Targeted Preparative Fraction Collection

Based on Analytical Mass of

Interest Retention Time

Technical Note
Table of Contents
Introduction
Targeted Preparative Fraction Collection Based on
Analytical Mass of Interest Retention Time . . . . . . . . . . . . . . . . . . 2

Hardware
GX-281 System Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
VALVEMATE® II Plumbing Schematic . . . . . . . . . . . . . . . . . . . . . . . 4

Software
Project Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Method Builder — Configuration . . . . . . . . . . . . . . . . . . . . . . . . . 7
Method Builder — Bed Layout . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Method Builder — Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Application Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Sample Quotation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
 Targeted Preparative Fraction
Collection Based on Analytical
Mass of Interest Retention Time
Gilson, Inc. is renowned for the ability to efficiently configure purification system solutions to exactly
meet application needs. These configurations range from single focused applications to systems that
can accommodate a wide variety of applications.

Virtually all purification labs utilize mass data. However coupling mass spectrometer with preparative
HPLC has numerous disadvantages; including splitters and maintenance issues with the splitters,
maintaining accurate split ratios and finding mass specs that can hold up to the extreme conditions
associated with preparative samples. The main goal of mass-based purification is a more targeted
purification that reduces the number of fractions collected to only the compound of interest. Using
Trilution® LC Software and data obtained from the analytical mass run, a targeted fraction collection of
the compound of interest can be achieved without the need for directly coupling the mass spec to the
preparative LC run. This technical note describes the configuration and software setup to achieve a
targeted mass-based collection without the need for directly coupling the mass spec to the preparative
HPLC system.

A commonly configured Gilson preparative system is the GX-281 Multi-column Purification System.
This is a system that accommodates a range of column applications ranging from 10 mm ID up to
50 mm ID. The system can accommodate up to six different columns with easy methods in place for
switching to the appropriate application, column and conditions. This system supports multiple users
each of which may require different separation of different size columns. This technical note provides
suggested hardware setup, software configuration and a sample quotation.


 Hardware
GX-281 Liquid Handler
The GX-281 offers unmatched versatility and
performance for liquid handling operations.
The efficient speed of the GX-281 allows
applications to be completed quickly and reliably.
Low Mount Fraction Collection Valve
The low mount collection valve enables fraction
collection to tubes and contains two separate
liquid paths which reduce cross-contamination.
GX Z-Injection Module or
GX Direct Injection Module
The GX Z-Injection Module allows sample aspiration
directly into the valve without a separate injection
port, eliminating a source of potential carryover.
GX Direct Injection technology features an injection
port that connects directly to the valve, minimizing
volume associated with connective tubing.
 GX Prep Solvent System
The GX Prep Solvent System features a dynamic
range of volumes and flow rates using syringeless
technology within a solvent delivery pump
capable of accessing five off-bed solvents.
333/334 Prep-scale HPLC Pumps
The 333/334 binary pumping system features
dual-piston reciprocating pump heads with a
flow rate range of 1 to 200 mL/min and a pressure
limit of 3,040 psi. The pumps have the ability to
access up to four solvents per pump and a built-in
solvent selection valve.
155 UV/VIS Detector
The 155 dual wavelength UV/VIS Detector with
preparative flow cell offers a 0.2 and a 0.05 mm
pathlength specifically designed for preparative
applications and high sample loads.
VALVEMATE® II Valve Actuator
Two VALVEMATE® II Valve Actuators with large-bore
6-position column section valves offer a 0.030” bore
size foraccommodating higher flow rates and also
reducing plugging.
RH1 Rack Heightener Quantity - 1
Rack Code 200 Quantity - 1
(holds 96 13 x 100 mm tubes)
Rack Code 207 Quantity - 5
(holds 75 16 x 100 mm tubes)

 Hardware

VALVEMATE® II
Plumbing Schematic

6 1
From Injection Valve
2
5
3 100 mm x 50 mm ID
4

150 mm x 50 mm ID

100 mm x 21.2 mm ID
150 mm x 30 mm ID

250 mm x 10 mm ID 50 mm x 21.2 mm ID

6 1
To Detector
2
5

4 3


Software

Trilution® LC can be set up in a simple-to-use, walk-up system that

is customized for every customer. The following sections provide
recommendations for controlling the GX-281 Purification System.

