External:

= Default setting.

Principle:

One or several standard substances of known concentration are analyzed separately. The values in the Amount column are used to establish a ratio between the area and the amount. On this basis, the amount of this substance in samples of unknown concentration is determined via the peak area.

Instructions:

Select External for all peaks that are to be calibrated with an external standard.

Internal:

Principle:

When calibrating with internal standards, area and amount ratios are used for the calculation, instead of absolute areas and amounts. For this procedure, it is necessary to inject a constant amount of the internal standard after sample preparation; for example, in a dilution series, the standard must not be diluted (Const. Internal Standard). Due to the identical amount of internal standard, the same result should be achieved for all samples. If the results vary, the ratio of the IS values provides information about the accuracy of the analysis and the actual result.

Due to the intense experimental procedure, this type of calibration is rarely used in HPLC.

Instructions:

If you want to calibrate a peak (peak A) on the basis of the Internal Standard method, you must determine the peak (peak B) that is used as Internal Standard.

Defining the ISTD Peak

Select peak B in the peak table and assign the ISTD Peak/Internal option in the F8 edit box. The corresponding line will be marked yellow.

Assignment

In the F8 edit box of the Standard column, assign peak A to the Internal option. Select the ISTD peak (peak B) in the Peak field. If you defined several internal standards for the Internal method, you can choose from among several different internal standards.

Internal/External:

Principle:

As an internal standard (IS), select a substance with a retention time behavior that is similar to the substances to be determined. Before sample preparation, exactly the required internal standard is added to all samples (unknown and standard samples), so that its concentration is identical in all samples. If the sample is diluted or if pre-column derivatization is performed, this will change the concentration of the internal standard. Both the internal standard and the substances to be determined will be calibrated.

Instructions:

If you want to calibrate a peak (Peak A) on the basis of the Internal/External Method, determine the peak to be used as the internal standard; that is, define which substance was added to the sample, such as Peak B.

Defining the ISTD Peak

Select Peak B in the peak table and mark it as the ISTD peak in the Standard column. (Press the F8 key to open the corresponding edit dialog box and select (ISTD Peak, Internal/External.) The yellow background allows easy recognition of the corresponding line in the peak table.

Assignment

Press F8 in the Standard column. Assign peak A the Internal/External option. Select the ISTD peak (peak B) in the Peak field. If you defined several internal peaks for the Internal/External method, you can choose from among several internal standards.