How to Read a Chromatogram?

Over the years chromatography has gained an enviable position in analytical laboratories involving separation and quantification of organic compound mixtures. However, a chromatogram is not a display of results in concentration units but rather a graphical display in real time of peaks generated as the separated components pass through the detector.

The chromatogram makes little sense to any layman as the peaks provide no information on the identity of the mixture components nor any knowledge on the amount present. That is why they aren’t able to learn much from the results. 

If you want to master the art of interpreting a chromatogram, you first need to know exactly what a chromatogram is. But before moving on to that, let’s first take a look at chromatography, its advantages, types, and other details that will further help in the understanding of a chromatogram.


As stated earlier, chromatography is used in laboratories to separate or quantify the mixtures of organic compounds. For this purpose, it utilises the polarity difference in molecules, and the compounds get divided based on their affinity towards the stationary phase. There is also one mobile phase that is used to carry the mixture over the stationary phase. 

This technique has been practised by experts ever since Mikhail Tswett found it in the year 1903. He only applied it to separate out different coloured pigments from plants with the support of a calcium carbonate column. However, chromatography proved effective for several components later on. It brought in different kinds of chromatography with time. 

The primary reason why experts opt for this method is that it provides proper separation and analysis. Moreover, it can work for purification of components as well.

This technique works for a wide range of mixtures, including the too complicated ones. On top of everything, it doesn’t require high quantities of sample for the purpose. Therefore, the work gets done without putting in high volumes of the mixture. 

Types of Chromatography

Before starting, you must know that different kinds of chromatography bring in different chromatographic analysis. That is why it is crucial to understand the major variations first. Based on the exact usage of the technique, chromatography can be divided into various types, such as:

  • Liquid Chromatography: Here, organic solvents are the mobile phase, while silica and alumina solve the purpose of stationary phase. 
  • Paper chromatography: The silica and alumina from the previous type get replaced with a paper soaked in liquid here. Plus, a liquid solvent is utilised in the place of the organic solvent. All the segregated particles can be observed as patches on the stationary phase after it dries out completely. 
  • Column Chromatography: In this sort of chromatography, the stationary phase, as well as the mobile phases are kept inside a given column. The disassociation of the sample takes additional time here. 
  • HPLC: This stands for High Performance/Pressure Liquid Chromatography. It is a more updated variant of the previous kind because high pressure is kept inside the column here to provide accelerated separation of the sample. 
  • Thin Layer Chromatography: This is almost the same to paper chromatography, except for the different phases used here. Instead of soaked paper, this one utilises alumina or silica-coated sheet, which is made of glass or plastic. The absorbent’s spots can be seen on the sheet after the process completes. 

A few more types of chromatography can be found in practice like gas and ion-exchange. In short, we can say that the techniques are almost the same in every kind, but their mobile and stationary phase differs. 

What Is a Chromatogram?

Now that you have understood everything about chromatography, let’s learn about chromatography and chromatographic analysis.

Chromatogram definition can be understood simply by studying what a chromatogram depicts. The chromatogram is a two-dimensional plot with the ordinate axis giving concentration in terms of the detector response, and the abscissa represents the time. The detector gives a response as a peak whose height should be ideally dependent on the concentration of the particular component.

Retention Time
Retention Time (tr)

However, due to analysis conditions peaks may deviate from ideal shape and peak height can no longer be a true measure of the concentration and instead the area under the peak is considered as a measure of component concentration.

Each peak represents a component present in the sample. Retention time is time interval between sample injection and the maximum of the peak. It is characteristic of the identity of the component under the operating conditions. Identity of the component can be confirmed by making injections of reference material under the same operational conditions. The matching of retention time of reference material and the component peak confirms the identity of the unknown sample component.

Now let us consider a sample which contains more than one sample component. Likewise each component will be eluted at different retention times depending upon solute – stationary phase interactions and mobile phase flow characteristics.

 Calculation of results
Calculation of results

 From the area measurements using simple arithmetic it is simple to calculate the concentration of each component as a percent of the total.

