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.
Pay Attention to Solvent Grade
As with any method, it’s important to use reagents with the correct purity – i.e. take note of the solvent grade. It’s not generally a risk to use solvents that are of higher grade than what you need, higher purity usually means a higher price tag and I’m not sure there are too many labs that will spend more money on solvents than they need to. In many cases, labs will use solvents that are of lower grade than what’s appropriate for the method and this is where you run into problems. Low grade solvents typically have higher concentrations of impurities. Whether the impurities exist as additional water content, additional analyte compounds or a mixture of isomers, these unwanted compounds can interfere with your ability to recover the analytes you’re looking for.
Here are a couple of examples from Method 1664B:
This needs to have a minimum purity of 85% and must consist of at least 99.0% saturated C6 isomers. Solvent grades with a purity of >95% are perfectly acceptable; however, you may find those solvents to be more expensive.
Hydrochloric acid (HCl)
This must be at least ACS grade. If you’re using a 1:1 solution of HCl instead of concentrated HCl, your reagent water must be pure as well (distilled water or water purified with activated charcoal is sufficient).
If you’re drying your extracts with sodium sulfate, purchase it as a granular anhydrous salt that is ACS grade.
Use the Whole Sample
Oil and grease compounds tend to be hydrophobic, which means they’re more likely to be found along the walls of the sample container or on the surface of the water sample and not distributed evenly throughout the sample. This uneven distribution reduces the homogeneity of the sample, making it very important to use the entire sample for each extraction. Dividing a sample into aliquots prior to extraction will produce results that don’t accurately represent the whole sample.
Develop a Good Rinsing Technique
If you read the section above, you already know that oil and grease samples aren’t very homogeneous. This is the reason to ensure you are properly rinsing all of the labware that your sample comes into contact with. The most important piece of labware is the sample bottle; however, rinsing should not stop there. Every piece of labware that the sample comes into contact with should be rinsed – this includes the collection flask, and any labware used for transferring the sample (including funnels and transfer pipettes).
Method 1664 provides rinsing guidance – which solvent to use, which volume to use, how many rinses to perform – but it does not emphasize the importance in having a good technique for performing your rinses. Oil and grease compounds are quite hydrophobic, so there are lots of surfaces they will cling to in order to avoid the water sample that’s being processed. Therefore, when rinsing your labware, you want to make sure your rinsing solution comes into contact with all the surfaces your water sample could have come into contact with. Sample bottles should be capped, inverted and vigorously swirled during rinsing. Collection vessels should be vigorously swirled at an angle to ensure the sides are well-rinsed, as close to the neck of the vessel as possible. Thorough rinsing will help ensure that all your target analytes ultimately end up in the final extract.
Temperature is More Important Than You May Realize
Temperature often plays a role in the stability of compounds in a sample. In the case of oil and grease compounds, temperature can influence the stability of the more volatile compounds, which affects their recovery. Samples must be stored at cool temperatures (≤6 ˚C) to help keep the target analytes – particularly the more volatile compounds – in solution. When samples have been extracted and the extracts are being evaporated, the evaporation temperature should be carefully monitored. A higher temperature will certainly speed up the evaporation process; however, the more volatile compounds will likely be lost and your overall recoveries will suffer.
Drying is Key
One of the keys to a successful evaporation is to have a dry extract to start with. If your solvent contains water, evaporating your extract will likely take the better part of a century to complete. That’s a bit of an exaggeration of course, but if you’re using a relatively low evaporation temperature such as 40 ˚C (for reasons explained in the previous section), you’re at a temperature that’s too low to effectively evaporate water. Even if you only have a few milliliters of water in your extract, it’s enough to cause you problems when you try to evaporate.
Most labs use sodium sulfate to dry their extracts; however, it’s not as simple as pouring your sample over a handful of sodium sulfate. After purchasing the correct grade (granular anhydrous, ACS grade), you need to dry it further in an oven or muffle furnace and store it in an airtight container to prevent it from absorbing any moisture from the air. Clean, dry extracts start with the use of clean, dry sodium sulfate.
Evaporate to “Almost” Dryness
When you get to the evaporation step in your procedure, there’s a tendency to evaporate the extracts to dryness. After all, the final HEM concentration is reported based on a gravimetric weight, so you want all traces of your solvent to be gone before you perform the weight measurement. Unfortunately, evaporating straight to dryness can cause some of the more volatile oil and grease compounds to evaporate with the solvent, resulting in low recoveries for those compounds. The key to improving recoveries is to evaporate your extracts until there’s only a thin visible layer of hexane left. The final evaporation can be done in a desiccator (once the samples are cooled to room temperature), where evaporation will take place more slowly.
As with most experiments, a few tweaks and adjustments often improve your performance and make it easier to generate high quality data that’s compliant with the requirements of your application. Feel free to share your tips and tricks in the comments below and don’t forget to share this post with others who may be struggling with low oil and grease recoveries!