Thermo Scientific HESI-II Probe User manual

HESI-II Probe
User Guide
70005-97000 Revision C April 2009

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Thermo Scientific HESI-II Probe User Guide v
C
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Safety and Special Notices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Precautions for Handling the HESI-II Probe . . . . . . . . . . . . . . . . . . . . . . . . . .viii
Contacting Us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .ix
Chapter 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Chapter 2 Functional Description. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Chapter 3 Removing and Installing the HESI-II Probe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
Removing the HESI-II Probe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Installing the HESI-II Probe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Adjusting the Probe Position . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Chapter 4 Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
Flushing the Sample Transfer Line, Sample Tube, and HESI-II Probe . . . . . . . 19
Replacing the Needle Insert. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Chapter 5 Replaceable Parts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25
Contents

Thermo Scientific HESI-II Probe User Guide vii
P
Preface
This HESI-II Probe User Guide provides you with information on using the
heated-electrospray ionization probe. It also provides procedures for installing and
maintaining the HESI-II probe.
Safety and Special Notices
Make sure you follow the precautionary statements presented in this guide. The safety and
other special notices appear in boxes.
Safety and special notices include the following:
CAUTION Highlights hazards to humans, property, or the environment. Each CAUTION
notice is accompanied by an appropriate CAUTION symbol.
IMPORTANT Highlights information necessary to prevent damage to software, loss of
data, or invalid test results; or might contain information that is critical for optimal
performance of the system.
Note Highlights information of general interest.
Tip Highlights helpful information that can make a task easier.

Preface
viii HESI-II Probe User Guide Thermo Scientific
Precautions for Handling the HESI-II Probe
When operating the mass spectrometer in the heated-electrospray mode, do not touch the
heated surfaces of the HESI-II probe or the Ion Max API source housing. Touching the
heated surfaces of the probe or the source housing can cause burns when the probe is
operating at typical temperatures of 350 to 450 °C.
When operating the mass spectrometer in the heated-electrospray mode, if you observe liquid
leaking from the sample inlet, place the mass spectrometer in Standby mode before you
tighten the fitting to eliminate the leak.
CAUTION AVOID BURNS. At typical operating temperatures (350 to 450 °C), the
HESI-II probe can severely burn you. Before removing the probe from the API source
housing, allow the probe to cool to room temperature (for approximately 20 minutes)
before you touch it.
CAUTION AVOID ELECTRIC SHOCK. Do not tighten the probe sample inlet fitting
to eliminate a liquid leak while the mass spectrometer is in operation. If you touch liquid
leaking from the probe sample inlet while the mass spectrometer is in operation, you
might receive an electric shock.
A
S
Hot surface caution
General caution
Hot surface caution
Avoid touching liquid leaking from
the sample inlet while the mass
spectrometer is in operation.

Preface
Thermo Scientific HESI-II Probe User Guide ix
Contacting Us
There are several ways to contact Thermo Fisher Scientific for the information you need.
YTo contact Technical Support
Find software updates and utilities to download at mssupport.thermo.com.
YTo contact Customer Service for ordering information
YTo copy manuals from the Internet
Go to mssupport.thermo.com and click Customer Manuals in the left margin of the
window.
YTo suggest changes to documentation or to Help
• Complete a brief survey about this document by clicking the link below. Thank you
in advance for your help.
• Send an e-mail message to the Technical Publications Editor at
[e-mail].
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Knowledge base www.thermokb.com
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E-mail [e-mail]
Web site www.thermo.com/ms

