How to determine the molecular size

Introduction	This section describes the technique for measuring molecular size in detail.

Before you start

Before you create the molecular size curve, you need to do the following:

Perform chromatographic runs with an appropriate standard with components of known molecular size. The standard should contain components of sizes that extend over the range that is expected in the sample. If you are using many components, it may be better to split them into two or more standard runs.
Peak integrate the curves to produce peak tables. The standard curves must all use the same X-axis base unit during the integration. Volume is the recommended unit for molecular size determination.
Save the results.

The Molecular size table dialog box

This dialog box is used to select the peaks that will be used to produce the molecular size curve. Each curve and its peak table name is color coded. All the available peaks for all the curves are listed together in the Retention/Mol.size table.

Triangles show that a peak has been selected. The name of its source peak table is shown above the curve window. This is useful when you wish to know which peak has been selected of two closely spaced peaks from different peak tables.

The illustration below shows the Molecular size table dialog box.

How to create and save a molecular size table

The table below describes how to create and save a molecular size table in the Evaluation module.

Step	Action
1	Open a result file and select Mol. Size:Edit Mol. Size Table:New. Result: The New Molecular Size Table dialog box opens.
2	Double-click the result file in the Select peak table(s) list. Select the source chromatogram on the Source chromatogram droplist.
3	Highlight a peak table that was prepared from the standard in the source Peak table(s) list and click the Select button.
4	Repeat step 3 to select more peak tables. Note: The runs must all have been made under the same conditions.
5	To deselect a table, highlight the table in the Peak table(s) list to the right and click the Remove button.
6	Repeat steps 2 to 4 to select peak tables from other result files.
7	Type the appropriate size measurement unit in the Molecular size unit label field (default kDa). Click OK when the Peak table(s) list to the right contains all the required peak tables. Result: The Molecular size table dialog box opens.
8	Use one of the following ways to select a peak: Click the peak in the curve. Click the peak entry in the Retention/Mol. size table.
9	Double-click in the Mol. size column cell and type the known molecular size from the standard.
10	Repeat step 8 and 9 for all components of known molecular size.
11	To remove unwanted entries, click the peak entry in the table and click the Exclude button.
12	Select the appropriate curve model in the Curve model field (see "The molecular size curve" below).
13	Click the Save as button. Result: The Save molecular size table dialog box opens.
14	Choose if the table is to be globally accessible to any user or restricted to your personal user ID. The default is global. Type a name in the Molecular size table name field. Click OK.

The molecular size curve

The molecular size curve shows the relationship between molecular size and the corresponding retention. The curve is plotted from the Retention/Mol. size data that you have typed in the table as described above. Before this can be done, a curve model is needed, which describes the relationship between molecular size and retention. Each of the peaks selected is represented by a point in this curve, which is drawn according to the best fit that can be achieved using the selected model. Select one of the available models in the Curve model field:

Linear
Linear (logMw) (Theoretically, this is the best choice.)
Quadratic
Quadratic (logMw)
Point to point
Point to point (logMw)

Molecular size Statistics

With the exception for the two point-to-point models, the molecular size curves can be expressed as mathematical expressions. The expressions and related items can be viewed in the Statistics dialog box.

Click the More button in the Statistics field of the Molecular size table to open the dialog box.

The expression is shown at the top of the window, followed by the values for the constant that it contains.

Statistical reference values

The correlation value (only for linear models) should be as close to -1.00 as possible.
The explained variance value should be as close to 100% as possible.

Note: Explained variance values are usually high. A value of 90% indicates a very poor model.

How to open an existing table

The table below describes how to open an already existing molecular size table for editing in the Evaluation module.

Step	Action
1	Select Mol. Size:Edit Mol. Size Table:Open. Result: The Open molecular size table dialog box opens.
2	Select a molecular size table from the Molecular size table(s) list. Note: By default the list will show the molecular size tables that are globally available. Click the Personal radio button to display the tables that are restricted to your own user ID. Click OK. Result: The Molecular size table dialog box opens.

How to rename a molecular size table

The table below describes how to rename an existing molecular size table.

Step	Action
1	Select Mol. Size:Edit Mol. Size Table:Rename. Result: The Rename molecular size table dialog box opens.
2	Select Personal to display the tables that are restricted to your own user ID, if needed. Select the molecular size table you wish to rename in the Molecular size table(s) list. Click in the Molecular size table name text box and type a new name. Click the Rename button.

Note: You must have Edit global list(s) rights to be able to rename global tables.

How to delete a molecular size table

The table below describes how to delete an existing molecular size table.

Step	Action
1	Select Mol. Size:Edit Mol. Size Table:Delete. Result: The Delete molecular size table dialog box opens.
2	Select Personal to display the tables that are restricted to your own user ID, if needed. Select the molecular size table you wish to delete in the Molecular size table(s) list. Click the Delete button. Click the Yes button to confirm.

Note: You must have Edit global list(s) rights to be able to delete global tables.

How to calculate the molecular size

The table below describes how the molecular size curve is used to calculate the molecular sizes of the components in the sample.

Step	Action
1	Perform a sample run and peak integrate the curve to produce a peak table. Note: The sample curve must use the same X-axis base unit as the standards. Use volume for molecular size calculations.
2	Select Mol. Size:Calculate Mol. Size in the Evaluation module. Result: The Molecular Size dialog box opens.
3	Select Global or Personal according to the location of the molecular size table. Select the molecular size table on the Molecular size table droplist. Select a chromatogram on the Source chromatogram droplist. Select a peak table on the Peak table(s) list and click OK. Result: The results of the molecular size calculation are shown in the Mol. size peak table column. (See illustration below)

The Mol. size peak table column

The illustration below shows the Mol. size peak table column.

When the result file is saved, it includes the molecular size table that was used for the molecular size determination. You can view the table that was used by selecting Mol. Size:Edit Mol. Size Table:View Current.

If the molecular size cannot be calculated

If the molecular size cannot be calculated, one of the following signs is shown in the peak table Mol. size column:

Sign	Function
>	This means that the molecular size is larger than the largest size in the molecular size curve, i.e. outside the valid range of the curve.
<	This means that the molecular size is smaller than the smallest size in the molecular size curve, i.e. outside the valid range of the curve.
-	This means that the retention value is negative.

Molecular size procedure instruction

The table below describes the new procedure instruction for molecular size measurement that becomes available when the Analysis module is installed.

Instruction	Description
MOLSIZE	The instruction calculates the molecular sizes from a molecular size curve. A Mol.size column will be added to the peak table.

2005-06-15