ITSP PAL Training - Advanced

ITSP PAL Advanced Course
for Method Developers

Instructor
2
ITSP PAL Advanced Course for Method Developers
for research purposes only
Rick Youngblood
Director of Technology
ITSP Solutions, Inc.
Ga Tech Electrical Engineer and Computer Scientist
Lean Six Sigma Green Belt
30 years experience in industrial automation and controls technology including 16
years with Honeywell, Inc.
Certified by CTC Analytics in PAL Service and PAL Programming
8 years experience in ITSP PAL lab automation

This course is intended to familiarize ITSP Method developers with the different
concepts, processes, and software programs used to create, modify, run, and
troubleshoot ITSP Methods.
Topics include:
1. Using CTC PAL Cycle Composer with Macro Editor (CC-ME) software to create,
edit, and test Macros and Methods from a PC.
2. Using CTC PAL Cycle Editor (CE) software to convert Cycle Composer Macros to
Cycles.
3. A discussion of important ITSP concepts related to creating PAL Macros and
Methods.
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The ITSP PAL Intermediate Course for Lab Personnel is prerequisite for this course.

Cycle Composer Control Levels Review
1. Sample List provides access to Sample Lists and pre-defined Methods (similar to
editing a MassHunter Sample List). Sample lists run Methods on assays of case
samples.
2. Method Editor provides access to pre-defined Methods and to create new
Methods with existing Macros (similar to MassHunter PAL Method Editor).
Methods run Macros with certain variables defined in the Method before the run.
3. Macro Editor provides full access with the ability to edit existing Macros and to
create new Macros (similar to editing MassHunter PAL Cycles with Cycle Editor).
Macros run a sequence of firmware Atoms on objects defined in a PAL.
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Sample Lists
Sample Lists can be used to perform ITSP PAL Methods on an assay of samples.
• Generally, three PAL parameters are accessible from the Sample List:
• SL.Index (which vial or well in the sample tray is to be used for the current
Cycle sample),
• SL.Volume (usually the injection volume to be injected into the LC injection
valve), and
• SL.Tray (usually the sample tray to be used for the current Cycle sample – in
ITSP Methods, this is the initial raw case sample tray before ITSP clean-up, it is
not the elution/injection sample tray).
• Additional “SL.” variables can be used in Macros and Methods by the Cycle
Composer Sample List but their ability to be easily transferred across platforms or
systems may be limited when Macros are converted to Cycles.
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Methods
Methods can be created to run pre-existing Macros and modifying the Macro variable
settings in order to make different Methods.
• Methods are not Macros, multiple Methods can call the same Macro with each
Method having different Macro variable settings.
• A single Method can call multiple Macros as sequential steps, but multi-step
Methods cannot be converted into certain LCMS control software Methods.
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Macros
Macros (or Cycles) are lists of one or more PAL firmware Atoms that will run in
sequence.
• Macros in Cycle Composer are directly analogous to Cycles in Cycle Editor and in
MassHunter.
• Macros can be converted into MassHunter Cycles using Cycle Editor.
• It is important to note that only 30 parameters can be controlled at the Method
level per Macro.
• The same Macro (or Cycle) can be called by different Methods using different
Macro variable settings and saved under different Method names.
• Macros can be either called alone by a single-step Method or combined into a
multi-step Method.
• However, multi-step Methods cannot be converted to MassHunter Methods.
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PAL Firmware Review
Firmware is the base level of control in the PAL autosampler.
• Within the PAL, firmware Atoms operate on objects.
• Atoms and objects have parameters.
o Atom parameters can be:
• Left undefined, or
• Hard-coded at the Macro level of control by the Method developer, or
• Passed 1 level up to the Method level of control as a Macro (or Cycle)
variable, or
• Passed 2 levels up to the Sample List level in some cases. (In ITSP Methods
those cases are SL.Tray, SL.Index, and SL.Volume.)
o Object parameters which are left undefined within a Macro can then still be
controlled from the local terminal.
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PAL PC File Extensions Review
• File Extensions
• PAL MEthods: *.pme
• PAL MAcros: *.pma
• PAL Sample List: *.psl
• PAL Object List: *.pol (latest_pal.pol and latest_pal.xml, which are the latest
PAL configuration, and other object files)
• PAL firmware backup files: *.sss
• Cycles: *.cyx
• When e-mailing ITSP Solutions for support with custom ITSP Macros, please include
the following attachments:
• [Macro_name].pma (or [Cycle_name].cyx for MassHunter Cycles)
• Latest_pal.pol (or latest_pal.xml for MassHunter Cycles)
• Method Macro (or MassHunter Cycle) parameter settings (e.g., *.pme)
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Cycle Composer Setup Review
Connection to PAL
• Cycle Composer accesses the PAL using the serial port or the Ethernet port.
When a Sample List is run, the program takes direct control of the PAL. The
software executes the sequence of steps defined in each Method assigned
to the current sample.
• When Cycle Composer is started, it checks for the presence of a PAL. If
found, objects on the PAL are uploaded.
• Running On-Line – If the PAL is connected and the correct COM port is selected,
then the current configuration of the PAL is uploaded to the computer and
saved to the file “latest_pal.pol” in the current Method folder.
• Running Off-Line – If no PAL is detected when the program starts, then the
previous configuration of the PAL is loaded from the file “latest_pal.pol” in the
current Method folder.
• If no PAL is detected and no “latest_pal.pol” file is found, then Cycle Composer
will not start correctly.
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Select File> Option Menu
• Port
• Reload Pal Configuration
• Choose Method folder
• Status Box
• Format Sample List
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• Reload PAL configuration
• If changes are made to the PAL configuration while Cycle Composer is running,
these changes are not automatically transferred to the PC and the program is
unaware of them.
• It is necessary either to close and re-start Cycle Composer or select “Reload PAL
configuration”.
• This is used when the syringe is changed on the PAL or any new objects are
created such as trays or even when re-training an object location.
• Choose Method Folder
• This allows a user to define or change a Method folder.
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• Status box
Displays a status window which can be
helpful for troubleshooting during Method
development.
• Format Sample List
This option allows the operator to select
which Sample List fields are visible for
editing in the Sample List table, as well as
which defaults are used for all fields.
13

