QTof1 Info For Engineers

Q-TOF
Information for Engineers

Document1 page 1 of 45 Kevin Howes

For additional information see:
Embedded PC Set-up 12804
Data System 21510
Pre-test Procedure 12046
MS1 Test 12805
MS2 Test 21285
INSIDE ........................................................................................................................................... 3
ION OPTICS ..................................................................................................................................... 3
VOLTAGES ....................................................................................................................................... 3
TOF DETECTOR .............................................................................................................................. 4
REFLECTRON AND FLIGHT TUBE ....................................................................................................... 6
PUSHER STACK ............................................................................................................................... 7
HEXAPOLE / QUADRUPOLE ............................................................................................................... 8
Z-SPRAY SOURCE ........................................................................................................................... 8
VOLTAGES (Z-SPRAY SYSTEMS) ....................................................................................................... 8
Z-SPRAY PIN-OUTS .................................................................................................................... 9
SOURCE CONNECTIONS ......................................................................................................... 10
HOUSING PIN-OUTS ................................................................................................................. 11
ELECTRONICS ........................................................................................................................... 12
MAIN ELECTRONICS UNIT MA3727 ................................................................................................ 12
BACKPLANE ................................................................................................................................... 14
LOW VOLTAGE CHECKS ................................................................................................................. 14
INSTRUMENT PUMPDOWN ............................................................................................................... 15
TURBO PUMP DRIVE MODULES....................................................................................................... 16
OPERATE SWITCHING .................................................................................................................... 17
PUMPING LOGIC PCB .................................................................................................................... 18
TOF PCB ..................................................................................................................................... 19
HEAD AMP PCB ............................................................................................................................ 19
ANALOG PCB ................................................................................................................................ 20
ANALOG CHANNELS PCB ............................................................................................................... 21
HEXAPOLE RF SUPPLY .................................................................................................................. 22
PUSHER PCB ................................................................................................................................ 23
20 DB ATTENUATOR ...................................................................................................................... 23
PUSHER UNIT ................................................................................................................................ 24
PUSHER VOLTAGES ....................................................................................................................... 24
MCP CONDITIONING ...................................................................................................................... 25
MCP RESISTANCE CHECK ............................................................................................................. 25
TRANSPUTER PCB ........................................................................................................................ 25
INSTRUMENT OPERATION ...................................................................................................... 26
MS MODE ..................................................................................................................................... 26
MS/MS MODE: ACQUISITION OF A FRAGMENT ION SPECTRUM ......................................................... 26
THE ANALYSER QUADRUPOLE LENS ............................................................................................... 27
THE TUBE LENS ............................................................................................................................. 27
COLLISION AND TRANSPORT RF ..................................................................................................... 27
TUNING CONTROL SLIDERS ............................................................................................................ 28
GRAMICIDIN-S TUNE SOLUTION ...................................................................................................... 29
TDC PARAMETERS ........................................................................................................................ 30
MASS TO TIME CONVERSION .......................................................................................................... 30
QUADRUPOLE CALIBRATION ........................................................................................................... 31
TOF CALIBRATION ......................................................................................................................... 32
DEAD-TIME CORRECTION ............................................................................................................... 33
Q-TOF


PUSHER RATES ............................................................................................................................. 34
RHEODYNE 7010 INJECTION VALVE ................................................................................................ 34
SOURCE AND PROBE GAS CONNECTIONS (Z-SPRAY) ...................................................................... 35
COLLISION GAS INLET SYSTEM ....................................................................................................... 35
CONNECTIONS TO PILOT VALVE ...................................................................................................... 36
APCI SENSITIVITY ......................................................................................................................... 36
NANOFLOW MS/MS SENSITIVITY .................................................................................................... 37
MASSES OF GLU-FIBRINOPEPTIDE B Y FRAGMENT IONS ................................................................. 37
CONTROL OF WATERS LC SYSTEMS .................................................................................... 38
CONTROL OF HP1100 LC SYSTEMS....................................................................................... 40
TROUBLE-SHOOTER ................................................................................................................ 42
INSTALLATION OF A US TRANSFORMER ............................................................................. 43
TEST REAGENTS ...................................................................................................................... 44
Q-TOF


Inside
Ion Optics
sampling
cone
RF
hexapole
extraction
cone
differential
pumping
aperture
analyser quad
pre-filter post-filter
gas cell
RF hexapole
transport RF
hexapole
differential
pumping
aperture
acceleration
lens
focusing
steering
tof entry
photomultiplier
anode
MCP
phosphor
conversion
dynode
flight tube
reflectron
pusher plate
3 rd grid
2 nd grid
2 nd ring lens
1 st grid
tube
1 st ring lens

Voltages
Vcone
1 150 V
wrt Vce
V
hex
0 -5 V
Vext
1 100 V
Vhex
+5 -10 V wrt V
ce
5 -15 V
wrt Vhex
5 -15 V
wrt Vhex
ground
0 -20 V
0 -20 V
0 -200 V
0 -200 V
Vent
-47 V
0 - 500 V
0 3 kV
wrt Vtof
V
tof
4 kV
Vtof
Vtof
-7200 V wrt Vent
1640 V wrt V
ent
0 V wrt Vent
0 V wrt Vent
0 200 V wrt Vent
1/3 Vtof
Vtof -7200 V wrt Vent
0.33 0.66
of Vent
2/3 Vtof

Note: voltages are w.r.t. ground unless otherwise stated. In negative ion mode, all voltages are
reversed except photomultiplier, anode and phosphor (which changes to +10 kV)
Q-TOF


TOF Detector
detector
PSU
solid cone
anode
Kapton insulator
(dielectric)
-ve +ve
signal to TDC
-7200 V
Vtof
grounded hollow cone
270 V Zener diodes
10 M
2 nF
MCP
100 K
Zener BZTO3C270 1820735
Resistor 100 K W24 2148103
Resistor 10 M VR68 2291108
x 200
preamp
20 dB
attenuator
union
BNC elbow

Note: A detector voltage of 3000 V puts about 2200 V across the MCP. The anode is
at + 270 V w.r.t. the MCP rear. The anode, Kapton insulator and solid cone together
have a capacitance of ~ 200 pF.
Wiring top view
to pin 2 of plastic feedthrough
to pin 3 of plastic
feedthrough
dynode
cone
Note: The zeners are
mounted vertically below
the feedthough. The
resistors are mounted
horizontally. Ensure that all
the detector wiring is kept
clear of other wires and
any grounded parts of the
assembly
Anode
MCP rear
10 M

Q-TOF


MCP Orientation - top view
pusher assembly
dynode
retaining ring
MCP rear connector
MCP
anode
connector
Galileo MCPs should be
installed with the
orientation marks to the
left and right.
Hamamatsu MCPs should
be installed with the
orientation marks to the
top and bottom.

Continuity Checks
MCP
Anode
Check for no continuity
between anode and MCP

Connections to Anode and MCP
anode MCP
spring contact
tag
washer

Q-TOF


Reflectron and Flight Tube
to reflectron feedthrough
reflectron plates are
linked together by a
resistor chain consisting
of fourteen 10 MO
resistors
Electrical connection between
reflectron and flight tube is via
spring contacts.
Flight tube
TOF lid assembly
spring contacts
grid

To test the internal wiring, connect a 500 V Megger between pin 2 of the 6-way plastic
feedthrough (on the lid), and the reflectron feedthrough (on the TOF housing base).
The indicated resistance should be about 140 MO.
Q-TOF


Pusher Stack

Clamp plate
M5 wavey washer

10 mm ceramic spacer
entrance aperture block

5 mm ceramic spacer
pusher pusher plate
0.7 mm lens plate

4 mm ceramic spacer
0.7 mm lens plate
3rd grid spacing plate / 70 L/in grid assy
0.7 mm lens plate
2 mm ceramic spacer
0.7 mm lens plate
2nd grid spacing plate / 1000 L/in grid assy
1.5 mm lens plate

