Calibration - Individual Compound tabs
The Individual Compound tabs contain a table for the displayed compound, several parameter sections, and its calibration curve.
Click on any area of the image below to get relevant information
Calibration - Individual Compound (malic)
Compound Table
The table is located in the top left corner of the individual compound tab. It contains a row for each calibration level, including up to 20 standard levels and the Blank (BL) level. The first column displays the level number, while the remaining columns represent the level-specific parameters for the given compound.
First column
Contains the level number of each displayed line.
Represents the signal intensity used for quantification, based on the selected Response Base. The Response value is read from the standard chromatogram during the (re)calibration process. Manual adjustment of Response is possible, but this option can be disabled in Calibration Options.
Amount of the compound at the selected level, in units specified in Calibration Options dialog. If the Amount or Response of a calibration level (1–20) is 0, the calibration point is not created.
The Response Factor at a given level is defined as the reciprocal of the slope of a straight line passing through the origin and the calibration point at that level. It is calculated automatically and cannot be manually edited. By default, it is determined as Amount / Response:
where:
Ai - Amount of the compound at level i.
Ri - Response (height or area) at level i.
The calculation can be switched to the inverse form (Response / Amount) by enabling the Response Factor as Response / Amount option in Calibration Options.
Indicates how many recalibrations with the Used checkbox enabled are included in the calibration, out of the total number of stored recalibrations for the given level. Clicking 
opens the Details of Calibration Point dialog, where you can view and manage individual recalibrations.
The total number of stored recalibrations is limited by the No. of Points setting in the Recalibration section of Sequence Options. If this limit is reached, adding a new recalibration permanently deletes the oldest one.
Determines whether a calibration level is included in the calibration curve. If the checkbox is cleared, the level is omitted from the calibration curve and is represented by a circle in the graph.
Represents the deviation of the entered Amount of the calibration point from the calculated Amount given by the calibration curve. This column is hidden by default, For more details see also chapter Setup Columns
where:
Amount - The entered Amount at the concentration level.
AmountC- The Amount calculated from the constructed calibration curve for the given concentration level.
Displays the relative standard deviation (RSD) of the averaged recalibrations used for the respective concentration level. This column is hidden by default, For more details see also chapter Setup Columns
Displays the calculated Response value, determined based on the option selected in Linearization Y. This column is hidden by default, For more details see also chapter Setup Columns
Displays the calculated Amount value, determined based on the option selected in Linearization X. This column is hidden by default, For more details see also chapter Setup Columns
Local menu of the Compound Table
In addition to the common commands for the majority of tables, the local menu contains the following items:
Erases all entered values on all levels of the given substance.
Erases all values on the given level.
Erases the value in the current field of the table.
Removes the current substance from the calibration.
Opens the Details of Calibration Point dialog, where you can view and manage individual recalibrations.
A section containing parameters used to identify the peak belonging to the given compound.
Specifies the type of the calibrated compound. See Calibration Summary Table section for more details.
Retention time of the compound. It is used for compound identification.
Defines whether the search window for a compound is specified in absolute values (Abs, set in minutes) or relative values (Rel, set as a percentage of the retention time).
Determines how the peak is identified when multiple peaks appear within the identification window. See Calibration Summary Table section for more details.
A section containing parameters related to the quantification of the given compound.
Sets which value will serve as Response. The Response is then used for calculating the calibration curve, calibration dependency equation, and response factors. This setting is specific to each signal. Available Options are:
- Area – Area is used as the response base.
- Height – Height is used as the response base. The height is measured as the vertical distance from the peak apex to the baseline, which may lead to imprecise values for small peaks on steep baselines.
- Area Percent – Calibration is based on the percentage of peak area relative to the total peak area of all compounds included in the calibration. This means that all relevant compounds must be added, even if their results are not needed. The recommended calculation type for this method is ESTD.
The selected Response Base affects other fields in the individual compound tabs and the calibration curve. Changing the Response Base will recalculate all dependent values accordingly.
Indicates whether the given compound is an Internal Standard (ISTD) peak and assigns an ISTD peak number. See Calibration Summary Table section for more details.
Specifies which Internal Standard (ISTD) peak is used for the calculation of a given compound. See Calibration Summary Table section for more details.
Determines the type of curve used to fit the calibration data. If the number of available points is insufficient for the selected curve type, the curve is automatically changed to the next lower type, while the Curve Fit Type selection remains unchanged. In Standard Addition Display Mode, only the Linear curve fit is available, and the Curve Fit Type selection is disabled.
Curve Fit Types available are:
Free Calibration
No calibration curve is constructed. The calibration equation is in the form:
Instead of values interpolated from a calibration curve, calculations use the global Response Factor from the global calibration table. That means the entered Amount value for the compound will not be used. Origin parameter is replaced by Manual Response Factor, in the Individual Compound tabs.
Point to Point
Calibration points are connected by a broken line, with no common calibration equation. Instead, each segment’s equation is used in calculations. The Point to Point fit type always includes the origin point (0,0) in the calibration curve, regardless of the Origin setting.
