you can not choose any “Instrument Mode” on the MS-tune.

(See next page)

Can not choose any mode

[Ion Source tab]

There is flange open/close sensor

at the flange.

Condition LED

5

RP TMP

①①①①

②②②②

③③③③

Orifice1 Orifice2

Ring Lens

Ion Guide

[Explain actual voltage of Orifice 1,2,and Ring lens]

Set point of Orifice 1,2 and Ring Lens Voltage is

①①①①,②②②②,③③③③ on right pictures

①①①① : Ring lens Voltage

(Potential difference between Orifice 1 and Ring lens)

②②②② : Orifice 1 Voltage

(Potential difference between Orifice 1 and Orifice2)

③③③③ : Orifice 2 Voltage

(Potential difference between Ion Guide Bias and Orifice2)

Actual Voltage is :

Ring Lens is Ion Guide bias Voltage[Analyzer tab] + Orifice 1 + Orifice 2 + Ring Lens Set Voltage.

Orifice 1 is Ion Guide bias Voltage[Analyzer tab] + Orifice 2 + Orifice 1 Set Voltage.

Orifice 2 is Ion Guide bias Voltage[Analyzer tab] + Orifice 2 Set Voltage.

[Analyzer tab]

Sensor in Ion Guide Power Unit.

Ion Guide Bias Voltage

Condition LED

6

[Detector Tab]

If you have never do MCP conditioning

after run evacuation,

Condition LED

[Exhaust Tab]

Condition LED

If you do not have ”More then 4950min”, you can not choose “Instrument Mode” on the MS-tune.

7

b. Detail of Force System to Quit

b-1. Click here

Get MassCenter Process

b-2 You can see running process on here

b-3 Click here Terminate MassCenter System (process stop)

b-4 Click Get MassCenter Process again then ascertain the following window. Then click OK

(If you have process again,

Click Terminate MassCenter System also again.)

b-5 Click Close (close window)

8

Data Reduction

MassCenter Main

(Data Acquisition(Data Acquisition(Data Acquisition(Data Acquisition））））

Agilent1100 Managet

CalibrationCalibrationCalibrationCalibration Sample Inlet ControlSample Inlet ControlSample Inlet ControlSample Inlet Control

Calib. MCalib. MCalib. MCalib. Manageranageranageranager

Chromatgrum Spectrum Viewer

●Chromatgram reduction

●Real Time Chromatgram Viewer

●Spectrum reduction

●Real Time Spectrum Viewer

Quantity Manager

●Calibration Data

●Make file of Calibration and Mass Drift Compensation

●Drift Compensation Data

●Mass Assign Information

●Make Quantity Project

●Quantity Data Reduction

TuningTuningTuningTuning

MS Tune Manager

●Tuning Parameter

●Single Acquisition

● Control Agilent1100

● Agilent1100 Parameter

PRO JECTPRO JECTPRO JECTPRO JECT

etc .

● MS Tune Parameter ● MS Acquisition setting

●Acquistion Data ●Quantity Data ●Quantity Project

● Agilent1100 parameter

●Analysis List

●Data measurement

Continuous Measurement

B. About MassCenter Software and opration.

[MassCenter Main]

MassCenter Main is fundamental system on AccuTOF Software.

Following applications are started from “MassCenter Main”.

MS Tune Manager,

Mass Calibration Manager

Agilent1100 Manager

Chomatgrum Viewer

Spectrum Viewer.

Each parameters are saved in Project Folder. You can create your own new project and save every

parameters and data files. You can administer every data file and parameter in each project folder.

[For example] If you change any parameter, not affect any parameters in another project folder.

Note: Mass calibration files are administered in MassCenterMain. Accordingly, Calibration files are

common files every projects.

9

1. Start Mass Center Main software

a. Start MassCenter Main from desktop. Double click

b. Create New Project

b-1. File > New

b-2. Open New window

Choose (double click) Project Wizard

Then OK

If you have had Created wizard files,

check Load Setting then choose from here

This case no choose then Next>

b-3. Create project name(TEST)

Choose or create save folder name

(C:/ACCUTOF)

then Next>

10

b-4. Choose “Create New Project”

Check “Create Analysis list”

(If you want create analysis list

same time, check here)

New Analysis List name

(Spec-test)

If you want to create new project from to have created project, check here and choose here.

(This mean, you want create new project almost same condition with you are having project.)

Then Next>

b-5. Choose your own parameter(Field)

Select here

This case(Spec-test) you should choose:

Sample Location,

Acquisition Data,

Acquisition Data Folder,

MS Acquisition Condition,

Agilent1100. (If customer has Aglent1100)

b-6. Finish Wizard

Click Finish

If you want save this setting condition, check “save setting” then put new name to here.

[Change Project]

Choose File > Open Project

11

If you have Agilent1100 LC-auto sampler and detector choose use “in Agilent1100”

Instrument > Configuration > Details tab

Module use in System

Auto Sampler : within Agilent1100

UV/VIS detector : within Agilent1100

2. Conditioning MassCenterMain Window

Analysis List

Project Name

Agilent 1100 status panel

Open Agilent1100 manager from here

Project View

MS Status Panel

Open MS Tune manager from here

Disk free space panel

Queue View

Displaying process in measurement

12

a. Open Analysis List

a-1-1 File > Open

a-1-2 Choose Analysis List File name

Analysis List

Other Window

Choose your own to show window from here

3. MS tuning

a-1. Open MS Tune manager from MassCrenterMain.

[MS Tune manager Window]

13

Check evacuation presser It will be 1.**x10-5Pa or less.(it will be green bar)

a-2. MCP conditioning (It need to do, when first using after pumping down.)

a-2-1

If you can’t choose MCP condition’s menu, you should check vacuum condition on the MS monitor.

a-2-2 Set MCP conditioning parameter.

Voltage =2500v

Time=120min

Than Click start

a-2-3 Close window after finish conditioning.