5 mm ceramic spacer
2nd ring lens ring lens

5 mm ceramic spacer
1st ring lens ring lens
1 mm ceramic spacer
2 mm ceramic spacer
1.5 mm lens plate
1st grid spacing plate / 1000 L/in grid assy

pusher support plate

Note: The grid assemblies should be mounted mesh-side up

Q-TOF


Hexapole / Quadrupole
Prefilter DC
Hexapole DC
Quad RF/DC
Quad RF/DC
47M
47M
100 pF
100 pF
62 pF
62 pF
4.7M
4.7M

Z-Spray Source
Hexapole
Extraction lens
Isolation valve
Cone
Capillary

Voltages (Z-Spray systems)
V
quad
= collision ion energy
V
hex
= collision RFlens
V
ext
= collision + extraction + RFlens
V
cone
= collision + cone + extraction + RFlens
Where V
quad
is the DC voltage on the quadrupole w.r.t. ground, V
hex
is the DC voltage
on the source hexapole w.r.t. ground, V
ext
is the voltage on the extraction cone w.r.t.
ground and V
cone
is the voltage on the sampling cone w.r.t. ground. Collision, ion
energy, RFlens, extraction and cone refer to MassLynx slider values.
Q-TOF


Z-Spray Pin-outs
Source
1
2
3
4
5
6
7
8
1 Sample cone
2 Extraction cone
3 Thermocouple +
4 Thermocouple -
5 Heater + 28 V
6 Heater return
7 not used
8 not used
1
2
3,4
6
5
Inside source support
thermal
fuse
Feedthrough

Probe Adjuster
1 2
4 5
3
6
7
8 9
10
pin 1 probe recognition
pin 2 probe recognition
pin 3 thermocouple
pin 4 thermocouple
pin 5 link
pin 6 link
pin 7 thermocouple screen
pin 8 not used
pin 9 probe heater return
pin 10 probe heater + 28 V
Gas panel
1 2
4 5
3
6
7
8 9
10
Plug

Source Hexapole Housing
1
2
4
3
pin 1 no connection
pin 2 no connection
pin 3 Aperture / hexapole bias
pin 4 no connection
Note: Connection to the hexapole is
via 4.7 M resistors
Feedthrough

APCI Probe
pin 1 Y probe recognition
pin 2 N probe recognition
pin 3 R thermocouple
pin 4 B thermocouple
pin 5 R thermal trip thermocouple
pin 6 B thermal trip thermocouple
pin 7 thermocouple screen
pin 8 not used
pin 9 B probe heater return
pin 10 R probe heater + 28 V
1 2
4 5
3
6
7
8 9
10
Plug

Q-TOF


Source Connections
Inner Source
exhaust gas out
drying gas in
counter electrode
APCI needle (via BNC)
sampling cone
heater
thermal fuse
thermocouple

Source Conduit Pin-outs
1 2
4
5
3
6
7
8 9
10
pin 1 code 1
pin 2 no connection
pin 3 thermocouple +
pin 4 thermocouple -
pin 5 heater +
pin 6 heater -
pin 7 code 2
pin 8 sampling cone
pin 9 counter electrode
pin 10 no connection
Fischer 10 way plug
in plug
1K1

Probe Socket Pin-out
8
2
3
4
5
6
7
1
pin 1 heater
pin 2 heater
pin 3 thermocouple
pin 4 thermocouple
pin 5 microswitch
pin 6 microswitch
pin 7 thermocouple
pin 8 thermocouple
pin 9 ground
9

Note: One of the thermocouples acts as a thermal trip operating at 60C above
ambient temperature.
Note: All Fischer connectors shown are viewed from the pin side (not the wire side).
Q-TOF


Housing Pin-outs
TOF Housing
Phosphor
6-way Fischer
pin 2 Pusher plate
pin 3 3rd grid
pin 1 Entrance
pin 2 TOF (flight tube)
pin 3 Anode
pin 4 2nd ring lens (LO)
pin 5 Guard (2nd grid)
pin 6 1st ring lens (HI)
(not shown) base of housing: Reflectron
2
3
1
4
5
6
6-way Fischer

Transfer Lens
1
2 3
4
5
6
7
8
9
10
11
pin 1 Transport
pin 2 Aperture 2
pin 3 AccV
pin 4 Focus / Steering
pin 5 Focus / Steering
pin 6 Tube
pin 7 Pre-filter
Fischer connector
internal connections
3
3
5
4
2 6

Source Housing
1
2
4
3
pin 1 Skimmer lens
pin 2 Skimmer
pin 3 Hexapole (dc offset)
pin 4 no connection

Analyser Housing
1
2
3
4
1
2 4
3
pin 1 collision cell hexapole RF
pin 2 collision cell hexapole RF
pin 3 ground
pin 4 collision cell
pin 1 transport hexapole RF
pin 2 transport hexapole RF
pin 3 transport (dc offset)
pin 4 collision cell front and rear

Q-TOF


Electronics
Main Electronics Unit MA3727
HT
supplies
+80 V for
turbos
+24 V for
vent valve
mains
filter
solid state relay
for rotary pump
circuit
breakers
electronics
pumps
Coutant
L V supplies
standby operate
pump logic
PCB
transputer
PCB
digital
PCB
analog
channels
PCB
analog
PCB
TOF PCB
QUAD
PCB
power
sequence
PCB
TOF
Phosphor
needle
HT
supply
reflectron
HV DC
external
connection
PCB

Pump logic PCB
N920210A
N920223A Vacuum monitoring. Phosphor, needle and
photomultiplier references.
Transputer PCB
N920200A
N920200A

Communication and control, MS1 detector ADC
Digital Scan PCB
N920204A
N920231A Control signals for mass, resolution, collision
energy
Analog PCB
N920202A
N920230A Source and APCI heaters, sampling cone,
skimmer lens, skimmer and source hexapole DC
offset
TOF PCB
MA3727-201
3727201DC1

TOF pusher timing, 200 V supplies for transfer
lenses, tube and TOF entry. Pot for tube lens.
Quad Control PCB
N920205A
N920232A Control of quad RF and DC, counter electrode
Analog channels PCB
N920212A
N920212A Four channel user input (e.g. UV detector).
Maximum input is 1 V, 12 bit ADC.
Power sequence PCB
N920214A
N920214A photomultiplier supply, operate switching
External connection
PCB N920206A
N920206A Input for 4 channels at rear of instrument, routes
signals to analog channels PCB
Applied Kilovolts K1/11 7990245 HV DC supply. 420 V for lenses and 1300 V
for quad DC
Applied Kilovolts
HP8/40
7990259 7200 V for flight tube (TOF)
Applied Kilovolts
KS20/39
7990241Q Phosphor supply
Applied Kilovolts
HP5/36
7990246 Probe and reflectron supplies

Q-TOF


Alpha 600W
5B28G24D15/15E
7990299 Standby supplies: 5 V, 28 V, 24 V, 15 V
Alpha 600W
24G28D28D28D
7990298 Operate supplies: 24 V, 84 V
Omega MML400
28D28D24D
7990288 Soft vent 24 V, Turbo pump 80 V supplies
Alpha 400
28/28H24N
7992202 used in place of Omega MML400
Elsewhere

Head amp PCB N922200A For photomultiplier
TDAT PCB A775200A For communication with transputer PCB
Time to digital
converter PCB (TDC)
7991605 For acquisition of TOF data
Embedded PC 8820117
3Com EtherLink card 8930110 For embedded PC
DE450 Network card 8900112 For host PC
ORTEC VT120A
preamp
7992111 Amplifies TOF MCP output by a factor of 200
Hexapole RF supply 3599001CC3 For collision and transport hexapoles
RF generator N925001A For instruments without z-spray (90.5 turn RF coil)
RF generator N925003A For z-spray instruments (81.5 turn RF coil)
RF generator PCB N920208A
Pusher PCB 3730200DC1 Pusher pulse for pusher plate and third grid
Spellman MI3P1/I15 3730025BC1 + 3 kV for TOF detector (unterminated PSU is 7992200)
Spellman MI1PN15 3730026BC1 1000 V for pusher pulse (unterminated PSU is 7992201)
Vacuum System