Thermo Scientific HESI-II Probe User Guide 1
1
Introduction
Heated-electrospray ionization (H-ESI) transforms ions in solution into ions in the gas phase
by using electrospray ionization (ESI) in combination with heated auxiliary gas. You can use
H-ESI to analyze any polar compound that makes a preformed ion in solution.
Basic compounds (for example amines) can form a protonated molecule [M + H]+, and acidic
compounds (for example sulphonic acids) can form a deprotonated molecule [M – H]-. In the
positive ion polarity mode, the protonated molecule produces a peak at an m/z value of
M + 1, where M equals the mass of the original molecule. In the negative ion polarity mode,
the deprotonated molecule produces a peak at an m/z value of M – 1, where M equals the
mass of the original molecule.
Because solution chemistry is the primary factor affecting mass spectra in ESI, other common
adducts include sodium ions (Na+), yielding an m/z value of [M + 23]+; potassium ions (K+),
yielding an m/z value of [M + 39]+; and ammonium ions (NH4+), yielding an m/z value of
[M + 18]+.
This chapter describes the principles of the heated-electrospray ionization technique through
use of the HESI-II probe (see Figure 1) and a Thermo Scientific mass spectrometer.
Figure 1. HESI-II probe

1Introduction
2HESI-II Probe User Guide Thermo Scientific
With H-ESI, a mass spectrometer can analyze a range of molecular weights greater than
100 000 u, due to multiple charging. H-ESI is especially useful for the mass analysis of polar
compounds, which include biological polymers (for example, proteins, peptides,
glycoproteins, and nucleotides); pharmaceuticals and their metabolites; and industrial
polymers (for example, polyethylene glycols).
In H-ESI, ions are produced and introduced into the mass spectrometer as follows:
1. The sample solution enters the ESI needle, which receives a high voltage.
2. The ESI needle sprays the sample solution into a fine mist of droplets that are electrically
charged at their surface.
3. The electrical charge density at the surface of the droplets increases as solvent evaporates
from the droplets. In heated-electrospray ionization, heated auxiliary gas aids solvent
evaporation.
4. The electrical charge density at the surface of the droplets increases to a critical point
known as the Rayleigh stability limit. At this critical point, the droplets divide into
smaller droplets because the electrostatic repulsion is greater than the surface tension. The
process is repeated many times to form very small droplets.
5. Electrostatic repulsion ejects sample ions from the very small, highly charged droplets
into the gas phase.
6. The sample ions enter the mass spectrometer through the ion transfer tube.
Figure 2 shows the steps in the formation of ions from highly charged droplets.

1 Introduction
Thermo Scientific HESI-II Probe User Guide 3
Figure 2. H-ESI process in the positive ion polarity mode
You can use the HESI-II probe in either positive or negative ion polarity mode. The ion
polarity mode of choice is determined by the polarity of the preformed ions in solution:
Acidic molecules form negative ions in solution, and basic molecules form positive ions.
Because the ejection of sample ions from droplets is facilitated when the ionic charge and
surface charge of the droplet have the same polarity, use the positive ion polarity mode to
analyze positive ions and the negative ion polarity mode to analyze negative ions.
Sample ions can carry a single charge or multiple charges. The number of charges carried by
the sample ion depends on the structure of the analyte of interest and the carrier solvent.
(In H-ESI, the buffer and the buffer strength both have a noticeable effect on sensitivity, so it
is important to choose these variables correctly.) In the case of higher molecular weight
proteins or peptides, the resulting mass spectrum consists typically of a series of peaks
corresponding to a distribution of multiply charged analyte ions.
Droplet size, surface charge, liquid surface tension, solvent volatility, and ion solvation
strength are factors that affect the H-ESI process. Large droplets with high surface tension,
low volatility, strong ion solvation, low surface charge, and high conductivity prevent good
electrospray.
–
++
+++
+
+
+
+++
++
+
––
–
–
––
+++
+
+
++
–
–––
––
–
+
–
Charged
droplet
Solvent
evaporates
from droplet
Ejected
positive ions
Ion transfer
tube
HESI-II probe
nozzle

