CEM liberty Operation Manual

Operation Manual

600178
Rev. 5

Contents

Section 1: Introduction

Introduction 7

About This Manual 7
Common Abbreviations and Units 8

How to Obtain Support 10

Applications Support 10
Technical Support 10
Requested Information 10
Contact CEM 11

CEM Corporation Headquarters 11
CEM International Subsidiaries 11
CEM Distributors 12

Safety Information 13

Safety Notations 13
Safety Information 14

Fume Ventilation 14
Waste Disposal 14

System Requirements 14

Bench and/or Fume Hood Space 14
Environmental Conditions 15
Inert Gas Source 15
Electrical Requirements 15

Section 2: Operation of the Liberty

Introduction to the Liberty 16

Components and Parts 18
Reaction Vessel Components 19

Introduction to PepDriver 20

PepDriver Main Screen 21

Control Buttons 22
Menu Buttons 22
Indicators 22

Tabs 23

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Editors 25

Microwave Editor 25

Microwave Method Folders 25
Microwave Method Types 25
Creating a New Microwave Method 26
Editing an Existing Microwave Method 27

Cycle Editor 28

Cycle Folders 28
Copying and Modifying an Existing Microwave Method 28

Cycle Types 29
Creating a Cycle 29

Editing an Existing Cycle 30
Copying and Modifying an Existing Cycle 31

Sequence Editor 32

Creating a New Sequence 32
Modifying a Sequence 33
Using the External Amino Acid Positions 33
Importing a Sequence 34

Method Editor 36

Creating a New Method 36
Modifying an Existing Method 38

Calculators 39

Usage Calculator 39
Reagent Calculator 40

Resin Calculator 40
Deprotection Calculator 41
Activator Base Calculator 41
Activator Calculator 42
Amino Acid Calculator 42
Cleavage Calculator 43

Setting up a Synthesis 44

Conguring Methods to Run 44

Loading a Method 44
Loading Multiple Methods 45

Preparing Reagents 45

Standard Concentrations 45
Preparing Activator Solution 46
Preparing Activator Base Solution 46
Preparing Amino Acids 46
Preparing Deprotection Solution 47
Preparing Capping Solution 47
Preparing Cleavage Solution 48

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Setting up the Liberty 48

Loading Reagents onto the Liberty 48
Loading Resin onto the Liberty 50

Changing the Reaction Vessel 50

125 mL Reaction Vessel 50
10 mL Reaction Vessel 50

Running the Liberty 51

Preparing the Liberty to Run 51
Running the Liberty 52

Generating Reports 54

Method Reports 54

Creating a Method Report 54
Sample Method Report 55

Run History Reports 56

Creating a Run History Report 56
Sample Run History Report 56

Section 3: Quick Setup Guide

Peptide Setup 57

Create and Load the Method 57
Prepare Reagents 58
Ensure the Liberty is Ready to Run 59

Section 4: Mainteance of the Liberty

Routine Maintenance 61

Daily Maintenance 61
Weekly Maintenance 61
Biweekly Maintenance 61
Monthly Maintenance 61
Semiannual Maintenance 62
Standby Procedure 62

Maintenance Screen 63

Cleaning Tab 63

Using the Change Bottle Command 65
Performing a Backush 66

Performing a System Check 67

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Sample System Check File 69

Volume Calibration Tab 70

Performing a Volume Calibration 70
Importing and Exporting Volume Calibration Values 72
Performing a Large Scale Calibration 73

Sensor Calibration Tab 76

Performing a Sensor Calibration 76
Importing and Exporting Sensor Calibration Values 79

Pressure Calibration Tab 79

Performing a Pressure Calibration 80

Section 5: Advanced Features

Default Cycle Editor 82

Changing Default Cycles 82

Options Menu 83

Program Options Tab 83
Defaults Tab 85
Run History Tab 85

Test System Screen 86

Pressure Adjustment 87

Main Pressure Adjustment 87

Low Pressure Adjustment 88
Leak Check 88
Test Rotary Valves 89

Internal, Nitrogen, and Liquid Delivery Tests 90

Setting Up the Liberty for a Delivery Test 90

Performing a Delivery Test 91
Test Sensors 91
Reagent, Amino Acid, and External Amino Acid Addition 93
Leak Check Reaction Vessel 94
Flow Performance 94

Diagnostics Screen 95

Commands Tab 95
Sensors Tab 95

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Valves Tab 96
Status Tab 96
Delay Times Tab 97

Section 6: Troubleshooting

Tips for Recovering from Errors 99

Section 7: Spare Parts and Consumables

Reaction Vessel Parts 100
Filters 101
Solvent Keg 101
Bottles 102
Bulk Tubing and Fittings 103
Bottle Tubing Assemblies 104
Bottle Caps 105
Dip Tubes 106
O-Rings 106
Valves and Valve Wiring 107
Liquid Sensors 108
Waste Container 109
Accessories and Tools 109
Computer Accessories 110
Circuit Boards 111
Regulators 111
Documentation 111

Section 8: Index

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Liberty™ Automated Microwave Peptide Synthesizer

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Section 1: Introduction

Introduction

About This Manual

This manual (PN 600178) describes the operation and maintenance of the Liberty™ Automated
Microwave Peptide Synthesizer. The manual is intended for use by both novice and experienced
users.

This introductory section contains a list of common abbreviations and units used throughout the
manual, as well as important information for the safe operation of the unit. The manual assumes

that the Liberty was installed by a CEM certied service technician.

This manual refers to PepDriver™ version 2.5.4 Build 2 for all software information, including

screenshots and technical information. The latest version of PepDriver can be downloaded from
CEM’s website at http://www.cem-technet.com. A registered account is required for download.

Additional information is available in the Appendicies (PN 600183), which can be found on the

included CD-ROM (PN 900105).

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7

Common Abbreviations and Units

Abbreviation Definition

°C degrees Celsius
°F degrees Fahrenheit
AA amino acid
ACP acyl carrier protein 65-74
Amp amphere
Boc tert-butyloxycarbonyl
tBu tert-butyl
DCM dichloromethane
DIEA diisopropylethylamine
DMF N,N’-dimethylformamide

DODT 3,6-dioxa-1,8-octane dithiol

E

a

energy of activation

EDT ethanedithiol

Fmoc 9-uoroenylmethyloxycarbonyl

g gram
h height
HATU N-((dimethylamino)-1H-1,2,3-triazolo-4,5-bi-

pyridin-1-yl-methylene) N-methylmethan-

aminium hexauorophosphate N-oxide

HBTU 2-(1 H-benzotriazol-1-yl)-1,1,3,3-
tetramethyl-uronium hexauorophosphate

HCTU O-(6-chlorobenzotriazol-1-yl)-N,N,N’,N’-

tetramethyluronium hexauorophosphate

HF hydrouoric acid

HOAt 1-hydroxy-7-azabenzotriazole

HOBt 1-hydroxybenzotriazole

Hz Hertz
ID inner diameter
L liter
l length
m meter

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Abbreviation Denition

meq milliequivalent
mg milligram
MHz megahertz
mL milliliter
mm millimeter
mM millimolar
mmol millimole
MW molecular weight

NMP 1-methyl-2-pyrrolidone

OAt 7-azabenzotriazole

OBt benzotriazole

OD outer diameter

PN part number
psi pounds per square inch
PyAOP (7-Azabenzotriazol-1-yloxy)

tripyrrolidinophosphonium

hexauorophosphate

PyBOP (Benzotriazol-1-yloxy)

tripyrrolidinophosphonium

hexauorophosphate

RV reaction vessel
s seconds
TBTU O-(Benzotriazol-1-yl)-N,N,N′,N′-

tetramethyluronium tetrauoroborate

TFA triuoroacetic acid

TIS triisopropylsilane
Trt trityl
VAC volts of alternating current
W Watts
w width

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How to Obtain Support

Applications Support

For the latest Liberty applications information, go to http://www.cem.com/bioscience. The CEM
Bioscience website contains downloadable applications notes, a listing of recent microwave
peptide synthesis publications, and more.

CEM is proud to provide applications support for any peptide synthesis related questions from
a team of trained chemists with a complete peptide synthesis lab. For applications support, call

[phone] (inside the US) or [phone] and ask for “Peptide Applications”, or email

CEM Liberty applications support at [e-mail].

Technical Support

For the latest technical support information, go to http://www.cemservice.us. The CEM Service
website provides access to the CEM Knowledge Base, which contains helpful troubleshooting
information. From the website requests for phone or email support can also be submitted.

CEM is proud to provide technical support for the Liberty from a team of specially trained Service

Technicians. For technical support in the US and Canada, call [phone] or [phone]
and ask for “Liberty Service”. For technical support outside the US and Canada, contact your

local CEM Subsidiary or Distributor.

Requested Information

When contacting CEM for support, please provide the following information about the instrument:

Liberty Serial Number•
Liberty Firmware Version•
Discover Serial Number•
Discover Firmware Version•
PepDriver Version Number•
A recent System Check report (see p. 67)•
The Run History report for the synthesis that was running when the error •

occurred (see p. 56)

Serial numbers, rmware versions, and software version can be found within PepDriver by

clicking the Help Menu (not the Help Button) and selecting About PepDriver.

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Contact CEM

CEM Corporation Headquarters

Toll-Free Phone (US/Canada): [phone]
Phone: [phone]

Service Hotline: [phone]

Fax: [phone]

Mailing Address: PO Box 200

Matthews, NC 28106-0200

Physical Address: 3100 Smith Farm Rd

Matthews, NC 28104

Email (Applications Support): [e-mail]
Email (Technical Support): [e-mail]

CEM International Subsidiaries

France: CEM mWave S.A.S.

Phone: [phone]

Fax: [phone]

Address: Immeuble Ariane Domaine Technologique de Saclay
4, rue René Razel
91892 ORSAY Cedex
France

Web Address: http://www.cemfrance.fr
Email: [e-mail]

Germany: CEM GmbH

Phone: [phone]

Fax: [phone]

Address: Carl-Friedrich-GauB-Str. 9

47475 Kamp-Lintfort
Germany

Web Address: http://www.cem.de
Email: [e-mail]

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Italy: CEM SRL

Phone: [phone]

Fax: [phone]

Address: Via Dell Artigianato, 6/8
24055 Cologno Al Serio (BG)
Italy

Email: [e-mail]

UK and Ireland: CEM Microwave Technology Ltd.

Phone: [phone]

Fax: [phone]

Address: 2 Middle Slade

Buckingham Industrial Park MK18 1WA
Buckingham
Great Britain

Email: [e-mail]

CEM Distributors

For a complete list of distributors of CEM products, including contact information, go to the CEM
website (http://www.cem.com), select Contact Us, and then select Distributors.

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Safety Information

Safety Notations

This manual uses three safety alert words at points in the documentation where the user should
be aware of potential hazards. The safety alerts are shown in color-coded boxes. The three

words–NOTE, CAUTION, and WARNING–indicate differing levels of observation or action as

described below:

NOTE

A NOTE is intended to provide emphasis of procedures that may be

misinterpreted or overlooked, or to otherwise clarify confusing situations.

CAUTION

A CAUTION is intended to provide essential information and to emphasize

procedures which, if not strictly followed, may result in improper instrument
operation.

WARNING

A WARNING is intended to provide emphasize dangerous or hazardous
conditions which may result in personal injury to the user and damage or
destruction of the instrument.

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Safety Information

Fume Ventilation

The Liberty operates as a semi-closed system, with minimal venting of any hazardous solvent
fumes through the vent line coming from the Waste Reservoir. The vent line from the Waste
Reservoir must be vented into a proper chemical fume hood or exhaust line no longer than six

feet (6’)/two meters (2 m) from the instrument.

In addition, adequate ventilation should be provided for preparation of reagents and solvents for
use on the system. All solvent bottles and the Waste Reservoir should be placed into proper
secondary containers to minimize the risk of exposure.

Waste Disposal

Waste produced by the Liberty can be hazardous. For detailed information on the safety
requirements for the chemicals used on the Liberty, refer to the appropriate MSDS documents.

WARNING

Handle all waste under a fume hood, and wear suitable protective clothing.
Dispose of all waste in accordance with all applicable local, state, and
federal health and safety recommendations.

Product Removal

When removing tubes containing cleaved peptide in TFA from the Product Manifold following
cleavage, wear suitable protective clothing. These tubes contain a mixture of TFA, DCM, and
peptide. The centrifuge tubes should be capped immediately after removal from the Liberty to
reduce exposure to solvent fumes.

System Requirements

Bench and/or Fume Hood Space

The Liberty should be positioned on the bench such that access to the electrical outlets for the
system is not restricted. The Liberty requires the following space for system components:

Liberty Instrument (Discover Module and Liberty Module):

30” (w) x 24” (d) x 33.5” (h) [76 cm (w) x 61 cm (d) x 85 cm (h)]

(Depth includes 3” (7.62 cm) clearance behind instrument for unimpeded airow at rear fan ducts)

CEM Supplied Controller:
Laptop:

12.5” (w) x 9.9” (d) x 15.5” (h) [32 cm (w) x 25 cm (d) x 39 cm (h)]

(Height includes 14.1” (35.8 cm) display)

Desktop:

Minitower: 7.4” (w) x 17.5” (d) x 16.1” (h) [19 cm (w) x 44.5 cm (d) x 41 cm (h)]

Monitor: 16” (h) x 8” (d) x 20” (h) [41 cm (h) x 20 cm (w) 51 cm (h)]

External Reagent Bottles:

16” (w) x 16” (d) x 15” (h) [41 cm (w) x 41 cm (d) x 38 cm (h)]
(Left side of instrument, facing front of instrument)

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Waste Reservoir:

10” (w) x 16” (d) x 18” (h) [25 cm (w) x 41 cm (d) x 45.72 cm (h)]
(Must be vented into fume hood)

Approximate overall dimensions for Liberty and external reagents:

48” (w) x 24” (d) x 33.5” (h) [122 cm (w) x 61 cm (d) x 85 cm (h)]

Environmental Conditions

The Liberty is designed for indoor used only.
Temperature Range: 50 °F – 85 °F (10 °C – 29 °C)

Relative Humidity Range: 0 – 85%

Inert Gas Source

The Liberty requires an inert gas source (either high purity grade nitrogen or argon) capable of

supplying 25 psi (20 L/min ow) within ten feet (10’)/three meters (3 m) of the right side of the

instrument.