  %A = [Latex]\frac{Area of Peak A  X 100}{Total Areas of Peaks (A + B + C + D)}[/latex]

Real Chromatogram

Let us now look at the actual chromatogram printout of HPLC separation of a mixture of vitamins A and E in a food matrix and see what the chromatogram represents

Actual Chromatogram

The ordinate is in units of volts and abscissa in minutes. The signals are recorded at a wavelength of 284 nm using a UV detector.

Retention time of each peak is marked above the peak and in the tabulated data below the chromatogram details of the retention time, area (as digital units), peak area%, height and height %. You can observe that due to non-ideal shape of peaks percentage area is different from percentage height for each component so area measurement is a more reliable measure of concentration.

You have been introduced to simple concepts on how to read a chromatogram. Please let us have your comments on the article and if you found it useful.

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  1. This clear article is base to further understanding chromatograms. It would be great if were it posible to put more examples, with clearest images to follow the practical issues in Reading thes graphics.

    Also there are an initial time where those components that pass without retention provide a small peak. So when Reading the full chromatogram, this could be confuse to understand its role in calculations.

      1. how can i calculate area of these graph peak manually,what is the unit of these area which is calculated by software

        1. Traditionally the practice was to cut the chromatographic peak from the chromatogram and weigh. The weight of sample peak was compared with that of of the standard peak.Otherwise approximations can be arrived at by using triangulation method or using graph paper and by counting the squares.Such methods gave approximate values only. Presently software provides area of each peak in terms of counts which are unitless figures.

  2. Dear Sir
    I recently joined your newletter and found it good reading. I am currently trying out various methods for HPLC, as we are trying to do some work with blood levels of levofloxacin. For this i have questions,
    I found a methods for seperation- it utilises acetonitrile – water (80:20) at pH 3.5, my samples however- are in methanol. can i inject the samples as they are or do i need to incorporate the mobile phase? The reason being – we used methanol to precipitate proteins out of serum.

    1. Hi Satish,
      First of all thank you for your comments, we are glad you are enjoying newsletter. You can inject samples in methanol, there should be no problem. Do let us know how it works out!

  3. Happy New Year Sir, I am one of your favourite blog readers. Sir, I can’t decode how to read and interprete the chromatogram as discussed above. Can you please elaborate more on the calculations.

    Thank you sir.

    1. Happy and prosperous new year to you as well.Please feel free to contact me on my mail id [email protected].I shall help you with calculations on getting details of your analysis and the data which is generated by your system.
      Best Regards

  4. I have seen some people calculate peak area using the triangle formula rather than from the area mentioned in the chromatogram. Is it a right way of calculating peak area?

    1. Hi,
      Chromatographic peaks are seldom perfect triangles so area calculation using the triangle area formula will not be representative of area under the peak.In the chromatogram shown in the article you will come across numerical units in area column which are proportional to area under the peak. You can use these units as representing the peak area for further calculations.

  5. Dear Dr.Bhanot,
    I find your article (How to read chromatogram) very useful, please can you elaborate more on it?

    1. Hi Auwalu,
      I am glad to note that you found the article useful.Please do forward your specific query on which you wish me to elaborate upon.I will like to clarify your doubts.

  6. Thanks for all your articles. Even the basic ones are useful as they lay the foundation of complex concepts to understand.

  7. thanks for such a simple and perfect explanation
    i am biotech passout
    i didnt stuidied seriously during classes , i hope i can get to know about HPLC enough to get a job
    thanks :)

  8. Hi,my shimadzu hplc machine just suddenly stopped printing out results.please what could have caused it? And what can i do?

    1. Hi samson,
      It appeares to be an electrical problem. The best option would be to inform the maintenance support of the supplier.

  9. Hello! I am currently doing an assignment on melamine detection within milk products. I am just learning how to read chromatograms as I need to investigate the HPLC-UV method (and ELISA and GC/MS). As I am trying to interpret some researched chromatograms however, there are several peaks but only some are marked *melamine*. How do you determine whether a spike is indicative of the analyte you are looking for or not? Thank you

    1. Hi Lauren, you will need to inject the pure standard to confirm the retention time and also spike it in the sample to confirm there is on shifting of the rt in the samples.