If do not increase voltage on this window,

you should check door-SW or HV-PB.

Actual voltage

Close window

a-3. After finish MCP conditioning increase High Voltage of Flight-Tube, Reflectoron, and “Pulser Unit” by manually.

a-3-1. Every voltage set to”0” then operate mode to “Warm Up” mode. (MCP=0v)

a-3-2. open “monitor window” and “vacuum window” watch base line and presser when increasing voltage.

If you have arcing in analyzer area, those may change. If you have arcing, decrease voltage immediately.

Or back to “Evacuation Ready” mode.

Also open MS monitor window then watch actual voltage.

a-3-3. Flight tube voltage: -300v step to -3000v. Leave and watch about 30-60sec each step’s voltages.

a-3-4. Reflectorn voltage: 50v step to 300v. Leave and watch about 30-60sec each step’s voltages.

a-3-5. +/- pulser voltages: 50v step to 300v. Leave and watch about 30-60sec each step’s voltages.







a-3-12. set every voltage to the “performance report setting” then leave 30 minutes and watch base line/presser.

Conform NO ARCING in the Flight Tube area.

14

a-3-13. increase MCP voltage slowly 100v step to 2500v.

Do same way on Negative mode before operating negative analysis.

a-4. Optimum of base line noise

a-4-1. Choose Tools > Noise Coefficient of Averager

It is optimum of base line noise on the FASTFLIGHT.

a-4-2. Start process. Click Start

It is only to do, when install, move and replace the FASTFLIGHT.

It is not daily routine.

a-5. Open Tuning parameter and MS monitor window.

[For example : Window layout]

15

a-6. Open tune parameter windows

a-5-1 right click on each function icon then choose Display or Display New Window

a-7 Open MS monitor window

a-6-1 Click here

a-8. About mode menu

You can choose operate mode

by two ways

If you did not do MCP conditioning , you can not choose “Warm-UP, Standby and Operate Mode”

Also, if you did not pass time 4095minutes or more after pumping down.

16

[Explanation of each mode]

Evacuation Ready >> Evacuation presser is 2x10-4Pa or less.

High volt (analyzer field and Ion Source) OFF

Temperature OFF

(Usually choose this mode , when the MassCenter is closing.)

Warm-UP >> High volt (analyzer field, Including MCP-voltage) ON

(Daily Rest position) High volt (Ion Source) OFF

Temperature OFF

(Choose this mode , when short time close the MassCenter.

and usual rest operation (night time))

Standby >> High volt (analyzer field, Including MCP-voltage) ON

(Daily Rest position) Temperature ON

High volt (Ion Source) OFF

Operate >> Every High Volt and Temperature ON

Notes: N2 Gas valves are separate with this mode. Any time can do open/close.

a-8. About MS monitor window

a-8-1. Change display MS Range from “Setup”

Right click then choose Setup

17

[Spectrum Monitor Parameter]

Start MS and End MS

Same as Scan speed on sector type MS

Averager Sampling Time. (ns)

(Same as data sampling points)

Short time is better for Mass accurate.

But can not available on wide scan mass range.

(Too much data point on one data)(too big data size)

Up to 2000 = 0.5ns (? not sure)

a-8-2. Zoon up MS monitor

There are three way for zoom up.

1. MS range and Intensity

Drag slanting then choose own zoom

up size.

2. Zoom Up Only Mass range

Drag horizontally

(Do not make window)

3. Zoom up Only Intensity

Drag Vertically

(Do not make window)

4. Double click in window to back to initial Size

18

b. Check Base line noise level (test-1)

b-1 set in each value as following.

Analyzer(Advanced) setting

Pusher volt.= 777.8v(or see performance sheet)

Pulling volt=-777.8v(or see performance sheet)

Flight Tube=-7000v

Detector Setting

= 2000v

ESI Ion Source Setting

Needle volt = 0v

Orifice 1 volt = 0v

Orifice 2volt = 0v

Ring Lens = 0v

Spectrum Monitor parameter

Range =100 to 1000

Spec. Recording Interval = 0.4 s

Data Sampling Interval = 0.5 ns

(other parameter will be any setting OK)

Take “Operate” Mode

Intensity count (up – low < 100)

Noise width is 100 counts or less.

(Usually 50 to 60 counts)

19

C. Resolution Test

R>6000

Sample : Reserpine m/z=609+ (using concentration 50to100ppb with Methanol )

On infusion mode = use syringe pump

Pump Flow : 0.05 to 0.2 ml/min

(Never use on customer’s LC-pump for Resolution test = Never pour a Reserpine to LC-bottles)

Usually high concentration is better for resolution. (High peak intensity)

c-1. Set up Syringe Pump and connect to ESI Probe.

Syringe Pump

Syringe (5 or 10 ml)

Connect between syringe

and probe.

c-2. Set syringe inside diameter if necessary.

(see Inside Diameter list on syringe pump)

c-3. Set Base Tuning condition that from a last page of the performance report.

c-3-1. Choose ESI+ mode

c-3-2. Set condition from performance report.

[Analyzer setting – General and Detail]

[Analyzer Advance Setting]

c-3-3. Set condition

[ESI+ Ion Source Setting Voltage/Current and Temperature/Gas]

Needle 1 Voltage [V] 2000v

Ring Lens Voltage [V] 10v

Orifice 1 Voltage [V] 80v

Orifice 2 Voltage [V] 32v

[Temperature]

Desolvating Chamber [C] 250c (check temperature control)

Orifice 1 [C] 80c

c-3-4 Take to [Standby] mode

20

c-3-5 Set Detector Voltage (step up)

1000v > 1500v > 2000v > 2500v

(finally to see performance sheet)

c-3-6 check Probe condition

Adjust needle position

0.1 to 1 mm (200ul/min)

3mm (10ul/min)

0.1 mm (1ml/min)

Pump flow on > Check probe tip to see sample coming.

c-3-7 Wait while increase temperature

c-3-8 Take [Operate] mode

Gas ON (ESI+ Ion Source Setting Menu)

Nebulizing Gas ON (Gas Flow rate 1cc/min)

Dringing Gas ON (Gas Flow rate 2.5-3.0 cc/min)

(Finally adjust gas flow by seeing ion intensity that increase highest)

c-9 Adjust Resolution and Intensity refer following Tune manual

AccuTOF: Peak Tuning

1. General

Tuning is intended to introduce to the detector samples ionized by the ion source as efficiently as possible without any loss.