Electrical Requirements

The Liberty requires four (4) dedicated, grounded electrical connections operating at 120 VAC
(110-140 VAC, 60 Hz, 10 Amp @ 120 VAC) (or 240 VAC [202-250 VAC, 50 Hz, 10 Amp @ 240
VAC] where applicable). Specic power requirements (120 VAC vs. 240 VAC) can be found on
the nameplate afxed to the rear of the Liberty instrument.

One (1) electrical connection is required for each of the following components:

Liberty Module power cord
Discover Module power cord
Router power cord
Controller (laptop computer) power cord

NOTE

Five (5) electrical connections are required when using the desktop
computer controller option: Liberty module, Discover module, router,
controller minitower, controller monitor.

NOTE

Optionally, the router can be powered directly from the Liberty Module

without the need for an external electrical connection (using Router-Serial

Power Cable, PN 243290).

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15

Section 2: Operation of the

Liberty

Introduction to the Liberty

The Liberty Automated Microwave Peptide Synthesizer is the rst and only automated microwave

peptide synthesizer. Built on CEM’s exible Discover microwave platform, the Liberty is capable

of synthesizing up to twelve peptides in sequence on scales ranging from 0.05 to 5 mmol faster

and more efciently than conventional synthesizers thanks to the system’s patented circular

microwave cavity.

Liberty Module

Discover Module

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3-Port

Manifold

Amino Acid

Manifolds

External

Amino Acid

Manifold

Resin

Manifold

Product

Manifold

Waste Lines

Solvent

Lines

Deprotection
and Capping

Power

Switches

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Filters

17

Components and Parts

Liberty Module: • The Liberty handles all of the uid transfer into and out of

the reaction vessel. In addition, the Liberty manages communications with the
computer controller and the Discover during peptide synthesis.

• Discover Microwave Reactor: The Discover microwave reactor holds the

reaction vessel and generates the microwave energy used to irradiate the
sample. The Discover is capable of operating independently of the Liberty.

Power Switches: • There are two power switches, one for the Discover and one

for the Liberty. Both switches must be on to operate the Liberty.

Solvent:• There are two bottles of Main Wash (DMF) and one bottle of Secondary

Wash (DCM) on the system (not shown). These positions are designed for use
with standard 4 L solvent bottles. The bottle caps and lines are color coded: black
for DMF and yellow for DCM. These bottles are pressurized at all times and the
Change Bottle command must be used to remove these bottles.

3• -Port Manifold: This manifold has three positions designated for activator,
activator base, and cleavage cocktail. These positions are designed for use with

250 mL glass bottles with GL45 thread.

Deprotection and Capping:• This shelf is designed to hold the Deprotection and

Capping bottles. The bottle caps and lines are color coded: blue for Deprotect
and purple for Capping. These positions are designed for use with 1 L glass
bottles with GL45 thread. These bottles are pressurized at all times and the
Change Bottle command must be used to remove these bottles.

Amino Acid Manifolds:• There are 20 positions on the manifold for amino acids,

each corresponding to a specic amino acid. Each position is labeled using the

3 letter abbreviations of the amino acids. These positions are designed for use

with 125 mL plastic bottles.

Resin Manifold:• Resin is added without liquid in standard 50 mL centrifuge

tubes to the twelve positions manifold. The resin will return to the same position
on the resin manifold after synthesis if cleavage is not done on the system.

Product Manifold:• If the peptide is cleaved on the system, the cleavage product

will be returned to an empty standard 50 mL centrifuge tube on one of the twelve

positions on this manifold. The spent resin will be ushed to waste.

• Reaction Vessel: The reaction vessel (not shown, see p. 19) is where the

synthesis takes place. Reagents are added to the vessel and washed out
throughout the synthesis of the peptide. There are different size vessels that can
be used depending on the scale of the synthesis.

Fiber Optic • Temperature Probe: The ber optic probe is inserted in the top

of the reaction vessel. It allows the system to monitor the temperature of the
reaction vessel.

• Waste Lines: The waste lines carry all of the system’s waste out into an external

waste container. The waste container is equipped with a level sensor and will
trigger the system to pause if the container is full.

Filters: • There are three in-line lters on the system. These lters should be

changed regularly as part of routine maintenance.

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Optional Features•

•

External Amino Acid Manifold: This manifold allows for the addition of non-

standard amino acids and other reagents from ve positions using standard

50 mL centrifuge tubes.

DMF • Keg: An optional 20 L steel keg (not shown) is available for the DMF

position. This keg allows for synthesis of longer peptides where total solvent
usage would otherwise be a limitation.

• 10 mL Reaction Vessel: A smaller reaction vessel assembly (shown below)

is available to allow synthesis on scales as low as 0.025 mmol.

Reaction Vessel Components

Fiber Optic

Probe

Spray Head

Thermowell

Resin Tube

Glass Frit

Filter

Drain Line

Quick

Disconnect

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Introduction to PepDriver

The operation of the Liberty is controlled through the PepDriver software package. The Liberty
includes an external computer controller (either a laptop or desktop Windows PC) for running
PepDriver. This computer is connected to the Liberty and the Discover through an ethernet
connection.

PepDriver Terminology

Throughout this manual, specic terms will be used to describe the various functions within

PepDriver.

• Microwave Method: The specic parameters used in a Microwave step

within a cycle. Microwave Methods can be created or modied using the

Microwave Editor (p. 25).

• Cycle: The specic steps used for each residue within a given sequence.

Cycles can be created or modied using the Cycle Editor (p. 28).

• Sequence: The specic peptide to be synthesized in a given method.

Sequences can be created or modied using the Sequence Editor (p. 32).

• Method: The specic parameters used to synthesize a peptide. For each

method, a sequence is selected, and then parameters (individual coupling

cycles for each residue, C-terminus type, nal deprotection, etc.) are also

selected as part of the method. Methods can be created or modied using

the Method Editor (p. 36).

• Run: A specic instance of a method being loaded and started in PepDriver.

A Run History le (p. 56) is recorded for each run.

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PepDriver Main Screen

Control

Buttons

Start

Pause

Stop

Peptide

Synthesis

Status

Resin

Positions

Menu Buttons

Methods

Sequences

Cycles

Setup

Maintenance

Calculator

Estimated Time

Remaining

Tabs

Current

Method Detail

System Status

Line

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Control Buttons

Start: • This button starts the rst method in the queue, or resumes a stopped

method.

Pause: • This button pauses or unpauses the current method.
Stop: • This button stops the current method. PepDriver will nish the operation it

is currently performing before stopping the method.

Menu Buttons

Methods:• This button opens the Method Editor. From the Method Editor, specic

parameters for each synthesis can be programmed.

Sequence:• This button opens the Sequence Editor, where peptide sequences

are entered to be used in methods.

Cycles:• This button opens the Cycle Editor. From the Cycle Editor, cycles can

be created or edited for use in any part of a method.

• This menu contains the Microwave Editor, Default Cycle Editor, Pepdriver

Setup:

Options Menu, and Communication options. From this menu, User Accounts can

also be accessed.

• Maintenance: From this menu, the Maintenance and Diagnostics screens can

be accessed. The Maintenance Screen allows for cleaning, volume calibration,
sensor calibration, and pressure calibration. The Diagnostics Screen contains
information about the sensors, valves, and delay times for the system.

• Calculators: This menu contains the Reagent and Usage calculators. The

Usage Calculator will calculate the amount of each reagent solution needed
for the currently loaded methods. The Reagent Calculator can then be used
to calculate how to make each of the stock solutions for the loaded methods
(including activator, activator base, deprotection, cleavage, and amino acids).

Indicators

Resin Positions:• The twelve positions on the screen correspond to the twelve

positions on the resin manifold of the Liberty. The resin should be loaded in the
same position on the manifold as the method has been loaded into in PepDriver.

Peptide Synthesis Status:• This displays the peptide sequence being

synthesized and indicates the status of the synthesis.

Current Method Detail: • This window displays the currently selected method.

When a method is running, the current step is indicated in yellow.

• System Status Line: The left box indicates the specic command the Liberty

is currently executing. The current temperature and pressure readings are
displayed next to the command indicator. Two indicators shows the computer’s
communication status with the Liberty and the Discover. The waste full and spill

tray warnings are also seen here. On the right, there is an indicator that shows

whether the microwave is running.

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Tabs

• Methods: From this tab, methods are loaded into one of the twelve resin

positions to be run.

• Queue: This tab shows all currently loaded Methods. From this tab, the order

that each Method is run in can be changed.

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23

• Current Run: This tab displays the Method that is currently running.

• Run History: From this tab, detailed logs of each Method that has been run can

be accessed. In addition, Method Reports can be generated for each Method
that has been run.

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Editors

Microwave Editor

The Microwave Editor allows for the control and customization of the microwave steps of any

cycle. By editing the power and duration of a microwave step difcult peptides can be synthesized

with higher purity and yield.
The Microwave Editor can be accessed by clicking the Setup Button on the PepDriver main

screen and selecting Microwave Editor from the menu.

Microwave Method Folders

The Microwave Editor contains separate folders for each type of microwave method. Within each
method folder, there are subfolders for each scale, allowing for the development of optimized
microwave methods for each scale. A microwave method will only appear in the Cycle Editor
when creating a cycle of the same scale.

PepDriver comes with optimized default microwave methods for deprotection, coupling, capping,
and cleavage.

Microwave Method Types

• Standard: A Standard method applies a set microwave power until the set

temperature is reached, and then turns off the microwave until the temperature
drops to 5 °C below the set temperature. The total agitation time can also be
selected.

• Multi-Step: Multi-Step methods allow for a single microwave method to use

multiple power, temperature, or time settings.

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25

Creating a New Microwave Method

Microwave methods allow for detailed control of the microwave heating and reaction time for
each step in the synthesis. The Microwave Editor allows the user to select the microwave time,
microwave power, maximum temperature, and sample agitation.

Open the folder of the appropriate method type, then open the subfolder for the 1.
appropriate scale.

NOTE

A microwave method saved in a specic scale folder will only be available

for use in Cycle Editor for cycles created in the same scale folder.

Click the New Method button.2.
The new method will appear in the selected folder. Enter a name for the method 3.

and press Enter.
Select the type of microwave method to make.4.

4.1. Standard

Enter the desired 4.1.1. microwave power setting (in Watts), maximum
temperature (in °C), and time for the microwave step (in seconds).

If 4.1.2. agitation is desired during the microwave step, check the box for
Bubbling During Microwave. Enter the amount of time to bubble and
the time between each bubbling. (Bubbling is enabled by default.

The default setting is 3 seconds On Time, 7 seconds Off Time, low

pressure)

High Pressure bubbling is not recommended as this can deposit resin on the
top of the reaction vessel. This can lead to poor synthesis quality, product
loss, and contamination between syntheses.

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4.2. Multi-Step

Enter the microwave power (in Watts), time (in seconds), and 4.2.1.

maximum temperature (in °C) for the rst step in the method.

Add the next step to the microwave method by clicking the Add Step 4.2.2.
button, and then enter the power, temperature, and time settings for
the second step.

Repeat step 4.2.2. for each step in the method.4.2.3.

If 4.2.4. agitation is desired during the microwave step, check the box for
Bubbling During Microwave. Enter the amount of time to bubble and
the time between each bubbling. (Bubbling is enabled by default.

The default setting is 3 seconds On Time, 7 seconds Off Time, low

pressure.)

CAUTION

High Pressure bubbling is not recommended. Bubbling at high pressure
can deposit resin on the top of the reaction vessel. This can lead to poor
synthesis quality, product loss, and contamination between syntheses.

Save the method by clicking the Save button.5.

Editing an Existing Microwave Method

The Microwave Editor allows for settings on existing methods to be modied and saved as

needed. The most common reason to modify an existing method is to increase or decrease the
microwave power to optimize peptide synthesis. To modify an existing method:

Open the folder of the appropriate method type, then open the subfolder for the 1.
appropriate scale.

Click on the microwave method to be modied.2.

Make any changes as needed.3.
Click Save to save the changes to the method.4.

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27

Copying and Modifying an Existing Microwave Method

Often, it is easier to copy and modify an existing microwave method rather than creating an
entirely new method. Methods can easily be duplicated and modied within the Microwave

Editor, and then be moved to different scale folders as needed. To copy an existing method:

Open the folder of the appropriate method type, then open the subfolder for the 1.
appropriate scale.

Right click on a microwave method and select Copy Method.2.
The new method will appear named “Copy of (method name)”. Type a new name 3.

and press Enter.
Any settings in the method can be changed. If the copied method was a Multi-4.

Step method, steps can also be added or deleted by using the Add Step and
Delete Step buttons.

When all changes have been made, click Save to save the modied method.5.
If the new method is to be used at a different synthetic scale, drag the method 6.

from the folder it was created in to the appropriate folder.

Cycle Editor

The Cycle Editor allows for the full control and customization of any step of a synthesis. This
allows for optimization of each step of a given peptide synthesis.

The Cycle Editor can be accessed by clicking on the Cycles Button on the main screen of
PepDriver, or by clicking the Cycle Editor button from the Method Editor.

Cycle Folders

The Cycle Editor contains separate folders for each type of cycle. Within each cycle folder, there
are subfolders for each scale, allowing for the development of optimized cycles for each scale. A
cycle will only appear in the Method Editor when creating a method of the same scale.

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PepDriver comes with optimized default cycles for resin transfer, amino acid addition, nal

deprotection, and cleavage for each of the available synthetic scales.

Cycle Types

• Resin: Resin cycles control how resin is transferred from the resin position into

the reaction vessel at the beginning of a synthesis, as well as the resin swelling
step.

• Amino Acid: Amino acid cycles control how the deprotection and coupling steps

of one amino acid in the sequence occur.

• Cleaving: Cleaving cycles control if and how the peptide is removed from the

resin following synthesis, including the return of either cleaved peptide to the
product position or peptide on resin to the resin position.