  10. Am very impressed with the unmeasurable knowledge I have learned from this tutorial. I am a master student and in my thesis i would like to identifiy some protein protein interactums. would this skill of HPLC help me to elucidate these interactions? thanks once again.

    1. Thanks Terry for your encourangement.HPLC holds great promise for applications in life sciences and biochemistry. You will certainly find useful references as you progress with your research

  11. Hi Dr. Deepak Really an informative article. Thanks a lot.

    I need more help on this to understand. I have some HPLC reports and by using the formula to find each component %age, the percentage is coming different than what HPLC is actually giving.

    We are using a carbohydrate column and we are seperating fructose, glucose, sucrose and maltose from a Honey sample. can you tell me that if we want to detect HMF, what can we do?

    Thanks in advance.

    1. Hi, the HPLC might be giving the % by area percentage and you might be calculating with standard areas thus the difference. As for HMF a separate method will be needed on a UV detector.

  12. What does difference in Retention time mean? In our undergraduate thesis, our sample Lycopene has RT of 3.937 and the standard has RT of 4.100. Does it mean that out sample is not lycopene?

    1. Hi Katrina, Slight variations in the RT are acceptable and the tolerance level is usually established during a method validation study. If you are working on HPLC it seems to be fine, you need to make sure you are injecting the sample and standard in the same diluent.

      1. Hello Sir,
        Its a great support for analyst through newsletter.
        In addition to this, RT variation criteria of different samples are not defined. It should be established during validation and limit must be justified. However RT variation criteria of same sample can be established (RSD up to NMT 0.2%)


  13. Much thanks,
    I have just obtained my chromatograms from analysis of bio- gas and bio-oils, and i do not know what to do with it.
    I am so relieved after reading this article.
    Thanks for post!

  14. I am glad to read through your write up Dr Deepak Bhanot. Indeed, they are very educative and I have learnt alot.

  15. Good morning Sir,
    The write up has being of tremendous useful, I appreciate your good work. Kindly assist me with an example or References on how to write the interpretation of the chromatogram on how it affect the seasons.
    I will appreciate your favourable response.
    Yours sincerely,

    1. Good to note that you found the information useful.I am not able to understand your query. Can you elaborate a little more on your requirement.Further clarify what you imply by seasons.

  16. Hi
    Thanks a lot for your articles. Its very helpful in understanding HPLC machine.
    Thanks again

  17. I need to find the resolution between second and third peak. How to get the peak width based on the graph? It is because the information state at the graph only have retention time, area, %area, height and %height. Thank you for reading my question sir.

    1. The best option available to you is to use the software capabilities available on your system. You have to first go through the software capabilities and then take help to get resolution between the selected peaks

  18. Hello Dr,
    Your brief and clearly composed HPLC chromatogram reading blog really impacts high on young researchers, well done.
    Sir, 1) where a peak area is wide, can one take the midpoint for the retention time or the point of emergence of the peak. 2) During Isolation of components of a mixture, we use to collect eluate from each peak immediately the peak appears and stop immediately it collapses, but I observed that the distance between detector and exit port has to take some seconds, how can we adjust this so as to avoid collection of impurity. thanks

    1. Try a slight increase in mobile phase flow rate keeping other operating conditions same. Hopefully it should solve both your problems. Your peak should get narrowed down and impurity content in collected portion should also decrease.

  19. i would like to subscribe to your newsletter. kindly provide me with the proper links both for PC and smart phone. I am in the beginning stages of learning the analytical experiments.

  20. Is this the % recovery?
    I have to calculate % recovery of butanol in my experiment, I know the retention time and area under curve and initial concentration of butanol too.

    1. The chromatogram will give the concentration in your sample.As you know the original concentration you can calculate %recovery.

  21. Hi Dr. Deepak.

    Thank you for providing these useful notes.
    I would like to ask you a question about the area of the peak. Do they represent amount or concentration of the compound we are trying to analyse?

    I understood that in order to do quantification of the compound, we should run the standards at different conc. for calibration purposes.

    Thank you.

    1. The area under the peak represents the amount of a component in the sample. You can calculate the concentration from the volume injected. Hope this clarifies yur query.

