“Do what you can, with what you have, where you are.”
In a never-ending list of chemical pollutants, a compound that is gaining a lot of attention is 1,4-dioxane. In fact, New Jersey just became the first state to set regulations on the quantity of 1,4-dioxane that can be present in drinking water.
1,4-dioxane, commonly called dioxane (the other two isomers – 1,2-dioxane and 1,3-dioxane are rarely ever seen), is an ether with the molecular formula of C4H8O2. Dioxane was previously used as a polar aprotic solvent. For those who remember their organic chemistry from college, SN2 reactions involve the use of polar aprotic solvents. Since its original use in laboratories, dioxane has been determined to be carcinogenic and, unlike many organic pollutants, it is completely soluble in water. Dioxane’s use as a solvent for industrial purposes has been mostly replaced with tetrahydrofuran, which has a higher boiling point and a lower toxicity. However, the story does not end there!
Have you ever been extracting samples for oil and grease compounds using solid phase extraction (SPE) and thought, “why do I have to use all these different solvents, when I’m just trying to get my compounds to retain on, and then elute from, an SPE disk?”
If you’ve been digging into the extraction method a bit, you’ve probably asked yourself “I wonder what the purpose of the methanol is” at least once or twice. If you’re processing samples for oil and grease, your goal is to determine the concentration of compounds that can be extracted in n-hexane (also known as HEMs), so it’s logical to think that you’d load your sample onto your SPE disk, then pour some hexane through it to elute your target analytes.
For the most part, that logic is sound; however, there’s more to the chemistry than that. I was trying to explain this chemistry to a colleague of mine recently and his eyes started glazing over about 3 minutes into my explanation. I tested my explanation on a few other colleagues and got the same response so I started to give up hope that anyone was going to share my excitement for chemistry and what’s going on within the SPE disk.
Then I stumbled across this graphic and my hope was restored. In this simple graphic, the overall extraction scheme is listed in the center and the addition of methanol and hexane are illustrated to the left and to the right, respectively. What I really like about this graphic is that it allows me to walk through the extraction, step-by-step, and see the impact of each solvent. Let’s walk through it and I’ll show you what I mean.
If you start at the top, the box labeled “1” shows you the state of your sample after it’s been passed through your SPE disk. The bulk of your water matrix has passed through the disk and been directed to waste, while your target analytes remain trapped in the disk. Some of your target analytes (the beige-colored circles) are present in solution with water molecules surrounding them. Water molecules are polar and their net dipole moments cause them to be attracted to each other (the positive dipole moment of one water molecule is attracted to the negative dipole moment of another water molecule). So, in their effort to cluster together, water molecules end up trapping a few compounds – compounds that you’d like to extract and quantify. Unfortunately, passing your sample through the SPE disk does not remove these water molecules.
In an ideal world, you would add some hexane, elute all your compounds, then evaporate off the hexane and record your HEM weight. Unfortunately, those pesky water molecules are going to prevent the hexane from reaching some of your compounds (don’t forget that hexane isn’t miscible in water). So you would add hexane to elute the analytes that are free from water molecules and accessible by the hexane (follow the first arrow to the right in the graphic). This will leave you with just the water-bound analytes (the box labeled “2).
Here is where methanol will come to your rescue. Methanol is a polar solvent and is soluble in water. Methanol isn’t as polar as water, but it’s still pretty polar. When methanol passes through your disk, the attraction between methanol molecules and water molecules becomes stronger than the attraction between water molecules and other water molecules. As water molecules seek out methanol molecules, the water molecule clusters break up and release the remaining target analytes you’re trying to extract (i.e. the box labeled “3). One more pass of hexane elutes those target analytes into your collection flask and now you’ve collected all your analytes of interest.
Skeptical of the chemical journey I’ve just outlined? Check out these data tables where the proof is in the numbers. I wanted to see if I could prove out my theory in the lab, so I obtained 6 liters (yes, SIX liters) of a real-world influent sample and divided the sample into six, 1-liter replicates. The six samples were extracted using an automated SPE extraction system. All six of the SPE samples were processed using the exact same system and the same extraction conditions, with one exception – three were extracted using methanol at the appropriate steps and the remaining three were extracted without methanol.