• Final Deprotection: Final deprotection cycles control the removal of the

N-terminal protecting group, and also allow for N-terminal acetylation of the
peptide before cleavage.

Creating a Cycle

Cycles allow for detailed control of each action taken in a given step of a peptide synthesis run.
PepDriver comes with default cycles of each type for each synthetic scale; however, cycles can
be customized to accomplish a number of non-standard chemistries. To create a new cycle:

Click the New Cycle button at the top of the Cycle Editor window and select the 1.
type of cycle to be created. For Amino Acid, Cleaving, and Final Deprotection the
synthesis scale must be selected.

NOTE

A cycle saved in a specic scale folder will only be available for use in

Method Editor for methods created at that scale.

The cycle will appear in the appropriate folder. Enter a name for the cycle and 2.
press Enter.

Step 1 will appear in the cycle window. 3.

Click the 4. Operation box and select the operation to be performed. (For a full list

of available operations, see Appendix 3: Operations in the Cycle Editor.)

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29

The default parameter and volume for the chosen operation will appear in the 5.
appropriate boxes. Click on the Parameter box to select a different parameter.
Click on the volume to enter the desired volume.

If the Drain box is checked, the reaction vessel will be drained using a ltered 6.
drain after the operation is carried out. Uncheck the box to leave any liquid
reagents in the vessel.

Enter the number of times the operation is to be carried out in the Cycles box.7.
If the 8. Pause box is checked, the method will be paused after the operation is

carried out. To insert a pause, check the box.

NOTE

The method will not resume until the user presses the Start button on the
main screen of PepDriver.

Click the Add Step button to add a new step to the cycle.9.

Repeat steps 3 through 9 until the cycle is fully programmed. 10.
Click Save to save the cycle.11.

Editing an Existing Cycle

The Cycle Editor allows for settings on existing cycles to be modied and saved as needed. To

modify an existing cycle:

Open the folder for the appropriate cycle type, then open the subfolder for the 1.
appropriate scale if necessary.

Click on the cycle to be modied.2.

Make any changes as needed.3.
Click Save to save the changes to the cycle.4.

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Copying and Modifying an Existing Cycle

Often, it is easier to copy and modify an existing cycle rather that creating an entirely new cycle.
Cycles can easily be duplicated and modied within the Cycle Editor, and then they can be

moved to different scale folders as needed. To copy an existing cycle:

Open the folder of the appropriate method type, then open the subfolder for the 1.
appropriate scale if necessary.

Right click on a cycle and select Copy Cycle.2.
The new cycle will appear named “Copy of [cycle name]”. Type a new name and 3.

press Enter.
Any setting can be changed. Steps may be added or deleted as necessary using 4.

the Add Step and Delete Step buttons.
When all changes have been made, click Save to save the modied cycle.5.
If the new cycle is to be used at a different synthetic scale, drag the cycle from 6.

the folder it was created in to the appropriate folder.

NOTE

When copying a cycle to use with a different scale, verify that the volumes
used are appropriate for the new scale before using the cycle.

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Sequence Editor

Peptide sequences to be synthesized are entered in the Sequence Editor. The Sequence Editor

allows for the use of all twenty standard amino acids as well as the ve external amino acid

positions. In addition, the Sequence Editor will automatically calculate the total molecular weight
of the peptide sequence.

The Sequence Editor can be accessed by clicking on the Sequences button on the PepDriver
main screen, or by clicking the Sequence Editor button from the Method Editor.

Creating a New Sequence

Open the appropriate folder for the new sequence.1.
Click the New Sequence button.2.
The new sequence will be created in the selected folder. Enter a name for the 3.

sequence (the default is “New Sequence”) and press Enter.

The 4. amino acids for the sequence can be entered in two ways:

Click the amino acid buttons:4.1.

Click the white Sequence box at the bottom of the Sequence Editor.4.1.1.

Click the button corresponding to rst (N-terminal) amino acid. The 4.1.2.

one letter abbreviation will appear in the Sequence box.
Repeat step 4.1.2. for the remaining amino acids.4.1.3.

Type in the one letter abbreviations:4.2.

Click the white Sequence box at the bottom of the Sequence Editor.4.2.1.
Type the sequence (N-terminus to C-terminus) using the one letter 4.2.2.

abbreviations.

Click Save to save the sequence.5.

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Modifying a Sequence

If a sequence is entered incorrectly, there are a few ways to correct the error.
To insert an amino acid:

Right-click on the amino acid that will come after the inserted amino acid.1.
Select Insert Amino Acid.2.
A box will pop up stating that the next amino acid entered will be inserted before 3.

the current amino acid. Click OK to close the box.

Click on the desired amino acid button or type the one letter abbreviation of the 4.
desired amino acid.

To delete an amino acid:

Right-click on the amino acid.1.
Select Remove Amino Acid.2.

To move an amino acid within a sequence:

Click on the amino acid to be moved.1.
Click the Move Left (<-Move) or Move Right (Move->) buttons at the bottom of 2.

the Editor window to move the amino acid one position in the indicated direction.
Repeat as needed until the amino acid is in the correct position.

Using the External Amino Acid Positions

There are ve external positions amino acid positions, labeled in the Sequence Editor as EX1
through EX5. The external amino acids can be used in a sequence by either clicking the

corresponding button or by typing the number of the external position into the sequence.
By default, the molecular weight of the external amino acids will not be included in the sequence

molecular weight calculation. To enable an external amino acid weight:

Click the MW box next to the appropriate external amino acid button. This will 1.

open the Amino Acid Weight conguration window for that position.

Check the Enable Ext Amino Acid Weights box.2.
Enter the protected and unprotected molecular weights for the amino acid being 3.

used.
Click OK. The Amino Acid Weight conguration window will close, and the 4.

unprotected weight will appear in the MW box next to the button.

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NOTE

The protected weight refers to the molecular weight of the fully protected
amino acid, and is used in the Reagent Calculator to determine how much of
the amino acid is required for the run.

The unprotected weight refers to the residue weight (the molecular weight
of the fully deprotected amino acid minus 18 g/mol due to the loss of water
from the formation of the peptide bond) and is used in the Sequence Editor

to calculate the total molecular weight for the nal peptide.

For example, Fmoc-Ala-OH has a protected weight of 312 g/mol, and a

residue weight of 71 g/mol:

312 (protected weight) - 223 (Fmoc) - 18 (water) = 71 (unprotected weight)

Importing a Sequence

PepDriver includes an option for importing sequences from external documents such as Microsoft
Word documents, PDF documents, or webpages. This is useful for longer sequences because it
minimizes the possibility of incorrectly entering the sequence.

Create a Microsoft Excel spreadsheet containing the sequence or sequences to 1.
be imported.

In Excel, create a new document.1.1.
In cell A1, enter the words SequenceImport (without a space between the 1.2.

words).
Highlight the sequence in the source document (PDF, webpage, etc.) and 1.3.

press Ctrl+C (or right-click and select Copy).
Paste the sequence into the rst open cell in column A of the Excel 1.4.

spreadsheet.
Enter a name for the sequence in column B.1.5.
Repeat steps 1.3 through 1.5 for any other sequences to be imported.1.6.

Save the le and close Excel.1.7.

NOTE

If using Excel 2007 or later, the le must be saved as an Excel 97-2003
compatible le (.xls, not .xlsx). The le name should be in English.

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From PepDriver, open the Sequence Editor.2.
Click Import Sequence to open the Import window.3.

In the Import window, open the destination folder for the sequences.4.
Click Import. In the window that appears, select the Excel document created 5.

earlier and click Open.

The Import window will be updated to reect the successful import of the 6.

sequences. The imported sequences are now available for use in the Sequence
Editor and Method Editor.

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35

Method Editor

The Method Editor is used to program the specic conditions to be used for a given synthesis.
Once a sequence is selected, several options for the run can be congured that will determine the
specic steps to be used in the synthesis.

The Method Editor can be accessed by clicking the Methods button on the PepDriver main
screen, or by clicking the Method Editor button from the Sequence Editor.

Creating a New Method

Open the appropriate folder in the Methods box for the new method.1.
Click the New Method button.2.
Enter a name for the method and press Enter.3.
Open the appropriate folder in the Sequences box, and click on the desired 4.

sequence. The sequence will then be loaded into the sequence box at the bottom
of the Method Editor window.

Select the synthetic scale (0.05 mmol to 5.0 mmol) from the Resin Information 5.
box.

NOTE

The scale must be selected before changing any other parameters, as each

scale has specic default options that will be loaded upon selection.

The remaining options can now be selected.6.

Skip Resin Load: This will skip the initial loading of the resin into the 6.1.
reaction vessel. Select this option only if the resin has been placed directly
into the reaction vessel. This will also skip the resin swelling step.

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C-Terminus: This corresponds to the type of resin used. Selecting 6.2. Acid

assumes the resin is preloaded (the rst amino acid is already attached)

and therefore skips that coupling. If the resin is not preloaded, Amide
should be selected.

6.3. Final Deprotection: If No Final Deprotection is selected, the N-terminal
Fmoc group will not be removed from the peptide prior to cleavage.

Default Cycles:6.4.

Resin: The resin loading cycle selected depends on the reaction 6.4.1.
vessel required for the synthesis.

NOTE

The 125 mL vessel must be used for syntheses above 0.25 mmol, and the

Large Vessel - Resin method should be selected.
The 10 mL vessel assembly must be used for syntheses below 0.1 mmol,
and the 10 mL Vessel - Resin method should be selected.

Cleaving: To cleave the peptide on the system automatically, select 6.4.2.
the Cleavage cycle. The cleaved peptide will be transferred to the
Product position on the Liberty. Selecting Non-Cleavage will skip
the cleavage step and return the peptide on resin to the same Resin
position from which the resin was loaded.

Final Deprotection: By default, the N-terminal Fmoc group is 6.4.3.
removed and left as the free amine. To perform N-terminal
acetylation, select the Acetylation method from the Final
Deprotection pull-down.

NOTE

Acetylation requires capping solution to be prepared and loaded on the
Liberty in the Capping bottle.

Enter the substitution value in the Resin Substitution box. This value is provided 7.
by the supplier of the resin, and is usually expressed in mmol/g or meq/g

NOTE

The substitution value entered here will not affect the operation of the
Liberty. It is only needed for the Usage Calculator to calculate how much
resin is required for the synthesis.

8. Assign cycles for each residue in the sequence that will not use the default cycle.

NOTE

Each amino acid is assigned an optimized default coupling cycle. By default,
the Double Arg cycle is used for arginine, and the Single 50 C cycle is used
for cysteine and histidine. The Single coupling cycle is used for all other
amino acids. To restore all amino acids in a sequence to the default cycles,
click the Restore Defaults button.

Click on the amino acid to highlight it.8.1.

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37

Click on the drop-down box above the sequence and select the desired 8.2.
cycle. This will change the cycle for that highlighted amino acid only.

To change the cycle for all amino acids to the selected cycle, click Apply to 8.3.
All button on the left.

Amino acids assigned cycles other than the default will be highlighted in
yellow in the Method Editor.

When the method is complete, click Save to save the method.9.

Modifying an Existing Method

Open the Method Editor.1.

Open the folder containing the method in the Methods window.2.

Click on the method to be modied. The sequence will automatically be selected 3.

and loaded.
Make any changes to the method as needed.4.
Click Save to save the changes to the method.5.

NOTE

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Calculators

PepDriver contains two calculators that allow for easy determination of reagent needs and
reagent preparation: the Usage Calculator and the Reagent Calculator. These calculators are
accessed from the Calculator Button on the PepDriver main screen.

Usage Calculator

Once a method has been loaded to run, the Usage Calculator can be used to determine the

amount of each reagent needed. If multiple methods are loaded, the calculator can calculate the
amounts needed for all runs together, or each run individually. To use the Usage Calculator:

Load any methods to be run by dragging them to the appropriate resin positions 1.
(see p. 46).

Click on the Calculator button, and select Usage from the menu.2.

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39

The calculator will display the volume of each reagent required for all methods 3.
currently loaded. To exclude a loaded method from the calculation, uncheck the
box next to the method name in the Current Methods window.

To print a Usage Report, click the Print button at the bottom of the Usage 4.
Calculator. This will open the report as a PDF which can be printed or saved for
future reference.

Reagent Calculator

The Reagent Calculator can be used to determine how to prepare all reagents to be loaded
on the system at the correct concentrations. There are six tabs: Resin, Deprotectors, Bases,
Activators, Amino Acids, and Cleavage.

NOTE

The Reagent Calculator contains a library of common reagents. The Liberty
is not limited to only these reagents; however, the Reagent Calculator can
only perform calculations using reagents in the library.

Resin Calculator

Enter the scale for the synthesis.1.

Enter the resin substitution value in the Resin 2. Substitution box. This value is
provided by the supplier of the resin, and is usually expressed in mmol/g or

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meq/g.
Click Calculate. The required mass of resin will be reported in the Grams of 3.

Resin box (in grams).

Deprotection Calculator

Click on the desired deprotection reagent from the list.1.
Enter the desired concentration (in percent volume).2.

The default concentration is 20%. Piperazine should be prepared at a 5%
solution due to its solubility in DMF.

CAUTION

Enter the total volume of deprotection solution required.3.
Click Calculate. The calculator will report the required amount of deprotection 4.

reagent in milliliters (for liquids) and grams (for solids).

Activator Base Calculator

Click on the desired base from the list.1.
Enter the desired concentration (in M)2.

The default concentration is 2 M. DIEA should be prepared using NMP as
the solvent because of its miscibility in DMF.

CAUTION

Enter the total volume of activator base solution needed.3.

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41

Click Calculate. The calculator will report the required amount of base in 4.
milliliters.

Activator Calculator

Click on the desired activator from the list.1.
Enter the desired concentration (in M). The default is 0.5 M.2.
Enter the total volume of activator solution needed.3.
Click Calculate. The calculator will report the required amount of activator in 4.

grams.

Amino Acid Calculator

To calculate a specic amino acid, click on that amino acid in the list so that it is 1.
highlighted and then click the Selected box. To calculate all amino acids at the
same volume, click the All box.