Linear
A straight line is fitted through the points.
Quadratic
A second-degree polynomial is used to interpolate the calibration curve.
Cubic
A third-degree polynomial is used to interpolate the calibration curve.
Sigmoid
A sigmoid function is used for curve fitting:
where:
c – Maximum Amount
d – Maximum Response
b = 1, a, k – Coefficients optimized through iteration ( <0,1 − 100 >) to best fit the calibration points.
The Ending Point field allows increasing c and d up to 125% (both parameters change simultaneously).
Half Sigmoid
A variation of the sigmoid curve that represents only one half of the function.
Logarithmic Functions:
ln – Interpolates using a natural logarithm function.
log10 – Interpolates using a decimal logarithm function.
Exponential Functions:
exp – Fits an exponential curve.
pow10 – Fits a base-10 exponential curve.
Hyperbola
Uses a hyperbolic function for interpolation.
Specifies how the zero point (origin) is handled in the calibration curve. This setting is individual for each signal. Available Options are:
- Ignore Origin – The origin is not included in the calibration curve. This is the only option where Blank level is included in calibration curve construction.
- Compute with Origin – The origin is treated as one of the calibration points. Blank level is ignored with this option.
- Curve passes through Origin – The calibration curve is forced to pass through the origin. Blank level is ignored with this option.
In Standard Addition Display Mode, blank is never used for construction of the curve. The Origin field is replaced by the Unknown field, which has the same options:
- Ignore Unknown – The Unknown point is not included in the calibration curve.
- Compute with Unknown – The Unknown point is treated as one of the calibration points.
- Curve passes through Unknown – The calibration curve is forced to pass through the Unknown point.
When using the Sigmoid curve fit, the Origin field is replaced by the Ending Point field.
This setting is only applicable when the Curve Fit Type is set to Sigmoid or Half Sigmoid. It allows the sigmoid interpolation to extend beyond the last calibration point. The last calibration point is taken as the limit value, and both its Amount (X-coordinate) and Response (Y-coordinate) can be increased by a selected percentage according to the Ending Point setting.
The available options range from Last Point (using the last calibration point as the limit) to Last Point + 25% (extending both coordinates by 25%).
This setting is useful when measuring calibration points within a sufficient range is not possible.
Used to enter the factor for calculating results for the compound when the Curve Fit Type is set to Free Calibration. It serves as a simple single-point calibration.
In the evaluated chromatogram, the amount is calculated as the response of the peak multiplied by this coefficient.
Determines how individual calibration points are weighted when calculating the interpolated calibration curve.
By default, higher concentration points (with larger Amount and Response) have a greater influence on the calibration curve than lower concentration points. This method helps improve calibration accuracy, especially when dealing with wide concentration ranges.
Weighting allows adjusting this influence:
- 1/x – Assigns the same relative influence to each point.
- 1/x² – Gives greater influence to lower concentration points.
Weighting can be applied based on:
- Amount – The amount of the compound at the given calibration point (1/Amount or 1/Amount²).
- Response – The response of the compound at the given calibration point (1/Response or 1/Response²).
Applies a transformation to the X-axis values (Amount) using the selected formula before calculating the calibration curve equation. This affects only the calibration curve equation itself, not how the curve is displayed. The X-axis and calibration points remain shown with their original (non-linearized) values, meaning the visual representation of the curve does not correspond to the linearized X-axis values, the modified calibration equation, or the linearized calibration points.
To display the curve according to the transformed equation and linearized calibration points, the Show Linearized Values checkbox must be enabled in the Graph Properties dialog.
Available Linearization Formulas:
- None – No linearization is applied.
- 1/Amount – Uses the reciprocal of the Amount.
- ln(Amount) – Applies the natural logarithm to the Amount.
- sqrt(Amount) – Uses the square root of the Amount.
- 1/sqrt(Amount) – Uses the reciprocal of the square root of the Amount.
- Amount² – Squares the Amount.
- 1/(Amount²) – Uses the reciprocal of the squared Amount.
Applies a transformation to the Y-axis values (Response) using the selected formula before calculating the calibration curve equation. This affects only the calibration curve equation itself, not how the curve is displayed. The Y-axis and calibration points remain shown with their original (non-linearized) values, meaning the visual representation of the curve does not correspond to the linearized Y-axis values, the modified calibration equation, or the linearized calibration points.
To display the curve according to the transformed equation and linearized calibration points, the Show Linearized Values checkbox must be enabled in the Graph Properties dialog.
Available Linearization Formulas:
- None – No linearization is applied.
- 1/Response – Uses the reciprocal of the Response.
- ln(Response) – Applies the natural logarithm to the Response.
- sqrt(Response) – Uses the square root of the Response.
- 1/sqrt(Response) – Uses the reciprocal of the square root of the Response.
- Response² – Squares the Response.
- 1/(Response²) – Uses the reciprocal of the squared Response.