Project Library
1. In Trilution LC , on the Liquid Chromatography menu, select
Project Library to begin creating the project and applications.


 Software

2. Click New Project to create a new project. The New Project

dialog is displayed. Name it Mass RT Targeted FC.

3. Right-click the Mass RT Targeted FC project and

select Create Application.

4. Right-click the Application and select Create Method.

The Method Builder opens.

Method Builder – Configuration

1. Click Scan. The software searches for Gilson GSIOC or
Ethernet instruments. All instruments detected will be
populated in the Available Instruments window.

continued...


2. To configure the available instruments, drag-and-drop them
onto the workspace. The instrument Properties page will appear
on the right, allowing values to be entered.

Tip: For easier identification, change the instrument

names on the General tab to the solvent that each
pump is delivering.


 Software

Method Builder- Bed Layout

1. After the configuration has been completed, click the
Bed Layout tab at the top of the screen.

2. Double-click the gray Rack footprints to browse for the racks

that are being used.

3. Double-click the Rinse Station footprints to select the rinse

stations being used.

4. Double-click the Direct Injection Module locations for

the 1/16" and 1/8" OD sample loop valve selection. If a
GX Riser Block is being used, select it prior to choosing the
type of injection valve installed.

A default sample zone is created, new sample zones can be

created by clicking the Add Zone button.


Method Builder- Control
There are two types of possible methods to control in the
GX-281 Purification System.

Generic method with variables that have to be entered

at run time.

A unique method for each column that automatically

sets the valves to the correct column position.


 Software

The below example and the example being shown in this

document is a generic method with the use of variables.

1. Under Mobile Phase drag-and-drop Variable Gradient Task.

If flow rate is required to be a variable, type in the dialog box
that appears #Flow Rate.

2. For the Organic Pump % enter the variable #Initial ACN or

equilivant solvent name. The other pump does not require a
variable to be entered, it will automatically be calculated at
run time when the ACN variable is defined.

The first Variable Gradient Task can be hard coded to time 0.

3. Drag-and-drop another Gradient Task with Variables onto the

workspace. For Time enter in #Gradient Time. For the 334 select
from the drop-down menu the #Final ACN variable. Copy the
Variable expression for Time.

10
4. Drag-and-drop another Variable Gradient Task onto the
workspace. Select #Flow Rate from the drop-down menu and
enter a time of 0.5 minutes. Select #Initial ACN from the drop-
down menu for the organic pump.

5. Under Mobile Phase drag-and-drop another Variable Gradient

Task onto the workspace. Select the variable #Flow Rate from
the drop-down menu. Select #Initial ACN for the organic pump
from the drop-down menu. For Time, enter in #Gradient Time +
0.5. This will allow for the Gradient Duration to be specified at run
time for each sample. For the ACN pump %, enter #Final ACN.

11
 Software

6. Drag-and-drop another Gradient Task with Variables onto the

workspace. For Time enter in #Gradient Time + Wash Time +0.5.
For the organic pump select from the drop-down menu the
#Final ACN variable. Copy the variable expression for Time.

7. Drag-and-drop another Gradient Task with Variables onto the

workspace. For Time copy in #Gradient Time + Wash Time.
At the end of the expression add + 0.5. For the organic pump
select from the drop-down menu the #Initial ACN variable.

12
The gradient has now been created. The variables for
the gradient conditions will need to be satisfied at run
time. This allows for one method to be used for all mobile phase
conditions.

The variables for mobile phase that will need to be

satisfied at run time are:

#Flow Rate
#Initial ACN
#Final ACN
#Gradient Time
#Wash Time
#Equilibration Time

continued...

13
 Software

8. Under Valve drag-and-drop the Switch VALVEMATE

Command onto the workspace at time 0.

