What are the differences between EPA Method 533 vs 537.1?

Per- and polyfluoroalkyl substances (PFAS) are a group of harmful organic compounds that are very persistent in structure. What this means is PFAS compounds accumulate in the environment over time as they do not break down easily. This makes it a concern to regulate and test these compounds as they have been shown to have adverse effects. One of the most common ways that someone would come in contact with PFAS is through drinking water. There are two notable EPA regulated methods that laboratories can use to analyze PFAS compounds, EPA method 533 and 537.1. When evaluating how to handle these methods in your lab there are some key differences in how to approach PFAS testing. See our earlier blog extracting perfluorinated compounds from drinking water – why is it so challenging?

EPA method 533 is a compliment to method 537.1, including an additional 11 compounds and excluding 4 compounds from 537.1. When used together, twenty-nine compounds can be tested in drinking water. All of these can be visualized in the table below, showing the acronyms for each compound and what methods they are tested in. Specifically, method 533 focuses on PFAS compounds that have short carbon chains, which are those with carbon lengths of C4 to C12. The first major difference between the two methods is the type of solid-phase extraction media that is used. Method 533 uses polystyrene divinylbenzene with a positively charge diamnino ligand and isotope dilution whereas method 537.1 uses styrene-divinylbenzene (SDVB) media. So, when it comes to preparing for the extraction of these compounds it is important to ensure that you are using the right type of cartridge to get the best results. The other major difference that goes hand-in-hand with the media type, is how the extraction techniques differ. With method 533; methanol and 2% ammonium hydroxide are used for extraction elutions, evaporated to dryness with a nitrogen blowdown and water bath, and then reconstituted with 20% water in methanol. However, with method 537.1, just methanol is used for extraction elutions and after it has been concentrated to dryness it is reconstituted with a 96:4 methanol:water mixture instead.

In summary, while the overall extraction process is similar, the media type, elution solvents, and reconstitution process differ between the two methods. These are the key things that you need to keep in line so that the similar extractions do not get mixed up. The easiest part to keep together is the fact that despite the differences in the extraction methods, both are analyzed on LC-MS-MS. Hopefully, this helps you to get started on understanding the key differences between the two methods and how to extract them.

If you are looking to certify or currently running EPA method 533 and or 537.1 in your lab I have included links to Biotage solutions that can help to get you started and improve your laboratory’s workflow.

Evaporation/Concentration System

TurboVap® LV Automated Solvent Evaporation System

Method 537.1

ISOLUTE® 101 SPE column

Method 533

EVOLUTE® EXPRESS Wax in a 6-mL format (150 mg) or

EVOLUTE® EXPRESS WAX 500 mg bed mass

Better Water Testing for Safer Produce

“Do what you can, with what you have, where you are.”
-Theodore Roosevelt

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.

Continue reading Better Water Testing for Safer Produce

The Importance of Methanol in Oil & Grease Extractions

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 IDStarting Weight (g)Final Weight (g)HEM Weight (mg)
Replicate #16.18316.213630.5
Replicate #26.20306.235932.9
Replicate #36.20156.235734.2
Avg. HEM Weight (mg)32.5

Extraction without methanol

Sample IDStarting Weight (g)Final Weight (g)HEM Weight (mg)
Replicate #16.24736.2656
Replicate #26.18016.204524.4
Replicate #36.25066.264313.7
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.