Edwards E2M28 6060122 Rotary pump
Edwards EXT250 6061944 Analyser and TOF turbo pumps
Edwards EXT250Hi 6061950 Source turbo pump
Edwards EXDC80 6061951 Drive module for EXT250 pump
Edwards EXDC160 6061952 Drive module for EXT250Hi pump
Edwards APG-L-NW16 6060932 Active Pirani gauge
Edwards AIM-PL-
NW25
6060933 Active Penning gauge
Edwards AIM-S-NW25 6060973 Active Penning gauge
Mechanical

MCPs (one pair) 6062659
Collision cell assy. M950046DC1

Q-TOF


Backplane
JPUM1
JMPU1
JDIG1
JANA1
JTOF1 JRF1
JPUM2 JMPU2 JDIG2 JANA2 JTOF2
JRF2
J1
JAC1
JDIG3 JANA3 JTOF3 JRF3

Low Voltage Checks
Remove all PCBs. Remove white Molex connector from top of right hand Coutant
Alpha power supply to enable the 84 V and 24 V operate supplies. Switch on
electronics circuit breaker. Measure voltages w.r.t. chassis (ground).
Supply Measure / V Connector Pin
+ 5 V + 5 0.05 JANA2 C32
DGND 0 0.05 JANA2 C30
+ 15 V + 15 0.05 JANA1 C2
AGND 0 0.02 JANA1 C6
- 15 V - 15 0.05 JANA1 C10
+ 28 V + 28 0.5 JANA3 C32
+24 + 24 0.5 JRF2 C16
+ 84 V + 84 0.5 JRF2 C12
+24 V operate + 24 0.5 J1 C30

Switch off breaker and refit Molex connector. Insert transputer and digital PCBs.
Switch on breaker, 0 0 should be displayed on transputer PCB. Measure TP9 w.r.t.
TP10 on transputer PCB. Adjust to 5.00 V using the pot at the top of the left hand
Coutant Alpha power supply.
5 V adjustment pot.
Pot is hidden from
view, near front
corner.

Q-TOF


Instrument Pumpdown
In normal use the pumps are switched on and off via MassLynx, but during instrument
test it is more convenient to use the pump toggle switch, which is located on top of the
main electronics unit. In the AUTO position, the pumps can be switched on and off via
the data system. In the ON position (nearest the tube unit), the pumps will come on
even if there is no data system. The ON position also allows the main electronics unit
to be powered off without the pumps going off.
When pumping down the instrument for the first time, monitor the 24 V on the soft vent
solenoid. This can be done on the rear of the fixed QM connector on the gas panel
interface PCB. The solenoid is a normally open type, i.e. it is closed when 24 V is
across it. In a vent state, both sides are at 24 V, i.e. zero volts across the solenoid.
Permanent 24 V from
Coutant Omega PSU
Switched 24V from
pumping logic PCB

The 24 V comes from the top of the three supplies within the Coutant Omega 400
PSU. It is adjusted via a pot on the right side of the power supply; its near the front
and level with the zero volts terminal. The other two supplies are at 28 V; all three are
connected in series giving a total of 80 V for the turbos.
0 V
24 V
terminals
pot

The minimum complement of PCBs necessary for MassLynx control and monitoring of
instrument pumping is pumping logic, transputer and digital. In the absence of the
digital PCB use EngCon.
Start the EngCon program, reboot then refresh using the appropriate EngCon
buttons. At the > prompt type gas. Move the cursor to TURBOS OFF, and press the /
key. This will turn the pumps on (if the toggle switch is in the auto position), and turn
the Penning gauges on. Type RD1 at the > prompt to display readbacks including
turbo speed, Penning and Pirani gauge pressures.
Normal operating pressure are: Analyser 1.0 x 10
-5
mbar
Backing 2.7 mbar
TOF < 8 x 10
-7
mbar
It will take a couple of weeks of pumping for the TOF pressure to reach this value for
the first time. Once this value has been achieved, complete recovery of vacuum will
occur overnight following an instrument venting.
Q-TOF


Turbo Pump Drive Modules
The green LED on the turbo pump drive module will come on when the pump is at
80% or more of full rotational speed. This should happen within a couple of minutes of
switch-on. Note: this LED will stay on when the pump is switched off, until the speed
falls to below 80%.
The three red LEDs indicate rotation of the pump. When the pump is switched on, the
three LEDs will flicker briefly as the speed of the pump increases. At higher speeds, all
three LEDs will stay on continuously. If, when the pump is switched on, only one red
LED lights, the motor is not rotating.
The 80 V supply to the turbo is always present when the vacuum breaker is on. The
start /stop control is via a contact closure on the pumping logic PCB. To start the
pump, the contact closes, pulling down the 24 V on the control line from the drive
module.
Drive Module Pin-outs
Pin Signal
1 start/stop control input link to pin 5 or 6 to start.
2 status output 15 V at < 80%, 0 V at > 80%
3 ground
4 speed analog output 0 to + 10 V
5, 6 0 V
7, 8 80 V allowable range 68 to 85 V

Caution
To avoid damage to drive module, switch off vacuum breaker and allow pump to stop
completely before disconnecting module from pump or main electronics unit.
Q-TOF


Operate Switching
The operate supplies, + 24 V for the Applied Kilovolts PSUs, and + 84 V for the quad
RF, originate in the RHS Alpha 600 supply. Operate switching is achieved by relays in
the power sequence PCB in the case of the 24 V, and for the quad, the RF drive is set
to zero.
There is an additional safety operate switch on the Alpha supply. There is a Molex
connector going to the top of the supply, which acts as an inhibit when the instrument
is in standby mode. When the Molex plug is disconnected, the 24 V and 84 V supplies
switch on. This inhibit circuit is shown below:

ALPHA 600 PUMPING
LOGIC PCB
AUX 5 V
0 V
INHIBIT
A26
A25
CN5
2
1
IC47
2
1
4
INHIBIT HIGH = ON
INHIBIT LOW = OFF
150 O

Pin 2 on the ALPHA connector is at 5 V w.r.t. pin 4. A 150 O resistor in the
Molex plug drags the inhibit line down to 0 V, switching off the ALPHA. When operate
is requested, IC47 connects the inhibit line to the 5 V, switching off the supply. The
state of IC47 is indicated by the LED D1, on the pumping logic PCB. This is on when
operate is requested and all interlocks are OK, i.e. the pumps are switched on, the
turbos are at least 80% of full speed, and the analyser vacuum is OK.

The Q-TOF can be operated with the Molex plug disconnected.
Q-TOF


Pumping Logic PCB

LK8
F1
RV4
Set phosphor to 4 kV (+ve ion mode)
RV3
Set phosphor to 10 kV (-ve ion mode)
LK7
Turbo speed (green LED)
LK4
LK1
D1
Safety interlock (green LED)
TOF Penning
Analyser Penning
Backing pirani
Links shown in normal positions
D6
Inlet pirani OK LED

Notes:
The safety interlock LED is on when the 84 V RF generator supply is enabled.
The Turbo speed LED is on when all turbo pumps are at greater then 80% of full
rotational speed.
F1 is a 2.5 A fuse on the 24 V input to the PCB
Analyser Penning Gauge Disable
Move LK7 to Q-TOF position
Move LK8 to QTB position
In this configuration, the operate interlocks originate from the TOF Penning only. The
analyser Penning will not come on. Collision gas pressure can be set by monitoring
the regulator pressure on the gas panel.
Q-TOF


TOF PCB
TP23
TP22
TP21
TP20
TP19
TP18
TP17
TP10
+5 V
D38
analog ground
digital ground
Tube lens pot

TP23 lens 1 Transport
TP22 lens 2 Aperture
TP21 lens 3 AccV
TP20 lens 4 Focus
TP19 lens 5 Focus
TP18 lens 6 Entrance
TP17 lens 7 Guard (second grid)

The focus lens consists of two half plates, one above and one below the beam. The
steering slider varies the voltage difference between these two plates.
The tube lens voltage is derived directly from the entrance supply. It has a range from
one third to two thirds of the entrance voltage. It is a high impedance supply; use a
high voltage probe to get accurate voltage readings.
Head Amp PCB
The output zero level is adjusted using RV1. Start EngCon. Reboot, Refresh, to give
the > prompt, then proceed as follows:
>t
>p (PPU Diagnostics)
>i (view input)
Adjust RV1 until the input min: is 0002. Input here refers to the input to the ADC on
the transputer PCB.