Enter the total volume of amino acid solution.2.
Click Calculate. The calculator will report the required amount of amino acid(s) in 3.

grams.

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Cleavage Calculator

The Cleavage Calculator allows for the custom calculation of each scavenger and additive for
whatever cocktail will be best for a given peptide. To calculate how to make a cleavage cocktail:

Enter the total volume required for the method in the Total Volume box at the top 1.
of the calculator.

Click on the rst reagent to be used. Enter the desired nal percent of that 2.

reagent in the Percent box on the right.
The percent and volume of 3. TFA required will automatically be updated. Repeat

step 2 for the remaining scavengers.
The calculator will automatically update the amounts required for each reagent 4.

as changes are made. When all of the percentages are correct, the required
amount of each reagent to be added will be displayed in grams (for solids) and
mL (for liquids).

NOTE

CEM recommends using 95% TFA, 2.5% TIS, 2.5% DODT, and 2.5% water

as the cleavage cocktail.

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43

Setting up a Synthesis

Conguring Methods to Run

Loading a Method

Once a method has been created and saved in the Method Editor, it is available from the

Methods tab on the main PepDriver screen. Methods can be loaded to run in two ways.
To load by clicking:

Open the appropriate folder in the Methods tab.1.
Right-click on the desired method, select Place Method, and then select the 2.

number of the appropriate resin position.

The resin position in PepDriver will turn green, indicating the method is loaded 3.
and ready to run.

Methods can be loaded to any of the twelve resin positions. The order of
the resin positions does not affect the order in which the peptides will be
synthesized.

To load by dragging:

Open the appropriate folder in the Methods tab.1.
Click on the desired method, and drag the method to the appropriate resin 2.

position.

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The resin position in PepDriver will turn green, indicating the method is loaded 3.

Reagent

Standard

Concentration

0.05 mmol Scale
Concentration

and ready to run.

Loading Multiple Methods

The Liberty is capable of running up to twelve peptide sequentially. When loading multiple
methods, the methods will be run in the order they were loaded. The run priority will be indicated
by the black number in the center of the resin position in PepDriver. The run priority can also be
viewed from the Queue tab.

To change the run priority:

Right-click on a green resin position and select Change Run Priority.1.

Select the new priority.2.

To remove a method from the queue, right-click on the green resin position and select Clear
Method. To empty the queue, right-click on a green resin position and select Clear All Methods.

Preparing Reagents

Standard Concentrations

The Liberty uses stock solutions of all reagents. The default cycles are designed to deliver
enough of each stock solution to give 5 eq of amino acid and activator and 10 eq of activator
base for each coupling. The table below details the standard concentrations used on the Liberty.

Activator 0.5 M 0.25 M

Activator Base 2 M 1 M

Amino Acid 0.2 M 0.2 M

NOTE

When working on 0.05 mmol scale, due to the size of the sample loops used
for reagent delivery, activator and activator base should be made at half the
standard concentration. Amino acids do not need to be diluted.

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45

Amino acid, activator, activator base, and deprotection solutions can be
used for up to two weeks. Some amino acids, notably His, will begin to
crash out of solution after two weeks.

Preparing Activator Solution

Weigh out the appropriate amount of the desired activator (as calculated using 1.

the Usage and Reagent Calculators) and transfer to a 250 mL amber glass

bottle.

Some activators, especially the aminium activators such as HBTU, are light
sensitive and should be prepared in an amber glass bottle.

Add the appropriate volume of DMF.2.
Put a cap on the bottle. Gently swirl the bottle to ensure that all of the activator 3.

has gone into solution.

Some activators, notably HBTU, may take up to 10 minutes to fully go into
solution.

NOTE

NOTE

NOTE

Preparing Activator Base Solution

Measure out the appropriate amount of activator base and transfer to a clear 250 1.
mL glass bottle.

Add the appropriate volume of NMP to give the correct nal volume.2.

NOTE

CEM recommends using DIEA as the activator base. When using DIEA,
NMP must be used as the solvent because DIEA is not miscible in DMF at

the standard concentration of 2 M. The nal concentration of DIEA in the

reaction vessel is low enough that miscibility is not an issue.

Put a cap on the bottle. Gently swirl the bottle to ensure all of the activator base 3.
has gone into solution.

Preparing Amino Acids

CEM offers a full line of pre-weighed amino acids in 5 mmol, 10 mmol, and 20 mmol sizes. It is

not required to use the pre-weighed amino acids.
To use CEM pre-weighed amino acids:

Open the bottles, breaking the safety seals.1.

Add the appropriate amount of DMF to each bottle (25 mL for 5 mmol bottles, 50 2.
mL for 10 mmol bottles, and 100 mL for 20 mmol bottles).

Replace the caps and shake vigorously to ensure all amino acid has gone into 3.
solution.

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To use bulk amino acids:

Weigh out the appropriate amount of the amino acid as calculated using the 1.
Usage and Reagent Calculators.

Transfer the amino acid to a clean 125 mL plastic bottle.2.

NOTE

If the amino acid is to be used on one of the external positions, instead
transfer it to a 50 mL centrifuge tube.

Add the appropriate volume of DMF to the bottle.3.
Place a cap on the bottle and shake vigorously to ensure the amino acid has fully 4.

gone into solution.

Preparing Deprotection Solution

Measure out the appropriate amount of deprotection reagent as calculated using 1.
the Usage and Reagent Calculators.

Transfer the deprotection reagent to a 1 L clear glass bottle.2.
Add the appropriate volume of DMF to give the correct nal volume as calculated 3.

in the Reagent Calculator.
Add HOBt if necessary.4.

NOTE

For sequences susceptible to aspartimide formation, CEM recommends
the addition of 0.1 M HOBt to the deprotection solution as this signicantly

reduces levels of aspartimide. The appropriate mass of HOBt can be

calculated in the Reagent Calculator under the Activators tab. Enter the
total volume of deprotection and change the concentration to 0.1, then click
Calculate.

Place a cap on the bottle. Swirl the bottle gently to ensure the deprotection 5.

reagent (and HOBt, if used) has fully gone into solution.

Preparing Capping Solution

Capping is optional, and is not necessary for most routine syntheses. The capping bottle
does not need to be in place unless capping is being used in the method(s) being run. CEM
recommends using 0.5 M acetic anhydride in DMF with 0.125 M DIEA and 0.015 M HOBt. To
prepare capping solution:

Determine the total volume of capping solution needed using the Usage 1.
Calculator.

Calculate the amount of acetic anhydride needed. Measure out the appropriate 2.
amount and transfer to a 1 L glass bottle.

Use the reagent calculator to determine the volume of DIEA and mass of HOBt 3.
required. Measure out the appropriate amount of each and add to the acetic
anhydride.

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47

Add an appropriate amount of DMF to reach the correct nal volume.4.
Place a cap on the bottle. Swirl the bottle gently to ensure all reagents have fully 5.

gone into solution.

Preparing Cleavage Solution

Cleavage is optional. There does not need to be a bottle on the cleavage position unless
cleavage is being performed as part of the loaded methods.

Measure out the appropriate volume of TFA as calculated by the Reagent 1.

Calculator into a 250 mL clear glass bottle.

Add the appropriate volume of the remaining reagents as calculated by the 2.
Reagent Calculator.

Place a cap on the bottle and swirl gently to ensure all the reagents have fully 3.
gone into solution.

Setting up the Liberty

Loading Reagents onto the Liberty

NOTE

Reagent positions that are not used in a given run do not require dip tube

lters or bottles.

1. Load amino acids onto the Amino Acid Manifolds.
Ensure each dip tube to be used has a dip tube lter in place.1.1.
Remove the caps from each bottle of amino acid. Screw the bottle onto 1.2.

the designated position for that amino acid.

2. Load activator, activator base, and cleavage (if needed) onto the 3-Port Manifold.

Ensure each dip tube has a dip tube lter in place.2.1.

Remove the cap from each bottle. Screw the activator bottle into the top 2.2.
port on the manifold. Screw the activator base bottle into the center port
on the manifold. Screw the cleavage bottle (if needed) into the bottom port
on the manifold.

WARNING

TFA should only be connected to the Cleavage position. The use of TFA on
any other position will cause serious damage to the Liberty.

3. Load deprotection and capping (if needed) using the Change Bottle command.

(For more information about the Change Bottle command, see p. 65.)

Click on the Maintenance button in PepDriver and select Maintenance.3.1.
Select Deprotect from the pull-down box next to the Change Bottle button, 3.2.

then click Change Bottle.

48

Liberty™ Automated Microwave Peptide Synthesizer

600178 • Revision 5 • October 2009

The Change Bottle window will appear. Follow the onscreen instructions to 3.3.
depressurize and remove the existing bottle.

Ensure the dip tube has a dip tube lter in place, then screw the cap onto 3.4.
the deprotection bottle.

Click Next to continue with the Change Bottle procedure. The Liberty will 3.5.

automatically pressurize the bottle and ensure the lines are lled and ready

to add.
Repeat steps 3.1 through 3.5 for capping if needed.3.6.

Check the level of 4. DMF remaining in the DMF bottles. If more DMF is required,
use the Change Bottle command (p. 65) to replace the bottles.

NOTE

The two bottles of DMF should have approximately equal amounts of
solvent.

Click on the Maintenance button in PepDriver and select Maintenance.4.1.
Select Main Wash from the pull-down box next to the Change Bottle button, 4.2.

then click Change Bottle.
The Change Bottle window will appear. Follow the onscreen instructions to 4.3.

depressurize the existing bottles.
Slowly loosen one of the DMF bottle caps to release any residual pressure, 4.4.

then remove both caps.
Ensure each dip tube has a dip tube lter in place, then screw the caps 4.5.

onto the new solvent bottles.
Click Next to continue with the Change Bottle procedure. The Liberty will 4.6.

automatically pressurize the bottles and ensure the lines are lled and

ready to add.

Check the level of 5. DCM remaining in the DCM bottle. If more DCM is required,
use the Change Bottle command (p. 65) to replace the bottle.

Click on the Maintenance button in PepDriver and select Maintenance.5.1.
Select Secondary Wash from the pull-down box next to the Change Bottle 5.2.

button, then click Change Bottle.
The Change Bottle window will appear. Follow the onscreen instructions to 5.3.

depressurize and remove the existing bottle.
Ensure the dip tube has a dip tube lter in place. Ensure the o-ring inside 5.4.

the cap is secure, then screw the cap onto the DCM bottle.
Click Next to continue with the Change Bottle procedure. The Liberty will 5.5.

automatically pressurize the bottle and ensure the lines are lled and ready

to add.

Liberty™ Automated Microwave Peptide Synthesizer

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49

Loading Resin onto the Liberty

Use the Reagent Calculator to determine the amount of resin required for the 1.
synthesis.

Weigh the appropriate amount of dry resin into a clean 50 mL centrifuge tube.2.

NOTE

Some resins will cling to the sides of the tube. If necessary, wash the resin
from the sides of the tube using a small amount (less that 5 mL) of DCM.

Connect the resin tube onto desired position on the Resin Manifold.3.
If cleavage is being performed, connect a clean, empty 50 mL centrifuge tube 4.

onto the corresponding position of the Product Manifold.

Changing the Reaction Vessel

When working on large or small scales, the reaction vessel will need to be swapped out for one of

an appropriate volume. The large (125 mL) vessel is necessary for reactions of 0.5 mmol scale

or higher. The small (10 mL) vessel is necessary for reactions of 0.05 mmol scale or lower.

125 mL Reaction Vessel

Remove the 35 mL (Standard) reaction vessel from the microwave cavity.1.

Disconnect the ltered drain line from the vessel body.2.

Disconnect the vessel body from the attenuator.3.

Connect the 125 mL vessel body to the attenuator.4.
Connect the ltered drain line to the 125 mL vessel body, being careful not to 5.

crossthread or overtighten the tting.

Place the vessel back into the microwave cavity, securing the attenuator.6.
Perform a Reaction Vessel Leak Check (see p. 94).7.

10 mL Reaction Vessel

Remove the 35 mL (Standard) reaction vessel from the microwave cavity.1.
Carefully remove the temperature probe from the thermowell.2.
Disconnect the four lines from the manifold on the front of the Liberty.3.
Place the 10 mL vessel into the microwave cavity, securing the attenuator.4.
Connect the four line from the 10 mL vessel to the manifold on the front of the 5.

Liberty, ensuring each line is connected to the correct position.
Insert the temperature probe into the thermowell, ensuring the probe is inserted 6.

to the bottom of the thermowell.
Perform a Reaction Vessel Leak Check (see p. 94).7.

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Liberty™ Automated Microwave Peptide Synthesizer

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Running the Liberty

Preparing the Liberty to Run

Verify the level of the waste container to ensure there is sufcient room before 1.
starting another synthesis.

Verify the nitrogen supply to ensure there is sufcient nitrogen to complete the 2.

synthesis.
Verify that all amino acids required for loaded methods are loaded in the 3.

appropriate positions.
Verify that the resins are loaded into the positions corresponding to the correct 4.

methods in PepDriver, and that tubes are connected to the corresponding
product positions if cleaving.

Verify the levels of both wash solvents (DMF and DCM) to ensure there is 5.

sufcient solvent.

Verify that all sequences and methods are correct and that they are loaded in the 6.
correct positions.

Verify that the appropriate reaction vessel for the scale of the loaded methods is 7.

connected, and that the ber optic probe is fully inserted into the thermowell.

WARNING

If the probe is not inserted all the way to the bottom of the vessel the Liberty
will not accurately measure the temperature, and signicant overheating of
the vessel will occur. This will result in poor synthesis quality and/or serious
damage to the vessel.

Verify that the method is correct by looking at the steps outlined in the Method 8.
box. If there are any errors, make corrections and reload the method.

Right-click on the green resin position where the method was loaded and 8.1.
select Clear Method.

To correct the method:8.2.

Open the Method Editor. In the Methods box on the left, open the 8.2.1.

folder where the method was saved. Click on the method to load it.
Make any corrections as needed (see p. 38). 8.2.2.
Click Save the save the method, then close the Method Editor.8.2.3.
Load the corrected method as described above.8.2.4.