The sample sprayed from the ESI needle is dried by the N2 gas to be ionized, introduced to the vacuum from the orifice

1, and directed to the ion guide in the transfer field through the ring lens and orifice 2. Ions are advanced to the analyzer

field by the energy of the bias voltage at the ion guide, to which the bias voltage and high frequency voltage are applied.

They are adjusted by the focusing lens to maximize the volume to be forwarded to the orthogonal ion accelerator. They are

accelerated by the orthogonal accelerator toward the flight tube, reversed by the reflectron, and forwarded to the detector.

The mass number is determined by the time of the flight from the accelerator (start) to the detector (goal).

The TOF-MS determines the mass number solely by the time when the ions arrive at the detector (goal). As a result, if

the ions spread widely at the orthogonal ion accelerator (start), ions with the same mass number will have different start

positions, and arrive at the goal at different times, compromising resolution and sensitivity. For the analyzer field alone,

tuning the TOF-MS is intended to minimize the spread of ions in the vertical direction at the orthogonal ion accelerator

(horizontal spread is unrelated to the distance to the detector with reference to the direction of ion acceleration (downward

direction in the figure).

To achieve that goal, adjusting the focusing lens, pusher bias voltage, and suppress voltage is essential. (See 2-2 below

for details.)

21

760 Torr 2 Torr 3x10－－－－3 Torr

Orifice1 Orifice2Ring Lens

Ion Guide

AccuTOF Model Diagram

2. Description of Components

2-1. Orifice, Ring Lens, Ion Guide

(Ring Lens Voltage)

The ring lens is located between orifices 1 and 2. When a voltage is applied to the ring lens, ions that have passed

through the orifice 1 will efficiently be directed to the orifice 2. (See the figure above.) Thus, the ring lens voltage will

determine the detection sensitivity for the target ions. Optimizing the ring lens voltage is essential in detecting the target

substance at high sensitivity. Normally, the ring lens voltage is set low to detect ions with smaller molecular weight, and

high to detect ions of high mass compounds.

22

(Orifice 1 Voltage)

The orifice 1 voltage is the difference in potential between orifices 1 and 2. If the difference is great, ions that have

passed through the orifice 1 collide more frequently with vapor molecules in the area between orifices 1 and 2, resulting in

a hard condition where fragment ions are easily detected (called In-Source CID). Setting the voltage difference between

orifices 1 and 2 low will result in a mild condition where molecular ions are easily detected.

(Orifice 2 Voltage)

The orifice 2 voltage is the difference in potential between the ion guide and orifice 2. The potential difference is

intended to efficiently direct ions to the ion guide. When the voltage is increased while keeping constant the potential

difference between orifices 1 and 2, and the potential difference between the orifice 2 and ion guide is increased, the CID

may sometimes occur near the inlet of the ion guide.

(Ion Guide)

A bias voltage and a high frequency voltage of 3 MHz are applied to the ion guide. The bias voltage carries the ions to

the next space (lens and orthogonal ion accelerator). The high frequency voltage contains ions within the pole. The

AccuTOF, a quadrupole system with a high ion focusing power, features enhanced sensitivity and resolution, compared to

other types (hexode or octal system). When the frequency voltage is set high, high mass ions go through the ion guide

efficiently; when the voltage is set low, low mass ions pass efficiently; and the ions are transferred to the focusing lens at

the speed of the bias voltage.

[Optimizing Orifices 1 and 2, Ring Lens, and Ion Guide Levels]

To detect quasi-molecular ions of a target compound at high efficiency, set the ring lens voltage to 10-20 V, and the

orifice 2 voltage to 5-10 V. Introduce the sample by infusion (continuous injection from the pump) or flow injection (from

the manual injector or auto sampler) in a few flows. Monitoring the target ion intensity on the ion monitor, adjust the

orifice 1 voltage to search for the conditions where the target quasi-molecular ions are detected at the highest sensitivity.

We recommend that you adjust the orifice 1 voltage first, then optimize the ring lens voltage and orifice 2 voltage, and

fine-turn the orifice 1 voltage again. If you introduce the sample by flow injection, and optimize the ion source level, you

may perform a single experiment from the MS Tuning Manager. For analysis like In-Source CID, follow the same

steps. Set the orifice 1 voltage and ring lens voltage higher than the level appropriate for detection of quasi-molecular ions.

The [General] tab of the [Analyzer-Setting] panel has an item [Ion Guide High-Frequency Peaks Voltage]. Setting this

low will facilitate detecting low mass ion, eliminating high mass ions. Setting this high (up to 2500 V) will optimize

detection of high mass ions, eliminating low mass ions. Adjust the voltage level according to your applications. We

recommend setting this voltage to 1000-2000 V and 2500 V when you detect background ions and reserpine respectively.

The bias voltage of the ion guide is theoretically 27 V, although it varies among units. Changing this a great deal will

change the ion speed in the horizontal direction, driving ions to incorrect locations, and reducing the sensitivity. (See the

figure below.)

23

You need not change the bias voltage at the user, since it is optimized at the Factory or at the time of

installation.

Ion Guide Bias Voltage - Optimized Ion Guide Bias Voltage - Inappropriate

The user needs to determine optimum voltage levels of the orifices 1 and 2, ring lens, and ion guide (RF) according

to the target samples and objective of analysis.

