ChromSword in action. Computer-assisted optimization of HPLC separations.

Examples for users 1

ChromSword Optimization

Beta-blockers

Optimization of the mobile phase methanol concentration

This example illustrates how suitable initial separation conditions can be predicted
without taking into account chemical structures of the analytes and column properties.

Dear chromatographer,

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ChromSword in action. Computer-assisted optimization of HPLC separations. Examples for users 2

This example illustrates how you can carry out a step-by-step optimization procedure of an HPLC
separation using your own PC. Acceptable separation conditions can be found after only a few runs.
Just follow the steps described below and you will see how easily you can solve your separation

problem using ChromSword . While carrying out the optimization procedure in this example,

you will gain valuable experience working with ChromSword in particular and in rapid
computer-assisted method development in general.

Objective: to optimize the separation of a mixture of 9 compounds

Compounds: beta-blockers

1. Metoprolol
2. Carazolol
3. Propranolol
4. Metipranolol
5. Bisoprolol
6. Alprenolol
7. Celiprolol
8. Carvendilol
9. Pafenolol

In this example 9 beta-blockers are separated using a methanol/water mobile phase. To

find the optimum concentration of methanol in the mobile phase, the following steps are
carried out:

1. Two initial separations using different methanol concentration are run (Step 1).
2. Retention, efficiency and peak area data are entered (Step 2).
3. Retention Models for the compounds are simulated (Step 3).

4. ChromSword finds the optimum separation conditions (Step 4).

STEP 1: Initial experiments

 Run 1


Column: LiChroCART Purospher RP18e (250 x 4 mm)
Mobile phase: 60% methanol/50 mM phosphate buffer; pH = 3.00
Temperature: 30°C

 Run 2


Column: LiChroCART Purospher RP18e (250 x 4 mm)
Mobile phase: 50% methanol/50 mM phosphate buffer, pH = 3.00.
Temperature: 30°C

STEP 2: Sample file creation

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ChromSword in action. Computer-assisted optimization of HPLC separations. Examples for users 3

Working procedure:


1. Start ChromSword (Fig. 1).

Click here

Fig. 1 ChromSword: The Sample module showing the blank Compounds page

2. Enter the compound names and save the sample file as BETABL9M.smp. See the on-
line Help (Tutorial, problem 4, method 2) on how to create a sample file.
3. Click the Open button, to open the Open file dialog box, select the BETABL9M.smp
file and click the OK button to open the sample file (Fig. 2).

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ChromSword in action. Computer-assisted optimization of HPLC separations. Examples for users 4

Fig. 2 The Compounds page with the BETABL9M.smp file opened

4. Open the Concentration module by clicking the Concentration button on the upper
button bar (Fig. 3).

Fig. 3 The Runs page of the Concentration module

 Input 1: Experimental zero time value (retention of 1 µl of 0,01% KNO3) (see

the experimental value in the Zero Time edit field).

 Input 2: Retention time, efficiency and peak area data for the each compound
of the mixture for two runs and Information about the column and the eluent
being used.

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ChromSword in action. Computer-assisted optimization of HPLC separations. Examples for users 5

5. You can edit or browse data of existing runs or add data for a new run. Browse the
data using the Current Run scroll box on the Runs page. To edit (or only to browse
experimental values) the retention time, efficiency and peak area data for the First
Run use the following procedure:

- Set the Run # to 1 in the Current Run scroll box.

- Click the Edit button to open the Run Editor dialog box (Fig. 4). (To enter data
for a new run, click the Add button).

Fig. 4 The Run Editor dialog box

- Enter (or only browse experimental values) the methanol concentration for Run
1, the retention time, efficiency and peak area data and click the OK button. (If
you are only browsing, click the Cancel button).
- To browse the data for the second run, set the Run # to 2 in the Current Run
scroll box.

STEP 3: Simulating the Retention models

1. Open the Model page in the Concentration module by clicking on the Model button
in the upper button bar. Linear retention models ln k = ln k 0 + a0 (C) will be simulated
for all compounds in the limits of 50 - 60% methanol (interpolated region).
2. Check the Extra check box to display the Retention Models plots in the extrapolated
region (Fig. 5).

