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ChromSword in action. Computer-assisted optimization of HPLC separations. Examples for users 1

ChromSword Optimization

Aromatic amines

1. Optimization of the mobile phase acetonitrile concentration

2. Temperature Optimization

This example illustrates how to optimize a separation starting from the structural
formulae of the analytes and data of the first run. The separation is then further
optimized using data of subsequent chromatographic runs.

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

Dear chromatographer,

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 15 compounds

Compounds: aromatic amines

1. 2,4-toluylendiamine
2. o-anisidine
3. o-toluidine
4. 4,4´-oxydianiline
5. 2,4,5-trimethylaniline
6. 2-aminonitro-4-toluene
7. 3,3´-dimethoxybenzidine
8. 2-naphthylamine
9. 4-chlor-o-toluidine
10. 3,4-dichloraniline
11. 4-aminobiphenyl
12. 4-aminoazobenzene
13. 3,3´-dichlorobenzidine
14. N-methylaniline
15. N,N-dimethylaniline

In this example the mobile phase acetonitrile concentration and the column temperature
are optimized. The following initial information about the sample was available before

starting the ChromSword optimization:

- structural formulae of the compounds

- retention data for the first test run
- separation conditions for the first run

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Column: LiChroCART Purospher RP18e (250 x 4 mm)
Mobile phase: 40% acetonitrile/3.4 mM phosphate buffer, pH = 5.0
Temperature: 22°C

 Problems:

1. The retention factors for the compounds are too low (experimental k = 0.4 - 5)
2. Overlapping peaks: 3,3’-dimethoxybenzidine/o-toluidine; N,N-dimethylaniline/
3,3’-dichlorobenzidine.

 Conclusion: Try to optimize of mobile phase acetonitrile concentration to improve

the separation.

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

STEP 1: Sample file creation

Input: Structural formulae of the compounds: See on-line Help (Tutorial, Problem 1)
on how to draw the structural formulae of the compounds and how to create a sample
file. The following structures were drawn and the sample file was saved as
ARAMTE3.smp:

NH2
NH2 NH2
O

NH2

2,4-toluylendiamine o-aninsidine o-toluidine

NH2

O
H 2N NH2

4,4'-oxydianiline 2,4,5-trimethylaniline

NH2 O

H 2N NH 2
NO2
O
2-aminonitro-4-toluene 3,3'-dimethoxybenzidine

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NH 2 NH2

NH2
Cl
Cl Cl
2-naphthylamine 4-chlor-o-toluidine 3,4-dichloraniline

NH2

4-aminobiphenyl

N NH2
N

4-aminoazobenzene

Cl Cl

H 2N NH2

3,3'-dichlorobenzidine

HN N

N-methylaniline N,N'-dimethylaniline

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Working procedure:

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1. Start ChromSword (Fig. 1).

Click here

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

2. Click the Open button, to display the Open file dialog box, select the
ARAMTE3.smp file and click the OK button to open the sample file (Fig. 2).

Fig. 2 The Compounds page with the ARAMTE3.smp file opened

3. Press F11 to open the Molecular Inspector and browse the structures in the file
(Fig. 3). To display a structure, click the name of a compound in the Compound list.

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Fig. 3 The Molecular Inspector with a selected compound

STEP 2: Evaluating compound parameters based on data for Run 1 and

conditions selected for Run 2

To select a suitable acetonitrile concentration for Run 2, data of Run 1 can be used. On
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the basis of structural formulae and Run 1 data, ChromSword evaluates the
compound parameters and predicts a suitable acetonitrile concentration to obtain the
desired retention. Two steps are carried out:

A. Evaluating compound parameters

B. Selecting separation conditions for Run 2

A. Evaluating compound parameters (The First fitting)

Note: If you are not using the file for the first time, see the Appendix

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Appendix:
If you are not using this file the first time, the compound parameters may already be corrected. To
determine this, open the Molecular Inspector (Press F11) and browse the compounds. The Parameters
field in the Molecular Inspector contains information on whether the compound parameters were
determined from structural formulae or from experimental data. Main. rul indicates that the compound
parameters were calculated from structural formulae. Eint Fitted indicates that the energy of interaction
of a compound with water (Eint) was corrected using experimental data. EintMolVolFitted indicates
that both Eint and the partial molecular volume of a compound in water (MolVol) were fitted, using
experimental retention data and column parameters.

