Volatile. Flammable. Skin irritant. Respiratory irritant. Possibly fatal if swallowed. For those of you processing samples according to EPA Method 1664B, you’ve seen these hazard descriptions before – on the safety data sheet (SDS) for n-hexane.For those of you who aren’t familiar with (or have forgotten about) the hazards related to n-hexane, those are just a few. It also smells unpleasant and could explode if heated. It’s a relatively unpleasant organic solvent to work with and it begs the question:

    Is there an alternative?

    The short answer is yes, but let’s break that question down.

    What’s the alternative?


    Aren’t cyclohexane and n-hexane different names for the same solvent?

    Not even close. Although they are both hydrocarbon alkanes with 6 carbons (hence, the word “hexane”), there is not much in common between these two solvents at all. How so? Here are a few examples:

    • -Molecular weight – n-hexane has 2 additional hydrogen atoms, compared to cyclohexane which makes it slightly heavier (approximately 2 atomic mass units heavier) than cyclohexane
    • -Density – the ring structure of cyclohexane gives it a higher density compared to the linear chain structure that n-hexane has
    • -Toxicity – there are several reasons as to why n-hexane is more hazardous to work with than cyclohexane. The relatively high vapor pressure and known neurotoxicity of n-hexane are two examples
    • -Boiling point – cyclohexane has a boiling point of 80.7 ˚C, compared to the boiling point of n-hexane which is 68.7 ˚C

    How do I know whether I can use cyclohexane for my extractions?

    Check with your lab protocols. If your data has to be compliant with a regulated method, check the method to see what it allows (or doesn’t allow).

    Are there any methods that allow cyclohexane to be used?

    There sure are – ASTM D7678, for example. This ASTM method is used for determining total oil and grease (TOG) and total petroleum hydrocarbons (TPH) with cyclohexane as an extraction solvent.

    Can I just take my existing method and substitute the n-hexane for cyclohexane?

    Methanol is a critical step in achieving the best oil and grease recoveries.Not if you’d actually like your extraction to be successful. Using cyclohexane to perform oil and grease extractions makes your methanol rinse step a whole lot more complicated. Here’s why:

    Methanol is a critical step in achieving the best oil and grease recoveries.

    Remember when I mentioned that cyclohexane has a higher density than n-hexane? Unfortunately, the density of cyclohexane (0.779 g/mL) is very close to that of methanol (0.792 g/mL), which creates a unique and challenging problem. The methanol rinse is used to eliminate residual water molecules on the inside of the bottle and on the SPE disk (don’t worry – the methanol goes to waste, so it won’t end up in your extract).

    Methanol is polar so it won’t elute the non-polar oil and grease materials, but it will do a great job removing residual water. However, a small amount of water and methanol will remain. It’s impossible to get every last drop of residual water and methanol to rinse down through the SPE disk and get pulled to waste. So how do you remove the remaining water/methanol mixture (you don’t want either of these to end up in your extract)?

    If you’re using n-hexane as your extraction solvent, you’ll get a very visible phase separation between the n-hexane and the water/methanol mixture inside your collection flask. So, separating the n-hexane is as easy as draining off the more dense methanol/water layer.

    If you’re using cyclohexane as your extraction solvent, your collection flask will look more mixed. Since methanol has essentially the same density as cyclohexane, you’ll get a suspension. The water/methanol mixture will get trapped within the cyclohexane and no clear phase separation occurs. In some cases, an emulsion of water, methanol and cyclohexane forms and breaking that emulsion can be incredibly challenging and time consuming to break. You could try to remove the water using sodium sulfate or physical drying techniques, but you’ll only get rid of the water. The methanol will still be there.

    Does it matter if there’s methanol in the extract if it’s just a small amount?

    Yes. It matters. Regardless of which method you’re following, it matters. EPA Method 1664 forbids methanol to be in your extract because it throws off your final gravimetric measurement. ASTM D7678 requires your analytical measurement to be with a Fourier-transform Infrared (FTIR) instrument and methanol will create a broad interference peak that prevents you from accurately measuring your oil and grease compounds. How do you get around this? Don’t use methanol in your extraction procedure.

    Did I just tell you NOT to use methanol?

    I sure did! But this is only for methods such as ASTM D7678 where your final measurements are done by FTIR. A small amount of water won’t cause you problems because the IR transmission peaks for the -OH bands in water are far enough away from where your analyte peaks will be that you shouldn’t have any issues quantifying them. If your final analysis is being done gravimetrically, you definitely want to get rid of that residual water.

    If you’re processing samples for oil and grease, look to see what your options are for extraction. Can you use cyclohexane as a safer alternative to n-hexane? Are you using a gravimetric method and, therefore, need to make sure all of your water has been removed from your extract? These are questions you should ask before you select your extraction solvent and fine-tune your method to ensure that you get the best recoveries from sample to sample and from day to day.

    If you have colleagues who could benefit from this information, feel free to share this post! Read more tips on improving your oil and grease recoveries.

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