9. Create the variable #Column Selector. This will allow the

user to select which column to inject onto at run time.

14
10. Drag-and-drop a second Switch VALVEMATE Task
onto the work space.

11. Select Valve (2) from the VALVEMATE drop-down menu

and from the Position drop-down menu select #Column.
This ensures that the both VALVEMATEs will be set to the
correct positions for each column.

15
 Software

12. Locate the Fraction Collection Settings Task also under

the Liquid Handler configured instrument. Drag-and-drop
in this task and enter a time of 0.04 minutes.

13. Fill in the necessary parameters.

A slope value of 30 is
recommended (see example below).

14. After this Main page has been completed, click the
Fraction Sites tab.

16
15. From the Fraction Well 1 drop-down menu select
#Fraction Well (see example below).

16. Under the Liquid Handler, select the GX-281 Prep Injection
with Collection Task. Drag-and-drop the task over to the
work space at time 0.06.

17
 Software

17. Fill in the necessary parameters for the Injection Task.

Remember variables can be used (see example below).

Recommended variables:

#Sample Well

# Injection Volume

18
18. Go to the Detector and drag-and-drop the 155 156 Detector
Settings Task onto the workspace. Enter in a time of 0.08.

19. For the Mode, select Dual. Set both sensitivity values to 0.1.
For Wavelength 1 enter in #WV 1. For Wavelength 2 enter
in #WV 2.

20. Under Auxiliary, drag-and-drop a Synchronize onto the

workspace. In the Task dialog box enter a time of 0.2 and
select the GX-281 Prep Injection with Collection as the task
to synchronize to and select to Synchronize to Synch 1.

Note: A message can be entered that will be displayed

on the Run screen while the synchronize has paused the
method, “Waiting for Injection” is recommended.

continued...

19
 Software

21. Go to the Detector Task list and drag-and-drop the

Detector Autozero Channel Task onto the work space.
Enter in a time of 0.35 minutes.

22. From the Detector Task list, drag-and-drop the

Start Data Collection Task onto the work space and
enter a time of 0.52 minutes.

23. From the Detector Task list, drag-and-drop the

Stop Data Collection Task onto the work space and enter
in #Gradient Time + #Wash Time + #Equilibration Time +
0.60 for the Time. This will allow your data collection to
match up with the end of your mobile phase conditions.

24. Go back to Auxillary and drag-and-drop another

synchronize onto the workspace at time 0.54 min.
Select to synchronize to the end of task for the
GX-281 Prep Injection with Collection Task.

20
25. From the Liquid Handler Task list, drag-and-drop the
Start Fraction Collection Task onto the workspace and
enter a time of #Mass RT – 1.0.

26. From the Liquid Handler Task list, drag-and-drop the

Stop Fraction Collection Task onto the workspace and
enter a time of Mass RT + 2.0.

Including these variables for Start and Stop Fraction

Collection will allow the user to enter in the sample list
the retention time of compound of interest from the
analytical mass spectrum run and have the fraction collection
windows be targeted only around the peak of interest.

Note: The window can be narrower or broader by adjusting

the (-) and (+) times.

21
 Software

Tip: Variables are used very frequently however many of them are usually
given the same values at run time. For example, most gradients are run
from 5 to 95% over the course of 10 minutes. Also, the wavelengths that
are used are commonly 210 and 254 nm. To allow for simpler start-up of
the application sample list but still allow for the flexibility of the variables,
default values can be assigned to each variable. These values will be
populated in the sample list but can be changed if desired.

1. Go to the Variable List button at the top of the workspace.

2. Right-click the variable #Final ACN and then select modify.

The Variable dialog box appears.

3. For the default value enter 95 and make sure that the Show In
Parent check box is checked.

4. Click OK. When the sample list is opened and this method is selected
a value of 95 will automatically appear in the cell for this variable.

5. Repeat for any other variables that are desired to have default values.

22
 The Global Method is now created and can be saved.

27. Click Save, the dialog box that appears requires you to select
an application to save the method to.

28. Name all of the components. This can be done by clicking

the component and typing in a unique name
(see example below).

