Yes , it is called calibration curve

Omar Gonzalez-Ortega

Thanks Dear Friend!

According to this valuable clue I found a handy and useful video on YouTube. Now I recommend it for who are looking for a way to calculate a concentration from peak area values easily.

https://www.youtube.com/watch?v=8efZRBCFpRg

The best

you must first create a standard calibration curve with khown concentration of you compound vs prak area

then simply you can get you conc. from equation resulted from standard curve

First thing you should do is find someone that knows something about analytical chemistry. Better yet take a course or two.

Yes. It is the linearity curve and is expressed as y = mx + b (a straight line!). y is the peak area. x is the concentration expressed as mg/mL , ug/mL or ng/mL. b is the y-intercept. m is the slope of the curve or response factor. All these items are determined by experiment!

To add one additional concept, in many cases it is useful to include an "internal standard" which is a compound that is spiked into the sample at a known (usually fixed) concentration. It is usually spiked into the sample at the beginning of the sample preparation process, and its purpose is to compensate for various sources of variability in the process, such as variable efficiency of extraction of a sample from a matrix, variations in instrument response, and so forth.

The internal standard should be chemically and chromatographically similar to the target compound. This works best in a mass spectrometer is the detection system for the chromatograph because one can then use isotopically labeled versions of your target compound as an internal standard. This allows the internal standard to give nearly (but not quite) perfect tracking of the target compound.

Here is how the internal standard is used in the data analysis. The peak area of the target compound is divided by the peak area of the internal standard. This number is then compared to a calibration curve, usually based on a linear equation, to obtain the concentration.

The calibration curve is prepared by preparing a set of samples containing several different concentrations of target analyte. The calibration samples all contain the same concentration of internal standard. These are then run on the instrument and the peak area ratio is calculated for each calibration sample. The concentration of the target compound is plotted on the x-axis and the measured response ratio is plotted on the y-axis. (In principle, you could swap the axis, but most people use the conventions just noted.)

Then, you run your unknown samples ("unknown" in the sense of unknown concentration of target compound, not in the sense of the chemical identity being unknown). You take the peak area ratio of an individual unknown sample, compare it to the y-axis of the calibration curve, and then drop down vertically from the calibration curve to read the unknown concentration on the x-axis.

You have to first run several concentrations of your target analyte standards. The standard concentration range should encompass the lowest and highest concentration you expected in your samples. Then plot the areas against the concentrations and fix the trendline (calibration curve). derive the equation of the straight line as well as the coefficient of determination (R2). From the equation, you can derive the concentrations of the target analyte in your samples.

*Caution: the equation must be considerably linear (i.e R2 above 0.98) for the calibration curve to be useful.

• Babatunde wrote: "*Caution: the equation must be considerably linear (i.e R2 above 0.98) for the calibration curve to be useful."

While a linear slope is ideal, it may not be a requirement (Remember, the poster did not provide any context to the question. Let us not assume). Some types of detectors do not provide a linear output. What is required is that the method follows good chromatography fundamentals and you document that the method is accurate and reproducible (i.e. proper retention, soluble, within scale, method is selective for the analyte, proper number of high purity standards are used for the range and they are bracketed "around" the sample, etc). A well developed method used with a multi-level calibration table correctly fitted to the points will work.

I'm staggered at the level of rudeness you have received from some posts for asking a simple question. From the following article https://www.japsonline.com/admin/php/uploads/2674_pdf.pdf the equation is :

c = A/(d × ε), (Eq. 1) where c = total protein (g/L), A = absorbance value, ε0.1% = extinction coefficient (mg−1 cm−1), and d = optical path (cm).

Peak area in mAu*mL is the integrated peak area and can therefore be used as "A" in the above equation. This is the calculation Akta Unicorn software uses and typically it is consistent (+/-1-2%) with a UV abs (via cuvette) derived concentration from the pooled peak. Keep in mind that the baseline correction is important and for an absolute value a std curve is advisable (but that's not what you asked).

I strongly agree with Bruce Neagle and Alan Rockwood ; it is the right procedure of results treatment.

What is unit of Peak Area inchromatography ?

As a practical matter the unit of peak area would probably best be described as "arbitrary units".

To amplify this response. Usually speaking the units for the y-axis of the chromatogram are basically arbitrary units. The x-axis is in units of time. Therefore, the units of the peak area are arbitrary units multiplied by time.

To explain this a bit more, if you really want to get technical the LC detector will have some kind of output, such milliamps (or some other variation of current measurement, such as microamps) or millivolts (or some other variation of voltage, such as microvolts). This will generally end up going through some kind of amplifying and signal conditioning electronics, and then an Analog to Digital converter. By the time the signal (i.e. the y-axis of the chromatogram) reaches the computer it is basically best considered to be arbitrary units.

The peak area (regardless of its units) ultimately ends up being converted to concentration via a calibration curve. If there is no internal standard (not usually a good idea) the calibration curve may be based on the raw peak area.

In many cases there is an intermediate step where the peak area of the target analyte is divided by the peak area of an internal standard, and in that case the ratio between the peak area of the target analyte and the internal standard is, technically speaking, a unitless number, but that unitless number ends of being converted to concentration via a calibration curve.

Dear Javaid Iqbal

With modern equipment, i.e. a so-called "digital detector," the detector response is available in the detection-specific unit. In old times, the detector response (analog) was converted (digital), processed, and sent to the recorder (analog). With those detectors, the area would be "mV/min" or "µV/min", depending on the detector settings. The last step (digital to analog) with the so-called digital detector is skipped, and the digital result is sent to the recording software.

In other words: A UV detector would show absorption in AU, a conductivity detector response in µS/cm, etc.

(Note: Part of modern qualification routines in chromatography is ensuring that the detector readings are consistent and comparable. Hence tests are performed to ensure the correct - within boundaries - and matching response. Modern detectors from different vendors - treated and maintained well - will give highly comparable readings even with different software packages, giving testament to this approach. )

As we integrate "over time," the peak area will be the detector unit "over time". As a convention, the "minute" is used as a base. So peak area of a UV detector would be "AU/min" (or "mAU/min" - depending on the setting), or (µS/cm)/min for a conductivity detector, or counts/min, e.g., for an MS-detector.

(mathematically speaking (µS/cm)/min could also be expressed as "µS min/cm", though I advise against it - it is creating too much irritation).

I hope this helps.

Good luck with your research,

Detlef.

To convert Peak Area to concentrations, a graph of Peak Area

(Y axis) from chromatogram and Concentration of calibrator (X- axis) from Insert is plotted. Concentrations of samples are determined from the curve using their respective peak values.