2-2. Focusing Lens, Orthogonal Accelerator

(Focusing Lens)

The focusing lens is designed to efficiently direct to the orthogonal accelerator the ions that have traveled at the speed of

the ion guide bias voltage. An orifice with a horizontal hole is located between the lens and orthogonal accelerator, and the

lens will push the ions efficiently through the horizontal hole, creating a thin, flat ion packet at the orthogonal accelerator.

24

[Optimizing Focusing Lens Setting]

Use the parameters in the Details tab of Analyzer-Setting to set the focusing lens voltage. The parameters were set to the

optimum levels at the time of installation. Since the lens and analyzer of the AccuTOF are designed to resist contamination,

the voltages do not change very much.

However, they may change slightly over time, and the user needs to fine-tune the voltages occasionally.

� Adjust the focus voltage in +/- 10 V steps to maximize the peak intensity and resolution.

� Adjust the voltages of the condenser lens and quadrupole lens in 1-2 V steps to maximize the peak intensity and

resolution.

� Adjust Right/Left in 0.5 to 1.0 V steps to maximize the peak intensity and resolution.

� Adjust Top/Bottom in 0.1 to 0.5 V steps to maximize the resolution.

� Right/Left and Top/Bottom are designed to introduce ions to the orthogonal ion accelerator. It is desirable to

adjust Right/Left and Top/Bottom within +/- 15 V and +/- 5 V respectively.

� The above adjustment is related to the pusher bias, suppress, and Reflectron voltages. Optimize the focusing

lens setting in combination with these.

(Orthogonal Ion Accelerator)

The ion packet directed by the focusing lens to the orthogonal ion accelerator will arrive at the center of the pusher and

pulling plate. A voltage of +/-777.8 V will be applied simultaneously to the pusher and pulling plate, pushing the packet to

the flight tube with the accelerating energy. The pusher and pulling plate voltages will be reset to 0 V after the orthogonal

ion accelerator has projected the ions, accumulating another ion packet for next acceleration. The next packet will be

accelerated to the flight tube in the same manner. This process is repeated every 0.5 to 1.0 ns.

Another packet of ions is accumulated in the orthogonal accelerator while the pusher and pulling voltages are 0 V.

Creating an ion packet as flat as possible at this point will greatly affect the resolution and sensitivity. In theory, ions will

be forwarded from the focusing lens in parallel when the pusher and pulling voltages are 0 V. In actuality, however, the

voltages are expected to be fully 0 V when switched from +/-777.8 V to 0 V at high speed. Therefore, the AccuTOF

allows the operator to adjust the pusher bias and suppress voltage to offset the voltage at 0 V. This will assure ions as

perfectly flat as possible at the orthogonal ion accelerator.

25

[Optimizing Orthogonal Ion Accelerator Setting]

To adjust the orthogonal ion accelerator, go to Pusher Bias in the General tab of Analyzer-Setting in

Analyzer[Advance]-Setting.

You need not adjust the following in Analyzer[Advance]-Setting at all, since they are basic settings for ion flight. (except

for polarity change for Pos/Neg analysis).

� Pusher voltage

� Pulling voltage

� Flight tube voltage

You need not adjust the suppress voltage most cases. When needed, adjust it in 0.05 to 0.1 V steps to maximize the

resolution. Adjust the voltage within +/-1.0 V at maximum.

Adjust Pusher Bias Voltage in the General tab of Analyzer-Setting when you adjust the focusing lens. Adjust it in 0.05 to

0.2 V steps to maximize the resolution and intensity.

2-3. Reflectoron

(Reflectoron)

Ion packets accelerated from the orthogonal ion accelerator to the flight tube will be reversed the direction by the

Reflectoron toward the detector. Functions of Reflectoron are:

1. The direction reversed along the way, ions with the same size will double the distance of flight (enhanced

resolution).

2. Passing through the Reflectoron, ions with the same mass number yet slightly different speed will be

simultaneously arrive at the detector (enhanced sensitivity and resolution).

26

Reflectoron Model Diagram

[Optimizing Reflectoron Setting]

To adjust the Reflectoron voltage, go to Reflectoron Voltage in the Details tab of Analyzer-Setting.

You need not adjust this in most cases, since it is optimized at the time of installation. When needed, adjust

the voltage in 10 V steps to maximize the sensitivity and resolution.

Changing the Reflectoron voltage will have ions reverse the direction in front or deep inside the Reflectoron, changing

the distance of flight (= time of flight). As a result, the mass number display will significantly change. Make sure to

perform Mass Calibration when you changed the Reflectoron voltage.

Adjust the voltage in combination with other items of Analyzer and Analyzer[Advanced] Setting.

As discussed above, you need not adjust Analyzer and Analyzer[Advanced] Setting frequently. Use a peak you

are familiar with (e.g., peak from LC solvent) as an indicator for tuning, and perform peak tuning when the

resolution and sensitivity have significantly declined.

The next section will describe the tuning process in detail with reserpine as a sample. You need not use

the initial values as used in the instructions. You may use the initial conditions of the user’s system.

3. Tuning from the factory setting

3-1. Set every parameter getting from performance sheet.(Analyzer setting, Ion source setting)

3-2. Tuning Procedure:

3-2-1. Tune the Top/Bottom potential in 0.1-0.5 V steps. Maximize peak height, not resolution (better keep

resolution ). The maximum should be within +/- 2V, otherwise there may be a problem.

3-2-2. Tune the Right/Left potential in 0.5-1.0 V steps. Maximize peak height, not resolution (better keep


3-2-3. Tune the pusher bias in 0.2 volt steps. Maximize both intensity and resolution. Decrease the step size to

0.1 volt steps and then to 0.05 volts steps.

If you have not good resolution following step

3-2-4. Adjust both the Focus and Deflector (Condenser/Quadrupole )lens voltages. These potentials are I

nterrelated. Maximize both intensity and resolution. to tune the Focus potential in 10 V steps; set the

Condenser and Quadrupole lens potential to 0 initially and then tune in 1 V steps.

