Dear Dania,

To calculation the pigments concentration without calibration curve is impossible. Because of at first you should calculate the equation (Y = aX + b) based on standards, then by using it you can gain the final data. 

In one way you can report your results in percentage and comparison between your samples. In another hand, you can use an alternative procedure such as a spectrophotometric method. I attached the original paper for the spectrophotometric method, Lichtenthlaer's protocol, for your reference.

Sincerely yours, Homayoon.

I think the best you can do is measure the relative proportion (% contribution) of each peak. That assumes the pigments have similar absorption and extinction coefficients, and I feel that is a shaky assumption unless all the pigments are related to one another.

Dear Jack,

It is very difficult to get accurate results without standard. try to extract the pigment and isolate and clean up using sutable technology. then you can use It as a referance to quantity unknown. But I Don't know wether your source is enough or not for such a thing.

piyal

The peak area represents the compound's concentration, but you can hardly quantify them by the concentration (mg/L) no matter they have a linear or other relationship.

I think actually it is a mathematic question. I think one possible way is adding a compound with known concentration as an internal standard sample. Even this way works, your pigment's concentration is still not accurate but an estimation related to this "internal sample".

Sorry about my poor English. I hope it is helpful. I am also going to do a similar quantification test on Gel Permeation Chromatography (a modified HPLC system).

Jack Silver and A. G. Piyal Aravinna are correct. It’s virtually (if not literally!) impossible to accurately quantitate compounds without standards. As noted, if they’re closely related, and have similar spectra, it’s possible to make perhaps rough estimates. As Di Ma noted, you need at least one internal standard for any kind of quantitation, but even then you can only measure relative changes as you go from one condition to another, and it doesn’t sound as if you’re interested in that as opposed to characterizing your pigments. With that being said, you can’t identify analytes with any confidence by only retention times without a standard for each one, in which case you’d also need something like tandem mass spectrometry, since it’s possible that more than one compound can co-elute. Can you clear up a bit about the objectives of your project, and what you mean by ‘recognizing’ a peak? Are you using a fixed-wavelength detector, or a diode-array? If fixed-wavelength, there could be even more quantitative uncertainty depending – as Jack Silver noted – on the UV characteristics of the analytes.

There are two possibilities to estimate the concentration of your pigments:

• RI (Refractive Index) detection. It is only possible using isocratic elution, the response factors are not exactly the same for related pigments, but the differences are small and not so great when using UV/VIS detection.
• ELSD (Evaporative Light Scattering Detection), is compatible with gradient LC (provided that there ar no salts or other nonvolatile components in the eluent). The differences in response for the pigments shall be small.
• Both detectors are less sensitive than UV/VIS so you have to inject more.

I agree with K Olieman, you can estimate the concentration of your pigments by ELSD, but you must take into consideration the fact that the ELSD is a non specific detector and you might see other compounds you extracted from your algae...

I agree with Jack Silver, the best you can get is a percentage proportion of each pigment. Even that may not be very accurate as the detector will not produce the same response for each pigment. You really need to do a calibration curve for each one, although internal standards would give you some idea.

WITHOUT STANDARDS IT IS IMPOSSIBLE TO QUANTIFY THE AREAS.

I would also be very cautious about identifying your peaks solely from the retention times and peak areas. Unless you have exactly the same conditions in your HPLC, including all the instrument parameters, running conditions, solvent mixtures, detector set up, it is almost impossible to match retention times accurately. Even this makes no allowance for the fact that more than one compound can elute at a given retention time under any set of conditions. The peak area is not a reliable indication of anything as it can vary as your column ages and with your exact detector conditions on each day. This is also why you need at least an internal standard with each sample to be able to convert peak areas into absolute concentrations. If you get co-eution of more than one compound at the same retention time, peak areas become entirely meaningless as you are adding two compounds together and you don't know the relative proportions.

Retention time is only really useful for giving a rough the identity of the molecule. As was pointed out you could have coeluting peaks. For that matter your column could be contaminated. Your eluent could be incorrectly prepared or your pumps malfunctioning. Internal standards are great if you have standards in the sample matrix and you which to determine the recovery of those standards as well as make assumptions for the samples and or ensure your retention times are correct. Furthermore just because a peak has the same retention time as the peaks in your reference it does not mean that it is the peak of interest. Mass spec can help you there. I am not sure why RI or ELSD was recommended. UV/Vis provides more information. RI really only works with larger molecules such as oligomers or polymers. It does not assist in the identification of the molecules. ELSD is relatively non linear so you need standards to set up a curve for the analysis. It does not assist in the identification of the molecules. So. You need a mass spec to identify the peaks and you need standards to validate the identifications and for quantitative analysis. Persio Ebner is absolutely correct.

Two things in play here.

One, you claim to know the identity of the peaks, and you state that you have Chl. A as a standard. If you know the compounds then you should be able to find the molar absorptivity constant for each peak (extinction coefficient) at the wavelength you are using. Given that you have a known concentration, peak area and known molar absorptivity for Chl. A you can then ratio the peak areas and correct for the difference in the molar absorptivities, which will allow you to quantify your other peaks.

Two, I would very carefully read Valerie's post (immediately above mine) before you make the enormous leap of claiming to know the identity of the compounds.

It is imposible to quntify It in mg/L without referances standard.

thanks everyone for the answers !

Without known concentration of standards, how can anyone calculate the concentration in sample. Standard must be required for quantification.

No,

it not possible to convert the peak area of pigments in mg/L with no standards using HPLC data.

it's not possible, you should make calibration curve with known standards to calculate the unknown concentration of samples

Dear Dania,

To calculation the pigments concentration without calibration curve is impossible. Because of at first you should calculate the equation (Y = aX + b) based on standards, then by using it you can gain the final data. 

In one way you can report your results in percentage and comparison between your samples. In another hand, you can use an alternative procedure such as a spectrophotometric method. I attached the original paper for the spectrophotometric method, Lichtenthlaer's protocol, for your reference.

Sincerely yours, Homayoon.