To correct the sequence:8.3.

Open the Sequence Editor. In the Sequences box on the left, open 8.3.1.
the folder where the sequence was saved. Click on the sequence to
load it.

Make any corrections as needed (see p. 33).8.3.2.

Liberty™ Automated Microwave Peptide Synthesizer

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51

Click Save to save the sequence, then close the Sequence Editor.8.3.3.
Open the Method Editor and correct the method as described above.8.3.4.

Running the Liberty

Click the Start button at the top of the PepDriver main screen.1.

If the Method is for 0.5 mmol scale or larger, a warning to verify that the 125

mL reaction vessel is connected will pop up. Verify the 125 mL vessel is
connected and then click OK to continue.

The Liberty will go through a series of initialization steps to ensure all valves are 2.

in the correct positions and that there is sufcient nitrogen pressure.

The rst method in the queue (indicated by a 1 inside the resin position on the 3.

main screen) will be loaded into the Current Run tab. The outline of the resin
position will turn yellow to indicate the method is running.

The Liberty will run a4. leak check on the reaction vessel to ensure the vessel is
properly connected. This option may be disabled from the Setup menu under

Options.

NOTE

The Liberty will perform a 5. sensor test to ensure all sensors are operational. This

option may be disabled from the Setup menu under Options.

Once all initialization and testing is complete, the Liberty will begin the Resin 6.
Loading step of the rst method.

During this step, solvent will be added to the resin tube and the resin will be
agitated with nitrogen bubbling to suspend it in solution before transferring to the
reaction vessel. This operation will be performed three times to ensure all resin

52

Liberty™ Automated Microwave Peptide Synthesizer

600178 • Revision 5 • October 2009

is transferred to the reaction vessel.
The status of the peptide is displayed above the resin positions. In this example,

the resin was preloaded with Fmoc-Gly-OH, so the C-terminal Gly is already

shown on the resin bead with the Fmoc attached. The estimated time remaining
is indicated in the top right. The current step of the method is highlighted in
yellow in the Current Method Display.

The Liberty will enter a wait state for 15 minutes following resin transfer to allow 7.
the resin to swell. The time remaining (in seconds) will be displayed on the
System Status Line.

After the swelling step, the vessel will be drained. The resin will be rinsed 8.

several times, and then the Liberty will begin the deprotection step for the rst

coupling.

Once all couplings are completed, the Liberty will perform the nal deprotection if 9.

one was selected in the Method Editor.
Following nal deprotection (if selected), the Liberty will begin the cleavage cycle.10.

For methods with 10.1. cleavage:

The resin will be rinsed with DCM, and then cleavage cocktail will be 10.1.1.
added from the Cleavage position.

Following the cleavage step, the cleavage solution containing the 10.1.2.
free peptide will be transferred to the corresponding centrifuge tube
on the Product Manifold.

The Liberty will wash the reaction vessel, sending the spent resin out 10.1.3.
to waste.

The Liberty will perform a neutralization procedure on the reaction 10.1.4.
vessel to ensure no residual TFA will interfere with subsequent
syntheses.

The Liberty will perform a reaction vessel cleaning cycle.10.1.5.

For non-cleavage methods:10.2.

Solvent will be added to the reaction vessel followed by bubbling, 10.2.1.
and the resin will be transferred back to the resin tube from which
it was loaded. This will be repeated once to ensure all resin is
returned.

The Liberty will perform a reaction vessel cleaning cycle.10.2.2.

The resin position on the PepDriver main screen will turn blue, indicating the 11.
method was successfully completed.

If multiple methods are loaded, the Liberty will load the next method in the queue 12.
into the Current Run tab, and begin the resin loading cycle for that method.

Liberty™ Automated Microwave Peptide Synthesizer

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53

Generating Reports

Method Reports

Method reports record all settings selected in the Method Editor when creating the method.

Method reports are created as PDF les, which can be saved and printed to allow for easy

recording of experimental parameters.

A method report consists of two sections. The rst section contains information about the

synthesis parameters: the sequence with calculated molecular weight, the selected C-terminus
(acid or amide), the resin parameters (loading, scale), and a list of the cycles selected for each
residue in the sequence. The second section shows every cycle used in the method, including

the nal deprotection and cleavage cycles. This section includes a full outline of each cycle,

providing a record of exactly what was performed at each step.

Creating a Method Report

From the Method Editor:

Open the Method Editor.1.

Open the folder containing the method in the Methods window.2.

Click on the method to be reported.3.
Click the Print Method button. The Report Viewer window will open, and the 4.

method report will be rendered as a PDF.

The Report Viewer window does not allow scrolling between pages. To
view the next page in the report before saving, click the Next Page button,
located next to the page number box.

Click Save As PDF to save the report.5.

From the Run History Tab:

Open the folder for the month in which the method was run.1.

Right-click on the specic run to be reported and select Display Method Report.2.

The Report Viewer window will open, and the method report will be rendered as 3.
a PDF.

NOTE

Click Save As PDF to save the report.4.

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Liberty™ Automated Microwave Peptide Synthesizer

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Sample Method Report

None

Method Comments:

0.10-Cleavage

Cleaving:

4/23/2005 4:41:29 PM

Modified Date:

Yes

Resin Load:

0.10

Final Deprotection:

Acid

C - Terminus:

0.6

Substitution:

0.10

Scale:

Sequence:

V Q A A I D Y I N G

1063.17Total MW:

ACP

Method

Resin

Cycles

Resin:

Standard - Resin

Amino Acid:

Specific Deriative:

Cycle:

Amino Acids

Gly - G

Standard - Resin

Fmoc-Gly-OH

Asn - N

0.10-Single

Fmoc-Asn(Trt)-OH

Ile - I

0.10-Single

Fmoc-Ile-OH

Tyr - Y

0.10-Single

Fmoc-Tyr(tBu)-OH

Asp - D

0.10-Single

Fmoc-Asp(OtBu)-OH

Ile - I

0.10-Single

Fmoc-Ile-OH

Ala - A

0.10-Single

Fmoc-Ala-OH • H

2

O

Ala - A

0.10-Single

Fmoc-Ala-OH • H

2

O

Gln - Q

0.10-Single

Fmoc-Gln(Trt)-OH

Val - V

0.10-Single

Fmoc-Val-OH

Resin Cycle - Standard - Resin

No

Operation

Parameter

Volume(mL) Drain Cycles Pause

1

Clean Reaction Vessel - Unfiltered

10.0

Yes1No2Add DMF to Resin

7.0No1

No

3

Add DCM to Resin

7.0No1No4

Transfer Resin to Reaction Vessel

10.0

Yes1No

5

Add DMF to Resin

7.0No1

No

6

Add DCM to Resin

7.0No1No7

Transfer Resin to Reaction Vessel

10.0

Yes1No

8

Add DMF to Resin

7.0No1

No

9

Add DCM to Resin

7.0No1

No

10

Transfer Resin to Reaction Vessel

10.0

Yes1No

11

Add Reagent

DMF

5.0No1No12

Add Reagent

DCM

5.0No1

No

13

Wait State

900 secsNo1No14

Drain - Filtered

Yes1No

Amino Acid Cycle - 0.10-Single

No

Operation

Parameter

Volume(mL) Drain Cycles Pause

1

Clean Resin Dip Tube

Yes1No

2

Wash - Top

DMF

7.0

Yes1No

3

Add Deprotection

7.0No1

No

4

Microwave Method

Initial Deprotection

Yes1No

5

Wash - Top

DMF

5.0

Yes1No6Add Deprotection

7.0No1

No

7

Microwave Method

Deprotection

Yes1No8Wash - Top

DMF

7.0

Yes1No9Wash - Bottom

DMF

7.0

Yes1No10Wash - Top

DMF

7.0

Yes1No

11

Clean Resin Dip Tube

Yes1No

12

Wash - Top

DMF

7.0

Yes1No13Add Amino Acid

2.5No1No14

Add Activator

1.0No1No15

Add Activator Base

0.5No1

No

16

Microwave Method

Coupling

Yes1No17Wash - Top

DMF

7.0

Yes1No

18

Wash - Bottom

DMF

7.0

Yes1No

19

Wash - Top

DMF

7.0

Yes1No

Final Deprotect Cycle - 0.10

No

Operation

Parameter

Volume(mL) Drain Cycles Pause

1

Clean Resin Dip Tube

Yes1No

2

Wash - Top

DMF

7.0

Yes1No

3

Add Deprotection

7.0No1

No

4

Microwave Method

Initial Deprotection

Yes1No

5

Clean Resin Dip Tube

Yes1No6Add Deprotection

7.0No1

No

7

Microwave Method

Deprotection

Yes1No8Wash - Top

DMF

7.0

Yes1No9Wash - Bottom

DMF

7.0

Yes1No

10

Wash - Top

DMF

7.0

Yes1No

Cleavage Cycle - 0.10-Cleavage

No

Operation

Parameter

Volume(mL) Drain Cycles Pause

1

Wash - Top

DCM

7.0

Yes4No

2

Wash - Bottom

DCM

7.0

Yes1No

3

Clean Resin Dip Tube

Yes1No4Wash - Top

DCM

7.0

Yes1No

5

Add Cleavage (TFA) - Sample Loop

8.0No1No6

Microwave Method

Cleaving 35minNo1

No

7

Transfer Product Cleaved

No1No8Add Reagent

DCM

5.0No1No9

Transfer Product Cleaved

No1No

10

Add Reagent

DCM

5.0No1No11

Transfer Product Cleaved

No1No

12

Clean Reaction Vessel - Unfiltered

10.0No3

No

13

Reaction Vessel Neutralization

10.0No3No14

Wash - Top

DCM

7.0

Yes2No

Liberty™ Automated Microwave Peptide Synthesizer

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55

Run History Reports

Run History

Action

Time

ACP

7/17/2009 9:35:06 AM

Pressurize - ALL

7/17/2009 9:35:09 AM

Pressurize DMF

7/17/2009 9:35:09 AM

Pressurize DCM

7/17/2009 9:35:12 AM

Pressurize Deprotect

7/17/2009 9:35:16 AM

End Pressurize

7/17/2009 9:35:21 AM

Starting Method

7/17/2009 9:35:21 AM

Initialization and Resin Loading

7/17/2009 9:35:21 AM

Clean Reaction Vessel - Unfiltered

7/17/2009 9:35:52 AM

DMF_Bottom (7.0/6.1)

7/17/2009 9:36:20 AM

Add DMF to Resin

7/17/2009 9:37:58 AM

Depressurize N2

7/17/2009 9:38:40 AM

Purge All to Resin

7/17/2009 9:38:46 AM

Add DCM to Resin

7/17/2009 9:39:35 AM

Depressurize N2

7/17/2009 9:40:12 AM

Purge All to Resin

7/17/2009 9:40:19 AM

Transfer Resin to Reaction Vessel

7/17/2009 9:41:07 AM

Drain Filtered

7/17/2009 9:41:23 AM

Add DMF to Resin

7/17/2009 9:42:03 AM

Depressurize N2

7/17/2009 9:42:45 AM

Purge All to Resin

7/17/2009 9:42:51 AM

Add DCM to Resin

7/17/2009 9:43:40 AM

Depressurize N2

7/17/2009 9:44:16 AM

Purge All to Resin

7/17/2009 9:44:23 AM

Transfer Resin to Reaction Vessel

7/17/2009 9:45:11 AM

Drain Filtered

7/17/2009 9:45:27 AM

Add DMF to Resin

7/17/2009 9:46:06 AM

Depressurize N2

7/17/2009 9:46:48 AM

Purge All to Resin

7/17/2009 9:46:54 AM

Add DCM to Resin

7/17/2009 9:47:43 AM

Depressurize N2

7/17/2009 9:48:20 AM

Purge All to Resin

7/17/2009 9:48:26 AM

Transfer Resin to Reaction Vessel

7/17/2009 9:49:15 AM

Drain Filtered

7/17/2009 9:49:31 AM

Add Reagent: DMF

7/17/2009 9:50:14 AM

DMF (5.4/5.9)

7/17/2009 9:50:34 AM

Add Reagent: DCM

7/17/2009 9:50:38 AM

DCM (4.1/4.7)

7/17/2009 9:51:05 AM

Drain Filtered

7/17/2009 10:06:22 AM

Amino Acid: Gly

7/17/2009 10:07:00 AM

Amino Acid: Asn

7/17/2009 10:07:00 AM

Clean Resin Dip Tube: DMF

7/17/2009 10:07:01 AM

Wash - Cycle 1 of 1

7/17/2009 10:08:05 AM

DMF (5.8/5.9)

7/17/2009 10:08:30 AM

Add Reagent: Deprotect

7/17/2009 10:09:11 AM

Deprotect (5.0/0.0)

7/17/2009 10:09:43 AM

Microwave: Deprotection - Power: 35 Watts

7/17/2009 10:10:30 AM

Final Max Temp:31.4 C at end of test

7/17/2009 10:11:06 AM

Total Microwave Time: 0:36

7/17/2009 10:11:09 AM

Drain Filtered

7/17/2009 10:11:09 AM

Wash - Cycle 1 of 1

7/17/2009 10:11:45 AM

DMF (5.5/5.7)

7/17/2009 10:12:06 AM

Add Reagent: Deprotect

7/17/2009 10:12:46 AM

Deprotect (4.9/0.0)

7/17/2009 10:13:19 AM

Microwave: Deprotection - Power: 35 Watts

7/17/2009 10:14:07 AM

ACP

7/17/2009 9:35:03 AM

Started:

Run History Reports record each command executed by PepDriver during a run with a date/time
stamp. In addition, a Run History Report records any system errors that occur, allowing for easy

diagnosis and troubleshooting of failed syntheses.

Creating a Run History Report

From the Run History tab:

Open the folder for the month in which the method was run.1.

Right-click on the specic run to be reported and select Display Run History 2.

Detail.

The Report Viewer window will open, and the method report will be rendered as 3.
a PDF.

Click Save As PDF to save the report.4.

