targeted preparative fc

8/7/2019 Targeted Preparative FC

1/30

Targeted Preparative Fraction Collection

Based on Analytical Mass oInterest Retention Time

Technical Note


2/30

IntroductionTargeted Preparative Fraction Collection Based onAnalytical Mass o Interest Retention Time 2

Hardware

GX-281 System Confguration 3

VALVEMATE II Plumbing Schematic 4

Sotware

Project Library 5

Method Builder Confguration 7

Method Builder Bed Layout 8

Method Builder Control 9

Application Run 24

Sample Quotation 28

Table of Contents


3/30

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 thatcan 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 ofthe 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-81 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 forswitching 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.

Targeted Preparative FractionCollection Based on AnalyticalMass of Interest Retention Time


4/30


5/304

Hardware

From Injection Valve

To Detector

250 mm x 10 mm ID

150 mm x 30 mm ID

50 mm x 21.2 mm ID

100 mm x 21.2 mm ID

150 mm x 50 mm ID

100 mm x 50 mm ID

1

4

5

6

1

4

5

6

VALVEMATE IIPlumbing Schematic


6/305

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-81 Purification System.

Project Library

1. In TriluTion LC , on the Liquid Chromatography menu, selectProject Library to begin creating the project and applications.


7/306

Software

3. Right-click the Mass RT Targeted FC project andselect Create Application.

4. Right-click the Application and select Create Method.The Method Builder opens.

1. Click Scan. The software searches for Gilson GSIOC orEthernet instruments. All instruments detected will be

populated in the Available Instruments window.

continued...

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

dialog is displayed. Name it Mass RT Targeted FC.

Method Builder Configuration


8/30

Tip: For easier identification, change the instrument

names on the General tab to the solvent that each

pump is delivering.

2. To configure the available instruments, drag-and-drop them

onto the workspace. The instrument Properties page will appearon the right, allowing values to be entered.


9/308

Software

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

2. Double-click the gray Rack footprints to browse for the racksthat are being used.

3. Double-click the Rinse Station footprints to select the rinsestations being used.

4. Double-click the Direct Injection Module locations for

the 1/16" and 1/8" OD sample loop valve selection. If aGX 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- Bed Layout


10/30

Method Builder- Control

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.

There are two types of possible methods to control in the

GX-81 Purification System.


11/3010

Software

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 orequilivant 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 theworkspace. For Time enter in #Gradient Time. For the 4 select

from the drop-down menu the #Final ACN variable. Copy the

Variable expression for Time.

The below example and the example being shown in this

document is a generic method with the use of variables.


12/3011

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

4. Drag-and-drop another Variable Gradient Task onto the

workspace. Select #Flow Rate from the drop-down menu andenter a time of 0.5 minutes. Select #Initial ACN from the drop-

down menu for the organic pump.


13/301

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 theworkspace. 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.


14/301

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...


15/3014

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


16/3015

10. Drag-and-drop a second Switch VALVEMATE Task

onto the work space.

11. Select Valve () from the VALVEMATE drop-down menuand from the Position drop-down menu select #Column.

This ensures that the both VALVEMATEs will be set to the

correct positions for each column.


17/3016

Software

12. Locate the Fraction Collection Settings Task also under

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

13. Fill in the necessary parameters. A slope value of 0 isrecommended (see example below).

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


18/301

15. From the Fraction Well 1 drop-down menu select

#Fraction Well (see example below).

16. Under the Liquid Handler, select the GX-81 Prep Injectionwith Collection Task. Drag-and-drop the task over to the

work space at time 0.06.


19/3018

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


20/301

20. Under Auxiliary, drag-and-drop a Synchronize onto theworkspace. In the Task dialog box enter a time of 0. and

select the GX-81 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 themethod, Waiting for Injection is recommended.

continued...

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 enter

in #WV .


21/300

Software

21. Go to the Detector Task list and drag-and-drop theDetector Autozero Channel Task onto the work space.

Enter in a time of 0.5 minutes.

22. From the Detector Task list, drag-and-drop theStart Data Collection Task onto the work space and

enter a time of 0.5 minutes.

23. From the Detector Task list, drag-and-drop theStop 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 anothersynchronize onto the workspace at time 0.54 min.

Select to synchronize to the end of task for the

GX-81 Prep Injection with Collection Task.


22/301

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

Start Fraction Collection Task onto the workspace andenter a time of #Mass RT 1.0.

26. From the Liquid Handler Task list, drag-and-drop theStop Fraction Collection Task onto the workspace and

enter a time of Mass RT + .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.


23/30

Software

Tip: Variables are used very frequently however many of them are usuallygiven the same values at run time. For example, most gradients are run

from 5 to 5% over the course of 10 minutes. Also, the wavelengths that

are used are commonly 10 and 54 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 5 and make sure that the Show InParent check box is checked.

4. Click OK. When the sample list is opened and this method is selecteda value of 5 will automatically appear in the cell for this variable.

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


24/30

TheGlobalMethodisnowcreatedandcanbesaved.

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

28. Name all of the components. This can be done by clickingthe component and typing in a unique name

(see example below).


25/304

Software

1. Go to the Project Library and click Refresh. The method createdshould 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.

Application Run


26/305

3. When the Application Run screen comes up select the method

from the drop-down menu that you want to run. All thevariables 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 havebeen entered so it can be opened again.


27/306

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 0 can be entered.

If 0 tubes are not available on the bed then no injection will

occur and the sample will be saved.


28/30

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.


29/30

Sample Quotation GX-81 Purification System

Quantity Product # Description

1 210631R20 TRILUTION LC Media v2.0

1 ORACLE10GXE ORACLE 10g Express Edition

1 21063130 TRILUTION LC License - Lietime

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 or 1/8" OD Sample Loops

1 26035411 GX Direct Injection Port or 1.5 mm OD probes

1 26035470 Plumbing Package, Direct Inject1 494400051 5 mL Sample Loop

1 49440010 10 mL Sample Loop

1 49440025 25 mL Sample Loop

1 499483602 30 mL Transer Tubing

1 2507216 Non-septum Piercing Probe (269 x 1.5 x 0.8)

1 2603614112 Lower Probe Holder or 1.5 mm OD probes

1 2603614216 Probe Guide Insert or 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

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

2 331052AB VALVEMATE II GSIOC

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

2 33035255 Plumbing Package (0.030"), Stainless Steel, 10 t

1 2504600 Code 200 Rack

1 25045514 RH1 Rack Heightener

2 250420065 RackCount Non-serpentine Back Let 1-96

5 2504607 Code 207 Rack

1 250420755 RackCount Serpentine Starting Back Let 1-75






30/30

targeted preparative fc

Documents