      1. Dear Dr. Deepak Bhanot,

        How do you calculate the concentration from the volume injected? does the area % mean that for instance 40% of the 15microliter sample is the component? Or is the area under the peak the amount of the component in mol and do you calculate the concentratie by deviding it by the volume injected? Or is the area under the curve the concentration?
        Please help

        1. Dear Famke,
          The area under the peak represents the amount of a compound present as a percentage of the total area of the peaks in the chromatogram. The areas are printed in tabulated format in numerical digits without any units.The area is calculated from these numerical figures with reference to the area count of the standard which is injected in between the sample runs.Please let me know if you are interested in the formula for concentration calculations which I can send on your e-mail id.

  22. Hi, I am a Food Engineering student and I am working in a presentation about HPLC. Congratulations for your website, the explanation is very clear!!

  23. Comment on the issue of integration when there is a shift in Retention time from expected. I understand this sometimes occurs when there is column deterioration.

    1. Shift in retentiontime can hppen due to several factors such as change i operating conditions, namely flow rate of mobile pahse, temperature of column,changes in composition of mobile phase,etc

  24. What does difference in Retention time mean?
    our sample has RT of 5.96 and the standard has RT of 6.56. Does it mean that out sample is not ?

    1. in HPLC analysis of my sample, I got a peak at 5.94 and my std gave a peak at 6.56 mins, having done the experiment under identical conditions can both the peaks be identified as the same compound?

  25. Hello sir
    I am very thank you for your helping me understand in which what way chromotopography works in a HCLP and I very muchly like to be read yuor articles as they are very help

  26. Hello sir
    I am very thank you for your helping me understand in which what way chromotopography works in a HCLP and I very muchly like to be read yuor articles as they are very help

  27. This contains is so much helpful. Thank You so much. And please try to give more example for completely understand.

    1. Most software give you the option to do this. If not you will have to add all of them up and calculate manually ore in excel.

  28. Very informative. I think I am smarter after reading that but believe it’ll take some time to process fully in order to apply to my situation of interest. I own a medical testing company and perform a lot of drug testing as a third party administrator. I have a question in regards to false positives dealing with GCMS, specifically with methamphetamine and cocaine, in particular with hair drug analysis. There seem to be 3 or 4 main reasons for false positives: inadequate sample washing process, environmental contamination, molecularly similar components from hair products or food and drug metabolites. With methamphetamine I believe I read there are around 12 known molecularly similiar ?neoisomers?? nanosomethings. other things that appear to have the same height/curve spectrum on a GCMS. DO you have any idea what I’m talking about or what other components that have been tested by GCMS/LCMS that appear to be identical to other components with similar spectrums or graphical appearance when tested??

    I am a business man that fell into the business. Not a scientist. However it seems that the majority of scientists and clinicians are not aware of this frequent occurrence of false positives specifically for methamphetamine. However, I deal with around 20 people a month that test positive and swear they have never even used the drug. They can’t all be calling my company lying since I don’t have anything to do with how they were originally tested. Several lawyers have contacted me and believe that a number of these people are probably telling the truth. This means that countless people are having their kids taken away and/or are being falsely imprisoned due to false allegations.

    Can someone with more knowledge of this topic give me some ideas of what could be or is likely happening?

    Much appreciation.

    Blake Brewer
    Founder and President
    Drug Testing Solutions Dallas
    Recovery Solution
    North Texas and Southern California

    1. Hi
      False positive rises from wrong interpretation of MS spectra when relying only on matching the spectra with authentic libraries. However such analysis must be confirmed by convolution as all positive falsies happened when both compounds eluted together and sum of fragmentations pattern match a third compound.
      It is very easy to clear out the results and match the ion ratios must be identical to those in the library without any additional ion.
      Other solution just extract the parent ion with 4 confirming ions if all appear in your sample this is positive result otherwise negative

  29. hi,
    i have a question…
    i did a hplc experment and added 4 compounds the sample, in the results i got 5 peaks 4 for my compounds and one little peak that is very close to the acetominophen peak.what could br the resone for that

    1. Hi there can be many reasons for the extra peak, it can come from impure compounds, contamination during sample prep, impure reagents, degradation of compounds.


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