Sample Run History Report

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Liberty™ Automated Microwave Peptide Synthesizer

600178 • Revision 5 • October 2009

Section 3: Quick Setup Guide

Liberty Setup

Create and Load the Method

1. Create a new sequence.
Open 1.1. Sequence Editor by clicking on the Sequence button at the top.
Click on the folder where the sequence should be saved, then click the 1.2.

New Sequence button.
Enter a name for the sequence and press Enter.1.3.
Enter the sequence either by clicking on the amino acid buttons or by 1.4.

typing the one letter abbreviations for the amino acids into the Sequence
box.

NOTE

The amino acids in the sequence should be entered from N-terminus to
C-terminus.

Click Save, then close the Sequence Editor.1.5.

2. Create a new method.
Open the 2.1. Method Editor by clicking on the Methods button.

Click on the folder where the method should be saved, then click New 2.2.
Method.

Enter a name for the method and press Enter.2.3.
Choose the desired sequence from the appropriate folder in the 2.4.

Sequences window. The selected sequence will be loaded into the
Sequence box.

Select all method parameters:2.5.

Resin Information: 2.5.1.

2.5.1.1. Scale: Select the scale for the synthesis of the peptide (0.05
mmol to 5.0 mmol).

CAUTION

The 125 mL reaction vessel must be used for syntheses above 0.25 mmol.
The 10 mL reaction vessel should be used for syntheses below 0.1 mmol.

Liberty™ Automated Microwave Peptide Synthesizer

600178 • Revision 5 • October 2009

57

Resin 2.5.1.2. Substitution: The resin substitution can be found on the
bottle of resin and it will be labeled either meq/g or mmol/g.

NOTE

The substitution value entered will not affect the operation of the Liberty.
It is only needed for the Usage Calculator to calculate how much resin is
required for the synthesis.

Skip Resin Load: Check this box only if the resin is already loaded 2.5.2.
into the reaction vessel.

2.5.3. C-terminus: If using preloaded resin (the resin already has the rst
amino acid attached), select Acid. Otherwise, select Amide.

Final Deprotection: Select Yes to remove the nal Fmoc from the 2.5.4.

N-terminus of the peptide.
Default cycles: 2.5.5.

Resin: After the scale is selected, the default Resin Transfer 2.5.5.1.
cycle will be loaded for the appropriate reaction vessel.
Ensure the correct vessel is connected to the Liberty.

Final Deprotection: Select the desired nal deprotection step 2.5.5.2.

(Final Deprotection or Acetylation).
Cleavage: To cleave the peptide on the system automatically, 2.5.5.3.

select the Cleavage cycle for the appropriate scale. Otherwise

select Non-cleavage.

Click Save to save the method. Close the Method Editor2.6.

From the main screen of PepDriver,3. load the method by clicking on the
appropriate method in the Method Tab and dragging it to one of the resin
positions at the top of the screen. Each position corresponds to one of the twelve
resin positions on the front of the Liberty. The resin position should turn green.

Prepare Reagents

To determine the amount of each reagent needed and how to prepare them, use 4.
the Usage and Reagent Calculators

Click on the Calculator button and select 4.6.1. Usage from the menu. The
amount of each reagent necessary for the synthesis is listed. Click
Print at the bottom of the calculator to generate a PDF that can be
printed or saved.

Click on the Calculator button and select 4.6.2. Reagent from the menu.

Resin: Enter the scale of the synthesis and the resin 4.6.2.1.
substitution to determine the mass of resin needed.

Other Reagents: For the other reagents, click on the 4.6.2.2.

appropriate tab (Activator, Base, Deprotection, Cleavage)
and then select the appropriate reagent from the list. Enter
the concentration and volume needed on the right and click

58

Liberty™ Automated Microwave Peptide Synthesizer

600178 • Revision 5 • October 2009

Calculate.

Load all reagents onto the Liberty.5.

Dissolve each 5.1. amino acid in the correct volume of solvent in a 125 mL
bottle. Connect each bottle onto the corresponding position for that amino
acid on the manifold.

Dissolve 5.2. activator in the correct volume of solvent in a 250 mL amber glass
bottle. Connect the bottle to the Activator position on the 3-Port Manifold.

Dissolve 5.3. activator base in the correct volume of solvent in a 250 mL clear
glass bottle. Connect the bottle to the Activator Base position on the 3-Port
Manifold.

If 5.4. cleaving the peptide on the system, prepare the cleavage cocktail

solution in a 250 mL clear glass bottle. Connect the bottle to the Cleavage

position on the 3-Port Manifold.
Add the correct volume of 5.5. deprotection reagent to a 1 L bottle, then add

the correct amount of DMF. Use the Change Bottle from the Maintenance
menu to replace the bottle.

Click on the Maintenance button at the top of the screen and select 5.5.1.
Maintenance in the drop down box.

On the right hand side of the Maintenance window, under Cleaning, 5.5.2.
choose the reagent in the rst box to be removed and then click

Change Bottle. Follow the onscreen instructions to replace the
bottles.

Check the 5.6. DMF and DCM bottles to ensure there is enough solvent to
complete the synthesis. If one or both solvents need to be replaced, use
the Change Bottle function from the Maintenance menu.

Click on the Maintenance button at the top of the screen and select 5.6.1.
Maintenance in the drop down box.

On the right hand side of the Maintenance window, under Cleaning, 5.6.2.
choose the reagent in the rst box to be removed and then click

Change Bottle. Follow the onscreen instructions to replace the
bottles.

Ensure the Liberty is Ready to Run

Verify that the waste container has sufcient capacity.6.
Verify that the ber optic probe is fully inserted into the thermowell.7.

WARNING

If the probe is not inserted all the way to the bottom of the vessel the Liberty

will not accurately measure the temperature, and signicant overheating of

the vessel will occur. This will result in poor synthesis quality and/or serious
damage to the vessel.

Verify that the method is correct by looking at the steps outlined in the Method 8.

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59

box. If there are any errors, make corrections and reload the method.

Right-click on the green resin position where the method was loaded and 8.1.
select Clear Method.

Open the Method Editor. In the Methods box on the left, open the folder 8.2.

where the method was saved. Click on the method to load it.
Make any corrections as needed. Click Save the save the method, then 8.3.

close the Method Editor.
Load the corrected method as described in step 3.8.4.

If the method is correct and all reagents are attached to the Liberty, press the 9.
Start button at the top of the screen to begin the synthesis.

NOTE

If the Method is for 0.5 mmol scale or larger, a warning to verify that the 125

mL reaction vessel is connected. Click OK to continue.

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Section 4: Mainteance of the

Liberty

Routine Maintenance

A routine maintenance protocol is vital for the long-term operation of the Liberty. Further details
about the maintenance procedures can be found in the Liberty Maintenance Procedures Manual

(PN 600119). The Liberty Maintenance Procedures Manual also contains twelve months of

maintenance logs for recording and maintaining maintenance records.

Daily Maintenance

Inspect the instrument for any visible signs of leakage in all spill trays, any at •

areas inside the Liberty, and inside the cavity of the Discover.
Clean the resin positions on the Resin manifold using methanol or another •

suitable solvent to remove any residual resin.
Check the level in the waste container and empty if necessary.•

Ensure the ber optic probe is fully inserted into the thermowell on the reaction •

vessel.

Weekly Maintenance

Replace the three • inline lters (PN 172105-M):

• F1 and F2 lters (front of Liberty)

• F3 (reaction vessel drain line)

Biweekly Maintenance

Replace • dip tube lters (PN 167485-M):

Amino acid bottles and external amino acid bottles•
Activator, activator base, deprotection, capping (if used), cleavage (if used) •

bottles
Wash solvent bottles (Main Wash/DMF, Secondary Wash/DCM)•

•

Backush all positions except Main Wash/DMF and Secondary Wash/DCM. (See
Performing a Backush, p. 66, for details.)

Monthly Maintenance

Perform a • sensor calibration on all sensors. (See Performing a Sensor
Calibration, p. 76, for details.)

Perform a• Volume Calibration for all additions except TFA. (See Performing a
Volume Calibration, p. 70, for details.)

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Verify the performance of the • ltered drain by adding 10 mL of DMF to the

reaction vessel and then performing a ltered drain. The reaction vessel

should drain 10 mL of DMF within 10 seconds. If the drain takes longer than 10

seconds, replace the F3 lter and try again. If the drain still takes longer than 10

seconds, replace the reaction vessel.
Inspect the reaction vessel for particulate accumulation, particularly on the top •

of the vessel. If necessary, clean the reaction vessel (from the Maintenance
Screen, on the Cleaning tab, click the Clean RV button).

Review recent • run history reports (see Run History Reports, p. 59, for details)
to ensure that the Liberty is heating properly. The temperature for a coupling
should reach 75 °C within 70 seconds. If the Liberty is not reaching temperature
in time, increase the power setting in the Microwave Editor (see Editing an

Existing Microwave Method, p. 29 for details). If the Liberty is routinely heating to

above 83 °C, decrease the power setting in the Microwave Editor.

Semiannual Maintenance

Replace the reaction vessel body.•
Verify the proper operation of the waste sensor.•

Standby Procedure

If the Liberty will be idle for a period of two weeks or more:

Remove all reagents (amino acids, activator, activator base, deprotection, •
capping, cleavage) from the system.

Backush all positions except Main Wash/DMF and Secondary Wash/DCM.•

Remove the wash solvents and perform a backpurge on those positions to empty •
the lines.

Prior to using the Liberty after it has been in Standby:

Connect the wash solvents using the • Change Bottle command (p. 65) to properly
prime the lines and pressurize the bottles.

Perform a backush on all positions except Main Wash/DMF and Secondary •
Wash/DCM. (See Performing a Backush, p. 66, for details)

Perform a • Sensor Calibration on all sensors. (See Performing a Sensor
Calibration, p. 76, for details.)

Perform a • Volume Calibration for all additions except TFA. (See Performing a
Volume Calibration, p. 70, for details.)

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Maintenance Screen

The Maintenance screen is accessed by clicking on the Maintenance button on the PepDriver
main screen and selecting Maintenance. From the Maintenance Menu, a number of important
maintenance procedures and manual system commands can be accessed.

Cleaning Tab

Clean•

•

Change Bottle: Clicking this button begins the Change Bottle procedure
for the reagent selected from the pull-down menu. The Change Bottle
procedure is used to remove and replace bottles not directly connected to a
manifold. (For more information about using Change Bottle, see the Using
the Change Bottle Command, p. 65).

• Add: Clicking this button will add the reagent selected from the pull-down

menu to the reaction vessel at the volume selected from the second pull­down menu.

• Depressurize: Clicking this button will vent any pressure from the reagent

selected from the pull-down menu.

• Clean All Manifolds: Clicking this button will begin a cleaning procedure for

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63

all manifolds on the system. This cleaning only cleans the manifolds and any
internal tubing; it does not affect the dip tubes or any bottles connected to the
system.

• Prime: Clicking this button will prime the lines for the reagent selected from

the pull-down menu.

• Clean RV: Clicking this button begins the reaction vessel cleaning

procedure, and sends all contents of the reaction vessel to waste.

• Neutralization: Clicking this button begins the vessel neutralization

procedure, washing the reaction vessel with deprotection solution followed by
a reaction vessel cleaning step. This command is intended to neutralize any
residual TFA in the vessel following cleavage.

• Resin Return: Clicking this button begins a resin return step, adding main

wash (DMF) and Secondary Wash (DCM) to the reaction vessel and then
transferring the contents to the resin position selected from the pull-down
menu.

• Clean Resin Tube: Clicking this button washes the resin dip tube of the

resin position selected from the pull-down menu to remove any residual
resin.

• Transfer from Resin Tube to RV: Clicking this button begins a resin

transfer step, adding Main Wash (DMF) and Secondary Wash (DCM) to
the resin tube selected from the pull-down menu and then transferring the
contents to the reaction vessel.

• Clean Product Tube: Clicking this button washes the product return tube of

the product position selected from the pull-down menu.

• Wash: Clicking this button adds the selected volume of the selected solvent

(Main Wash/DMF or Secondary Wash/DCM) to the reaction vessel from either

the top or the bottom, then performs a ltered drain.

Drain•

•

Filtered: Clicking this button drains the contents of the reaction vessel
through the glass frit on the bottom of the reaction vessel and through the

inline lter out to waste. Any solid reagents will remain in the reaction vessel.

• Unltered: Clicking this button drains the contents of the reaction vessel

through the resin dip out to waste. This will completely empty the reaction
vessel of all contents (solid and liquid).

• Backush

Backush:• Clicking this button will perform a backush on the reagent(s)

selected from the list. For more information about using the Backush
command, see Performing a Backush, p. 65.

• Backpurge: Clicking this button will perform a backpurge on the reagent(s)

selected from the list.

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System Tests•

•

System Check: Clicking this button begins a System Check diagnostic
procedure. For more information about the System Check procedure, see
Performing a System Check, p. 67.

• Leak Test: Clicking this button beings a full leak check of the system. For

more information about the leak testing procedure, see Test System, p. 86.

• Test Sensors: Clicking this button begins a full sensor test of the system.

For more information about the sensor test procedure, see Test System, p.

86.

• Test System: Clicking this button opens the Test System menu, which

contains a number of diagnostic tests and troubleshooting procedures.

Using the Change Bottle Command

Click on the pull-down menu to select the reagent to be changed, then click the 1.
Change Bottle button.

The Change Bottle window will appear. Click Next to begin the Change Bottle 2.
procedure.

The Liberty will clear the line to the bottle, and then vent the pressure to the 3.

bottle.
The Change Bottle window will indicate it is safe to remove the bottle. Remove 4.

the old bottle from the system and connect a fresh bottle.
Click Next to continue. The Liberty will pressurize the bottle and prime the lines. 5.

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65

Once the lines are primed, click Close to close the Change Bottle window and

return to the Maintenance screen.

Performing a Backush

For detailed instructions on performing a backush, see the Liberty
Maintenance Procedures manual, PN 600119.

Remove all bottles and dip tube lters from the positions to be backushed.1.

Connect empty bottles to all positions to be backushed.2.