- log(Response) – Applies the base-10 logarithm to the Response.
Used to enter a correction factor for Amount, for example, to account for lower recovery. The Amount - whether entered directly, derived from another compound using Calculate By, or calculated from Manual Response Factor based on the response - is multiplied by the Correction Factor to obtain the final Amount.
Sets a compound whose Calibration Curve will be used for Amount calculation of a given compound in the evaluated chromatogram. See Calibration Summary Table section for more details.
Calibration Curve section
This section displays calculated parameters of the calibration curve, including the calibration equation and other derived values. All values in this section are computed based on the settings and data entered in the previous sections. These parameters are read-only and cannot be modified directly.
Represents a value in the range <0, 1> that characterizes the linearity of data. The value is only relevant if the Curve Fit Type is set to Linear. In all other cases, the value is displayed but does not reflect the quality of the calibration curve.
where:
xi - The actual (measured) response at level i.
yi - The calculated (predicted) response at level i.
X - The mean of all actual responses (including the zero point if Compute with Origin or Curve passes through Origin is selected, Blank level is ignored when one of these options is selected).
Y - The mean of all calculated responses (including the zero point if Compute with Origin or Curve passes through Origin is selected, Blank level is ignored when one of these options is selected).
Determination Coefficient (R^2)
Also referred to as coefficient of determination. It describes how well the independent variable(s) explain the variability of the dependent variable.
where:
xi - The actual (measured) response at level i.
yi - The calculated (predicted) response at level i.
X - The mean of all actual responses (including the zero point if Compute with Origin or Curve passes through Origin is selected, Blank level is ignored when one of these options is selected).
Represents the calibration curve fit residuum, which quantifies the average deviation of actual responses from the calculated calibration curve. A lower value indicates a better fit, meaning the calibration points closely follow the curve.
where:
xi - The actual (measured) response at level i.
yi - The calculated (predicted) response at level i.
n - The number of calibration points (including the zero point if Compute with Origin or Curve passes through Origin is selected, Blank level is ignored when one of these options is selected).
This value helps assess how well the calibration curve represents the measured data.
Displays the equation of the calibration curve, which defines the relationship between Response and Amount based on the selected Curve Fit Type. This equation is used to calculate the Amount of an unknown sample from its measured Response.
The displayed equation provides a mathematical representation of the calibration curve, ensuring consistent quantification of unknown samples.
Note:
In case the calibration curve of the compound cannot be constructed because the relevant information is missing (e.g. ISTD peak has no response on any level or is disabled), a warning is displayed instead of the Equation (ISTD peak unavailable in the mentioned case).
Displays the calibration curve linearity check result, performed only if enabled in Calibration Options. To activate the check, enable Deviation and/or Correlation and set the corresponding limits.
The result can be:
- OK – The calibration curve meets the selected Deviation and/or Correlation criteria.
- Error – At least one of the applied Curve Check tests has failed.
If failed, it is also indicated in evaluated chromatograms in the Chromatogram window (Peak Type column as Error (Curve Check)).
Calibration curve graph
The calibration curve for each compound is displayed in the right-hand section of the compound tab. It represents the relationship between Amount and Response.
Name of Compound and Retention Time
Displayed above the graph, this section contains identifying data for the compound, including its Compound Name, Retention Time, and the Name of the active signal.
Graph Overview
The graph shows calibration points for the selected compound along with the calibration curve. Its appearance can be modified using the Graph Properties dialog.
Graph Symbols and Interpretation
- Calibrated points (levels with only a single calibration value) are displayed as crosses ⑤.
- Recalibrated points (levels with multiple recalibrations) are displayed as asterisks ⑥, with a range indicating the recalibration span.
- If the Used checkbox is unchecked ① for the entire level in the Compound Table, the calibration level is excluded from the calibration curve and displayed as a large circle ②.
- If a single recalibration is excluded from the calibration point calculation by unchecking the Used checkbox ③ in the Details of Calibration Point dialog, it is displayed as a small circle ④.
Representation of different points in the graph
Standard Addition (STDADD) Calibration Curve
In the STDADD calibration type, the Blank sample is represented as a vertical line with a multiplication cross sign. This indicates that the sample amount is not determined from the intersection of the calibration curve with the X-axis at zero response, but rather from the intersection of the calibration curve with the blank horizontal line.
Internal Standard (ISTD) Calibration Curve
When using the ISTD method, the calibration curve is displayed as a relative calibration curve, depending on whether the same or a different amount of the internal standard was used:
- Same Amount of Internal Standard
The relative calibration curve represents the dependence of the response ratio of the compound and the internal standard on the amount of the compound injected:
Ri - Response of the compound
RISTD - Response of the internal standard
Ai - Amount of the compound
- Different Amount of Internal Standard
The relative calibration curve represents the dependence of the response ratio of the compound and the internal standard on the ratio of their respective amounts:
Ri - Response of the compound
RISTD - Response of the internal standard
Ai - Amount of the compound
AISTD - Amount of the internal standard