There’s nothing more satisfying than successfully extracting a really challenging sample. Solid phase extraction (SPE) is a powerful technique for extracting semi-volatile organic compounds and hexane-extractable materials (HEMs). When the chemistry is tailored to meet the requirements of the application, literally hundreds of compounds can be extracted with a single pass of solution through an SPE disk.
On the surface, EPA Method 1664B seems pretty straightforward – use n-hexane to extract compounds (commonly referred to as “oil and grease”) from an acidified water sample. Evaporate the hexane from the extract, weigh the residue that gets left behind, and report that weight in terms of a concentration (often as mg/L of HEM). Yet many laboratories have found themselves looking at data which indicates that their spikes aren’t being recovered at levels that are compliant with the method. Unfortunately, there are a few details in the method that can cause trouble, regardless of whether you are extracting your samples using liquid-liquid extraction (LLE) or solid phase extraction (SPE). Keep reading for some tips to improve your analyte recoveries when doing oil and grease extractions.
“I’m so tired of doing dishes!”
Between the dishes I wash at home and those I wash in the lab, that phrase leaves my lips no fewer than 3 times a day. If I were to add up the number of hours I’ve spent washing dishes over the past year, I’d….well…it’s too upsetting, so I try not to do that calculation. Let’s just say I’d have had time to become a seasoned marathon runner and to backpack across both Europe and parts of Australia.
If you’re a laboratory that’s processing drinking water samples using solid phase extraction, you’ve inevitably gotten to the step in your procedure where you’ve eluted your analytes from your SPE media and you find yourself saying “How do I dry my extracts?”
What’s the best way to dry my extracts?
This is a question we get quite frequently and it’s a reasonable question to ask. Unfortunately, the answer is – it depends. Solvent drying (not to be confused with solvent evaporation) is an important step in your extraction process when you’re using organic solvents to elute your target analytes. Residual water in your solvent can cause issues if your target analytes extract back out of the solvent and into the water while you’re trying to evaporate or analyze your sample. Water can also damage your chromatography system, so if you’re quantifying your extracts by GC/MS or LC/MS, you want your solvent extracts to be dry prior to analysis.
So, given the importance in solvent drying, I thought I’d share some of the commonly asked questions that come up under this topic.
Q: I’m processing my samples against EPA Method 525.3. Does it matter how I dry my extracts?
A: If your lab is being audited against EPA Method 525.3, you need to dry your extracts per the recommended procedure – in this case, sodium sulfate. Make sure you purchase the recommended grade of anhydrous sulfate and store it appropriately.
If your lab processes a large volume of samples, you may have sought out alternative approaches to solvent drying, such as phase separation membrane. While sodium sulfate is readily available for purchase in bulk quantities and is pretty easy to learn how to use, it has some potential downsides to it.
- It has to be dried and carefully stored, which is time-consuming and requires you to have adequate drying and storage equipment
- It has to be disposed of as hazardous waste
- It’s a chemical that dries your solvent by reacting with water to form a hydrated salt, which means it can retain some of your target compounds (particularly those that are highly water soluble)
- It can contaminate your extract, particularly if it’s stored incorrectly or purchased at a lower grade than is recommended
- It can be saturated. What that means is, if you didn’t calculate the mass of sodium sulfate you needed, given the volume of water you needed to remove, you could exceed the capacity of the salt and end up with a solvent that’s not completely dry
Phase separation membranes physically separate the water from your solvent, which eliminates all of the challenges you face with a chemical drying agent such as sodium sulfate. Plus, it’s compact and easy to store, intuitive to use and easy to dispose of.
There are a handful of benefits to using a phase separation membrane over sodium sulfate – just make sure you check the method you’re following and adhere to the drying method outlined there (if there is one). Check out the method summary in this app note for an example protocol that adheres to EPA Method 525.3 guidelines.
Q: Since EPA Method 525.3 specifies that I use sodium sulfate, can I put sodium sulfate on top of phase separation membrane to dry my extracts?
A: While clever, this is an idea that you would want to run past your auditor first. Since the method specifies the use of sodium sulfate but does not specify the physical separation of water (using a phase separation membrane, for example), physical separation isn’t forbidden, but it’s also not specifically allowed. Yep, this one is a gray area so have a conversation with your auditor before cleverly devising a drying setup that includes both chemical and physical solvent drying.
Q: I’m running samples against EPA Method 525.2. Do the same rules apply to me?
A: Yes. As with Method 525.3, this method specifies the use of sodium sulfate.
Q: I’m not processing samples against an EPA Regulated Method and my protocol doesn’t specify a protocol for extract drying. What should I do?
A: If your lab is not reporting results against a method that specifies an extract drying method, you should have the option to decide whether you want to dry your extracts using physical or chemical separation (double check your laboratory’s established protocols to make sure your SOP allows you this flexibility).
If this decision were up to me, I’d order myself a huge stack of DryDisk® Disks and wave goodbye to sodium sulfate forever!
Do you prefer physical drying over chemical drying? If so, let us know in the comments and share this post to spread the word!
“Do what you can, with what you have, where you are.”
As the seasons change, I’m reminded of this quote and its significance to the air, land and water that sustain us. As the weather gets warmer and winter transitions into spring, I love listening to the sound of birds chirping in the morning and watching new flowers blossom. I look forward to the coming weeks and months of picking strawberries, raspberries, cucumbers and a myriad of other fresh fruits and vegetables. There’s nothing like the feel of the warm sun and a gentle breeze as you pluck a fresh apple from a tree and bite into it.
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.
If you’re like most laboratories that are responsible for processing samples for organic compounds, you are on a constant quest to improve efficiency and operating costs while maintaining regulatory compliance and technician safety.
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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!