For bottles not connected to manifolds (Deprotection and Capping), the
Depressurize command should be used before removing the old bottle

and connecting the empty bottle prior to the backush. The Change Bottle
command should to remove the backush bottle and connect a fresh bottle

of reagent to ensure the bottle is properly pressurized and the lines primed.

From the Cleaning Tab, check the box next to each position to be backushed.3.

NOTE

NOTE

Main Wash (DMF) and Secondary Wash (DCM) should not be backushed.

66

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NOTE

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Click the Backush button. A warning box will pop up. Verify that dip tube lters 4.

have been removed from all positions to be backushed and that empty bottles
have been connected and tightened. Click OK to continue.

For each position, a progress window will pop up showing the progress of that 5.

backush command. Once that window closes (and the window for the next

position pops up) it is safe to remove and empty the bottle from that position.

As each position is backushed, the date will be updated to the current date.6.

NOTE

When backushing Cleavage (TFA), DCM will rst be added to the reaction
vessel, and then backushed into the cleavage bottle. This is done to avoid

mixing DMF with TFA.

Performing a System Check

A System Check is a basic diagnostic test of the Liberty’s ow paths and valves. When a System

Check is run, a PDF containing information about the Liberty as well as the results of the various
tests is generated. This document can be submitted to CEM’s Service Department to aid in
diagnosis and troubleshooting of problems with the Liberty. To perform a System Check:

Before beginning the System Check, ensure that there is a bottle in place on all 1.
amino acid positions, including the external amino acid positions.

From the Cleaning Tab, click System Check.2.

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67

A warning box will pop up about the reaction vessel. Verify that the reaction 3.

vessel is properly connected to the Liberty, and then click OK to continue.

A warning box will pop up about the contents of the reaction vessel. Verify that 4.

there are no solid reagents in the reaction vessel, and then click OK to continue.

The Liberty will perform an unltered drain on the reaction vessel to ensure it is 5.

empty, and then it will purge all liquid from all manifolds. This process will take
approximately 5 minutes.

The System Check window will open. As each test is completed, the values 6.

will be recorded in the window. The System Check will take approximately 20

minutes to complete.

The results of the7. System Check will be rendered as a PDF. Click Save to save
the PDF.

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Manifold Check

-0.1

Nitrogen:

-0.1

Ext1:

0.0

AA1:

0.0

AA2:

0.0

AA3:

-0.1

Ext2:

0.0

AA4:

Flow Performance

Flow Path

15.9 PSI

Nitrogen:

Reading(PSI)

Delta

Ext1

10.9

4.9

Flow Path

Reading(PSI)

Delta

AA2

12.9

3.0

AA1

12.9

3.0

AA3

12.9

3.0

Ext1ToSprayhead

11.3

4.6

AA4

12.1

3.8

Ext1toFilter

11.6

4.2

Ext1_Ext2ToSprayhead

12.7

3.2

Ext1toExt2

12.7

3.1

Ext2

10.7

5.2

Ext2ToFilter

11.4

4.5

Ext2ToSprayhead

11.1

4.8

FilterTop

10.8

5.1

SprayHead

10.9

5.0

FilterBottom

11.4

4.5

ROT3

11.1

4.8

V2

5.1

10.8

TFAToRV

12.1

3.7

Ext1_2ToFilter

12.8

3.0

0.0

RV:

Ext2Bypass

10.3

5.5

0.0

Ext2 Bypass:

Valve Check

-0.1/-0.1

RV2:

-0.1/-0.1

RV3:

0.0/-0.1

RV4:

0.0/0.0

RV6:

0.0/0.0

RV7:

-0.1/0.0

M2:

0.0

M1:

-0.1/-0.1

M3:

(Normally Closed/Normally Open)

4/24/2009 9:27 AM

4/24/2009 9:52 AM

Start:

End:

Valve RV7(3.1, 3.7) Failure: Valve did not open

CEM Liberty System Check

207A16

Liberty S/N:

DP7094

Firmware:

PepDriver Version:

2.5.4.2

201A11

Discover S/N:

Firmware:

Please email this file to: [e-mail]

Sample System Check File

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69

Volume Calibration Tab

The Volume Calibration tab allows for the calibration of the timed delivery of reagents to
the reaction vessel. Reagent calibration should be performed monthly as part of routine
maintenance.

In addition, the Volume Calibration tab allows for the calibration of amino acids at large scale.
For additions of more than 11 mL, the Liberty switches from a sample loop to a timed addition to
reduce the time required for each addition.

Performing a Volume Calibration

NOTE

For detailed instructions on performing a volume calibration, see the Liberty

Maintenance Procedures manual, PN 600119.

Place the reaction vessel into the 1. volume calibration stand (PN 576250).

Remove the 1.1. reaction vessel assembly from the cavity of the Discover.
Set the volume calibration stand on top of the cavity opening, with the 1.2.

graduations on the right.

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Insert the reaction vessel into the calibration stand so that the drain line 1.3.

from the bottom of the vessel ts into the slot on the left of the stand.

From the Volume Calibration tab, click on the Delivery pull-down to select the 2.
reagent to calibrate.

CAUTION

When calibrating reagents, do not add Deprotect immediately before or after
DCM. Mixing DCM and Deprotect will cause a solid precipitate to form in
the reaction vessel that can damage the Liberty.

CAUTION

Cleavage should only be calibrated if Boc chemistry is being performed
on the Liberty. After performing a cleavage volume calibration, perform a
reaction vessel neutralization by clicking Neutralization on the Cleaning tab
of the Maintenance screen.

Once a reagent has been selected, click Calibrate to begin the calibration 3.
procedure.

Enter the volume to be used for calibration, then click Add. Typically, 10 mL of 4.
reagent are used for calibration.

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71

The selected reagent will be added to the reaction vessel through the sprayhead. 5.

Once the addition is nished, measure the volume delivered to the reaction

vessel using the graduations on the calibration stand.
Enter the amount of reagent delivered to the reaction vessel. 6.

Click Calibrate. Two values will be displayed below the Calibrate button, New 7.
Rate and New Flow. Click Save to save the new calibration values.

Click the Filtered Drain button to empty the reaction vessel.8.

NOTE

When calibrating Add DMF to Resin or Add DCM to Resin, the Liberty
will add reagent to a centrifuge tube connected to position 1 on the Resin

Manifold. One the reagent has been added, remove the centrifuge tube

and transfer the liquid to a graduated cylendar to accurately measure the
delivered volume.

Importing and Exporting Volume Calibration Values

To create a backup copy of the current volume calibration values:

From the Volume Calibration tab, click Export.1.

Enter a name for the backup le, and then click Save.2.

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To restore previous volume calibration values from a backup le:

From the Volume Calibration tab, click Import.1.

Select the backup le to be restored, and then click Open.2.

NOTE

When importing values from a backup le, the current calibration values for

all reagents will be discarded.

Performing a Large Scale Calibration

Place the reaction vessel into the 1. volume calibration stand (PN 576250).

Remove the reaction vessel assembly from the cavity of the Discover. 1.1.
Set the volume calibration stand on top of the cavity opening, with the 1.2.

graduations on the right.
Insert the reaction vessel into the calibration stand so that the drain line 1.3.

from the bottom of the vessel ts into the slot on the left of the stand.

From the Volume Calibration Tab, click on the Large Scale Addition Tab.2.

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73

Select the desired reagent and click Calibrate.3.

Click the rst Add button to begin the calibration procedure.4.

A warning box will pop up to conrm that Main Wash (DMF) has been added to 5.

the appropriate position for the calibration procedure. Ensure sufcient DMF
(approximately 30 mL) is connected and click OK.

A prime volume of DMF will be added to the reaction vessel. 6.

Once the addition is nished, measure the volume delivered to the reaction

vessel using the graduations on the calibration stand.

Enter the delivered volume in the Prime Volume box, and then click the second 7.
Add button.

The solvent will be automatically drained from the reaction vessel. Following the 8.

ltered drain, the Liberty will add a delivery volume of DMF to the reaction vessel

(approximately 20 mL).

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Once the addition is nished, measure the volume delivered to the reaction

vessel using the graduations on the calibration stand.

Enter the delivered volume in the Delivered Calibration Volume box, and then 9.
click Calibrate. Two values will appear below the Calibrate button: New Rate and
New Prime Volume. Click Save to save the new calibration values.

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Sensor Calibration Tab

The Sensor Calibration tab allows for the calibration of the sensors used to monitor the addition
and transfer of reagents in the Liberty. Sensor calibration should be performed monthly as part of
routine maintenance.

Performing a Sensor Calibration

NOTE

For detailed instructions on performing a sensor calibration, see the Liberty

Maintenance Procedures manual, CEM PN 600119.

Open the Sensor Calibration tab. PepDriver will load the current calibration 1.

values for each sensor.

Check the Select All box. This will select all sensors except LS2/3.2.

NOTE

LS2/3 do not need to be routinely calibrated. A separate calibration

procedure is required for these sensors. Contact CEM Service for help if
these sensors require calibration.

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Click Calibrate to the begin the sensor calibration procedure.3.

A series of warning boxes will pop up to ensure sufcient DMF is loaded on the 4.

Liberty for the test. Verify there is sufcient DMF in the Main Wash bottles and

click OK.

NOTE

Although the calibration procedure only requires 75 mL of DMF, each bottle
should have a minimum of volume of 150 mL to ensure accurate delivery.

The instrument will pressurize the DMF bottles and then ll all manifolds and 5.

tubing with DMF. Once this is complete, the calibration procedure will begin. As

each sensor is calibrated, the updated calibration value is displayed in the New
Threshold column. The entire process should take approximately 5 minutes.

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77

When calibration is complete, a window will pop up stating that the procedure is 6.

complete. Click OK to close the window.

Click Save to save the new calibration values. As each value is saved, the 7.
New Threshold column will be cleared and the Current Threshold values will be
updated with the new calibration value.

Once all the new calibration values are saved, a window will pop up to purge the 8.

lines of DMF. Click Yes to empty the lines, or click No to leave the lines lled.

NOTE

The rst step of a Sensor Test is to ll the lines. To conserve DMF, click No
to leave the lines lled if a Sensor Test will be performed.

A window will pop up suggesting running a 9. Sensor Test. Click OK to close the
window.

Click the Test Sensors button to begin a Sensor Test.10.

For information on performing a Sensor Test, see Test Sensors, p. 91.

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Importing and Exporting Sensor Calibration Values

To create a backup copy of the current sensor calibration values:

From the Sensor Calibration tab, click Export.1.

Enter a name for the backup le, and then click Save.2.

To restore previous sensor calibration values from a backup le:

From the Sensor Calibration tab, click Import.1.

Select the backup le to be restored, and then click Open.2.

NOTE

When importing values from a backup le, the current calibration values for

all sensors will be discarded.

Pressure Calibration Tab

The Pressure Calibration tab allows for the calibration of the pressure sensor on the Liberty.
Pressure calibration is not necessary for routine maintenance; a calibration should only be done
as part of a troubleshooting protocol as advised by CEM Service.

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79

Performing a Pressure Calibration

Click Calibrate to begin the calibration procedure. A warning will pop up about 1.

performing a backpurge. Click Yes to backpurge the system before calibrating.

NOTE

Put empty bottles on all positions before performing a backpurge.

Adjust the pressure on the main pressure regulator in the Liberty to 16 psi, and 2.

then click OK to continue the calibration.

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The Liberty will vent the pressure on the entire system to get a zero pressure 3.
reading, and then apply pressure to get a main pressure reading.

Look at the pressure regulator on the Liberty and enter the current reading. Click 4.

OK to continue the calibration.

The software will save the calibration values automatically. Click Verify to test the 5.
pressure readings.

The Test System screen will automatically open and begin performing a Pressure 6.

Adjustment to conrm the pressure is within the correct range.

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81

Section 5: Advanced Features

Default Cycle Editor

The Default Cycle Editor is accessed by clicking the Setup button and selecting Default Cycle
Editor. The Default Cycle Editor allows the user to assign a default Amino Acid Cycle to each
amino acid. Different cycles can be assigned for different scales, allowing for the use of higher
power or more agitation at higher synthesis scales.

Changing Default Cycles

Open the Default Cycle Editor by clicking the Setup button and selecting Default 1.
Cycles.

Select a scale from the Scale pull-down menu.2.
Click on the cycle next to the an amino acid and select the desired cycle from the 3.

pull-down.

Click OK to save the new default cycles.4.

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Options Menu

The Options Menu can be accessed by clicking the Setup button from the PepDriver main screen
and selecting Options. The Options menu allows conguration of a number of important system

functions.

Program Options Tab

Method Options•

•

Test Sensors at Start: When this option is enabled, the Liberty will perform

a full Sensor Test before starting the rst method in the queue. This option is

enabled by default.
Automatic • Leak Check RV at Start: When this option is enabled, the

Liberty will perform a leak check of the reaction vessel before starting the rst

method in the queue. This option is enabled by default.
Automatic • Clean RV at Start: When this option is selected, the Liberty will

perform a reaction vessel cleaning step before starting the rst method in the

queue. This option is off by default.

NOTE

The default resin loading cycles include a reaction vessel cleaning step as

the rst step that is sufcient for routine operation of the Liberty.

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83

Microwave Options•

DeltaT Available in• Microwave Editor: When this option is enabled, the

delta T value can be changed when programming a microwave method in the
Microwave Editor.

NOTE

During a microwave method, microwave power will only be applied until the
temperature reaches the programmed temperature. At that point, the power
will be turned off until the temperature drops to below the delta temperature
value. This reduces the risk of overshooting the temperature and improves
the quality of the synthesis. The default value for delta T is 5 degrees.

Cooling Available in • Microwave Editor: When this option is enabled,
air cooling can be enable when programming a microwave method in the
Microwave Editor.

NOTE

During a microwave method, if air cooling is enabled, and the air cooling line
is connected to the Discover and to a nitrogen source, a stream of air will
be blown over the reaction vessel. This will decrease the time needed for
the temperature to drop below the delta T and for the microwave power to
resume. Air cooling is not recommended for peptide synthesis.

System Options•

Pause When Running on Battery Power: • When this option is enabled,

PepDriver will pause the current method in the event the laptop computer
switches to battery power. This is critical in the event of power failure. This
option is enabled by default.