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ChromSword in action. Computer-assisted optimization of HPLC separations. Examples for users 6

Fig. 5 The Model page in the Concentration module in extrapolation mode

STEP 4: Selecting the optimum conditions

1. Open the Pairs resolution page to simulate the Pair Resolution Map (Fig. 6) and
analyse it.

Fig. 6 The Pairs Resolution page of the Model page showing the simulated Pair
Resolution Map

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ChromSword in action. Computer-assisted optimization of HPLC separations. Examples for users 7

From the Pair Resolution Map we can see that a resolution of more then 1.5 can be
obtained for a methanol concentration region lower than 50%. In practice, a resolution
value of 2 is preferred in order to develop robust methods. A resolution of more then 2 can
be obtained in a methanol concentration region lower than 47% (Fig. 6).

2. Move the cursor (use the Left and the Right buttons of the keyboard, or just drag the
cursor) to the 45% methanol position, where the pair resolution value is 2.25 (Fig. 6)
and then open the Chromatogram page to simulate the chromatogram (Fig. 7).

Fig. 7 The Chromatogram page showing the simulated chromatogram

 Conclusion: The simulated chromatogram is acceptable.

3. To find separations condition for which the peaks in the chromatogram are evenly
spaced, simulate the Overall Resolution Map (The Overall Resolution Page) (Fig.
8).

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ChromSword in action. Computer-assisted optimization of HPLC separations. Examples for users 8

Fig. 8 The Overall Resolution page

 Conclusion:

- The Overall Resolution function has a maximum value in the region of 40 -

46% methanol.
- A 45% methanol concentration can provide acceptable resolution, retention, as
well as a regular chromatogram.

FINAL CONDITIONS SELECTED


Column: LiChroCART Purospher RP18e (250 x 4 mm)
Organic modifier: 45% methanol
Water Component: 50 mM phosphate buffer, pH = 3.0
Isocratic elution
Temperature: 30°C
Flow rate: 0.5 ml/min
Analysis time: 22 - 25 min

 Experiment

Run 3 is now carried out using the following conditions:

Mobile phase: 50 mM phosphate buffer, pH = 3.00, 45% methanol

Temperature: 30°C

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ChromSword in action. Computer-assisted optimization of HPLC separations. Examples for users 9

1.0 7 2 3

5
1 8
9
4 6

0.5

0.0
0 10 20
R E T E N T I O N (min)

Fig. 9 The experimental chromatogram

 Conclusion: The compounds have been well separated. The predicted and
experimental chromatograms show no substantial differences (compare Figs. 7 and 9).

We can also use the data for Run 3 to obtain more reliable linear models and also to
simulate quadratic retention models. The data have already been entered and you only
need to actualize them to compare the two types of models.

To actualize the data for the third run, carry out the following steps:

1. Open the Runs page of the Concentration module (Fig. 3).

2. Set the Run # to 3 in the Current Run Scroll box.
3. Click the Edit button to open the Run Editor dialog box (Fig. 4).
4. Open the Actualize page in the Run Editor dialog box (Fig. 10).
5. Click the Actualize All button and click the OK button to actualize the data for
Run 3.

Fig. 10 The Run Editor Actualize page

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ChromSword in action. Computer-assisted optimization of HPLC separations. Examples for users 10

6. Click the Model button to open the Model page (Fig. 11)
7. Set the power 2 in the Power list box to fit the data by the quadratic models.

Fig. 11 The Model page showing the plots of the quadratic models

8. Open the Pair Resolution page (Fig. 12).

Fig. 12 The Pair Resolution Map for the quadratic retention models

9. To compare the two models, set the power to 1 in the Power scroll box (Fig. 13).

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ChromSword in action. Computer-assisted optimization of HPLC separations. Examples for users 11

Fig. 13 The Pair Resolution Map for the linear retention models

Conclusion: The quadratic model predicts that the region of the optimum condition is less
wide than predicted by the linear model and 45% methanol is therefore the optimum
concentration.

Dear colleague!

We have guided you through this optimization procedure. In time, when you gain

more experience in computer assisted method development using ChromSword
you will find other ways and other separation conditions using the experimental
data for this example. The steps described here is not a definitive procedure but
rather a set of guidelines to get successful results quickly, working with a stand-alone
PC rather then using an HPLC data system.

MERCK