Q-TOF


Analog PCB
TP52 analog ground
TP 30 28 26 24 22
F1 probe heater
F2 source heater
Z-Spray instruments:
TP22 cone
TP24 extraction
TP26 not used
TP28 hexapole
TP30 not used
non Z-spray instruments:
TP22 cone
TP24 skimmer lens
TP26 skimmer
TP28 hexapole
TP30 counter electrode

Non Z-Spray instruments only:
The collision energy slider adds an equal voltage to the cone, skimmer lens,
skimmer, hexapole and counter electrode.
The cone slider adds an equal voltage to the cone and skimmer lens.
The output of the counter electrode circuit goes to the RF generator PCB where it is
converted from a 350 V range to a 0 to 700 V range.
Q-TOF


Analog Channels PCB
This PCB is connected to the external connections PCB, at the rear of the instrument.
There are three types of connections:
EVENT OUT
Only event out 1 is operative at present. It provides either a contact closure output (S1
up) or voltage output (S1 down), at the end of an acquisition function. The voltage
output is a 5 V pulse. The pulse duration is a fraction of a second - making it too fast
to see on some voltmeters. An event out can be used to trigger an autosampler.
CONTACT CLOSURE INPUTS
Inputs IN1 and IN2 can be used to start an acquisition externally, e.g. from an
autosampler. In the acquisition control panel, Configure, Select Inlet, Contact
Closure. Start the acquisition from MassLynx; the acquisition will then wait for the
contact closure.
ANALOG CHANNELS
The analog channels PCB accepts up to four 0 - 1.0 V positive inputs (e.g. from a UV
detector). It has 12 bit DACs. The input channels have autoranging amplifiers, with
gains of 1, 10, 100 or 1000. MassLynx displays a 1.0 V signal as an intensity of 10
6
.
Channel numbering is as follows (as viewed on the external connections PCB):
2
1
4
3

For isolated inputs, link the negative terminal to the ground terminal. Ground link plugs
should be left in unused channels.
Q-TOF


Hexapole RF Supply

J2
TP4 TP0 TP6
FS1
500 mA
A/S
RV1
TP7
TP12
TP13
TP14
TP1
R14

TP0 0 V
TP4 + 15 V
TP6 15 V
TP7 24 V operate
TP12 15 V enable
TP13 w.r.t. TP14 0 to 10 V RF reference
RV1 Adjusts RF frequency (~1.1 MHz)
R14 top RF output feedback signal
Tuning the supply
Set RF offset to 0.1, adjust RV1 until the voltage on TP1 w.r.t. TP0 is a minimum.
Increase RF offset to 0.5 and repeat. Note: there may be more then one minimum.
The brightness of the neon lamp indicates reflects the amplitude of the RF. It will only
illuminate at RF offset values greater then approximately 0.4.

RF offset RF reference TP1 w.r.t. TP0 R14 top
0.1 1.0 2 4 -1.0
0.5 5.0 8 9 -5.0

Q-TOF


Pusher PCB
TP6 TP3
TP2
TP1
TP5
TP4
TP10
J1
J2
J3
Ground + 15 V
+ 24 V
Third grid
Pusher PSU high
Pusher PSU low
Pusher plate
current monitor
(vs. TP5)
Pusher PSU
connection
CV1
RV1
TP8
TP7

The voltage produced by the Spellman can be monitored on TP1 w.r.t. TP5. The
current drawn from this supply is monitored on TP10 w.r.t. TP5 (or on issue C PCBs,
across R17). At 1000 V, the current drawn should not exceed 10 mA, which
corresponds to 1V on TP10 w.r.t. TP5.
The pusher pulse is best monitored using a x 100 probe at the rear of J1.
Non-overlapping pulses of approx. +15 V, repeating at 100 S, should be on TP7 and
TP8.
When the pusher pulse is off, the third grid and pusher plates should be at the
entrance voltage.
20 dB Attenuator
The output from the TOF detector goes to an ORTEC preamp via a 20 dB BNC
attenuator. To check the state of this attenuator, measure the resistance between the
centre pin and case at each end. Both readings should be ~ 53 O. In the event of a
high voltage arc to the detector anode, the attenuator acts as a fuse, saving the
preamp and TDC. A blown attenuator will give resistance readings of ~ 62 O and 370
O.
Q-TOF


Pusher Unit
TOF detector HT supply
Spellman MI3P1/I15
Pusher HT supply
Spellman MI1PN15
Pusher PCB
Ring lens
potential
divider
Third grid output (BNC)
Pusher plate output (BNC)
TOF HT input (SHV BNC)
CV1
RV1
ring lens
adjustment
pots

Guard ring potential
divider box
first ring adjust
second ring adjust
MCP (to negative side
of TOF detector
power supply)
TOF voltage out
ENT (entry voltage in)
HI (to first ring lens)
LO (to second ring lens)
TOF voltage in

Pusher Voltages
The voltages shown below are present during the pusher pulse, in positive ion mode.
Between pusher pulses, and when in MS mode, the pusher plate and third grid are at
0 V. All voltages are w.r.t. the entry voltage. The voltage on the second grid can be
varied using the guard slider.

977 V
410 V
0 V
-2636 V
-5180 V
-7200 V
pusher plate
third grid (70 L/in)
second grid (1000L/in)
second ring lens
first ring lens
first grid (1000L/in)

Q-TOF


MCP Conditioning
The MCP must be conditioned before first use, by increasing the applied voltage
gradually over a long time period. This is necessary to allow the escape of all
absorbed water from within the microchannels. To do this access other, MCP
Conditioning... A suitable program for overnight conditioning is:
Start = 100 V, Stop = 2700 V, Duration = 600 minutes, Step = 5 minutes
This program can be accelerated up to 200 minutes, but only if the MCP has been
previously conditioned, and the instrument has been under fine vacuum for at least 12
hours. MCP conditioning has to be repeated after every instrument venting (or loss of
fine vacuum).
MCP Resistance Check
The resistance across the MCPs can be measured directly using a 500 V Megger.
Disconnect the leads going to pins 2 and 3 of the 6-way peek feedthrough. Connect
the positive Megger lead to pin 2, and the negative lead to pin 3. The measured
resistance should be within the range 12 - 25 MO. A resistance that is higher than this
could indicate faulty MCPs (possibly ones which exhibit low gain). An infinite
resistance (>999 MO) would indicate either destroyed MCPs (subjected to excess
voltage) or disconnected internal wiring.
Transputer PCB
This should show 0 0 when the instrument is not acquiring. During TOF acquisitions
the display alternates between 3 5 and 2 A. The transputer PCB is reset every time
MassLynx is started - this will reset most error conditions. A complete listing of error
codes is given in the MassLynx Knowledge Base. Look under Knowledge base
topic menu, Miscellaneous. The presence of decimal points on the display indicates
a transputer crash. Recovery from this condition will require powering off the
electronics.
Q-TOF


Instrument Operation
The Entrance voltage should always be set at around 47 eV. This is necessary to
ensure the TOF beam hits the MCP. High resolution is normally selected; this
switches on the quad DC, mass analysing the ion beam. When High resolution is
deselected, the quad DC is set to zero. Cone Ramp should not be selected.
MS Mode
Collision energy should be set to 4 eV. During the acquisition the quad DC goes to
zero, and the RF is used for ion transport. Ions over a wide mass range are
simultaneously transmitted, with a gradual fall-off in transmissions at higher masses,
and with a sharp cut-off at about 0.78 of the RF mass i.e. the mass that would be
transmitted if the DC were on. The RF mass is determined by the amplitude of the RF.
In order to collect a spectrum over a wide mass range it is necessary to scan the quad
RF during each TDC integration period. This is controlled under MS profile. A
possible RF program is represented below. As a general rule, it is not necessary to
scan the quad RF higher than one half of the acquisition high mass.
mass 3
mass 1
ramp time
dwell time
scan time
mass 2