1Introduction
4HESI-II Probe User Guide Thermo Scientific
Organic solvents, such as methanol, acetonitrile, and isopropyl alcohol, are superior to water
for H-ESI. Volatile acids and bases are good, but salts above 10 mM concentration and strong
acids and bases are extremely detrimental.
Follow these rules for achieving a good electrospray:
• Keep salts out of the solvent system.
• Use organic/aqueous solvent systems and volatile acids and bases.
• Optimize the pH of the solvent system.
Tabl e 1 shows initial heated electrospray settings for different liquid flow rates for a 50 percent
aqueous solution. These initial settings provide a starting point for optimizing system
performance. The optimal settings for your application depend on the compounds of interest,
the solvent matrix, and the chromatographic conditions. For information on optimizing these
settings, refer to the getting started guide for your mass spectrometer.
For an LTQ Series mass spectrometer, the allowable range for the auxiliary gas flow depends
on the version of the instrument control software and the vaporizer temperature setting:
• For LTQ 2.5.0 or lower, the allowable range for the auxiliary gas flow is 5 to 60 units.
• For LTQ 2.5.5 or higher, the allowable range for the auxiliary gas flow is 0 to 60 units for
vaporizer temperatures up to 100.00 °C. For vaporizer temperatures above 100.00 °C, the
minimum auxiliary gas flow is 5 units.
For a TSQ Series mass spectrometer, you can set the auxiliary gas flow from 0 to 60 units.
Tip For best results, avoid operating the HESI-II probe at elevated temperatures without
solvent flow from the LC system or the syringe pump. Allowing the HESI-II probe to run
dry at elevated temperatures can cause blockage of the replaceable metal needle (see
“Replacing the Needle Insert” on page 20).
Tip For best results, set the auxiliary gas flow to a minimum of 5 units for vaporizer
temperatures above 100.00 °C.
Table 1. Initial heated electrospray settings
Liquid
flow rate
(μL/min)
Ion transfer
tube
temperature
(°C)
H-ESI
vaporizer
temperature
(°C)
Sheath gas
pressure
(psi)
Auxiliary gas
flow
(arbitrary units)
Spray voltage
(V)
Typical
nitrogen gas
consumption
(L/min)
5 240 Off to 50 5 0 +3000 (-2500)*<1
200 350 250 to 350 35 10 +3000 (-2500)8
500 380 300 to 500 60 20 +3000 (-2500)13
1000 400 500 75 20 +3000 (-2500)17
*Negative ion mode

Thermo Scientific HESI-II Probe User Guide 5
2
Functional Description
The HESI-II probe produces charged aerosol droplets that contain sample ions. The HESI-II
probe accommodates liquid flows of 1 μL/min to 1 mL/min without splitting.
The removable components of the HESI-II probe are the end cover that is secured to the
probe body with socket head screws and the needle insert that screws into the probe body
(see Figure 3). The end cover includes the high voltage feedthrough and the sample inlet port.
The external components of the probe body include the grounding union holder, vaporizer
cable connector socket, probe sleeve with depth markers, and probe nozzle (see Figure 3 and
Figure 4).
Figure 3. HESI-II probe removable components
Socket head screws
Probe body
Needle insert
End cover
Probe sleeve with
depth markers

2Functional Description
6HESI-II Probe User Guide Thermo Scientific
Figure 4. HESI-II probe views
Sample and solvent enter and exit the HESI-II probe through the needle insert, which
protrudes from the sample inlet port at the back of the probe and from the nozzle at the front
of the probe (see Figure 4). The needle insert (see Figure 3 on page 5) includes an adjustable
union, a needle guide fitting and ferrule, an ESI needle, and a metal needle. The adjustable
union is used to adjust the protrusion of the needle insert tip from the probe nozzle (see
Figure 4).
Sheath gas inlet (S)
Aux gas inlet (A)
High voltage socket for the
8 kV cable connector
Sample inlet
Grounding
union holder
Vaporizer cable
connector socket
Probe nozzle
ESI needle
Metal needle
1.5 mm

