ChemStation External and Internal Calibration Calculations

Answer

This Information Applies To: Agilent OpenLab ChemStation Edition Software 

Issue

There are different quantitation calculation types available in the ChemStation software. The most commonly used quantitation methods include (but are not limited to):

• External standard (ESTD)
• Internal standard (ISTD)

Background

The quantitative calculation produces a raw result that is adjusted for the following correction factors, which are entered into the sample table before analysis: 

• Multiplier (M): Converts unit to amounts e.g. μg/μL to mg/kg. This value is entered in the Calibration Settings or the Sample Table
• Dilution factor (D): Adjusts for changes in normal sample preparation or any other fixed factor purpose. This value is entered in the Sample Table.

External standard (ESTD)

The ESTD calculation determines the absolute amount of the target analyte in the sample:

1. The calibration standards, blanks, quality control, and sample are run under the same instrument conditions and the responses for each target analyte (height or area) are added to the calibration table

2. The program calculates the response factor (RF) for each compound at each level, stores the values in the method file:

   RF = Amount of component in sample / Response of component in the calibration standard (area or height)
   
   

3. The RF information is plotted, and the selected curve fit calculation equation creates the calibration curve (Figure 1). The curve is the response factor (RF): 

   
   Figure 1. Calibration curve
   1. Response, 2 Amount, 3 Response factor

4. Absolute component amount in the unknown sample = Response of the target component in the sample * RF * M * D       

   Where:

   The response of the target component is measured in the same units as the units used to calculate the RF (area or height)

   RF: Calculated as above

   M (multiplier): Converts unit to amounts e.g. μg/μL to mg/kg. This value is entered in the Calibration Settings or the Sample Table    

   D (dilution factor): Used to scale the results for changes in sample preparation, or any other purpose that requires use of a constant factor. 

Internal standard (ISTD)

The ISTD calculation determines the absolute amount of the target analyte in the sample corrected for sample size variation, instrument drift, and chemical interactions during sample preparation. A known and constant concentration of internal standard is added to all prepared standards, blanks, quality control, and unknown samples. 

Tip: For the best results choose an internal standard that does not occur in the sample matrix and is chemically similar to the target analyte. Multiple internal standards can be used across a long method that targets multiple analytes, and the closets eluting internal standard can be used for target analytes.

1. All solutions are run under the same instrument conditions

2. The program calculates the calibration standard ISTD method relative response (RR) and amount ratio (AR) for each compound at each level and stores the values in the method file:

   RR = Response of compound / Response of the internal standard (area or peak height) 
   AR = Amount of compound / Amount of internal standard

3. The internal standard method RR and AR information are plotted and the selected curve fit calculation equation creates the calibration curve (Figure 2). The curve is the internal standard method response factor (RF_ISTD method):

   
   Figure 2. Calibration curve
   1. Response ratio, 2 Amount ratio, 3 ISTD method response factor

4. Response ratio unknown (RR_Unknown) = Unknown component response / unknown ISTD response. The response of the target component is measured in the same units as the units used to calculate the RF (area or height)

5. Absolute component amount in unknown = RF_ISTD method * RR_unknown * actual amount of ISTD (known added amount) * M * D       

   Where:

   RF_ISTD: Internal standard response factor 

   RR_Unknown: Response ratio for the target analyte in the unknown sample

   M (multiplier): This value is entered in the Calibration Settings or the Sample Table and multiplies the result for each component by a set number. It may be used to convert units to express amounts. This value is entered in the Calibration Settings or the Sample table

   D (dilution factor): Used to scale the results for changes in sample preparation, or any other purpose that requires use of a constant factor. 

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