23
 Software

Application Run
1. Go to the Project Library and click Refresh. The method created
should now appear under the Mass RT
Targeted FC Application.

2. To run a method select the Mass RT Targeted FC Application.

Go to the lower left corner of the Project Library and click the
Run button.

24
3. When the Application Run screen comes up select the method
from the drop-down menu that you want to run. All the
variables that need to be satisfied will appear as columns.

4. After all the parameters have been created, click Run to start.
Note: The sample list can be saved after all the parameters have
been entered so it can be opened again.

25
 Software

Tip: The Minimum Fraction Sites button on the Application

Run screen can be used to ensure that fraction tubes are always
available before performing the injection. If there are typically
15 fractions per injection a safety factor of 20 can be entered.
If 20 tubes are not available on the bed then no injection will
occur and the sample will be saved.

26
Tip: Sample selection can be done graphically by placing the cursor in
the Sample Well variable cell, going to the Bed Layout tab and selecting
the wells to add. After all the wells have been lassoed, click the Add Well
button and the samples will be added to the sample list.

Tip: Three other key functions are Manual Advance and Divert to
manually collect fractions or switch to divert, and Gradient Hold for
changing mobile phase conditions mid-run. They are located at the
top left corner of the screen.

27
Sample Quotation –
GX-281 Purification System
Quantity Product # Description

1 210631R20 TRILUTION® LC Media v2.0

1 ORACLE10GXE ORACLE 10g Express Edition
1 21063130 TRILUTION® LC License - Lifetime

1 38103331 333 H3 Primary Solvent Pump

1 38103341 334 H3 Secondary Solvent Pump
1 38073215 PEEK Binary Plumbing Package

1 261030 GX-281 Liquid Handler

1 261355 GX Direct Injection Valve for 1/8" OD Sample Loops
1 26035411 GX Direct Injection Port for 1.5 mm OD probes
1 26035470 Plumbing Package, Direct Inject
1 494400051 5 mL Sample Loop
1 49440010 10 mL Sample Loop
1 49440025 25 mL Sample Loop
1 499483602 30 mL Transfer Tubing
1 2507216 Non-septum Piercing Probe (269 x 1.5 x 0.8)
1 2603614112 Lower Probe Holder for 1.5 mm OD probes
1 2603614216 Probe Guide Insert for 1.5 mm OD probes 3-way
1 26034555 GX-281 Inside/Drain Rinse Station (Tall)
1 26034551 GX-281 Outside Rinse Station (Tall)
1 2617725 GX-281 Low Mount Fraction Collection Pkg

1 1010561 156 Dual Wavelength UV/VIS Detector

Cell/Accessory Kit for Preparative Applications ,
1 104443 0.05 pathlength

2 331052AB VALVEMATE® II – GSIOC

High Pressure 6-position (0.030"),
2 494C53006 Stainless Steel, 1/16" OD tubing
2 33035255 Plumbing Package (0.030"), Stainless Steel, 10 ft

1 2504600 Code 200 Rack

1 25045514 RH1 Rack Heightener
2 250420065 RackCount Non-serpentine Back Left 1-96
5 2504607 Code 207 Rack
1 250420755 RackCount Serpentine Starting Back Left 1-75
1 250420756 RackCount Serpentine Starting Back Left 76-150
1 250420757 RackCount Serpentine Starting Back Left 151-225
1 250420758 RackCount Serpentine Starting Back Left 226-300
1 250420759 RackCount Serpentine Starting Back Left 301-375
Gilson, Inc. World Headquarters | 3000 Parmenter Street | P.O. Box 620027 | Middleton, WI 53562-0027, USA | Tel: 608-836-1551 or 800-445-7661 | Fax: 608-831-4451
Gilson S.A.S. | 19, avenue des Entrepreneurs | BP 145, F-95400 VILLIERS LE BEL, FRANCE
www.gilson.com | sales@gilson.com | service@gilson.com | training@gilson.com

©2007 - 2008 Gilson, Inc. | LT319238-02 | Specifications subject to change without notice.