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 the layman as the peaks provide no information on the identity of the mixture components nor any information on the amount present.

First of all it is necessary to understand what a chromatogram depicts. The chromatogram is a two-dimensional plot with the ordinate axis giving concentration in terms of detector response and the abscissa represents the time. The detector gives response as a peak whose height should be ideally dependent on 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 = \frac{Area of Peak A  X 100}{Total Areas of Peaks (A + B + C + D)}

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

Chromatogram

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.

About Dr. Deepak Bhanot

Dr Deepak Bhanot is a seasoned professional having nearly 30 years expertise beginning from sales and product support of analytical instruments. After completing his graduation and post graduation from Delhi University and IIT Delhi he went on to Loughborough University of Technology, UK for doctorate research in analytical chemistry. His mission is to develop training programs on analytical techniques and share his experiences with broad spectrum of users ranging from professionals engaged in analytical development and research as well as young enthusiasts fresh from academics who wish to embark upon a career in analytical industry.

Comments

  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.

    • Dr. Saurabh Arora says:

      Hi Hector

      We will add more examples in future posts covering this topic, thanks for your suggestion!

    • i completly agree :)

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

        • 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.
    Regards
    Satish

    • Dr. Saurabh Arora says:

      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. Nureni Bashiru says:

    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.

  4. GITANJALI ROY says:

    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?

    • 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. Auwalu Bala says:

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

  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 b.tech biotech passout
    i didnt stuidied seriously during classes , i hope i can get to know about HPLC enough to get a job
    thanks :)

  8. onuoha samson says:

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

  9. Lauren Crawford says:

    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

    • Dr. Saurabh Arora says:

      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.

    • 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. Mohammad Farrukh says:

    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.

    • Dr. Saurabh Arora says:

      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?

    • Dr. Saurabh Arora says:

      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.

      • 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%)

        Thanks.

  13. Peter Keliona says:

    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. Ukanah Suleiman Pendo says:

    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,
    Olusola.

    • 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. Anna Lucas says:

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

  17. Anna Lucas says:

    Hello sir,
    Thank for your article

  18. 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.

    • 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

  19. Adisa MJ says:

    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

    • 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.

  20. jawad nadeem says:

    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.

  21. Vertika says:

    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.

  22. 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.

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