Preparative HPLC and flash chromatography are two of the most utilized techniques for purifying molecules post synthesis. To monitor sample purity and elution times, detection techniques such as UV/UV-Vis, mass identification, and evaporative light scattering detection (ELSD) have been used to illustrate separations in real-time. Each of these detection technologies leverage a different feature of the molecules to “see” it during the purification process.  In today’s post, I want to highlight how Evaporative Light-Scattering Detection (ELSD) can be used to find compounds and how that signal can change with different types of compounds. 

    So how does ELSD work? We have discussed in detail how ELSD works in a previous post. To summarize, a solution of dilute sample is aerosolized into the detector. This aerosol is then heated to drive off volatile solvent leaving behind solid particles or oil droplets depending on the sample. These particles can then scatter light (through both reflection and refraction) and this scattered light can then be detected. 

    My first question after hearing the ELSD process was – is there a difference in ELSD signal from a compound that is a liquid oil droplet vs a solid particle? As expected, this also created several other questions about what affects this signal. So, I set out to learn more about how the ELSD works and what I can expect in terms of a signal from different compounds. 

    I had a class of compounds at my disposal known as fatty acid methyl esters (FAMEs) that have variable UV absorption, molecular weights, and states of matter. Below is a summary table of all these compounds (Table 1). 


    Molecular Weight (g/mol)

    Melting Point (˚C)

    UV Absorbance*


    Methyl octanoate





    Methyl laurate





    Methyl oleate





    Methyl palmitate






    Table 1: Characteristic properties of FAME compounds. * Relative absorbance compared to methyl oleate at UV-all wavelength with range 200-220 nm.

    One of the first things I ran was my liquid FAMEs in a neat mixture: octanoate, laurate, and oleate in a 1:1:1 ratio by mass (Figure 1).

    Figure 1-1

    Figure 1: Biotage® Selekt chromatogram of liquid FAME solution. Biotage® Sfär C18; methanol in water. Black trace is UV-All wavelength with range 200-220 nm; Tan trace is the ELSD response

    As you’ll notice, there is a significant difference in ELSD response between each of these three compounds even though they are similar types of molecules and loaded in similar quantity. Why is that? Well, ELSD response is proportional to molecular weight (MW). In this instance we created droplets that are not detectable by the ELSD for octanoate. The response does increase with molecular weight, as seen with the laurate and oleate compounds. 

    ELSD response is also proportional to particle size. To increase particle size, I need to reduce the evaporation rate of solvent by reducing the evaporation temperature.  With the Biotage® Selekt ELSD I can simply reduce the evaporation temperature of my method, Figure 2. This increased sensitivity for laurate 3-fold.

    Figure 2: ELSD response of FAMEs at 25 ˚C temperature.

    I also compared samples that were of similar MW: palmitate 270 g/mol and oleate 296 g/mol, Figure 3. Not surprisingly the MW proportionality holds true with compounds of similar MW. At the same concentration, I have a greater ELS detection of oleate compared to palmitate due to the molecular weight differences.

    Figure 3: Comparison of similar MW oils by ELSD. Top) Methyl-Palmitate dissolved in DCM 167 mg/mL, 100 μL injection Bottom) Methyl oleate dissolved in DCM 167 mg/mL, 100 μL injection.

    What about my big question solid vs liquids? Methyl palmitate is a great sample for this test because at room temperature it is a waxy solid and above 30 ˚C it is an oil. I can therefore eliminate the molecular weight effect on ELSD signal and focus on the difference between oil droplets and solids. Interestingly, when I reduce the ELSD temperature from 36 ˚C to 25 ˚C for the palmitate sample I get an increase in signal, Figure 4, Table 2. At this temperature, palmitate should form solid particles rather than oil droplets.

    With this seemingly small change, we see a huge increase in signal, which to me indicates that solid particles have increased light scattering responses compared to oils. My assumption is that when the compound changes from a liquid to a solid, the density increases. The amount of mass traveling to the detector should remain constant under these two conditions, therefore the particles I create would have a smaller size. So why is there an increase in signal? Digging into the literature particle size plays a large role. The mechanism of light scattering changes based on the ratio of D/λ where D is particle size and λ is the wavelength of light. At D/λ < 0.1 the predominant mechanism of light scatter is Rayleigh scattering which has the highest intensity of scattering compared to Mie and refraction-reflection type scattering (see here for more details).

    Figure 4: Methyl palmitate chromatogram and ELSD signal at 25 ˚C.


    ELSD Response 36 °C

    ELSD Response 25 °C

    Methyl oleate



    Methyl palmitate




    Table 2: ELSD response of high MW FAMEs and comparison between methyl-palmitate response at 36 ˚C and 25 ˚C.

    Overall, I learned some interesting effects of sample variation on ELSD responses. The lower the molecular weight the lower the ELSD response. If the sample has a low ELSD signal, decreasing the evaporation temperature in ELSD will generally increase that sample response. Finally, palmitate as a solid, has a higher response compared to an oil due to a decrease in particle size. If you want to learn more about the Biotage® Selekt ELSD check out more information here. If you have questions or comments about the work, you see here, feel free to message me at the link below!

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