To repeat the procedure from the very beginning you must restore the compound parameters (if
they were corrected). To do this, carry out the following steps:
1. Open the Fit Structure Parameters page.
2. Click the Recalculate button.
3. Select „All“ from the menu in the Recalculate dialog box.

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4. Click the OK button to recalculate compound parameters to the initial values from structural
formulae
______________________________________________________________________

1. Open the First guess page (Fig. 4).

Cursor

Fig. 4 The First Guess page

2. Click the Sorbent button, to open the Choose Sorbent dialog box (Fig. 5). The
Choose Sorbent box contains a data base of a large number of reversed phase
columns in combination with acetonitrile- or methanol-based eluents and the specific
characteristics for each column/eluent combination.

Fig. 5 The Choose Sorbent dialog box

3. Select the column type: Purospher RP18e A (Purospher RP18e/acetonitrile/buffer

eluent combination) and click the OK button. Parameters for the Purospher

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RP18e/acetonitrile-buffer combination will be loaded as the current selection and

displayed in the Sorbent and the Eluent fields.
4. Move the cursor on the plot of ln k versus modifier concentration to the position of
40% acetonitrile (Fig. 4), which corresponds to the modifier concentration used in
the Run 1. (To move the cursor, use the Left and the Right buttons of the
keyboard).
5. Click the Send to fit button (the lower right-hand button) to open the Select Run
dialog box (Fig. 6).

Fig. 6 The Select Run dialog box

6. Set the Run # to 1 in the Select Run scroll box, click the OK button to send column
parameters for the first correction.
7. Open the Fit Structure Parameters page (Fig. 7).

Fig. 7 The Fit Structure Parameters page

 Input 1: Experimental zero time value (retention of 1 µl of 0.01% KNO3) (you

can see an experimental value in the Zero Time edit field).

 Input 2:Retention time, efficiency and peak area for the each compound of the
mixture.

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You can edit data of existing runs or add data for a new run. To edit (or only to browse
experimental values) the retention time, efficiency and peak area data for the first run
continue the procedure as follows:

8. Click the Edit button to open the Run Editor dialog box (Fig. 8). (To enter data for
a new run click the Add button.)

Fig. 8 The Run Editor dialog box

9. Enter (or only browse experimental values) data for the retention time, efficiency
and peak area and click the OK button. (If you are only browsing, click the Cancel
button).
10. Check the Eint check box to select the energy of interaction of the
compounds with water, as the first parameter to be corrected (Fig. 7).
11. Click the Fit button to display the Fit Compounds dialog box (Fig. 9).

Fig. 9 The Fit Compounds dialog box

12. Click the OK button for fitting the Eint for all compounds. A Table with the
evaluated compound parameters will appear (Fig. 10).

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Fig. 10 The Fitted Parameter(s) values table

13. To accept fitting data click the OK button and then confirm the fitting (click the Yes
button).
14. Return to the First Guess page (Fig. 4).

B. Selecting separation conditions for Run 2

To select suitable separation conditions for Run 2, try to find the mobile phase
acetonitrile concentration that will provide values of k in the range of 0.5 - 15. To do
this, carry out the following steps:

1. Move the cursor to the position 30% acetonitrile concentration (Fig. 4).
2. Click the Select button to display the simulated chromatogram in the Methods Base
page (Fig. 11).

Fig. 11. The Methods Base page showing the simulated chromatogram.

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3. Click the first peak to display the predicted retention factor (k = 0.49).
4. Click the last peak to display the predicted retention factor (k = 12.09).

 Conclusion: A mobile phase containing 30% acetonitrile is acceptable for Run 2

(k value range: 0.45 - 12).

 Experiment:

Run 2 is carried now out under the following conditions:

Mobile phase: 30% acetonitrile in 3.4 mM phosphate buffer, pH = 5.0.

Temperature: 22°C.

