Calibration Examples
Internal/External Calibration: Calculation

In a calibration with the internal/external method, external calibration is adapted to the corresponding sample, by using an internal standard substance, i.e., an Internal Standard (= ISTD):

Each standard sample and each unknown sample is added exactly that amount of internal standard substance to make sure the concentration is identical in each vial. The internal standard substance and the substance to be determined are calibrated using known standard solutions; that is, the Calibration Coefficients are determined from the Amount values of the standard sample and the corresponding peak area values by means of the Calibration Function. Thus, the amounts of all substances (including the ISTD) can be determined. As the concentration of the internal standard substance is identical in all samples, the same ISTD amount should result.

If this is not the case, an error occurred in the chromatography system (sample preparation, injection, carry-over, etc). The deviation of the actual ISTD amount from the nominal ISTD quantifies of the error. If the substances to be determined and the internal standard substance are similar, it can be assumed that the values of the remaining contents of the sample deviate in the same way; that is, they are incorrect. A correction by the deviation of the nominal and the actual internal standard substance supplies the actual values.

Example:

You want to determine the concentration of alanine and glycine in two samples. One standard sample is available. The internal standard substance norvaline is added to all three sample vials, so that the final concentration is 10 mmol/l. During the subsequent pre-column derivatization, 10 ml sample + 20 ml OPA reagent + 20 ml stop reagent are pipetted together. A constant concentration of 2 mmol/l is added from the norvaline. The chromatographic separation follows the derivatization of the amino acids in OPA derivatives. 10 and 20 ml of the standard solution (Dilution Series) and 10 ml of each sample (autosampler position 2 and 3) are injected.

a) User Input:

Sample List

QNT Method/Peak Table Tab

Only one standard concentration (STD1; same autosampler position in the sample list) is required to perform the calibration of alanine and glycine. Therefore, only one amount value is required for each peak. Afterward, the internal standard substance is defined.

How to define the Internal Standard substance:

• In the Norvaline line, select the Standard column and open the F8 edit box.

• Select Use this peak as internal Standard and thus define norvaline as the internal standard substance.

A light yellow background and the ISTD: Internal entry indicate that the assignment is correct.

• In the Alanine line, change to the Standard column and open the F8 edit box again.

• Select the option Internal/External. In the Associated ISTD Peak field, select the standard substance serving as Internal/External Standard (here: norvaline).

• Repeat this operation for each peak that should be calibrated using the Internal/External method (here: glycine).

After completing the input, the following occurs: In the Standard column, alanine and glycine are labeled Int/Ext Norvaline. In addition, norvaline is labeled as the internal standard for the Internal/External calibration. The ISTD: Internal description is changed to ISTD: Int/Ext and the yellow coloring of the norvaline line intensifies.

 Note:

In addition to the color changes from light yellow to dark yellow, there are two other possible colors. If the retention time is expressed depending on a selected reference peak (see Retention Time), a light blue background highlights this reference peak in the peak table. If this reference peak is also used as the internal standard peak, the corresponding line is displayed in green (blue + yellow = green).

QNT Method/General Tab

The Total mode is selected. This ensures that the calibration of all samples (Samples I and II) is performed based on all standard samples (STD 1).

QNT Method/Calibration Tab

The page shows all standard samples (of a sequence) that are used for calibrating the current sample.

Press the F4 key or the SHIFT+F4 key combination to open the samples of a sequence one after the other. The standard samples forming the basis for calibration are shown for each sample.

Due to the selected mode, samples I and II appear as follows:

If you notice that an error occurred during the analysis of the standard sample, you can "exclude" this standard sample. Remove the standard in the Enable column on the Calibration tab page of the QNT Editor. Only the standard samples labeled X are considered for the calibration.

b) Analysis Structure:

Injection is four times. During the first run, the first calibration point of the calibration curve is determined, and during the second run, the second point is determined. Run three serves to determine the concentration of alanine and glycine in sample I. In the fourth run, the concentrations of alanine and glycine in sample II are determined.

Chromeleon determines the following area values:

The determined area values of the internal standard substance norvaline reflect the ratio of the injected volumes (amounts), except for minor inaccuracies.

c) Calibration Points

From the known amount values and from the determined area values of the standard samples, the value pairs of the individual calibration points can be established:

Chromeleon determines all calibration coefficients, depending on the selected calibration function.

d) Calculation of the Calibration Coefficients

Only one calibration coefficient (c1) is required to describe a linear calibration curve through the origin (calibration type Linear). If the example is selected so that the calibration points in each calibration curve are located exactly on a straight line, that is, for example, in an exact measurement, c1 results as the x/y-quotient of each value pair (= slope of the calibration curve; also see RF Value).

If the calibration points are not located exactly on one line, Chromeleon calculates an optimized c1 approximate value for each substance. If a different calibration type were selected, Chromeleon would also calculate the remaining calibration coefficients (c0 and c2) according to the calibration function.

e) Amount Calculation: Internal Standard Substance in Unknown Samples

If the area values of the internal standard substances from samples I and II are known, the amount of the internal standard substance norvaline can be determined in the two samples by means of the calibration coefficient c1 (here = 0.5) established for the calibration curve of the norvaline.

The ratio between the (nominal) amount value entered in the peak table and the ISTD Amount (Amount of the Internal Standard) of the internal standard in the corresponding sample is referred to as ISTD factor.

The following values are resulting:

The result states that an error was made by 1.026 (sample 1) or 0.976 (sample 2). The actual amounts of alanine and glycine deviate in all probability by 2.6 or 2.4% from the "real" values. They are corrected by this amount.

f) Amount Calculation: Alanine and Glycine

The Formula for Amount Calculation is used to calculate the amount values of glycine and alanine. In contrast to an external calibration, the ISTD Factor, the results are corrected by the calculated ISTD factor.

The alanine or glycine amount values corrected by the norvaline deviation are resulting.

For an overview of the different calibration possibilities provided by Chromeleon, refer to How to …:  Calibrating.