Macros
• A Macro is a sub-unit of a Method. It contains the instructions for the
sampling protocol.
• The “Standard Macro Set” is a group of small Macros which provide a range of
PAL operations which can be used in combination like building blocks to
construct a complete sampling protocol:
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Macro Editor
To create a Macro:
• Start with an existing similar Macro
• Simplify the Macro
• Insert needed Atoms
• Document the Macro
• Save the Macro
• Test the Macro
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Atoms
• An Atom is the lowest level command available to the Cycle Composer
software.
• Clicking the "F" button beside the Atom list will cause the Atoms to be listed in
alphabetical order. The "F" (frequent) will change to an "A" (alphabetic).
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Atoms
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There are 53 available Atoms. Some examples are:
Note that there is sometimes more than one way to perform a specific task.
INJ_SAMPLE, PUT_SAMPLE, DISPENSE_SYR, and EJECT_SYR are four Atoms that
dispense an aliquot from the syringe, each with different parameters available.

Examples of Atoms
Some Atoms are more complex than
others:
• Complex: Get Sample (GET_SAMPLE)
Fill syringe from a selected tray, vial or
wash station. Many details as to how
the syringe is filled can be controlled.
• Simple: Move Relative (MOVE_REL)
Move relative to the current position.
If a value is left empty (default=0mm)
then the injection unit is not moved
along that axis.
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Atom Sequence Example in the LC-Inj Macro
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Macro Variables Defined as a Numeric Value
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Macro Variables Defined as an Object
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Making Macros independent of the Method Syringe
• SYR.Min Volume μl Minimum volume allowed for the selected syringe
• SYR.Max Volume μl Maximum volume allowed for the selected syringe
• SYR.Fill Volume μl Fill Volume of the selected syringe
• SYR.Min Speed μl/s Minimum plunger speed allowed for the selected syringe
• SYR.Max Speed μl/s Maximum plunger speed allowed for the selected syringe
• SYR.Fill Speed μl/s Fill Speed of the selected syringe
• SYR.Eject Speed μl/s Eject Speed of the selected syringe
• SYR.Inject Speed μl/s Inject Speed of the selected syringe
Because Macros should be written so they can be used in Methods with different
syringes, limits of syringe dependent variables can be defined using symbolic values:
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Integrating a Macro with the Sample List
• SL.Index Sample number taken from the “Vial” field of the Sample List
• SL.Volume Volume taken from the “Volume” field of the Sample List
• SL.Tray Tray name taken from the “Tray” field of the Sample List
• SL.Injector Injector name taken from the “Injector” field of the Sample List
• SL.Volume2 Volume taken from the “Volume 2” field of the Sample List
• SL.Tray2 Tray name taken from the “Tray 2” field of the Sample List
• SL.Injector2 Injector name taken from the “Injector 2” field of the Sample List
ITSP Solutions recommends limiting the use of
“SL.” variables to only:
• SL.Index (as the current sample, i.e. well or
vial number),
• SL.Volume (as the injection volume), and
• SL.Tray (as the initial raw sample tray).
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Algebraic Macro Parameters
Numeric parameters can also be calculated:
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Synchronization With Input Signals
A PAL Method can be forced to wait for any incoming Sync Signal using the Atom
WAIT_SYNC_SIG. See the PAL user manual for a complete list and descriptions.