The following general MS profile can be used in most situations:
mass1 = LM dwell = 20 ramp = 60
mass2 = HM/2 dwell = 20 ramp = 0
mass3 = HM/2
where LM = acquisition low mass, HM/2 = half of acquisition high mass.
MS/MS mode: Acquisition of a Fragment Ion Spectrum
- Tune up as in MS mode, i.e. collision energy = 4 eV, collision gas off.
- Select required resolution. For best sensitivity this is set to a minimum, in which
case MS/MS is performed on an isotopic cluster, not a single isotope..
- Set up a TOF MS/MS experiment, with the required precursor ion mass entered.
- Select a suitable collision energy e.g. 30 eV. There is no need to retune.
- Switch on the collision gas (usually argon). Adjust the regulator to give ~ 6 x 10
-5

mbar analyser pressure. This will require about 25 psi on the regulator.
- Start the acquisition.
- Multiple collisions lead to the formation of fragment ions of equal energy. These
emerge from the collision cell with energies similar to that used in MS mode. Any
precursor ions that pass straight through the gas cell without colliding will emerge
with a higher energy, and as a result miss the TOF detector. Hence the precursor
ion peak in the fragment ion spectrum will be very small.
- Note: The quad DC is set to give the required resolution. The mass (m/z)
transmitted depends on the magnitude of the RF.
Q-TOF


The Analyser Quadrupole Lens
V
off
- V
res
- V
RF
V
off
+ V
res
+ V
RF
V
off
+ V
res
+ V
RF
V
off
- V
res
- V
RF
V
off
= offset voltage
V
res
= resolving DC
V
RF
= RF ( -V
RF
implies a
180 phase difference)

The Tube Lens
This is set for maximum beam intensity by adjusting a trimpot mounted on the TOF
PCB.

Collision and Transport RF
These are set under Other, RF Settings... The gain factor scans the RF during
acquisitions. Typical values are:
Transport RF gain 0.0
offset 0.30
Collision RF gain 0.0
offset 0.30
Where it is important to detect low mass ions, e.g. immonium ions in peptide fragment
spectra, do not use a higher values than given above. Low mass sensitivity will be
improved slightly if the offsets are reduced to 0.20, but at the expense of higher mass
sensitivity.
For best sensitivity at masses above 2000 Da, set RF offset to 0.5.
Q-TOF


Tuning Control Sliders
Capillary (Needle)
Sets the absolute voltage on the electrospray probe or APCI corona needle. Typically
needs to be around 3 kV.
Counter (non z-spray only)
Sets the absolute voltage on the counter electrode. Set to 600 V to start with.
Cone
Sets the voltage on the sampling cone relative to the extraction lens. It is compound
and charge state dependent. Set to about 46 eV for doubly charged gramicidin S.
Extraction
Sets the voltage on the extraction lens (replaces skimmer in z-spray).
Skimmer Lens (non z-spray only)
This adjusts the voltage on the skimmer lens relative to the skimmer. Has little effect .
Skimmer (non z-spray only)
This adjusts the voltage on the skimmer without affecting any other voltages. It is
generally left at 1 V.
RF Lens (Hexapole)
This sets the offset voltage on the hexapole and the first differential pumping aperture.
It usually optimises at about 1 V.
LM Res and HM Res
These set the resolving DC on the quad. The two sliders are set to give constant
resolution across the mass range. When the quad has been set up, 15 on both sliders
should give unit resolution.
Collision
This sets the collision energy, i.e. the energy of the ions have when they reach the
collision cell. It adjusts the voltage on the cone, extraction lens, source hexapole, first
differential pumping slit and quad simultaneously. Set to 4 eV. Do not use to maximise
peak height.
Ion Energy
This sets the quad offset DC w.r.t. the collision energy, i.e. the energy of the ions
travelling through the quad. This should be set between 1.2 and 1.8 V.
IE Ramp
This programs the ion energy to increase with mass. It can be left at zero.
Steering
This adjusts the voltage difference between the top and bottom half plates of the
steering / focus lens. It acts as a z-deflect, directing the beam into the pusher. It
should optimise at close to zero.
Entrance
This sets the voltage on the pusher entrance and exit. The theoretical value is 47 eV.
It should be optimised when looking at a TOF beam, not on the first detector.
Q-TOF


Multiplier
This sets the photomultiplier voltage. Leave at 550 V.
MCP
This sets the voltage on the TOF detector. The voltage across the MCP is 800 V less
than this value. Ensure MCP has been conditioned before applying high voltage. The
TOF is usually operated with this detector at 2700 V.
Transport
This sets the offset DC on the transport hexapole. About 4 V is OK.
Aperture 2
This sets the voltage on the second differential pumping aperture. About 15 V is
typical.
AccV
This sets the voltage on the accelerating lens; it is usually at maximum.
Focus
This adds an equal voltage to both the top and bottom steering / focus lens halfplates.
This typically optimises at 80 - 130 V, but for maximum resolution set to zero.
Guard
This adjusts the voltage on the second grid. It should not affect the beam in MS mode.
It can be used to compensate for field penetration through the third grid, but is usually
set equal to the entrance.
TOF
This sets the voltage on the flight tube. It is normally set to -7200 V.
Reflectron
This is set to a predetermined value (one that gives a reflectron voltage of 1640 V at
-7200 V on the flight tube). It is expressed as a percentage of the flight tube voltage.
Pre-filter
This sets the offset voltage on the quad pre- and post- filters. Start at around 10 V.
Time (s)
This set the time between pusher pulses when manual pusher is set.
Gramicidin-S Tune Solution
The recommended tune solution is 2 ng/L in 50/50 water/methanol with 1 % acetic
acid. This should be introduced at 5 L/min. With the photomultiplier set to 550 V and
the entrance set to 47 eV, the unresolved doubly charged gramicidin-S cluster should
give an intensity of ~ 10
6
.
species m/z
(M + H)
+
1141.71
(M + 2H)
2+
571.36
Q-TOF


TDC Parameters
Start (mV)
This is the size of trigger signal that is necessary to trigger the TDC i.e. start the clock.
A typical value is 1500. Varying this value will change the start time and hence the
TOF masses.
Stop (mV)
This is the size of pulse needed to register as being an ion i.e. to stop the clock. It
should be set to a value high enough to prevent electronic noise being detected as
ions. To do this acquire TOF data with the TOF detector turned off, i.e. at zero volts.
The instrument must be in operate. Use an integration time of about 2 seconds. At a
stop value of 50 mV hundreds of ions will be detected as noise. Increase the stop
value in 20 mV increments during the acquisition. The number of ions will fall until a
value is reached where only a few (< 10) ions are detected per integration. This
value should be used in all subsequent acquisitions. A typical value is 100.
A stop value that is set too low will result in high background noise, as well as
distorted peak shapes and isotopic distributions.
Threshold
This should always be set to zero.
Bunching
This should always be set to 1.
Limit (Da)
This should be set to 8000.
Lteff
This is used to make the TOF mass measurement nominally correct without a
calibration. 2096 is the default value.
Veff Factor
This should be set to 1.096.
Mass to Time Conversion
The mass of an ion (strictly the m/z) is given by the following equation:
m
T V
L
eff
teff
=
2
2
5184
where L
teff
= 2096 and V
eff
= 7893 and T is in ns
Simplifying we get T m = 1700 where T is in ns.
e.g. for doubly charged gramicidin, m = 571.4 T = 40600 ns
When considering a peakwidth, differentiate to give
o
o
T
m
m
= 850

where oT is the peakwidth in ns, om is the peakwidth in Da, and m is the mass in Da.
Q-TOF


Quadrupole Calibration
The quadrupole electronics are set-up to give 0.3 Da accuracy at 1 Da peakwidth. A
software calibration can be used to improve this calibration.
Tune up on sodium iodide, and set up the peak display to monitor m/z 172.88, 622.57,
1072.25 and 1971.61. Adjust LM Res and HM Res to give a peak width of 1 Da at half
height.
Select Other, Manual Pusher. Set Time slider to 255 s.
Call up Acquisition Control Panel.
Select Edit, Calibration parameters and set to:
perform auto peak matching
peak window 1
Initial error 2
Intensity threshold 2
polynomial order 2
calibrate display
Select Instrument, Calibrate, select reference file NAICS.REF
Press Start...
Select Quad calibration and Acquire & calibrate
Press Acquisition Parameters, then default
Set Run Duration 2 minutes
Static span 4 amu
Static Dwell 0.3 s
InterScan Delay 0.3 s
OK, OK, follow progress in calibration status window
When the calibration is complete, the peaks will reappear in the calibration window,
and then shift as the calibration is loaded.
Save Instrument Calibration when prompted.
To review calibration, select Process, Calibration from file... (data is in STATMS1)
Change Calibration parameters if necessary.
When satisfied with calibration, press OK
MassLynx will ask Are you sure you want to update the current calibration?
Answer YES. This will overwrite the current calibration file.

