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

How to plan for dirty samples for both extraction and analysis

When juggling the responsibilities of working in a sample preparation lab as well as working as an analyst, it is very easy to get caught up in a never-ending cycle of samples. There is no situation “more frustrating” then when you have a bunch of wastewater samples that need to be extracted and analyzed ASAP and there is that one sample that is so much more challenging to extract than the others. After struggling all-day-long, you finally get the batch of rush samples set up to run on your gas chromatography (GC) system overnight only to come in the next morning to find that your mid and closing check standards are low and the data is effectively useless!

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Overcoming drying and concentrating bottlenecks in the lab

Working in an environmental lab requires a lot of concentration, both mentally and for the samples that you are working with. When New England finally begins to thaw and local companies rush to get their samples completed, a bottleneck that is usually experienced is the drying and concentration of so many samples.  This bottleneck is partly due to ensuring that samples are extracted within their holding times. There have been many times I have had to multitask while concentrating samples on the TurboVap® classic, leading to some extra work when that rare sample was overconcentrated.  Many of my past coworkers brought up the challenge they faced with the extraction of water and soils. In my opinion, the bigger issue was drying and concentrating.  My main complaint with these steps was it was never efficient enough and I always had to baby each step so that all of my hard work (shaking the sample) did not go to waste.  What I strived for most in the lab was an efficient and streamlined workflow for this part of the process.

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Automating EPH Fractionation in the Lab

Anyone familiar with Extractable Petroleum Hydrocarbons (EPH)  methods such as those developed by Massachusetts DEP, New Jersey DEP, or one of the other various state agencies that regulate EPHs is familiar with the long and grueling process of fractionation. These methods require you to split the initial sample extract into two distinct fractions, the aromatic and aliphatic portions, which allow you to better characterize hydrocarbons that may be affecting the environment (for more info read out previous blog post). It is most commonly achieved through a manual method which is driven by only gravity that can cause quite a bottleneck in the lab. This process can be particularly finicky requiring you to determine the exact volumes needed so that you do not elute one fraction’s compounds into the wrong fraction by mistake. On top of this, the traditional procedure involves the use of gravity to elute the fractions through a cartridge which requires a lot of hands-on time to ensure that the cartridge does not go dry and that it is moved at the correct time. All in all, this process can cause many a headache when it does not run smoothly.

Biotage Extrahera

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Common Mistakes in the Lab

When working in a contract lab or any analytical testing lab, you may be prone to periods where it seems like there is never going to be a light at the end of the tunnel, as the samples just keep on coming in. For me, I always dreaded when spring rolled around and the whole world thawed out because I knew samples would start coming in nonstop since everyone and their mother wanted to get their quarterly testing done. When faced with what seems to be such an insurmountable workload some of your normal good lab practices might take a hit if you are rushing to extract before a sample’s hold time expires. One such good lab practice is properly cleaning the glassware or anything else that might come in contact with your samples.

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Extractable Petroleum Hydrocarbons (EPH) Fractionation and Bottlenecks in the Laboratory

Anyone familiar with EPH methods such as those developed by the Massachusetts or New Jersey Department of Environmental Protection is familiar with the long and gruelling process of fractionation. For those unfamiliar, with EPH or Extractable Petroleum Hydrocarbons it is an extraction that essentially occurs in two distinct parts: the initial extraction & concentration and then the fractionation of that initial extract into the aromatic and aliphatic fractions followed by concentration again.  EPH is a method that replaces the TPH (Total Petroleum Hydrocarbons) or 8015 methods and allows for the calculation of specified carbon ranges giving you a more accurate assessment of potential health risks.

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