Standard Assignment for GC PAL
Injection Cycles
Description Default
Events
INTERFACE
1 Pin #
Start JobQueue (Sync Signal) Start a Job Queue (Job). Prerequisite is
a defined “Job” with a Method
assigned. Useful for automated,
unattended timed sampling.
Immediat
Start (Sync Signal) Start a Cycle (Input from GC or data
system) Immediat
1 7
8(GND)
Start2 (Sync Signal) Continue the Cycle “GC-Dual” for the
second sample
Immediat
(Ignore)
Inject (Sync Signal) Inject
READY to INJECT
Immediat
(Ignore)
Inject2 (Sync Signal) Inject the second sample for “GC-Dual”
Cycle
Immediat
(Ignore)
Injected (Out Signal) Is activated at the moment when the
sample has been injected
SW-Out1 3
4
Injectd2 (Out Signal) Is activated at the moment when the
second sample has been injected
Off
Running (Out Signal) Active as long as the Job Queue is
being processed and the PAL is not in
an error state
Off
1) If used to synchronize with Sync cable,
change the setting “Immediat” to “TTL-In1”.
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Synchronization With Output Signals
A PAL Method may send any of the Output Signals at any time using the Atoms
SET_OUT_SIG or INJ_SAMPLE. See the PAL user manual for a complete list and
descriptions.
Standard Assignment for GC
PAL Injection Cycles
Description Default
Events
INTERFAC
E1 Pin #
Start JobQueue (Sync Signal) Start a Job Queue (Job).
Prerequisite is a defined “Job” with
a Method assigned. Useful for
automated, unattended timed
sampling.
Immediat
Start (Sync Signal) Start a Cycle (Input from GC or
data system) Immediat
1 7
8(GND)
Start2 (Sync Signal) Continue the Cycle “GC-Dual” for
the second sample
Immediat
(Ignore)
Inject (Sync Signal) Inject
READY to INJECT
Immediat
(Ignore)
Inject2 (Sync Signal) Inject the second sample for “GC-
Dual” Cycle
Immediat
(Ignore)
Injected (Out Signal) Is activated at the moment when
the sample has been injected
SW-Out1 3
4
Injectd2 (Out Signal) Is activated at the moment when
the second sample has been
injected
Off
Running (Out Signal) Active as long as the Job Queue is
being processed and the PAL is
not in an error state
Off
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Using Repeat Loops
REPEAT: Starts a section of
Atoms (stopping at the next
“END” Atom) which is repeated
a programmable number of
times.
Count: defines the quantity of
times the section is repeated.
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Editing Macros
ITSP Solutions recommends that you do not edit Macros with Microsoft Editor or any
other text editor. It would be too easy to corrupt the file. Cycle Composer has a very
good syntax checker and lexicon checker built into the Macro Editor.
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Training Exercises
Instrument preparation
• Equip an HTS PAL-xt with tray holders, vials, wash station, a 100µL
syringe, and the injection valve.
• Make sure all objects are installed correctly and taught.
• Load a 96-well plate on the PAL.
• Prepare 2 wells A1 and A2 with 300µL water (sample A and B).
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Exercise 1
Create and document a Cycle Composer Macro for the following actions:
1. Aspirate 100 µL sample A from well A1.
2. Dispense 100 µL sample A into well A3.
3. Repeat steps 1) to 2) two times.
4. Clean the syringe in a wash station.
5. Add 50µL of sample B from well A2 to well A3 and mix.
6. Inject a 25µL aliquot from well A3 into the LC injection valve.
7. Clean the syringe in a wash station.
8. Add a Method description of the purpose of the Macro.
9. Save the Macro.
Training Exercises
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Exercise 2
1. Create a Method with the Macro from exercise 1.
2. Document the Method.
3. Test the Method stepwise.
Training Exercises
31