Q-TOF


TOF Calibration
The mass to time relationship for TOF is (m/z)
1/2
= aT + b , where a and b are
constants defined by the instrument. These may be expected to show small variations
from one instrument to another. Deviations from this relationship can be
accommodated by the use of higher order terms.
Two types of calibration exist in MassLynx: instrument and post processing. An
instrument calibration is automatically applied to data during an acquisition. A post-
processing calibration may be applied to data at any time after acquisition.
Post-Processing Calibration
- Acquire spectra of a suitable reference compound.
- Combine several scans, Center spectrum.
- Click on Tools, Make Calibration. Select the correct reference file. Click on OK.
- If any of the peaks are incorrectly assigned then use right clicks to manually
assign. Click to the right of the reference peak then click to the right of the
corresponding spectrum peak.
- Use File, Save As, to save the calibration file in the CAL folder, then File, Exit.
- This calibration can be applied to other spectra using Tools, Apply Calibration.
Note: post processing calibrations cannot be used as instrument calibrations.

Instrument Calibration
- Acquire spectra of a suitable calibration compound.
- Combine spectra, Process, Center selecting Centroid top 70%, minimum
peakwidth 3 channels. Select create centroid spectrum. OK.
- File, Save Spectrum...
- On the acquisition control panel, click on Calibrate
- Use File, Save Calibration As... to save the current calibration under a different
name, if desired. The current calibration will be overwritten if a new calibration is
accepted.
- Select a suitable Reference File.
- Process, Calibration from file... Select calibration type TOF, use Browse to
select data file.
- Click on History, select centered spectrum, OK the History Selector, OK, OK
- In the Calibration window, Edit, Calibration Parameters, to set Polynomial order
as required.
- Exit the calibration window
- MassLynx will ask Are you sure you want to update the current calibration?
Answer YES
- Calibration status should now indicate that a TOF calibration is loaded
Q-TOF


Notes:
Save calibration saves both the current quad and TOF calibrations in the same file.
The file produced by Spectrum, Tools, Make Calibration is a post processing
calibration. The file produced by Instrument, Calibrate, Save Calibration is an
instrument calibration. Both files have the same extension, but are of incompatible
format. Be judicious in your choice of locations and naming conventions for these files.
Recommended Locations:
Instrument calibration: MassLynx\default.pro\ Acqudb
Post processing calibration: MassLynx\cal
To remove instrument calibration, use Process, Delete all calibration..., File, Save
Calibration As... (e.g. uncal). To restore calibration File, Load calibration.
For the Best Mass Accuracy
- Peaks should have intensities not greater than 500 counts per second, in order to
avoid dead-time effects.
- The instrument calibration should be acquired at the same pusher rate as will be
used for a specific accurate mass determination. The pusher rate changeover
masses are: 900, 1800, 3600 and 7200.
- MS profile should be set to:
mass1 2, dwell 10, ramp 5, mass2 M, dwell 85, ramp 0, mass3 M
where M is the lowest mass that will be measured.
Dead-time Correction
Due to the dead-time of the TDC, high ion currents result in peak centroids shifting to
lower masses. There is dead-time correction software within MassLynx that will allow
accurate mass measurement at higher ion currents. This requires a value for the Np
Multiplier, which must be determined for each instrument.
- Acquire a spectrum of the standard PEG mix, with high peak intensities and a
resolution in excess of 5000.
- Create a centered spectrum, with Resolution, Np Multiplier and Lock Mass set to
zero.
- Select Tools, Make Calibration, using the peghnh4 reference file. Set polynomial
order to 1.
- The residual errors will reveal a difference between high and low intensity peaks
due to dead-time distortion.
- Press Cancel.
- Re-center the data using the actual Resolution and an Np Multiplier of 1. The
residual errors between adjacent peaks of low and high intensity should reduce.
- Repeat this process using lower and lower values of Np Multiplier until the
residuals are minimised (try 0.9, 0.8, 0.7, 0.6....)
A typical value for Np Multiplier is 0.7. The resolution figure used should be that
measured at m/z 500. Once evaluated, the Np Multiplier and a typical resolution can
be left active in the TOF... menu.

Q-TOF


Pusher Rates
The time period between pusher pulses is variable; one of five available values is
selected automatically according to the acquisition high mass. High mass acquisitions
require a longer period between pulses because of the longer flight times involved.
Shorter time periods are used for lower masses in order to maximise sensitivity.
Periodic Time / s Acquisition high mass
51 s 900
72 901 - 1800
102 1801 - 3600
144 3601 - 7200
204 > 7200

Rheodyne 7010 Injection Valve
The injection valve mounted in the gas handling panel can be used for loop injections
when doing megaflow electrospray or APCI.
A 20 L loop is supplied. Always use an excess of sample when filling the loop. About
three loop volumes are needed for good precision.
To minimise sample carryover, flush out the loop with solvent when in the load
position before making an injection.

Port
1 sample loop
2 pump
3 probe
4 loop
5 waste
6 inject

Q-TOF


Source and Probe Gas Connections (Z-Spray)
There are five different gas connections to the gas panel: nanoflow, desolvation and
nebuliser to the front, and purge and curtain gas to the rear.
Nanoflow is used with borosilicate nanovials. It can be used to increase sample
flowrate or overcome blockages.
Desolvation is connected to the probe adjuster, for use in microflow electrospray and
APCI. It is usually set to 200 - 300 L/hour.
Nebuliser is connected to microflow electrospray and APCI probes. For electrospray it
is set to about 10 L/hour, for APCI to maximum.
Purge can be used to supply purge gas to the source. It is connected via a 1.0 mm
restrictor (black), and its flowrate is set by the desolvation flow controller. It is not
normally used.
Curtain gas is used in nanoflow, for improved sensitivity, and reduction of solvent
clustering. It is connected via a 0.3 mm restrictor (white), and its flowrate is set by the
desolvation flow controller. Set to about 50 L/hour. The desolvation gas output at the
front should be blanked off.

