ACD/Method Development Suite

Version 8.0 for Microsoft Windows

Tutorial

Hands-on Chromatographic Method Development Using ACD/Labs Software

Advanced Chemistry Development, Inc.

Copyright 20012004 Advanced Chemistry Development, Inc. All rights reserved. ACD/Labs is a trademark of Advanced Chemistry Development, Inc. Microsoft and Windows are registered trademarks of Microsoft Corporation in the United States and/or other countries Copyright 2004 Microsoft Corporation. All rights reserved. IBM is a registered trademark of International Business Machines Corporation Copyright IBM Corporation 1994, 2004. All rights reserved. Adobe, Acrobat, PDF, Portable Document Formats, and associated data structures and operators are either registered trademarks or trademarks of Adobe Systems Incorporated in the United States and/or other countries Copyright 2004 Adobe Systems Incorporated. All rights reserved. All the other trademarks mentioned within this tutorial are the property of their respective owners. All trademarks are acknowledged. Information in this document is subject to change without notice and is provided "as is" with no warranty. Advanced Chemistry Development, Inc., makes no warranty of any kind with regard to this material, including, but not limited to, the implied warranties of merchantability and fitness for a particular purpose. Advanced Chemistry Development, Inc., shall not be liable for errors contained herein or for any direct, indirect, special, incidental, or consequential damages in connection with the use of this material.

Table of Contents
Introduction .................................................................................................................... 1 STEP 1. Opening Database.......................................................................................... 3 STEP 2. Drawing Structures ......................................................................................... 4 STEP 3. Searching a Database .................................................................................... 4 STEP 5. Calculating the Prediction Equation................................................................ 7 STEP 6. Predicting New Compounds ........................................................................... 8 STEP 7. Seeking the Best pH..................................................................................... 10 STEP 8. Archiving Successful Methods...................................................................... 11 STEP 9. Selecting Another Column............................................................................ 12 Conclusion ................................................................................................................... 13

ACD/Method Development Suite

Tutorial

Introduction
The combination of searchable database and retention time prediction constitutes a powerful aid to method development. Chromatographic databases of successful separations, no matter how extensive, rarely contain the exact mixture you would like to separate. In spite of this, such databases still contain valuable chemical information that can allow you to propose good initial methods to try. For example, you can select a method based on functional group similarities to your mixture. Better yet, ACD/Labs predictions allow you to simulate in minutes how your compounds would behave under this method. Together, ACD/Labs database and prediction software can dramatically speed up method development by allowing you to make chemically intelligent choices. How can you leverage information for a few chemical compounds to maximize returns by reducing method development time? A great deal of HPLC method development is still done by trial and error; you can save time by reducing the amount of guesswork. ACD/Labs software uses stored chemical information and fundamental properties predictions to guide your selection toward a viable separation method on your first try. The procedure is outlined below.
Important To carry out the following method development tutorial, you MUST have

ACD/LC Simulator and ACD/SpecManager (CHROM module) installed in the same ACD/Labs software folder. Advanced Chemistry Development (ACD/Labs) allows you to easily use previously developed methods, or to develop new chromatographic methods using chromatographic application database and our liquid chromatography simulator. This document is organized as a tutorial. Throughout this tutorial, we will use Phenoxymethylpenicillin and some related impurities whose contents are regulated by British Pharmacopoeia (version 5.0, 2001). The goal will be to find a method that achieves good separation between Phenoxymethylpenicillin and the five impurities shown below.

O O

NH

H S N

H2N O

H S N O

O O HO

HO
(2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia1-azabicyclo[3.2.0]heptane-2-carboxylic acid

Phenoxymethylpenicillin

O O

OH

O O

NH O

S N H OH O HO

Phenoxyacetic Acid

(2R,4S)-2{carboxy[(phenoxyacetyl)amino]methyl}-5,5dimethyl-1,3-thiazolidine-4-carboxylic acid

ACD/Method Development Suite

Tutorial

HO NH O O H S N O HO
(2S,5R,6R)-6-{[(4hydroxyphenoxy)acetyl]amino}-3,3-dimethyl-7oxo-4-thia-1-azabicyclo[3.2.0]heptane-2carboxylic acid O O H NH

S N H HO O

(2R,4S)-5,5-dimethyl-2{[(phenoxyacetyl)amino]methyl}-1,3thiazolidine-4-carboxylic acid

This tutorial will start with the traditional approaches, but then will go beyond these approaches by using ACD/Labs prediction software. By integrating experimental results with our high quality octanol-water partition coefficient algorithm (logD), we will provide chemically reasonable information suited to the method of interest. This will allow you to tweak your experimental parameters even before you run your first chromatogram. This should reduce the number of runs required before finding an optimized method.

ACD/Method Development Suite

Tutorial

STEP 1. Opening Database

First, let us open a database of successful chromatographic methods. We will use a database built from methods proposed by the chromatographic column manufacturers themselves. The APPL_DB.ND5 applications database is included in ACD/ChromManager. But we also allow you to easily build your own application database. ACD/Labs databases are searchable according to chemical structure and many other parameters.
To open the chromatographic applications database 1. Run ACD/SpecDB.