(Condenser and Quadrupole lens voltages are not big change the ion intensity.)

3-2-5. Repeat 3-2-1 to 3-2-3.


3-2-5. Tune the reflectron in 20 volt steps. Look for good resolution.

3-2-6. Repeat 3-2-1 to 3-2-4.


3-2-7. Set the Ion-Guide Bias voltage +/- 1v step then repeats 3-2-1 to 3-2-4.

27

Probably you can get resolution > 6000 by this procedure.

If you get resolution > go to C-10 at page 28

4. Tuning from initial condition.

4-1 Setup (Analyzer):

• Peaks Voltage (RF): 2000 (maximum level).

• Bias: 27 (or Performance sheet)

• Pusher bias: 0.

• Focus: -120

• Condenser: 0

• Quadrupole 0

• Left/Right: 0

• Top/Bottom: 0

• Reflectron: 900v

Setup (Ion Source):

• Ring lens: 5 to 10v (for reserpine)

• Orifice 1: 30v (use 50 to 85V for Reserpine tuning -- see note 2)

• Orifice 2: 5v

• Needle: 2000 V. Current will be tens of nanoAmperes. If corona discharge occurs, the current will increase

to hundreds of nanoAmperes.

• Drying gas: 2.5-3 on bubble meter.

• Nebulizing gas: ~ 0.5 on bubble meter.

• Needle position: inner needle protrudes 0.5 mm for flow rate of 200 ul/minute (see Note 3).

4-2. Tuning procedure

4-2-1. Tune the reflectron in 20 volt steps. Look for good resolution. (Not big reduce intensity)

4-2-2. Tune the Top/Bottom potential in 0.1-0.5 V steps. Maximize peak height, not resolution (better keep


4-2-2. Tune the Right/Left potential in 0.5-1.0 V steps. Maximize peak height, not resolution (better keep


4-2-2. Tune the pusher bias in 0.2 volt steps. Maximize both intensity and resolution. Decrease

the step size to 0.1 volt steps and then to 0.05 volts steps.

If you have not good resolution following stepIf you have not good resolution following stepIf you have not good resolution following stepIf you have not good resolution following step

4-2-3. Adjust both the Focus and Deflector (Condenser/Quadrupole )lens voltages. These potentials are I nterrelated.

Maximize both intensity and resolution. to tune the Focus potential in 10 V steps; set the Condenser and

Quadrupole lens potential to 0 initially and then tune in 1 V steps.

(Condenser and Quadrupole lens voltages are not big change the ion intensity.)

4-2-5. Repeat 3-2-1 to 3-2-3.


4-2-5. Tune the reflectron in 20 volt steps. Look for good resolution.

4-2-6. Repeat 3-2-1 to 3-2-4.


4-2-7. Set the Ion-Guide Bias voltage +/- 1v step then repeats 3-2-1 to 3-2-4.

28

Peaks Voltage (RF)

The peaks voltage affects the high-pass character of the RF ion guide. Decreasing this potential affects the low-m/z

cutoff. A very rough guide to the peaks voltage is given below (an approximation for easy recall):

Peaks

Voltag

e

Lowes

t m/z

500 50

1000 100

1500 150

2000 200

2500 250

A value of 600 volts is useful for small-molecule work (up to m/z 1000).

In-Source Fragmentation:

Reserpine fragments

For reserpine, drop the Peaks Voltage to 1000 and increase the orifice 1 potential from 30 V to 80 V. Adjust the ring lens

potential slightly to optimize fragmentation.

Definitions:

• Orifice 1 refers to the potential difference between orifice 2 and orifice 1.

• Orifice 2 refers to the potential difference between the ion guide and orifice 2. Do not adjust under normal

operation because this can induce fragmentation in the ion guide region.

• Ring lens refers to the potential difference between the ring lens and orifice 1.

Coordinate system:

• The x-direction is the direction of time-of-flight extraction (into the reflectron)

• The y-direction is the direction of ion injection.

• The z-direction is side-to-side (left and right when facing the ion source)

Focusing lenses:

The first lens is segmented into quadrants to steer the beam up and down and left and right:

+

+

- -

Direction of ion motion into

reflectron region

1 2 3

29

The second lens is the einzel focusing lens, and the third lens is grounded.

• Condenser: Average value of all elements of lens 1.

• Quadrupole: Quadrupolar potential applied to lens 1 (+ on top/bottom, - on left/right: see above figure)

• Right/left: Balance between top and bottom segments, steers beam vertically.

• Top/Bottom: Balance between left and right segments, steers beam horizontally.

Use C:¥Program Files¥JEOL¥MassCenter¥Utilities¥MS Monitor to see actual voltages on each segment (for

troubleshooting).

English Database:

Resides in c:¥Program Files¥JEOL¥Mass Center¥Applications¥StringTable.mdb

Edit with: C:¥Program Files¥JEOL¥MassCenter¥Tools¥MdbViewer¥MdbViewer.exe

Notes:

1. The quadrupole resonating frequency is set with the configuration tool and the value is stored in the registry.

The correct value is set for each instrument and is listed in the parameter list in the final test document. If software is

reinstalled, this value will have to be set for each machine.

2. Use an orifice 1 voltage of 50 V for tuning to give the strongest signal for the [M+H]+ on reserpine, but this

potential is high enough to induce fragmentation. For typical small molecules, an orifice 1 potential of approximately 30

V results in minimal fragmentation.