Collision Gas Inlet System
The gas bottle pressure should be set to about 40 psi. The collision gas regulator
should be set to about 25 psi. The solenoid valve will only operate correctly when
there is at least 10 psi of collision gas pressure at the valve.
collision
cell
solenoid valve
regulator
to collision gas
inlet
to rotary pump

Q-TOF


Connections to Pilot Valve
The Z-spray source exhaust is vented to the rear of the instrument via a pilot valve.
This valve is designed to isolate the source from the exhaust line when the API gas
flow is turned off. This prevents the possibility of contamination of the source with any
substances which may be present in the external (building) exhaust system.
For correct operation of the pilot valve, ensure it is connected as shown below:

The pilot valve is located behind the gas panel. The API gas flow should be switched
off before changing the position of the 4 mm gas line connection.
APCI Sensitivity
Tune up on a solution of 0.001 % 3-picoline in 50/50 acetonitrile/water. Use an LC
pump set to 1 mL per minute. The nebuliser gas flowrate should be set to maximum.
Set desolvation gas to 300 L/h. The peak at m/z 94 should have an intensity of about
1E6 with the photomultiplier at 550 V.
Typical settings: Corona needle 3000 V
Cone 30 V
Source temp 120C
Probe temp 600C
Acquire TOF data using : Scan time 5 s
High mass 500 Da RF gain 0
RF offset 0.2 MS profile 90
The peak at m/z 94 should have an ion count of about 1.5E4.
Loop Injections
Tune up on 10 ul injections of 250 pg/L reserpine at a flow rate of 1ml/min. Acquire
over the range 100 to 800 Da, with a 0.5 s scan time. Make 10 L injections of
50 pg/l reserpine solution through the loop injector. Display the mass chromatogram
of the (M + H)
+
ion at 609.28 Da. Each injection should give a peak with a signal to
noise ratio of >20:1.
1
3
2
10 mm PTFE tubing
from source
10 mm PTFE tubing
to rear of instrument
4 mm gas line from
solenoid valve
no connection

Q-TOF


Nanoflow MS/MS Sensitivity
The signal to noise from a consumption of 2 fmol of [Glu
1
] -Fibrinopeptide B (1569 Da)
from a solution of 500 fmol/L concentration in MeOH/H
2
O + 0.2% formic acid
solution, using glass micropipettes with 1 or 2 m tips, will be greater than 10:1 on the
most intense y sequence ion from the MS/MS spectrum of the doubly charged
precursor ion. The integration period per spectrum will be about 5 sec and data will be
summed over a period appropriate for the required consumption of sample.
Conditions: Acquisition range 50 - 1700 Da
Scan time 2.4 s
RF gain 0
RF offset 0.4
Cone 48 eV
Collision energy 32 eV
Capillary 700 - 1000 V
Nanoflow pressure 2 psi
Load several microlitres of sample into the borosilicate nanovial. This can be done
using either a syringe fitted with a fused silica needle, or an automatic pipetter fitted
with a GELoader tip. When using GELoader tips, cut the nanovials in half (score with
fused silica cutter), to allow the pipette tip to reach the end of the nanovial.
Measure sample consumption by measuring distance travelled by meniscus (4 mm =
1 L). Initiate sample flow by distressing tip and/or increasing nanoflow pressure
briefly, if necessary. Acquire data for at least 30 minutes. Set the argon pressure to
give ~ 6 x 10
-5
mbar in the analyser. The doubly charged precursor ion is at m/z 785.8.
Smooth the data (mean, 1 x 3). The y ions are: 175.12, 246.16, 333.19, 480.26,
627.33, 684.35, 813.39, 942.43, 1056.47, 1171.50, 1285.54, 1384.6, 1441.6
The sample consumption per spectrum, Q, is given by:
Q
Vtc
T
=

where V = volume of sample consumed, t = scan time, c = concentration, T = duration
of spray (acquired time).

Masses of Glu-Fibrinopeptide B y Fragment Ions
(M + 2H)
2+
= 785.8427
175.1195 627.3254 1056.4750 1441.6348
246.1566 684.3469 1171.5020
333.1887 813.3895 1285.5449
480.2570 942.4321 1384.6133

Q-TOF


Control of Waters LC Systems
MassLynx uses a GPIB card for control of Waters LC systems. A typical system is
represented below.
MassLynx
PC
Waters 2690
Separations Module
Waters 996
Photodiode
Array
Detector
IEEE cable
Contact Closure
Q-TOF

The Waters 2690 Separations Module includes an LC pump and an autosampler. The
output from the Waters 996 Photodiode Array Detector is sent to MassLynx via the
IEEE link. A contact closure is used to trigger acquisition starts.
Loading Software
Load the MassLynx CD-ROM and run setup.exe. Deselect software. Select the
appropriate Waters option from the lists of inlets, autosamplers and detectors. Start
MassLynx, and from the Acquisition Control Panel, set the Inlet to ACE.
Configuration of MassLynx
Call up the Inlet Editor from the tune page or from Acquisition Control Panel -
Instrument - Set Up Inlet. Select Waters IEEE tab, set Waters Alliance 2690 ID = 2,
PDA Detector ID = 4.
Configuration of Waters 2690
Begin at Main menu; check firmware is revision 1.2 or later. Select Config. Use arrow
keys to highlight System. Press Enter to call up option list, use arrow keys to highlight
Controlled by MassLynx, press Enter to confirm choice.
Use arrow keys to highlight IEEE 488 Address. Press Enter, select 2, press Enter.
Press Exit to return to Main menu.
Note: use Exit to go up menu tree, use Enter to get pull-down menu. Menu/Status
toggles between menu tree and status display.
Connection of Contact Closure Cable
Connect contact closure input IN1 on rear of Q-TOF to terminals B1 and B2 (Inject
Start) on the rear of the 2690. The operation of the contact closure can be verified as
follows:
In MassLynx, set inlet to contact closure and start an acquisition from the sample list.
The message Waiting for injection should be produced.
From the 2690 Main menu, press Diag
Press Other Tests
Select Inputs and Outputs using arrow keys
Press 1 on numeric keypad to toggle the Inject Start output to ON.
This should start the acquisition.
Q-TOF


Configuration of Waters 996
The IEEE 488 Address is set via switches on the rear of the unit. Set to 4.
Checking Communications
From the Inlet Editor, select the LC tab, then Reset Communications. There should
be no error messages. The PDA Detector Mode will show Initialising at first. This will
change to Idling after a couple of minutes.
An error message such as There is no device present at address 4 on board 0 may
be produced. The board 0 referred to is the GPIB card, and the device defined as 4
within MassLynx is the PDA Detector. Check that the PDA detector is switched on.
If all is well, the Ready and OK status indicators in the Inlet Editor will be green. The
OK refers to the state of the communications. If an indicator is red, click on it for
further information.
For successful communications, the addresses declared within MassLynx must agree
with the actual addresses set on the devices. In the above example, the 2690 has
been given an ID of 2, and the 996 has been given an ID of 4.
Getting Started
If the pump is dry, a Dry Prime is necessary. This is done from the 2690 control
panel. Exit Inlet Editor (in MassLynx) to allow manual control of 2690. Press
Menu/Status to get Status(1) menu. Press Direct Function, to gain access to dry
prime control.
Restart Inlet Editor. Press Wet Prime button.
Press Start or Stop Pumping button to flush out the lines with solvent. There is a
delay of several seconds before this button has any effect.
Note: a flashing status light on the 996 PDA detector indicates the presence of air
bubbles. Restart the 996 after flushing out the lines with solvent.
The Inlet Editor can now be used to create a method. This involves the setting of:
Pump Initial Conditions
Pump Gradient Timetable
Autosampler Initial Conditions
PDA Parameters
When in doubt, stick to default values. The External Event Timetable and PDA
Analog Channels are not normally used. Set flows (initial and gradient) to 0.2
mL/min. Use the Load Method button to transfer parameters to 2690. Higher solvent
flowrates require the use of higher desolvation heater temperatures - try 250C.
Do not switch off the desolvation gas flow without first allowing the heater to cool down
to not more than 100C.
Put some standards in the autosampler, and set up the sample list, remembering to
specify the correct Inlet File.
Q-TOF


Control of HP1100 LC Systems
MassLynx uses an HPIB card for control of HP1100 LC systems. A typical system is
represented below.
G1315A
Diode Array Detector
MassLynx PC
G1316A
Column Compartment
G1313A
Autosampler
G1312A
Binary pump
G1322A
Degasser
Solvent tray
Q-TOF
G1351 BCD
Remote
CAN
CAN
CAN
HPIB
G1103-
61611
cable
Remote
Control
CAN
82341C
Contact
closure
input