2. From the Database menu, choose Open. The Open Database dialog box appears. 3. Specify the location of APPL_DB.ND5 (similar to \\ACD8\DATABASE\), and click Open:

Note

The number of records in the ACD/Chromatography Applications Database is constantly being updated.

For more information about the options available in the Database window, and on the basic operations, refer to the ACD/Spec DB Reference Manual and Tutorial.

ACD/Method Development Suite

Tutorial

STEP 2. Drawing Structures

To draw chemical structures for the mixture of compounds of interest, follow these steps: 1. Click ChemSketch window. on the Window Switching bar to switch to the ChemSketch

2. Draw the structures of Phenoxymethylpenicillin and its impurities. A full description of ChemSketch options is available in the separate Reference Manual and Tutorial.

STEP 3. Searching a Database

Does the chemical structure of Phenoxymethylpenicillin exist in the application database? Performing a structure search will answer that question. 1. On the Structure toolbar, click Select/Move Phenoxymethylpenicillin to select it. 2. On the Window Switching bar, click Database , and then click next to .

3. In the Database Window dialog box that appears, select the Search Structure in the Database(s) option and click OK. The search yields 6 hits in the database. We will restrict the number of records by looking for the records containing prediction equation.
Tip Multiple databases can be searched. You can search your own database at the

same time you are searching the ACD/Chromatographic Applications Database. From the Search menu, choose Define Multiple DB List, and define the list of databases to be searched. For more information, refer to the ACD/SpecDB Reference Manual. 4. From the Search menu, choose User Data. 5. In the Search Data dialog box that appears, specify the following options:

6. Click OK to start searching and retrieves 4 hits. 7. From four chromatograms, select the one having a quite good prediction statistics (R = 0.9641, StD = 0.12) and the greatest number of structures (n = 9) used for calculation of the prediction equation. This is a record with ID = 26981.

ACD/Method Development Suite

Tutorial

8. To view the structures attached to each eluting peak and obtain a detailed view of the separation conditions, in the Database window, double-click the Plot subwindow to transfer the data to the chromatogram processor:

9. In the Processor window, point to each peak in turn. Note that the chemical structures belonging to the peaks appear in the Structure subwindow. (To display the Structure window, on the General toolbar, click Show Chemical Structure 10.From the Edit menu, choose Chromatogram Parameters. OR Click Edit/Show Chromatogram Parameters on the General toolbar. )

ACD/Method Development Suite

Tutorial

The HPLC Parameters dialog box appears. It contains sample data, instrumental data, and elution data, as well as the column parameters:

Note that this data is very close to the requirements of the British Pharmacopoeia for Phenoxymethylpenicillin, version 5.0, 2001: The chromatographic procedure may be carried out using:
a column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 m), as mobile phase at a flow rate of 1.0 ml/min: Mobile phase A. Mix 10 volumes of phosphate buffer solution pH 3.5 R, 30 volumes of methanol R and 60 volumes of water R, Mobile phase B. Mix 10 volumes of phosphate buffer solution pH 3.5 R, 35 volumes of water R and 55 volumes of methanol R, as detector a spectrophotometer set at 254 nm.

ACD/Method Development Suite

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If you are satisfied with the chosen method, then we can find out how your sample mixture will separate in the selected conditions. Otherwise, you can switch back to the database and choose another method.
Tip If you do not have the GROM-SIL ODS-7 pH column at hand, you can use

ACD/Column Selector to find a column similar to the aforementioned one for your experiment. For more information, refer to Step 9.

STEP 5. Calculating the Prediction Equation

Prior to predicting retention times for new compounds, it is required to transfer the experimental chromatography data from ACD/ChromManager to ACD/LC Simulator and generate a prediction equation: 1. On the General toolbar, click Transfer to LC Simulator . The program switches to the Optimizations window of ACD/LC Simulator and displays the Select Mode dialog box. In this dialog box, select the Prediction (Single) option:

2. Click OK, this will transfer the experimental structures, retention times, peak width, dead time, and pH to ACD/LC Simulator and open the Data Input (Prediction) dialog box with calculated prediction equation (this equation was stored in APPL_DB.ND5):

The calculated prediction equation will be used to extrapolate retention times for our target compounds, and uses property calculations based on their chemical structures.

ACD/Method Development Suite

Tutorial

3. Click OK in the Data Input (Prediction) dialog box, the chromatogram will be displayed in the Optimizations window.

STEP 6. Predicting New Compounds

So far, we have dealt only with structures from the archived method, but we have not yet dealt directly with the chemicals of interest: Phenoxymethylpenicillin and its impurities. We will now use the prediction equation to calculate the retention time of our new compound in the selected conditions. 1. On the Window Switching bar, click ChemSketch window. 2. Select the structures of Phenoxymethylpenicillins impurities. 3. On the Window Switching bar, click Optimizations . The structures will be transferred to LC Simulator, and their retention times will be calculated according to the prediction equation: to switch to the ChemSketch

4. To focus just on the peaks belonging to Phenoxymethylpenicillin and its impurities, we are going to exclude the original peaks from view. Click Data Input .