Training Exercises
Exercise 3
Modify the Macro from exercise 1 to allow the operator to access the
following parameters from the Method level:
1. Volume for Injection.
2. Number of Syringe Washes.
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Training Exercises
Exercise 4
1. Modify the Macro from exercise 3 to take the sample tray, sample volume
and sample position from the Sample List.
2. Incorporate the changes into a Method.
3. Test the Method step-wise.
4. Incorporate the Method into a Sample List with 2 samples.
5. Run the Sample List.
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Training Exercises
Exercise 5
1. Make a version which waits for a start signal after mixing and before injection.
2. Incorporate the changes into a Method.
3. Test the Method step-wise.
4. Incorporate the Method into a Sample List with 2 samples.
5. Run the Sample List.
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ITSP Macros
• ITSP Solutions includes with ITSP startup kits a complete suite of examples to
demonstrate how to create ITSP Macros and Methods for your own Methods
and assays.
• ITSP Solutions also includes with ITSP startup kits a complete suite of examples
of Macros and Methods to demonstrate automated Method development of
ITSP SPE Methods.
• These “ITSP SPE MD…” examples demonstrate how to experimentally determine
the sample capacity of a selected ITSP SPE cartridge and how to experimentally
determine optimal organic strengths of reverse phase wash and elution
solvents.
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ITSP Macro and Method Examples
There are almost 100 Macro and Method example included with ITSP startup kits.
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Example of an ITSP Conditioning Macro
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Example of an ITSP Elution Macro
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Atom Sequence Example of "ITSP Wipe"
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1. First, retract the needle from the cartridge.
2. Second, move the PAL injection unit up and
back to wipe the cartridge off the needle.
ITSP cartridge transport is similar to what
happens at the end of a “magnetic vial
transport” action; but with the addition of
retracting the needle, as is seen with “needle
vial transport”.
This sequence is imperative for robust ITSP PAL
performance.
Be aware that the PAL will lose track of the
location of the PAL injection unit once a
“MOVE_REL” Atom is executed and will stay
lost until the PAL injection unit is moved to a
different PAL object using any Atom other than
“MOVE_REL”, e.g. “MOVE_TO_OBJECT”.

Training Exercises
Exercise 6
1. Create and document a Macro that solvates an ITSP cartridge in the ITSP
prep tray and then flushes the cartridge with air.
2. Incorporate the Macro into a Method and test stepwise.
3. Create and run a Sample List of 2 samples with the Method.
40

Training Exercises
Exercise 7
1. Create and document a Macro that elutes an ITSP cartridge to the elution
tray and then flushes the cartridge with air.
2. Incorporate the Macro into a Method and test stepwise.
3. Create and run a Sample List of 2 samples with the Method.
41

CE Cycle Editor
• The CTC Cycle Editor software is a
stand-alone application to generate
Cycles to be used in the data
system application using ICC –
Instrument Control Components.
• Cycles are the basic components of
Methods to control PAL
instruments from the data system.
• Each Cycle consists of a number of
single steps, called Atoms.
• Cycle Editor is very similar to Cycle
Composer Macro Editor.
• Atoms are characterized by parameters and their values, which are user-definable,
just as in Cycle Composer.
42

CE Menu Bar – Data Location
The Data Location is the instrument-specific folder that contains the relevant files of the
instrument including the Cycles. The shortcut key is Crtl+D.
43

CE Menu Bar – Object Configuration
In the Object Configurator users can add references, so-called User-Defined Object
Names (UDON) to objects used in Cycle Editor and Method Editor. Be aware that if you
create custom UDON’s, you may affect the portability of the Cycle between systems. The
3 default UDON’s: SL.Tray, SL.Index, and SL.Volume and are the only 3 recommended for
use by ITSP Solutions in ITSP MassHunter Cycles.
44

CE Graphical User Interface
• Description: lets the user enter a description for the current Cycle.
• Cycle Variables: numeric value or object class, similar to Cycle Composer Macro Editor.
45

CE Graphical User Interface
• Atom Sequence: The Atom Sequence lists all Atoms that have been selected to be
included in the current Cycle, similar to Cycle Composer Macro Editor.
• Selected Atom: The selected Atom can be added to the Atom Sequence to be
included in the current Cycle. Its parameters can be changed in the associated list.
46

CE File Formats
*pma: File format for Macros
that are used in Cycle Composer.
47
*.cyx: File format for Cycles
that are used in ICC.