HPIB Card
This must be type 82341C, with SICL software version B.03.11. Type 82341D (no dip
switches) will not work with this software.
Contact Closure PCB
A G1351-66500 BCD board must be installed in the pump module. It is not standard in
HP1100 systems. Connection to the Q-TOF contact closure inputs can be made using
a G1103-61611 general purpose cable. The wires corresponding to relay 1 are white
(pin 1) and brown (pin 2). From the MassLynx Inlet Editor, select Inlet System,
External Events. Set as follows:
Time Event State
initial contact 1 off
0.00 contact 1 on
0.10 contact 1 off

Q-TOF


HPIB Comms
Call up Inlet Editor. Select Tools, Instrument Configuration.
On HPIB communication tab set PC connection to Detector. The default address
for a DAD module is 26.
The HPIB link should always be made to the DAD module (when present).
Status Indicators
The Ready status indicator in the Inlet Editor must be green before an acquisition can
start. If it is red, click on it to reveal what is not ready.
The OK status is red if an error is detected in one of the LC modules, or if there is a
communication error. Click on it to reveal the error. Check all LC modules are
switched on, check HPIB communication is configured correctly. Try LC - Reset
Communications.
Pump Module Types
Binary G1312A
This type contains two pumps, with high pressure solvent mixing taking place after the
pumps. A solvent selection valve allows A1/A2 and B1/B2 solvent selection, but this
feature is not supported by MassLynx.
Quaternary G1311A
This pump type contains one pump, with low pressure solvent mixing taking place
before the pump. It can be used to mix up to four different solvents: A, B, C and D.
Further Information and Part Numbers
20848 Controlling the HP1100 LC system from MassLynx
20954 HP1100 HPLC system Set-up
21510 Q-TOF Data System (includes installation of HPIB card)
6666574 MassLynx NT Guide to Data Acquisition
8930800 HPIB card
7992114 HPIB cable

Q-TOF


Trouble-shooter

Beam on MS1 detector but not on TOF
- Check pusher is operating by selecting manual pusher then varying time slider
whilst observing the beam on the first detector. Compare intensities at 255 and
40 s. If intensity is the same, there is a pusher fault.
- The presence of a pusher pulse can also be detected by acquiring TOF MS data
with a low mass of 10. A large peak should be observed at approximately m/z 28
due to the end of the pusher pulse.
- If the pusher can be observed on the MS1 detector but not on the TOF then check
wiring from MS1 detector to TDC.
- If pusher is operating normally, check MCP and reflectron readbacks. Check
attenuator - is it blown? (should be 53 O from pin to case)
Pusher is not operating
- Check 977 V on TP1 vs. TP5 on the pusher PCB
- If voltage is low or absent, disconnect Spellman from pusher PCB and measure
977 V directly on D-type connector.
- If voltage is OK on supply then replace or repair pusher PCB: check Pickering
relays are not welded closed, replace FETs.
- If no voltage on Spellman output then replace Spellman.
Resolution varies with pusher frequency
- RV1 on pusher PCB is not set-up correctly
- Tune up CV1 and ring lenses at 150 s, switch to 40 s, and then adjust RV1 to
give good resolution / peak shape. RV1 has a greater effect higher pusher
frequencies; the use of 40 s will exaggerate any tuning errors on RV1.

Q-TOF


Installation of a US Transformer
Installations in the USA and Canada require the use of an ET/VGA-237 auto-
transformer. The local supply should be rated at 25 A minimum. Wiring connections
should be made according to the following diagram:

E
0 V 240 V
120 V 115 V 110 V 0 V
L N E
ground
(green)
neutral
(white)
hot
(black)
to Q-TOF
from local supply
connect hot wire to
one of these three
terminals

Check output voltage before connecting to Q-TOF. The live terminal should be at
~ 230 V w.r.t. ground, and the neutral at 0 V w.r.t. ground. The hot wire from the local
supply should be connected to the input terminal which gives an output voltage closest
to 230 V.

Q-TOF


Test Reagents
[Glu
1
]-Fibrinopeptide B Sigma F-3261 1569.6 Da
Sequence: GluGlyValAsnAspAsnGluGluGlyPhePheSerAlaArg
Contents of bottle = 0.1 mg = 64 nmol
Stock Solution
Add 2000 L of water, to give a solution of 32 pmol/L. Store in freezer.
Intermediate Solution
Take 100 L of the stock solution and add 3100 L of 50/50 MeOH/water + 1% acetic
acid, giving a solution of 1 pmol/L.
Spec Solution
Take 100 L of the intermediate solution and add 900 L of 50/50 MeOH/water + 1%
acetic acid, giving a solution of 100 fmol/L.
Note: The intermediate solution and spec solution should be discarded at the end of
the day. Make up fresh solutions from the stock solution in the freezer.

Horse Heart Myoglobin Sigma M1882
(mass = 16952 Da)
Stock Solution
Weigh out 5 mg (295 nmol) in a reagent bottle, and add 60 mL of water, to give a
solution of 5 pmol/L. Store in refrigerator.
Spec. Solution
Take 100 L of the stock solution and add 2400 L of 50/50 water/acetonitrile + 0.2 %
formic acid, giving 200 fmol/L. Store in freezer.

D(+)-Raffinose Sigma R-0250 C
18
H
32
O
16
.5H
2
O FW = 594.5 (M - H)
-
= 503.16
Stock solution
200 ng/L
Tune/Spec. solution
Dilute stock solution 40 times with 50/50 acetonitrile/water, giving a solution of
5 ng/L. e.g. put 1250 L of stock solution in a 50 mL volumetric flask and fill to the
mark with 50/50 acetonitrile/water. Store in refrigerator.
Q-TOF


Gramicidin-S Sigma G 0900 C
60
H
92
N
12
O
10
FW = 1214.4 (hydrochloride)

Stock solution
Dissolve 5 mg in 50 mL of 50/50 methanol/water + 1% acetic acid giving 100 ng/L.
Tune solution
Dilute stock solution by a factor of 50, e.g. to 1 mL of stock solution add 49 mL of
50/50 methanol/water + 1% acetic acid giving 2 ng/L.
PEG Mix
25 ng/L PEG 200, 50 ng/L PEG 400, 75 ng/L PEG 600 and 150 ng/L ammonium
acetate in 50/50 acetonitrile/water + 0.2% formic acid
Quad test mixture
Poly(propylene glycol) 25 ng/L average mol. mass 1000 Aldrich 20,232-0
Reserpine 2 ng/L C
33
H
40
N
2
O
9
(M + H)
+
= 609.28 Aldrich R17-1
Triacetyl-|-cyclodextrin 50 ng/L FW = 2017.79 Aldrich 33,262-3
Ammonium acetate 300 ng/L
Sodium Iodide
Dissolve 200 mg in 100 mL of 50/50 2-propanol / water giving a solution of 2 g/L.
Store in refrigerator.
Haemoglobin (Human) Sigma H 7379
Dissolve 3.5 mg in 20 mL of 50/50 water/acetonitrile + 0.2 % formic acid, giving
10 pmol/L.
ACTH fragment 18-39 Sigma A 0673 C
112
H
166
N
27
O
36

(M + 2H)
2+
= 1233.1
Melittin Sigma M 7391 C
131
H
229
N
39
O
31
monoisotopic mass = 2844.7541
Stock Solution
Add 2 mL of water to the solid (5 mg), to give 2.5 mg/mL.
Spec. Solution
Add 16.8 L of stock solution to 3 mL of 50/50 water/acetonitrile + 0.2 % formic acid,
to give 5 pmol/L.
(M + 2H)
2+
= 1423.3850 (M + 3H)
3+
= 949.2593
(M + 4H)
4+
= 712.1964 (M + 5H)
5+
= 569.9587
Sequence: GlyIleGlyAlaValLeuLysValLeuThrThrGlyLeuProAlaLeuIleSerTrpIleLys-
ArgLysArgGlnGln-NH
2

Erythromycin Sigma E 6376 C
37
H
67
NO
13

M - H
2
O + H
+
= 716.4585
5 ng/L in 50/50 acetonitrile/water + 0.2% formic acid
Leucine Enkephalin Sigma L 9133 C
28
H
37
N
5
O
7
M + H
+
= 556.2771
TyrGlyGlyPheLeu

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