separtaion of amino acids

7/27/2019 Separtaion of Amino Acids

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ACD/LC Simulator Tutorial 12

2. Separation of Amino Acids

2.1 Objectives

This chapter will familiarize you with the automatic selection of optimal separation conditions.

Practically all analytical techniques, whether they are spectroscopic or otherwise, depend on purityof compound. This means that prior to applying those other techniques, the components must beseparated. An effective separation depends on a large number of parameters. Chromatographicsoftware from Advanced Chemistry Development allows you to modify virtually any parameters toget an effective separation.

2.2 Loading Experimental Data

In this section well work with five separations of amino acids recorded in differentchromatographic conditions.

1. Run ACD/SpecManager (Chrom module).

2. On the General toolbar, click Open/Import and, in the Import dialog box that appears,

specify the location of AAA-Q1.ESP file (placed in the ACD/Labs example folder,\\EXAMPLES\HPLC\GRADIENT\). Click OK.

3. On the general toolbar, click Full and New Window Mode .

Repeat step 2 to load the following files in the separate windows: AAA-Q2.ESP, AAA-Q3.ESP,AAA-Q4.ESP, and AAA-Q5.ESP. Note that the separations are no satisfactorythere are fourchromatograms with two compounds eluted as a single peak (AAA-Q1.ESP, AAA-Q3.ESP, AAA-Q4.ESP, and AAA-Q5.ESP):

AAA-Q1.ESP AAA-Q3.ESP AAA-Q4.ESP AAA-Q5.ESP


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Separation of Amino Acids


And one chromatogram with two poorly resolved peaks (view the Thrand Pro peaks on thechromatogram from AAA-Q2.ESP):

AAA-Q2.ESP

Rs = 0.333

4. Let us make a series of these chromatograms. Select all the chromatographic windows byclicking their tabs holding down SHIFT:

Tip You can also use Select All from the Windows menu to select all the subwindows atonce.

5. From the Series menu, choose Collect Chromatograms and all of them will be placed into

the active window (for the current example, it is aaa-q5.esp).

6. From the Windows menu, choose Unselect All to clear the selection of chromatographicsubwindows.

7. On the Edit menu, choose Chromatograms Locations. In the Set Chromatogram Positiondialog box that appears, specify the offset for chromatograms along the Y axis to 0.07. ClickOK.

8. On the General toolbar, click Show Gradient to display the gradient line for the activechromatogram in a series:

Tip To make a chromatogram in a series active, click it.


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2.3 Transferring to ACD/LC Simulator

Now we can transfer a series of chromatograms to ACD/LC Simulator for optimization of thegradient program:

1. On the Tools menu, choose Transfer to LC Simulator.

2. In the Select Mode dialog box that appears, select the Other Mode option and click OK.

3. In the Modes Manager (Select Mode) dialog box, click New .

4. Create a new optimization mode using linear model for dependence of retention on solvent Bconcentration: in the Modes Manager (Add New Mode) dialog box, specify the followingsettings:

New to 8.0!

5. Click OK in the Modes Managerdialog boxes.


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6. In the Data Input dialog box that appears, switch to the HPLC Parameters tab and view thetransferred chromatograms and their parameters, note that the dwell volume and dead timevalues are the most important parameters for building a model:

The gradient program for each experiment can be viewed in the Experimental Parameters areaon the 1st, 2nd, 3rd, 4th, and 5th tabs:

Note that for each chromatogram the gradient program slope is different: 42/33.6; 54/32.4;63/27.1; 100/28; 100/14.


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2.4 Combining Chromatograms

In this section we are going to combine experimental chromatograms:

1. Switch back to the Combined tab, click Combined and, in the CombineChromatograms dialog box that appears, specify the required options for combining theexperimental chromatograms automatically according to the structures assigning the names ofthe chromatogram whose peaks are completely separated:

2. Click OK to start combining. After it is finished, scroll down the table of peaks.

Note that the nearest of the maximal peaks that have no structures cannot be combinedautomatically so well combine it manually.

3. On the Combined tab, click Combined again and, in the Combine

Chromatograms dialog box that appears, select the Manual option and click OK.

4. The program prompts you whether you want to completely rebuild combined chromatogram.Click No.

5. To combine experimental data for the last peak, click the eighteenth row of the table, and thenclick the nineteenth one. Repeat this step until the data from all of the experimental

chromatograms is combined.

6. Click End Combining .


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7. Click OK in the Data Input dialog box and the combined data appears in the Optimizationswindow:

Note that the minimal resolution is very small for the 1314 pair of peaks:

8. Save the combined data: on the File menu, choose Save and specify the name (e.g.,LC_GRAD_AAA.LC7) and location to which the combined data is to be saved.

2.5 Viewing Correlation

Now well view how the experimental chromatographic data correlates with the data calculatedwith the program, we can also try to improve the correlation.

1. From the Tools menu, choose Peak Model. (Or, on the Top toolbar, click Show/Hide Peak

Model Window .)


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2. Switch to the Experimental/Calculated tab, then point to the chromatogram peaks and notethat experimental and calculated data is not well correlated for all the peaks:

3. On the Top toolbar, click Modes Manager .

4. In the Modes Managerdialog box that appears click Edit and change theretention time model from linear to quadratic one that is more accurate:

5. Click OK in the Modes Managerdialog boxes.

6. Point to the chromatogram peaks, note that the correlation became better though the minimalresolution on the Experimental Conditions panel is indicated as 0.06.

7. On the Top toolbar, click Show/Hide Peak Model Window to hide the Peak Model

window.