3. At lower flow rates (5-10 microliters per minute), extend the needle to about 2 mm. If the desired flow rate is

lower than this, you will need to replace the ESI needle with the optional microspray needle or switch to the optional

nanoESI source.

c-10 Zoom up around m/z=609

c-11 Hard Copy on PC’s monitor

d. Disconnect Syringe pump then connect LC with test-column. (For calibration and sensitivity test)

Test Column:(HP-LC column Zorbax Eclipse XDB-C18 2.1x50mm %-micron (HP P/N=960967-902))

30

Set flow rate to 0.2ml/min (Use LC controller or Agilent1100 controller on MS-Center)

Set Solvent B (Methanol) 100%

d-1. Agilent1100 controller operation.

Open Agilent1100 Menu

If you do not see Agilent 1100 status panel on

the desk top, open from : Instrument >

Instrument Status Panel > Agilent 1100

Click here

[Status Menu]

Click here to operation menu

[Binary Pump menu]

[General]

Flow rate setting

(set 0.1 or 0.2) in this case

Solvent %-rate setting

Set 100% now

(later , will set to 80 to 100% for

sensitivity test)

Different by sensitivity condition

31

[Details]

Set Solvent name etc…

Unnecessary full in these menus for specification test

[Auto Sampler Menu]

[General]

Injection Volume [ul]

(Set 2.0 for sensitivity test)

Needle Wash [yes/no]

Use which vial for wash

(Check here and set 91 then

put in MeOH sample bottle at 91 location on Auto sampler)(for sensitivity test)

[Details]

32

Unnecessary change these menus for Test

[Detector Menu]

[General]

Detector wave setting

And Step

(Set 100-950 step=0.2

for detector work test)

Check pre-run

[Details]

(no need set any thing for test)

[Column Compartment]

[General]

(set 35 for test and check same as Left)

33

[Details]

(no need set any thing)

[Time Table Menu]

Parameter Window

Time Table view menu

a. Time table

b. Solvent graph

c. Flow/Presser graph

d. Detector Wave graph

Create/Add/Delete the time of tables. (Right click)

Set Stop time 10 to 30 minute for test. Different by pump flow and solvent A&B %.

(same as time on retention time in acquisition setting)

Another module are set to “same as pump”.(for test)

[ON/OFF Menu]

[Standard]

Check here is the module is “ON’.

All On/Off

34

[At Power on]

Set All on (for test)

Other : Instrument > Configuration..

Select each module.

Add/Delete

Recognize/Details

35

e. Calibration

Sample : TFA Na (Sodium Trufuoroacetate) (m/z > 3000) 100ppm with MeOH/H2O=70/30 %

PEG mix (m/z>2000) (10 to 100ppm with MeOH)

Use manual injection (Disconnect remote cable from manual-injector to LC)

LC flow 0.2ml/min (Set by Agilent1100 manager or manual set using LC module)

Tune condition of AccuTOF :

Same as Resolution test. (If necessary, adjust orifice 1,2 and ring lens for PEG.)

e-1 Acquire PEG(or TFA Na) that using single acquisition.

Acquire > Start Acquisition

e-2 Start Wizard

Next >

(If you have the acquisition setting file,

You can check and load here.)

e-3 Create new data name(acquisition Data)

If necessary, you can put in some

sample information and comment here.

Choose data saved folder.

(If there is no folder name,

you con create new folder at here now)

Next >

e-4 Select Acquisition Setting

This case choose

“Specify new MS acquisition settings”

Must blank Agilent1100 settings

36

NEXT >

e-5 Data Acquisition Setting

[m/z=range]

start = 100 end = 1000

[Stop Time]

2.00 min

[Spectrum Recording Interval]

0.5 s

[Data Sampling Interval]

0.5 ns

NEXT >

Never check “Use Data Compression”

e-6 Data Reduction

NEXT >

No need to do anything.

e-7 Choose real time display mode

TIC

Mass Spectrum

NEXT >

e-8 Finish then tart acquisition data.

e-9 If you have never had folder, you will get the message.

37

OK

e-10 Appear this window on the MS tune window.

Then Open Chromatgram , Spectrum Viewer and Start-Run Window automatically.

e-11 Click Start Run then inject PEG(or TFA Na) immediately. (5 to 10ml)

Check PEG (or TFA Na) that is detecting on spectrum monitor.

e-12 Wait Run time (2.0min) or

If you want to stop in the run time, choose MassCenter Main Window then Stop button.

1 Or 2

38

Choose Quit Current Acquisition

OK

e-13. Open Calibration Maneger Menu

Instrument > Mass Calibration

(Mass Center Main Menu)

e-14.

Choose (1-1000)line

(This time)

Start Calibration Wizard

e-15. [Start Wizard]

NEXT >

39

e-16. [Type]

Set file name that is going to make a calibration “New File”.

Or you have made file name, if you want update file. Choose from browse.

New File name (create here)

or choose “Old file name”

Choose 1000 or less

NEXT >

e-17. [How to create]

Choose “Use Existing Data”

Select you measured data name.

(See above “k a-3”, this case “cal-1”)

Set PEG(or TFA Na) detected time

NEXT >

e-18. [Calibration Parameters]

Select “Mass Reference”

(Used sample “PEG”

> Mass Reference “PEG+Na+”)

Used sample “TFA Na”

> Mass Reference “YOKUDERUNA”

Choose “Auto”

40

Curve Approximation “4 or 5”

(See later explanation)

NEXT >

e-19. [Centroided spectrum]

Smoothing “3”

Single Moving Average Method

Set Peak threshold level

(low and high mass side)

NEXT >

e-20. [Finish]

If you want to save this parameter setting,

check then create name to here.

Finish

e-21. See small window then open Calibration Window automatically

[See Next Page]

41

e-21. [Mass Calibration Viewer]

To be assigned Real data with in Reference date Automatically

Then, change parameter to minimum value here

Choose best setting for calibration value(above) to

minimum.