ACD/Method Development Suite

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5. In the View column, click the next to the experimental values used in the training to change it to the x (except for Phenoxymethylpenicillin). The peaks marked with x will be hidden from view:

6. Click OK. So now only the peaks of the Phenoxymethylpenicillin and its impurities are shown. The following figure shows the results. Peak named Phenoxymethylpenicillin is sufficiently resolved with the nearby peak structure 5 (view Pre Rs/Post Rs data, the resolution with previous and next peaks, outlined with red):

This separation is considered suitable.

ACD/Method Development Suite

Tutorial

STEP 7. Seeking the Best pH

As we are interested only in pure Phenoxymethylpenicillin, we can plot resolution on the resolution map for the Phenoxymethylpenicillin peaks. Click Table of Peaks in the Upper subwindow and in the Resolution Map Control column designate the peak belonging to Phenoxymethylpenicillin as a peak of interest () and all of the other peaksas interference peaks (x):

As you switch to the resolution map display in the Upper subwindow, ACD/LC Simulator calculates minimal resolution (resolution for the least-resolved pair of peaks) and displays it in the Chromatogram Parameters panel, only for Phenoxymethylpenicillin and its nearest neighbors:

Looking at the resolution map (the curve expressing dependence of minimal resolution on pH), one can notice that a slight shift in column pH (this can happen during the experiment) will not dramatically influence the separation of Phenoxymethylpenicillin from its impurities and the minimal resolution will still remain reasonable. The next step in the method development process is likely the optimization of the experimental chromatogram; please see the LC Simulator online Help for a description of this feature. Once you are comfortable with the interface, the selection of a starting point for method development should take less than five minutes to do. This is certainly a shorter time period than equilibrating a system and obtaining an actual chromatogram.

ACD/Method Development Suite

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STEP 8. Archiving Successful Methods

Once you have achieved a satisfactory resolution, the last step is to archive the method for later retrieval. The predicted chromatography data can be saved, updated to the user database, or printed and then used for comparison with the experimental data. To archive the chromatogram you should first transfer it to ACD/ChromManager by clicking Copy
to SpecManager

on the Top toolbar to transfer predicted data to ACD/ChromManager:

Important The New Window mode should be activated to display the optimized

chromatogram in a new window.

To save the results on disk: From the File menu, choose Save and specify the name and location to which the predicted chromatogram is to be saved. OR From the File menu, choose Export to export the obtained chromatography data to ASCII (.TXT) and NetCDF (.NC, .CDF) formats, if desired. To update the chromatogram to the database: First open or create a user database, switch to ACD/ChromManager, and then click Update Database on the General toolbar. For more information, see the ACD/Spec DB Reference Manual and Tutorial.
Tip User data fields are shown to the right in the Parameters subwindow of the Database

window. You can define your own fields, and you can semi-automate the introduction of these fields using ACD/Forms Manager (for more information on creating database forms refer to the separate guide).

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To create a professional report for the method: From the File menu, choose any of the Print commands, and specify either the objects that should be printed (chromatogram, tables, user data, etc.) for the standard report, or specify an .SK2 file where the objects that are to be included in the report (based on a template) are specified. For more information on printing reports, refer to the ACD/ChromManager online Help.
Tip The report can also be exported to PDF format using the Export to PDF command

from the Edit menu.

STEP 9. Selecting Another Column

Let us assume that you have no GROM-SIL ODS-7 pH column at hand. Then, you can use ACD/Column Selector to find a column similar to the aforementioned one for your experiment. To find the columns similar to GROM-SIL ODS-7 pH, follow the steps: 1. Switch to the ChemSketch window. 2. On the General toolbar, click Column Selector
Note

ACD/Column Selector should be previously installed. For more information, refer to the ACD/Column Selector Users Guide. 3. In the Choose Mode dialog box that appears, select the Compare Column vs List option and click OK. 4. In the Pick the Target Column from the List box of the Select Column to Compare Against the Rest of the List dialog box that appears, select GROM-SIL ODS-7 pH and click OK. The program displays the list of columns that most closely match your selected column. The columns with the lower column difference factor (CDF) are those that best match your chromatographic column:

Tip You can not only locate the most similar columns, but you can also find the most

dissimilar one.

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Conclusion
One of the greatest benefits of ACD/LC Simulators Prediction mode is that you do not have to attempt an injection in order to assess the value of a method. Upon predicting the retention times of the compounds, you can make some changes immediately (you can find optimal pH value for sufficient separation of compounds or view how pH influences separation). Using application databases is a good way of keeping method information on your old separation, and to retrieve this knowledge in the future. In this tutorial, we have not covered all of the ACD/Labs tools available to help with your separations. For example, if you run two trials on your instrument at different gradients, we can help you predict the best gradient to separate your compounds. Optimizing your chromatogram parameters to achieve a desired separation is a natural extension to the method development process. Using Optimization mode can help you develop faster and more robust separations. For more information on the options available in ACD/Method Development Suite, refer to the Reference Manuals of ACD/LC Simulator, ACD/GC Simulator, and ACD/ChromManager.

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