Results:

1. Experimental k range: 0.45 - 11

2. Overlapping peaks: 2,4,5-trimethylaniline/4-chloro-o-toluidine

 Conclusions:

1. Retention is acceptable. Predictability is OK.

2. Further optimization of the organic modifier concentration is needed.

STEP 3: Selecting conditions for Run 3

To select a suitable acetonitrile concentration for Run 3, data for Run 1 and Run 2 can
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be used. On the basis of these data, ChromSword will correct the evaluated compound
parameters (the Energy of Interaction with Water, Eint and the Molecular Volume,
MolVol) and will precisely predict separation under different conditions. The next two
steps are carried out:

A. Correcting compound parameters (step 4)

B. Selecting separation conditions for Run 3 (step 5).

STEP 4: Correcting compound parameters (The Second Fitting)

1. Return to the First guess page (Fig. 4).

2. Check Sorbent and Eluent dialog boxes: these must show the Purospher RP18e
column and the acetonitrile eluent that you have used both for Run 1 and Run 2.
3. Move the cursor on the plot of ln k versus modifier concentration to the 30%
acetonitrile position that corresponds to the modifier concentration used in Run 2.
4. Click the Send to fit button to display the Select Run dialog box (Fig. 6).
5. Set the run # to 2 in the Run scroll box, click the OK button to send the column
parameters for the next correction.
6. Open the Fit Structure parameters page ( Fig. 7).
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7. Click the Add button to open the Run Editor dialog box (Fig. 8).

 Input: Retention, efficiency and peak area data for Run 2 (30% acetonitrile). To
browse the data for Run 2, set the run # to 2 in the current Run scroll box.

8. Return to the Fit Structures Parameters page.

9. Check the Mol Volume check box to select the partial molecular volume of the
compounds as the second parameter to be corrected. (Now both the Eint and the
MolVolume check boxes must be checked) (Fig. 7).
10. Click the Fit button and then click the OK button for fitting both the Eint and the
Mol Volume for all compounds. A Table with evaluated compound parameters
appears (Fig. 12).

Fig. 12 The Fitted Parameter(s) Values table

11. To accept the fitting data, click the OK button and then confirm the fitting (click the
Yes button).
12. Return to the First Guess page (Fig. 4).
13. Click the Model button to open the Model page. (Fig. 13).

Fig. 13 The Model page

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14. Open the Pair Resolution page to simulate the Pair Resolution Map (Fig. 14) and
analyse it.

Fig. 14 The Pair Resolution page, showing the simulated Pair Resolution Map

 Conclusion: The predicted pair resolution values in the region of 0 - 100%

acetonitrile at 22°C are not high enough for complete separation (max. value is -
1.29).

 Recommendation: Try optimizing the temperature to improve the separation.

STEP 5: Temperature optimization

1. To find the optimum temperature, an additional run at 43 °C is carried out. To

browse retention and efficiency data for two temperatures used as Input open the
Sample Compound page (Fig. 2). Open the Temperature module (Fig. 15) by
clicking the Temperature button on the upper button bar.

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Fig. 15 The Runs page of the Temperature module

 Input: Retention and efficiency data for two runs at different temperatures and
constant acetonitrile concentration (30%):

1. 22°C (Run 1). Retention data of Run 2 from the Concentration page were
used as Run 1 data for temperature optimization.
2. 43°C (Run 2).

2. Open the Model page in the Temperature module. Check the Extra check box to
display the plots of the retention models in the extrapolated region (Fig. 16).

Fig. 16 The Model page opened from the Temperature module

3. Open the Pairs Resolution page and analyse the Pair Resolution Map (Fig. 17).

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49°C 38.3°C

Fig. 17 The Pairs Resolution page showing the Pair Resolution Map

 Conclusion: The best resolution can be obtained in the temperature range of 38

- 49°C where the resolution is higher than 1.5.

4. Move the cursor to the 40°C position and then open the Chromatogram page to
simulate the chromatogram (Fig. 18a)

 Conclusion: The simulated chromatogram is OK. A test run should be carried

out using these separation conditions.

Simulated Chromatogram
1,100 4 6 10
1,00 7 14 15

0,75
5
11
0,50 1 8 13
2 3 12
9
0,25

0,000
0 5 10 15 20 25 30
Time, min

Fig. 18a

FINAL CONDITIONS SELECTED

Optimized parameters:

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Column: Purospher RP18e, 250 x 4 mm

Mobile phase: 30% acetonitrile/3.4M phosphate buffer, pH=5.0
Isocratic elution
Temperature: 40°C
Flow rate: 1.0 ml/min
Analysis time: 30 - 35 min

Experimental chromatogram:

Fig. 18b

Final conclusion:
The compounds have been well separated. Predicted and experimental chromatograms
do not show any substantial differences. (Fig. 18a and 18b).

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.

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