42

e-22. [Manual Peak Assignment]

1. Remove Assignment

Calibrate > Remove All Assignment

2. Choose you own peak

Choose some peak

(For example)

3. Assign correct peak

Calibrate > Assign Peaks > Choose “Correct Mass Number”

4. One or two more assign peaks by same way above 1 to 3 process.

5. Re-Assignment

After Assignment 2 or 3 peaks, Choose Calibrate > Automatic Assignment

6. Save New Calibrated name (or update calibrate)

File > Save As then “Create New File”

or

File > Update

43

7. File > Exit then Return

Finish calibration

8. Other : Change to other Calibration file on parameter

Choose you want to change calibration file’s line

Calibration > Register Data > Then choose you own calibration file.

44

f. Sensitivity Test (with continuity analysis function check)

Sample : Reserpine

Specification : 10pg s/n= 10 or better ( 5ppb 2ul inject 3 injections)

f-1 Set LC-MeOH=90% first to try to get data by this value then if necessary change 80 to 100%

f-2 If necessary re-tuning, connect syringe pump again, then do tuning best condition for Reserpine.

Then save tune condition.

(Use Reserpine 50 to 500 ppb)

(MS Tune Manager)

f-3 Make Acquisition Setting

1. Instrument > MS Acquisition Setting

2. Full in these setting menu.

1. Click TOF

2. Set Retention time

20 min

(Reserpine detect time)

3. Data Acquisition

Start : 100

End : 1000

Spectrum Recording Interval : 0.5 s

Data Sampling Interval : 0.5 ns

4. MS tune setting

Set “MS tuning” file that made as “Resolution test” or above “a-1”.

Browse > choose MS tune file

45

5. Monitoring

Chromatogram : TIC

Mass Chromatogram : Start : 609.00

End : 609.50

Spectrum : Mass Spectrum

3. Save Acquisition Setting name

f-4 Make LC setting for sensitivity test.

1. Open Agilent 1100 manager

2. Full in each parameters

[Binary Pump]

Flow rate : 0.2 ml/min

Solvent ratio MeOH : H2O

90 : 10

(Set B = 90%)

[Auto Sampler]

Injection Volume [ul] : 2 ul

Enable Automatic Needle Wash

Wash Vial Number : 81

46

[DAD detector]

Wavelength Range [nm] : 190-950

Step [nm] : 0.2

Auto Balance : Pre-run

(These are not necessary setting for

sensitive test.)

Enable analysis at UV Lamp off

Enable analysis at VIS Lamp off

[Column Compartment]

Left Heater : Control

Temperature : 35 c

Right Heater : Same as Left

[Time Table]

Stop Time and Post Time

Binary Pump : Unrestricted

Or

Measure Time

(same as Acq setting time)

[ON/OFF]

All ON

3. Save LC condition file

Make your own name.

47

(For example: ReserpineLC)

f-5 Make Analysis List

1. Open or Create Analysis List.

Open Analysis List from “Project View”

Choose Analysis List then Right click Open

If necessary do refresh viewer window

If you did not create “Analysis List”, create from File > New

Then create “Analysis List” by seeing

“b Create New Project”

at the “4. Start Mass Center Main software ”

f-6 Create Analysis List Parameter.

Right Click then choose “Add Analysis”

Number of Analyses

3 (this time)

48

Fill out each parameter (For example following)

(Your own, sample location, Acquisition Data, Acquisition Data Folder, MS Acquisition Conditions,

Agilent1100, each parameters.)

f-7 Start measurement

Analysis > Start

If you don’t have any problem in parameter, you have this window.

Then start acquisition wizard.

NEXT >

Made Analysis list

Analyze Sample

Acquire MS Data

Acquire UV/VIS Data

49

Analysis line

NEXT >

Delete this process

Monitor Acquisition

NEXT >

Finish

Then LC Auto sampler pick sample up,

Start Acquisition automatically with

Agilent1100.

Stop acquisition after three times

measure samples.

or

Stop from Mass Center main window

50

g. Reduction

Open from “Mass Center Main” window

1. Choose Data Tug

2. Select “Data name” then right click

Choose “Open” , to open data

After that “Spectrum Viewer” and

“Chromatogram Viewer” are opened automatically.

If you can not see, select “Refresh”

[Spectrum Viewer]

Maximum

51

[Chromatogram Viewer]

[Basic Operation on Viewer]

[Zoom up] Left drag for intensity, Mass Number or both

[Back to original size] Double click

[Peak label by manual] Alt + click peak (spectrum & chromatogram viewer)

[Delete Peak label] Alt+ click labeled peak (Only labeled peak by manually)

[Peak label by auto] Tool > Project Settings > Peak Label-tab

Then choose labeling menu

Check > Label automatically

> Use threshold ….. then set %

Also there are some display setting in “Project Settings”

As necessary you can change this setting. Printer header label etc…..

52

[Create (Mass) Chromatogram]

1. Create from Chromatogram Viewer

Chromatogram > View > Chromatogram > open Chromatogram papameter

Select parameter

Set Mass Number range

Apply than Close

2. Create From Spectrum Viewer

Right button drag that you want to create mass number range.

53

[Create Mass Spectrum]

1. Create From Spectrum Viewer

Spectrum > View > At… >

Open Spectrum Parameter

Select Retention Time

or Scan number

Set you want to create Spectrum Range

[If you want to display subtraction

background Spectrum] Check here

and set Range.

Apply then Close [Subtraction Spectrum]

Manual Peak m/z-number labeling

Alt+Click on Peak top you can move label using drag the label

Remove created label>>Alt+Click the Label

Background subtract from chromatogram viewer.

1. Shift + Right button drag (or click) at sample area (This operation does not create MS pectrum)

2. Then Ctrl + Right button Drag (or click) at background area.

3. Display Background subtracted Spectrum.

54

2. Create from Chromatogram Viewer

Right button drag that you want to create R.T. number range.