Extraction with methanol
|Sample ID||Starting Weight (g)||Final Weight (g)||HEM Weight (mg)|
|Avg. HEM Weight (mg)||32.5|
Extraction without methanol
|Sample ID||Starting Weight (g)||Final Weight (g)||HEM Weight (mg)|
|Avg. HEM Weight (mg)||18.8|
Under the exact same conditions, the use of methanol produced an HEM weight of 32.5 mg. Without methanol, the average weight of the hexane extractable material was 18.8 mg – a difference of 42% by weight!
Seems like methanol plays a pretty important role in oil and grease extractions.
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If you are processing environmental samples then you’ve probably dealt with contamination at some point. If you haven’t, then you should be congratulated for creating the only laboratory on Earth that has ever been completely free of all sources of contamination! There are many (in some cases, many many many) sources of contamination and the severity of your contamination issues can vary significantly depending on what types of samples you run, the cleanliness of your laboratory, the systems that are running, and the care with which samples are being collected, stored, prepared, run and disposed of.
EPA Method 8270 is one of the 8000 series methods that outlines the preparation of wastewater samples. It is one of dozens of methods for processing wastewaters for semivolatile organic compounds (SVOCs), all of which fall under Method SW-846. Expand the graphic below to see the breadth of the method.
As you can imagine such a complicated method generates a lot of questions. It is a regular occurrence for me to get various questions about EPA methods but recently I have had quite a few about EPA Method 8270 and I wanted to share them in case someone else has the same questions. You can find a great summary of Method 8270 in this blog that one of my colleagues recently wrote – Extraction of Polyphenols in Tea with Lemon Juice.
Q: Do I need a carbon cartridge for my 8270 extraction?
A: The answer to this question is – it depends. The purpose of the carbon cartridge is to collect the light end compounds that are not retained by the Atlantic® One Pass disk. Which compounds are on this list? There are a handful, but some of the more interesting compounds are those related to NDMA, benzyl alcohol, as well as surrogates like phenol-d5 and 2-fluorophenol. When I analyze samples using Method 8270, I look at quite a few classes of compounds and some of them require a carbon cartridge to ensure that my recoveries are acceptable.
As a side note, NDMA is a known carcinogen and has made news headlines because the recalled blood pressure medication, Valsartan, was found to be tainted with NDMA. The EPA also put out a technical fact sheet on NDMA in 2014 that provides some interesting facts.
Q: What is the purpose of the acetone in the elution steps using the Atlantic® One Pass disk and carbon cartridge using the method in your application note?
A: Well, one reason to use acetone is for elution. That’s not the main reason for using this solvent, however. Samples that are processed using Method 8270 are typically dirty samples with high levels of particulates or suspended solids. That means a decent amount of sediment will need to be filtered by your Fast Flow Disk Holder. In the process of filtering out the solid material, some of your water could get trapped in the disk holder. If we moved straight from the aqueous phase to using dichloromethane (DCM) – which is immiscible with water – the residual water would form a barrier, preventing the DCM from passing through the disk. Acetone removes any residual water before DCM is used.
Once the DCM step is complete, acetone is used again to remove any residual DCM. The next step in the elution process involves the use of 1% ammonium hydroxide (NH4OH) in water so all remaining DCM must be gone before proceeding to this step. Believe it or not, acetone is used once again after the dilute ammonium hydroxide solution has passed through the disk because DCM is used to elute analytes from the carbon cartridge. When you think about it, acetone is pretty integral to the elution process!
Fun fact: The carbon cartridge is loaded from the bottom up in the sample processing stage so you don’t have to use as much solvent to elute your target analytes.
What are your burning, or more likely evaporating, questions about 8270?
Solid phase extraction is a powerful technique – it can be used to clean up the most challenging samples, and extract and preconcentrate hundreds of semivolatile organic compounds. When performing the extraction, the goal is to get the entire sample to run through the extraction disk. But in order to do that, the disk must have the chemical and physical capacity to handle your sample matrix. If your disk becomes overwhelmed or clogs, you risk losing your sample and the chance to complete your extraction.
How do you prevent the disk from clogging? Prefilters!