Welcome to check the blog about ” How to read HPLC Chromatogram.”
For many students who are new to liquid chromatography, the Chromatogram is
a part that must be learned to read, so today, we will also briefly talk about how to view the Chromatogram
First, please check the right, and This is how a typical chromatogram looks Like
And then, you can see The Y(vertical) axis represents ‘mAu.’
mAu = milli absorbance unit
mAu indicates the absorption of analytes.
mAu is also used to determine the concentration of the analyte
Ok, now let’s check The X (horizontal) axis
represents retention time.’
Retention time is the time between the injection of the analyte to the detection of the analyte.
And now you can check Compound has a retention time of 6 minutes.
Retention time can also be used as an identification tool.
Reference list of compounds and retention times:
- Acetaminophen; 2 minutes
- Caffeine; 6 minutes
- Benzoic acid; 7 minutes
- Aspirin; 9 minutes
So What is the identity of Compound X?
Then let’s check the Caffeinecan.
The concentration of Caffeine can be determined with a
Then let’s check the Example calibration curve:
Prepare sample and runHPLC analysis as follows
Sample C1 – 5o mg/l
Sample C2- 100 mg/l
Sample C3- 150 mg/l
Sample C4- 200 mg/l
Sample C5 – 250 mg/l
As concentration increases, the peaks become bigger and higher.
Then we can do a simple calculation as the diagram.
The Peak Area represents the amount of Compound that has passed the detector.
So a Higher Peak does not mean the concentration is higher
Peak A has a higher concentration compared to peak B as the
peak AREA of peak A is larger!
Then The computer software can automatically calculate the area of the peak.
Let’s have a look at the calibration curve.
So with these numbers, you have to work with to
calculate the concentration of ‘compound X ‘
The computer software will also calculate the peak area of ‘compound x’
With this information, you should be able to calculate the concentration.
Compound x = 207.757 mg/l
So if the software calculates everything, why bother looking at the Chromatogram?
You want to make sure your analysis is done correctly by looking at the Chromatogram.
May you also have questions like, Is this correct retention time ? Is there any absorbance ?
and Is the baseline straight ?
This is an example of a chromatogram with poor baseline
I hope this is a helpful video; now you know How to read chromatograms.
Thanks for watching, and see you in the next video!
And by the way, have any questions about HPLC and the requirements for the HPLC Column?
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