Converting Macros Into Cycles
1. Open a *.pma file and
click the OK button.
2. Save as a Cycle to run in
MassHunter.
48
Cycle Editor can be used to convert Cycle Composer Macros into MassHunter ycles.

The first Atom of a Macro that will be converted to a MassHunter Cycle must be
LOCK_TERMINAL with Terminal Locking turned on.
49

The second Atom of a Macro that will be converted to a MassHunter Cycle must be
CLEANUP with the parameter settings exactly as shown below.
50

The next to last Atom of a Macro that will be converted to a MassHunter Cycle
must be CLEANUP with the parameter settings exactly as shown below.
51

The last Atom of a Macro that will be converted to a MassHunter Cycle must be
LOCK_TERMINAL with Terminal Locking turned off.
52

Important ITSP Concepts
• When solvating the ITSP Cartridge during SPE conditioning or washing, the
solvent volume aspirated by the syringe is not critical, so simple, quick PAL Atoms
and parameters can be used.
• ITSP sample load, elution, and injection volumes, however, are much more
critical and more care should be taken to deliver repeatable good recoveries. The
GET_SAMPLE and ASPIRATE_SYR Atoms have parameters which can be used to
improve performance, e.g.:
• Fill strokes
• Aspiration speed
• Pullup delay
• Consider syringe needle ID,
solvent and sample viscosity,
carryover, and LC run time
during Method development
when choosing Atoms and
parameter values.
53

Important ITSP Concepts
• The average ITSP SPE Macro/Cycle has between 80 and 120 Atoms in the Atom
sequence. Most Atoms have around 3 or 4 parameters. That means there are a total
of several hundred variables to consider when writing ITSP Macros which will be
converted into MassHunter Cycles, but the limit which can be exposed to the Method
level is 30; these 30 are in addition to the 3 which can be exposed to the Sample List
level (SL.Tray, SL.Volume, and SL.Index).
• Carefully consider which parameters should be exposed to the Method or Sample
List level both for Method development and later for production runs which will be
used by lab personnel.
• This will affect both your selection of which Atoms to use when more than one could
be used, as well as which parameters that you either will leave blank to be controlled
by the local terminal, or will set to a fixed constant value.
It is highly recommended that both the Method developer and the lab personnel running ITSP
Methods become familiar with the various user manuals and other reference documents provided
by CTC Analytics, your instrument vendor, and ITSP Solutions.
54

Certification Test
55
1. Create an ITSP Macro that will:
1) Condition the cartridge with an organic solvent to activate the sorbent functional group(s) and
clean any residual interference compounds from the manufacturing process which might affect
chromatography.
2) Condition the cartridge with an aqueous solvent to equilibrate the sorbent bed to the polarity
of the sample matrix.
3) Load the sample onto the cartridge.
4) Clean the syringe.
5) Wash loosely bound sample matrix interference compounds to waste with an aqueous solvent
and flush the cartridge with air.
6) Elute the cartridge with an organic solvent to an unused well and flush the cartridge with air to
evacuate dead volume in the cartridge.
7) Discard the spent cartridge.
8) Inject an aliquot of the eluate into the LC injection valve.
9) Clean the syringe.
10) Clean the valve.
2. Create a Cycle Composer Method to run the Macro and test it once stepwise.
3. Create a Cycle Composer Sample List to run the Method and test it on 2 samples.
4. Convert the ITSP Macro to a Cycle that can be used in a MassHunter Method.

Additional ITSP Resources
The USB memory stick included with ITSP startup
kits include:
• ITSP Application Notes
• ITSP Objects
• Sample ITSP Macros and Methods
• ITSP Automated Method Development Methods
• ITSP Setup Guide
• ITSP Method Development Guide
• Other resources such as catalogs, videos, and
presentations with more information about ITSP.
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Instructor Contact Information
Rick Youngblood
Director of Technology
ITSP Solutions, Inc.
706-395-8300
855-395-8300 toll free
Rick.Youngblood@ITSPsolutions.com
www.ITSPsolutions.com
http://www.linkedin.com/in/rickyoungblood/
Mailing Address Shipping Address
212 Northlake Drive 10 S. Carolina Street
Hartwell, GA 30643 Hartwell, GA 30643
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