Optimizing Gradient Program Manually

Now well optimize the second point of the gradient program for the combined chromatogram.

1. On the toolbar of the Upper subwindow, click Display Gradient Edito r and Resolution Map

Subwindows .

2. On the Options menu, choose Gradient and, in the Gradient Options dialog box thatappears, specify the required settings for optimizations of the second gradient point:


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3. Click OK.

4. On the toolbar of the Upper subwindow, click Move Point Horizontally and move the

second point to approximately 22 minutes.

5. On the toolbar of the Upper subwindow, click Move Point Vertically and move the

second point to approximately 11%.

Note If you cannot approach the integers, edit time and concentration in the gradientprogram table on the Experimental Conditions panel.

6. On the Chromatogram Parameters panel, point to the minimal resolution value, note thatminimal resolution is considerably improved:

On the Top toolbar, click Copy to Report Editor and, in the Report Page Setup dialog

box that appears, specify the parameters to be output. In the ChemSketch window, you canedit the report.

3530252015105

Time, min

0.05

0.10

0.15

0.20

100%

0%

impLys

PheLeuIle

CysMetVal

Tyr

Pro

ThrAla

ArgHis

GlySerGlu

Asp

Min.Rs 0.92 (14, 15 peaks)Min.k' 2.97 (1 peak)Run Time 32.92 minSolvent Used 46.09 ml

No. T, min % B

1 0 0

2 22 13.41

3 35.1 100

4 38.1 100

5 41.1 0

Selecting the optimal conditions manually is a time-consuming process. ACD/LC Simulator allowsyou to specify the suitable conditions for the separation of your compounds and find the optimalgradient program. Later the report we have just created will be used to compare the results ofmanual and automatic optimization.


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2.7 Specifying Suitable Conditions

We can specify the conditions we consider suitable for separation of our mixture. To specify thesuitability options, from the Options menu, choose Suitability. In the Suitability Options dialogbox that appears, specify the minimal and suitable resolution, robustness showing the possibleerror while mixing solvents A and B, run time, retention factor (k), and gradient time (tG):

Note that after clicking OK the general suitability coefficient is equal to zero, i.e., the gradientprogram we have optimized manually does not suit our compounds:


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2.8 Generating Gradient Programs

ACD/LC Simulator allows you to find the optimal gradient program automatically according to thespecified conditions.

1. To specify the options for generation of all the possible gradient program variants, from theTools menu, choose Generate Gradient Programs.

2. Specify the required options in the dialog box that appears. The most important options areincrement step for isocratic, increment step for maximal gradient, gradient maximal, andgradient parts to divide. Note that the more segments for the desired gradient program youspecify, the more time it takes to generate suitable gradient programs:

3. Click O K. After the list of gradient programs is generated, the program informs you that58 gradient program(s) ensuring suitability from 0.6 to 0.6177 were found.

Note The program generates and picks out only those gradient programs that lead to theseparation conforming to all our requirements. At that, in the list, the gradientprograms leading to better separations replace those leading to the worseseparations.

4. Click OK, and a list of generated gradient programs appears in a separate subwindow. Clickany suitability value in the list to view the corresponding gradient program in the GradientEditor subwindow.

2.9 Selecting the Best Gradient Program

In this section the optimal gradient program for separation of our compounds will be selected fromthe list of the automatically generated gradient programs.

1. On the toolbar of the gradient programs list, click Sort Gradient Programs List to display

the corresponding dialog box.


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2. In the Sort By list of the Sort Gradient Programs List dialog box, select Minimal Resolution.

3. In the Parameters area, select the Descending option.

4. In the Naming area, specify the following options:

Note For more information on creating patterns, refer to the online Help.

5. Click OK to apply the specified settings.

6. In the list of generated gradient programs, select the last gradient program with minimalresolution 1.36 and on the toolbar of the gradient programs list, click Delete All Gradient

Programs After Current to keep all the gradient programs with the best minimal

resolution.

7. On the toolbar of the gradient programs list, click Sort Gradient Programs List and in

the dialog box that appears, specify the required options to sort the gradient programs in theascending order of their run times.

8. Select the minimal run time and view the results on the Chromatogram Parameters panel:

9. On the Top toolbar, click Copy to Report Editor and, in the Report Page Setup dialog

box that appears, specify the parameters to be output. In the ChemSketch window, you canedit the report.


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10. Compare the reports for the gradient programs:

!" Manually optimized:

3530252015105

Time, min

0.05

0.10

0.15

0.20

100%

0%

impLys

PheLeuIle

CysMetVal

Tyr

Pro

ThrAla

ArgHis

GlySerGlu

Asp

Min.Rs 0.92 (14, 15 peaks)Min.k' 2.97 (1 peak)Run Time 32.92 min

Solvent Used 46.09 ml

No. T, min % B

1 0 0

2 22 13.41

3 35.1 100

4 38.1 100

5 41.1 0

Manual Optmization

!" Selected from a list of automatically generated gradient programs:

30252015105

Time, min

0.1

0.2

0.3 88.81%

0%

imp

Lys

PheMet

ValTyr

Pro

ThrAla

ArgHis

Gly

SerGluAsp

No. T, min % B

1 0 0

2 18.38 8.88

3 23.53 28.84

4 26.96 38.81

5 29.46 88.81

Min.Rs 1.36 (14, 15 peaks)Min.k' 3 (1 peak)Run Time 31.47 minSolvent Used 44.06 ml

Auto Generation

Note that the minimal resolution is half as better and the separation takes less time for theautomatically generated gradient program. Moreover, the peaks are distributed more evenly onthe chromatogram with automatically generated gradient program.

separtaion of amino acids

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