Please mention all chromatographic conditions before anyone make a suggestion.

Based on this figure, it is very clear that your peak is eluting at the dead time, which means there that the analyte is not interacting with the stationary phase. There are several reasons for shoulder development, but before that you have to ensure that the peak is retained longer.

The stationary phase: C-18 column.

Mobile phase: Methanol : H2O : Acetic acid (60:30:10).

Elution rate: 1 ml/min.

Sample concentration: 1 mg/ml of 100 % methanol.

I followed the protocols in two different papers with different mobile phases as well as two types of C-18 columns, but each time I get the peaks earlier than expected.

I have attached the solvent chart as well.

Do you have 10 % acetic acid? There is something wrong with the chromatography. Could you show the published methods?

The paper is attached (page: 6). The ratio for Methanol : H2O : Acetic acid (7:3:1), but I modified it to be 60:30:10 %.

C18 column are different from company to company in terms of pore size, surface area and end-capping. You cannot expect same retention time even if the mobile phase is same. Also keep in mind that you cannot use such a large percentage of acids. Acids wash off the bonded C18 phase. What is the pore size of your column? This information should be available on the manufacturer's website. The column used by the authors of the paper is BetaBasic C18. The pore size is 150 Ao , this pore diameter is suitable for small and medium sized molecules.

https://www.labcompare.com/1518-LC-Columns-HPLC-Column/5191584-BetaBasic-18-HPLC-Columns-5-m-Particle-Size/

Thanks Dr. Wahab for your reply. I will check the details of my columns.

The retention time is very low, I would suggest to lower the methanol content to 30-40 % in the mobile phase as well as to lower the acetic acid concentration to 0,1% and compare the results.

regards,

G

So are you using the 250 mm or the 150 mm column for the UV work? Assuming you are using the appropriate column for the analysis are you using a quaternary pump and allowing that to set your methanol, H2O, acetic acid ratio or are you premixing and using one channel? The latter method is more appropriate.

remember about the degassing of the mobile phase.

It does appear that there are several problems with the chromatography and the setup.

(1) It is true there is little retention, and greater retention is recommended so that some separation of components in the mixture can be achieved. This is a very basic idea in chromatography,

(2) the patterns are not the same at the 2 wavelengths, indicating some co-elution of components of the mixture which have different spectral properties

(3) The sharp negative going far edge looks to be a refractive index effect of something else eluting. (Notice it goes below 'baseline")

(4) The periodic 'rumble in the baseline is indicative of another problem-either hardware, or perhaps you are making the eluent composition via gradient maker, in which case the precolumn mixing volume is not large enough , compared with the proportioning rate of the solvent sources.

This is easy to check in a separate experiment: put 1/1000 acetone in methanol and water on the other side. Use the gradient maker to generate the mixtures, and track the composition via a uv detector. See if there is steady signal from the detector at each desired composition, and also check the true composition versus desired composition over the range of values of interest. There are a lot of crummy gradient makers in commercial instrumentation-both for accurate composition, and for stable composition.

Dr. Michael

I am using the 250 mm column. Does it make difference in the analysis other than the change in the retention time?

I use also the quaternary pump. How can the premixing process be better than the mixing through the channels?

Dr. Grzegorz

The mobile phase components are already degassed.

I have tried methanol with the 40 and 30 % concentrations, but got the same results as shown in the question figures whether using acetic acid solutions of 1 or 10 %. However, the shoulder disappeared in the new cases.

Dr. Scot

I have checked the details and previous work done with the type of column I am using on the company's website and they showed early retention time for eluted compounds with similar molecular weight to heme ( some Rt are 1.5 min). However, I am still worried about my result.

After trying other concentrations of methanol and acetic acid, I got very similar chromatograms at the both wavelengths with no or weak shoulder. Although the sample is pure heme, but I don't understand what's the cause of these changes just with changing the mobile phase components.

(1) The separation mechanism is surely not by molecular weight or molecular size, since it elutes after the solvent front. chromatography based on Separations based on Molecular size in solution (sec/gpc) result in elution of the larger molecules before the solvent front. In general, the mechanisms involved in bonded phases are partition like mechanisms, and that translates into relative soklutilities (partition coefficients) of materials to be separated.

(2) The different behavior may be an instrument artifact, but. It is likely real information, indicating some impurity.

(3) Solvent composition generated by multichannel systems are never as good as a one pump system pumping a well mixed solution of solvents and additives. This has been proven for over and over during my time (nearly 50 years) of using HPLC and developing instrumentation.

(3) It is not good practice to have so little retention and rely on the results. I'd interpret your experience to say the Heme is not attracted to the stationary phase, so there is little retention. Not a surprise, based on the chemical nature of a quite non polar and non polarizable stationary phase..

One more suggestion to the original poster: Do benchmark experiment to confirm if the column is still alive. Do a standard C18 separation, if you have the QC paper from the manufacturer (or check their website). If you have lost retention of simple compounds, chances are that the stationary phase is damaged/old.

Dr. Scot and Dr. Wahab

Thanks for your notes and clarifying some concepts of chromatography for me.

I have tested my column with a standard solution with known Rt according to the manufacturer's protocol, but got the same peak as in heme, so it seems that there's a problem with my column.

I tried another column with the same mobile phase, but got the same results as in the first one. However, I started to follow the protocol in another paper using a different mobile phase, and some retention takes place. Anyway, I am still optimizing the conditions of that protocol.