[Show Mass Tables]

View > Table View Mode

Next or back page of Mass Tables

[Mass Spectrum Resolution Display]

1. Zoom up Mass spectrum

2. Spectrum > Mass Resolution

55

Apply then Close

[S/N calculate on chromatogram]

1. Create TIC or Your own Mass Chromatogram

2. Select Chromatogram > Signal to Noise Ratio

Select Noise Range

Apply then Close

56

[Save Disk Space]

If you need only to be shown spectrums and chromatograms, you can save just displayed data

then remove raw (huge) data for save disk space.

(No need whole raw data)

File > Save As > View Data

[Reduction Sensitive data]

1. Open sensitive data.

2. Make Mass Chromatogram (Mass range 609.00 to 609.50)

3. Calculate S/N.

4. 3 times getting data then print out

(Make good window layout that looks like to same with factory data)(using Print Screen)

5. If you can’t reach to specifications, re-select noise range on “Signal to Noise Ratio”

6. If you can’t reach to specifications; re-tune , change LC setting or etc…

Then re-measure data again.

Note: Slow LC rate better chromatogram.

Much water rate better peak shape chromatogram.

57

h. Exact Mass Test

Sample : PEG600 with Reserpine ( Mix100ppb each) (inject from manual injector)

Spec. : 5ppm or less (Each mass number and Flight Tune voltage)

(At Flight Tube Voltage 7000v, 6999v, 7001v)

h-1. Tuning Best condition if necessary on PEG or Reserpine

(If necessary save tune setting)

Create tune file just only change Flight Tube Voltage on 6999v and 7001v

Change Flight Tube Volt.

Finally create three Tune files (Flight Tube Voltage at 6999v, 7000v and 7001v)

h-2. Measure Sample using Flight Tune Voltage at 6999v, 7000v, 7001v.

(Use “Single acquisition” will be useful)

Single acquisition >>>> see “e Calibration page 34” process measurement.

h-3. Set(Open) 6999v(tune file) then measure. (Do not change other parameter)



h-6. Reduction for exact mass test. (Little be deferent of user exact mass analysis)

1. Open Data

Choose data name then Open

If necessary, do Refresh

58

2. Choose sample then make sample spectrum.

[Chromatogram Viewer]

Right drag then create (PEG & Reserpine) spectrum.

[Spectrum Viewer] (PEG with Peserpine spectrum)

3. Internal Calibration

a. choose M/Z Determine

Spectrum > M/Z Determine

b. Set parameter

Usually only change Threshold level

Apply then Close

59

c. Display Determine spectrum [looks bar type spectrum]

Then close parameter window

d. Keep choosing Determine spectrum (to be surround by blue rectangular)

e. Choose “Change Mass Calibration”

Tools > Change Mass Calibration

f. Click “Change” on Mass Drift Compensation

Then choose “Internal Mass Drift Compensation”

60

g. Start Internal Mass Drift Compensation Wizard

NEXT >

Choose Mass Reference Table or m/z Number

(This time choose File=Reserpine or 609.2811)

Automatic

Finish

h. Open “Intarnal Mass Drift Compensation Data” window

Then select Reselpine Peak (Change peak color to Red)

61

i. Assign m/z number

Calibrate > Assign Peaks > 1: 609.2811

Then Reserpine Peak color change to blue

j. Finish Mass Drift Calibration

File > Exit and Return

Confirm to be Changed Mass Drift Compensation

Internal Mass Drift Compensation Data

Then Close Change Mass Calibration window

Now m/a of “Mass M/Z Determined Spectrum (Bar graph)” has been changed

(If Sample with Internal reference are not same scan times,

reduction process will be little deference. See> AccuTOF Basic Operatin=other paper manual)

k. Compare PEG Table between Theoretical and Measured number.

Open measured Table and Printout

View > Table View Mode

62

Table View Mode

File > Print

Theoretical PEG m/z number

437.23625

481.26246

525.28867

569.31488

613.34109

657.36730

701.39351

745.41972

789.44594

Calculate between measured m/z number and theoretical m/z number

(Measured m/z – Theoretical m/z) divide theoretical m/z = error m/z

Then do test (reduction) on Flight Tube Voltage –6999v and –7001v by same way.

If not good value, try to re-tuning then calibration again.

Then measure again

63

Other

Function TEST 1. ESI NEG

1. print out ESI pos tune condition.

2. Change to ESI neg On Tune Manager

Instrument > Ionization Mode > ESI -

Compare ESI pos tune file then set every voltage opposite priority (+/-)

except ion-source temperature, MCP voltage and Peak-frequency(Ion Guide volt.)

(same setting with POS)

3. Connect syringe pump then monitor reserpine m/z=607 (M-H-)

Usually you can get resolution 5000 or good ( but, Negative mode not has a specification value)

4. If necessary tuning then to save tune file.

2. Option (APCI, Nano-ESI, Dual-ESI etc.)

1. Every Ion mode may be almost same setting with ESI mode except ION-source setting.

2. APCI needle volt. = 4000 to 4500v, vaporizer temp 400 to 450C

3. Nono-ESI pos needle volt=2000v, orifice 1 temp.=80C (for reserpice)

Then negative mode needle volt=1000v, orifice 1 temp=80C (for reserpine)

4. Dual-ESI same as ESI (each needle)

[How to Back up data to DVD+RW device]

1) Set DVD+RW media

2) Format DVD

2-1. Open control panels then right-click on DVD drive.

2-2. Select Format (Format will be 2 minutes or less)

3) Create a Folder using by windows explore

4) You need to have Project folder in the DVD, when copy a data.

4-1. If you do not have project create project by New project wizard

in Mass Center.

or

You need to copy whole project each.

(Can not copy only data, analysis list, tune file etc…)

5) Open “Mass Center Data Manager” software on desk top.

6) Copy your own data to DVD using Mass Center Data Manager.

6-1. Select data then click [copy]

6-2. Select file of destination folder or project name.

6-3. Paste (files will stat copying to

basic operation of accutof

Documents

evacuation presser

sw vent sw led

ms tune manager page

evac sw led

check evacuation condition

evac led

base line noise check

tune file page