2 Functional Description
Thermo Scientific HESI-II Probe User Guide 7
A large negative or positive voltage is applied to the ESI needle (typically ±3to±5kV),which
sprays the sample solution into a fine mist of charged droplets. The probe nozzle directs the
flow of sheath gas and auxiliary gas at the droplets. The probe body houses the probe nozzle
and needle insert, and includes the sheath gas and auxiliary gas plumbing.
The probe body has inlets for the sheath and auxiliary gases. The sheath gas is the inner
coaxial nitrogen gas that sprays (nebulizes) the sample solution into a fine mist as it exits the
metal needle sample tube (see Figure 5). The heated auxiliary gas is the outer coaxial nitrogen
gas that assists the sheath gas in the desolvation of sample solutions (see Figure 6). The
auxiliary gas heats as it passes through a vaporizer. The vaporizer is thermally insulated from
the sample tube to prevent direct heating of the sample solution. You can control the
vaporizer temperature from the Xcalibur™ data system. The temperature range is from
ambient room temperature to 600 °C. For recommended operating temperatures and gas flow
settings, see Table 1 on page 4.
Figure 5. Sheath gas plumbing
Sheath gas fitting (blue)
in gas inlet (S)
Metal needle
(inner needle)
Sheath
gas
ESI needle
(outer needle)

2Functional Description
8HESI-II Probe User Guide Thermo Scientific
Figure 6. Auxiliary gas plumbing
The angle of the HESI-II probe is fixed at approximately 60 degrees. To help optimize the
spray stability, use adjustment screws on the Ion Max source housing to make small changes to
probe position. The fixed angle, off-axis spraying affords long-term signal stability
(robustness) for most solutions that contain non-volatile matrix components, mobile phase
buffers, or ion-pairing reagents. For information on adjusting the probe position, see
“Adjusting the Probe Position” on page 17.
Auxiliary gas fitting (green)
in gas inlet (A)
ESI needle
(outer needle)
Auxiliary gas
Metal needle
(inner needle)

Thermo Scientific HESI-II Probe User Guide 9
3
Removing and Installing the HESI-II Probe
This chapter describes how to set up the API source with the HESI-II probe. You must
remove the HESI-II probe to perform maintenance on the probe.
Removing the HESI-II Probe
YTo remove the HESI-II probe from the API source housing
1. Place the mass spectrometer in Standby mode, and allow the probe to cool to room
temperature.
Wait approximately 20 minutes for the probe to reach room temperature when it has
been operating at high temperature. If the mass spectrometer is connected to an LC
system, leave the solvent flow from the LC pump on while the probe is cooling to room
temperature.
2. If the mass spectrometer is connected to an LC system, turn off the solvent flow from the
LC pump, and then disconnect the tubing from the left side of the HESI-II probe
grounding union (see Figure 7 on page 10).
Note To install the APCI probe, refer to the Ion Max and Ion Max-S API Source Hardware
Manual.
Contents
•Removing the HESI-II Probe
•Installing the HESI-II Probe
•Adjusting the Probe Position
CAUTION AVOID BURNS. At typical operating temperatures (350 to 450 °C), the
HESI-II probe can severely burn you. Before removing the probe from the API source
housing, allow the probe to cool to room temperature (for approximately 20 minutes)
before you touch it.

3Removing and Installing the HESI-II Probe
Removing the HESI-II Probe
10 HESI-II Probe User Guide Thermo Scientific
Figure 7. Front view of the connections to the HESI-II probe
3. Disconnect the 8 kV cable from the HESI-II probe high voltage receptacle as follows (see
Figure 8 and Figure 9):
a. Unlock the cable by twisting the locking ring counterclockwise.
b. Unplug the 8 kV cable from the HESI-II probe high voltage socket.
Figure 8. Enlarged view of the 8 kV cable connector
Sheath gas line
Vaporizer cable
To LC outlet
8 kV cable
Auxiliary gas line
Connection between
grounding union and
sample inlet
Probe locking knob
Locking ring
High voltage socket
(for the 8 kV cable
connector)