Test System Visible:• When this option is enabled, the Test System button

is visible on the Cleaning Tab of the Maintenance Screen. This option is
enabled by default.

Vent Rotary 3 Valve on Start:• When this option is enabled, pressure to

Rotary 3 will be vented before starting the rst method in the queue. This

option is disabled by default.
Omit Check for • 125 mL Reaction Vessel: By default, if a method of 0.5

mmol scale or higher is loaded into the queue, PepDriver will prompt the user
to check that the correct reaction vessel is installed.

CAUTION

The 125 mL reaction vessel must be used for syntheses above 0.25 mmol.
Using the 35 mL reaction vessel for large syntheses will result in overlling

of the vessel, loss of product, and possible damage to the instrument.

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Defaults Tab

The Defaults Tab is used to select the default settings for all options in the Method Editor. To
change the default settings:

Select a scale from the Resin Information box.1.
Make any changes to the other settings.2.
Click OK to save the changes.3.

Run History Tab

The Run History tab is used to select the format for naming the folders into which run history les
are saved. Folders can be named in either American (Month-Year) or European (Year-Month)

format.

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Test System Screen

The Test System screen contains a number of diagnostic tests that can be used to troubleshoot
the performance of the Liberty. The Test System screen is accessed by clicking the Maintenance
button, selecting Maintenance, and then clicking the Test System button from the Cleaning tab of
the Maintenance menu.

There are four Quick Test buttons at the top of the Test System screen: Leak Check, Test Valves,
Test Sensors, and Test Additions. Clicking any of these buttons will automatically select the
appropriate categories and begin the necessary tests.

To perform a specic test:

Check the box next to the desired test category in the Category window.1.
Click the name of the category. The tests within that category will appear in the 2.

Tests window.
Click Test to run all the tests in the selected category, or double-click on a 3.

specic test to run only that test.

Once a test is complete, a green check mark will appear if the test was passed, or a red X will

appear if the test was failed. Details about the results will be recorded in the Status Log window.

When testing a valve, if a failure is reported, clicking on the red X will open a picture of the valve’s

location.

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Pressure Adjustment

The Pressure Adjustment category contains two tests: Adjust Main Pressure and Adjust Low
Pressure. These two tests are used to ensure the high and low pressure regulators are adjusted
to within the standard operating parameters.

Main Pressure Adjustment

Select the Pressure Adjustment category, and then double-click on the Adjust 1.
Main Pressure test.

The current pressure reading (in psi) will be displayed in the status box. The 2.
reading will be displayed in green if the value is within the standard operating
parameters (15-17 psi), and in red if it is outside the standard parameters.

If the pressure reading is outside the range, the status box will display 3.
instructions for adjusting the pressure regulator. The pressure reading will be
displayed in real time. Adjust the regulator until the reading is between 15 and
17 psi.

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Low Pressure Adjustment

Select the Pressure Adjustment category, and then double-click on the Adjust 1.
Low Pressure test.

The current pressure reading (in psi) will be displayed in the status box. The 2.
reading will be displayed in green if the value is within the standard operating

parameters (2.5-4.5 psi), and in red if it is outside the standard parameters.

If the pressure reading is outside the range, the status box will display 3.
instructions for adjusting the pressure regulator. The pressure reading will be

displayed in real time. Adjust the regulator until the reading is between 2.5 and

4.5 psi. Contact CEM Service for help adjusting the low pressure regulator.

Leak Check

The Leak Check category contains tests to ensure none of the manifolds on the system are
leaking. There are eight manifolds that are tested in the Leak Check category. To perform a Leak
Check:

Select the Leak Check category, and then click Test.1.

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The Liberty will purge all manifolds, and then apply pressure to the manifold to be 2.
tested.

The pressure will be monitored for a set amount of time, during which the current 3.
drop in pressure at the manifold being tested will be displayed in the status box.

If the pressure drops more than 0.4 psi, the manifold will fail the Leak Check. For 4.
help with troubleshooting a leaking manifold, see the Troubleshooting section of
this manual or contact CEM Service.

Test Rotary Valves

The Test Rotary Valves category contains individual tests for each of the three rotary valves. The
tests are designed to check the rotary valves for leaks.

Test Rotary 1

Select Test Rotary Valves and then double-click on Test Rotary Valve 1 in the 1.
Test window.

A warning box will pop up stating that a centrifuge tube should be connected to 2.
position 1 on the resin manifold. Ensure a tube is connected and properly sealed

and then click OK to continue the test.

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The test will check the valve in the open position rst, and then in the closed 3.

position. If the valve fails the test, contact CEM Service for troubleshooting help.

Test Rotary 2

Select Test Rotary Valves and then double-click on Test Rotary Valve 2 in the 1.
Test window.

A warning box will pop up stating that a centrifuge tube should be connected to 2.
position 1 on the product manifold. Ensure a tube is connected and properly

sealed and then click OK to continue the test.

The test will check the valve in the open position rst, and then in the closed 3.

position. If the valve fails the test, contact CEM Service for troubleshooting help.

Test Rotary 3

Select Test Rotary Valves and then double-click on Test Rotary Valve 3 in the 1.
Test window.

The test will check the valve in the open position rst, and then in the closed 2.

position. If the valve fails the test, contact CEM Service for troubleshooting help.

Internal, Nitrogen, and Liquid Delivery Tests

There are three types of delivery tests available in Test System: Internal Delivery, Nitrogen

Delivery, and Liquid Delivery. Each category corresponds to a specic ow path within the

Liberty, and each contains individual tests of each valve in that path. The delivery tests are
intended to test the function of each valve on the system.

There are three categories of Internal Delivery tests: the RV valves, the M valves, and the EN/EX

Valves. There are three categories of Nitrogen Delivery tests: the P valves, the PE valves, and
the R valves. There are two categories of Liquid Delivery test: the L valves and the LE valves.

Setting Up the Liberty for a Delivery Test

Before beginning a delivery test, certain precautions need to be taken to ensure accurate test
results:

Remove all reagents (amino acids, external amino acids, activator, activator 1.
base, cleavage, deprotection, capping, Main Wash/DMF, Secondary Wash/DCM)
from the system.

Remove the dip tube lters from all dip tubes.2.

Perform a backpurge on all positions to ensure the lines are free of liquid. 3.
Loosen or remove any bottles connected to the reagent positions (amino acids, 4.

external amino acids, activator, activator base, cleavage, deprotection, capping,
Main Wash/DMF, Secondary Wash/DCM).

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If leaving bottles in place, ensure the bottles are loose enough to allow
pressure to escape. Tightening the bottles will result in false failures.

Disconnect the spray head line from the reaction vessel manifold on the front of 5.
the Liberty and connect a plug (PN 167800) to that position.

Performing a Delivery Test

Select the category of delivery test, and then double-click on the individual 1.
valve to be tested in the Test window (or click Test to test all valves within the
category).

CAUTION

The Liberty will vent all pressure from the lines. The valve will be closed and the 2.
line pressurized, then the nitrogen turned off. The Liberty will wait to see if there
is a decrease in pressure. A decrease in pressure indicates the valve is leaking,
and will cause the test to fail. If the test fails, contact CEM Service for help with
troubleshooting.

The valve will then be opened and the pressure should vent. Failure to vent 3.
pressure indicates the valve is not opening or the line is restricted, and will cause
the test to fail. If the test fails, contact CEM Service for help with troubleshooting.

Test Sensors

The Sensor Test is the most commonly used category in the Test System screen. A Sensor Test

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should be performed after every sensor calibration. To perform a Sensor Test:

NOTE

A Sensor Test can also be started from either the Cleaning tab or the Sensor
Calibration tab by clicking the Test Sensors button. Clicking Test Sensors
from either tab will open the Test System screen and automatically select
and begin a Sensor Test.

Select the Test Sensors category, and then click Test.1.

A warning will pop up to conrm that there is sufcient DMF for the test and that 2.

the waste container is connected. Verify the DMF supply and that the waste

container is not full, then click OK.

The Liberty will purge the lines. The sensors for the rst test will be displayed in 3.

the status window.
The Liberty will ll the line with DMF. As each sensor detects liquid, the 4.

corresponding indicator in the status window will turn bright green.

The Liberty will turn off the DMF ow and empty the line. As each sensor stops 5.
detecting liquid, the corresponding indicator in the status window will dim.

If a sensor fails the test, a red X will appear next to the test in the Test window, 6.

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and the specic sensor that failed will be recorded in the status log. If any

sensors fail, contact CEM Service for troubleshooting help.

Reagent, Amino Acid, and External Amino Acid Addition

There are three types of addition tests available in Test System: Reagent, Amino Acid, and
External Amino Acid. The addition tests are intended to verify that the sample loops for each of
these positions are delivering the correct volume.

To perform an Addition Test:

Disconnect the sprayhead line from the manifold on the front of the Liberty. 1.

Connect a piece of 1/8” OD tubing (PN BR199116) to the manifold using a nut

(PN 167810) and yellow ferrule (PN 164315).
Place the open end of the tubing into a 10 mL graduated cylinder.

Select the category of addition test, and then double-click on the individual 2.
position to be tested in the Test window (or click Test to test all additions within
the category).

The Liberty will begin adding one sample loop of the selected reagent. The 3.
progress of the addition and the target volume will be displayed in the status
window.

When the addition is complete, verify the volume delivered to the graduated 4.
cylinder. If the delivered volume is more than 0.3 mL different from the target,
contact CEM Service for troubleshooting assistance.

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Leak Check Reaction Vessel

The Reaction Vessel Leak Check should be performed any time the reaction vessel has been
disconnected from the system. In particular, a leak check should be performed when switching

between the 125 mL, 35 mL, and 10 mL vessels, or after any test that requires disconnecting the

spray head line.
To perform a Reaction Vessel Leak Check:

Select the Leak Check Reaction Vessel category, and then click Test.1.

A warning will pop up to conrm that the reaction vessel is connected and secure. 2.
Verify the reaction vessel is properly connected, then click OK.

The Liberty will pressurize the reaction vessel.3.

The pressure will be monitored for a set amount of time, during which the current 4.
drop in pressure at the manifold being tested will be displayed in the status box.

If the pressure drops more than 0.4 psi, the reaction vessel will fail the Leak 5.
Check. (For help with troubleshooting a leaking reaction vessel, see p. 115 or
contact CEM Service.)

Flow Performance

The Flow Performance test is intended to verify there are no restrictions in the lines of the Liberty

by testing the rates of nitrogen ow through all lines and manifolds. Typically, this test is only

performed on the advice of CEM Service to aid in diagnosing a problem. To perform a Flow
Performance test:

Select the Flow Performance Test category, and then click Test.1.

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The Flow Performance window will open. As each pathway is tested, values will 2.
be recorded in the window.

When the test is complete, report these values to your service technician.3.
Click OK to close the window.4.

Diagnostics Screen

The Diagnostics Screen contains a number of reports and manual controls that are not intended
for normal use. In general, the Diagnostics screen should only be used under the advice of a
trained CEM Service Technician.

Commands Tab

The Commands tab allows for the manual execution of all system commands. In addition, the

Read Discover Errors button will detect any rmware errors in the Discover.

Sensors Tab

The Sensors tab displays the current status (on or off) of each sensor on the system. Sensors
are on when they are seeing liquid, as shown by the bright green indicator.

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Valves Tab

The Valves panel allows for the manual control of each valve on the system. Checking the box

next to a valve opens that valve. Unchecking the box closes the valve. Clicking the All Off button

unchecks all valves.
The Rotary Valves are controlled by selecting the desired position from the pull-down box and

then clicking the button corresponding to that valve (1 for Rotary 1, 2 for Rotary 2, 3 for Rotary 3).

Status Tab

The Status tab contains diagnostic information about the current status of the Liberty.

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Delay Times Tab

The Delay Times tab is used to change the delay times for various system commands and
parameters.

To change a delay time:

Click on the delay time to be changed in the list.1.
Enter the new delay time in the Delay Time (sec) box on the right.2.
Click Update to save the new delay time.3.

CAUTION

Do not change a delay time without consulting with a trained CEM Service
Technician. Incorrect delay times can result in poor synthesis quality and
instrument malfunction.

CAUTION

Before changing any delay times, click the Backup button to save a backup

le of all current delay times.

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Section 6: Troubleshooting

Problem

Possible Solution

Replace the empty bottle with fresh reagent.•

Empty Amino

Acid Bottle

Error

Empty

Activator,

Activator Base,

or Cleavage

Bottle

If the bottle is not empty, ensure bottle is securely connected •
to the manifold.

If the bottle is not empty, replace the dip tube lter.•

If the error still occurs, perform a Sensor Calibration (p. 76).•
If the error still occurs, contact CEM Service.•

Replace the empty bottle with fresh reagent.•
Ensure the bottle is not cracked.•
If the bottle is not empty, ensure bottle is securely connected •

to the manifold.

•

If the bottle is not empty, replace the dip tube lter.

If the error still occurs, perform a Sensor Calibration (p. 76).•
If the error still occurs, contact CEM Service.•

F1/F2

Restriction

Error

F3 Restriction

Error

Waste Full

Error

Nitrogen

Pressure Errors

Reaction
Vessel Fails
Leak Check

Replace the F1 or F2 lter.•
Replace the dip tube lter for the reagent being added.•

If the error still occurs, contact CEM Service.•

Replace the F3 lter.•

If the error still occurs, replace the reaction vessel.•
If the error still occurs, contact CEM Service•

Empty the waste container.•
If the waste container is not full, ensure the sensor is not •

engaged (in the up position).
If the error still occurs, contact CEM Service.•

Verify the nitrogen source is functional.•
Verify the pressure setting on the Main Pressure Regulator is •

between 15 and 17 psi.
Perform a Leak Check (p. 88), then contact CEM Service.•

Verify the tubing connections to the manifold on the front of •
the Liberty are tight and not cross-threaded

•

Verify the vessel body is tightly connected to the attenuator

Verify the PEEK tting on the drain line is tightly connected to •

the bottom of the reaction vessel
Verify the quick disconnect on the drain line is tightly secured•
If the vessel still fails the Leak Check, contact CEM Service•

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