To Be or Not to Be Water Miscible

“Oh my!  This is crystal clear!” – said nobody who has ever read through an EPA Method.

For anyone who processes samples in an EPA-regulated laboratory, you know that these methods can be very specific in some spots, and incredibly vague in others.  The complexity worsens if you’re following one method for sample cleanup and another method for sample preparation and data collection.  Consult this handy infographic to make sure you’re following the right methods for sample cleanup, processing and analysis.

The EPA provides guidance when possible to navigate the methods that are available for processing samples – EPA Method 3500 is a great example.  Whether you are extracting your analytes from a water sample using liquid-liquid extraction (LLE), continuous liquid-liquid extraction (CLLE), or solid-phase extraction (SPE), EPA Method 3500 provides guidance for selecting which method to use for sample preparation and organic extraction.  This method outlines the steps that must be followed to ensure your extraction is compliant, regardless of whether you’re working with water, soil or waste samples.

Choosing Your Dilution Solvent

One of the parts of this method that I find most helpful is Section 7 which provides guidance for preparing reagents and standards.  This section tells you which solvent (or solvents) must be used to prepare your stock standards.  It’s important to use this method for guidance so you don’t get misled by your concentrated standard.  What do I mean by that?  Here’s an example:

Let’s say you’re processing samples per EPA Method 8270 and you’re working with a concentrated standard that’s shipped and stored in methylene chloride.  Method 8270 itself references the use of methylene chloride in the reagents and standards section.  Your instincts (plus the information you read in Method 8270) might lead you to dilute the standard in methylene chloride to match the solvent in the undiluted stock standard.

But sections 7.4, 7.4.1, 7.5.2 and 7.6 in Method 3500 instruct you to use water-miscible solvents to prepare your standards and surrogates.  Last time I checked, methylene chloride wasn’t miscible in water.

How do you proceed?  Do you use methylene chloride as a dilution solvent?

If you want your extractions to be EPA compliant, you should follow the guidelines in Method 3500 and prepare your standards in a water-miscible solvent.

The Importance of Water-Miscible Solvents

Does it matter whether your dilution solvent is water-miscible?

It does matter, and the success of your extraction depends on it.  Remember, you’re trying to make standard solutions for use as spikes and surrogates.  The purpose of these is to measure the efficiency of your sample preparation – in this case, your extractions.  You can’t measure your extraction efficiency if you can’t blend your spikes and surrogates and recover them as you would your sample analytes.

How do you blend spikes and surrogates into a water sample if they’re in a solvent that isn’t water-miscible?

Let’s see how that plays out with an example using liquid-liquid extraction.

Let’s say you prepare your standard in methylene chloride and you add it to your water sample as a spike.  The standard goes into the top of the separatory funnel, races through the water sample and plummets to the bottom, where it remains as a separate layer from your sample.  As you process the sample, the analytes in your standard partition into the organic solvent you use during the extraction.  When you complete the extraction and perform your data analysis, you should have recoveries around 100% for every analyte.  Unfortunately, that near-perfect dataset provides no information about your extraction efficiency.  The analytes never entered into your water sample which means you never extracted them.

The same scenario also happens if you prepare your standard and surrogates in hexane.  If you add this standard into your sample-containing separatory funnel, the hexane layer will float on top of your water sample.  As you proceed with your extraction, you’ll likely be adding methylene chloride as part of your procedure.  Given the choice between water and methylene chloride, the analytes in the hexane-based standard will enter into the methylene chloride layer and you’ll never have the chance to extract these analytes from your sample.

Now let’s see how this plays out with an example using solid-phase extraction.

You prepare your standard in methylene chloride and add it to your sample bottle, prior to loading your sample onto your SPE disk or cartridge.  The immiscible solvent containing your analytes will sink to the bottom of your sample and remain as a separate layer in the bottle.  As your sample loads, your spiked analytes pass through the SPE media and get routed straight to the water waste, along with the sample.  The analytes in your sample will be retained on the disk or cartridge, but your spiked analytes aren’t captured.  Just like in the previous LLE example, you can’t extract what you never captured in the first place; however, the difference with this example is with your analytical results where you’ll measure recoveries near 0% for your spiked analytes.

Here’s why:

In the LLE example, the spiked analytes went straight into the organic solvent that was used to complete the extraction.  In the SPE example, the spiked analytes went straight through the disk and got sent to waste along with the sample matrix.  After the sample was loaded into the disk (and the spiked analytes had been lost), a separate organic solvent was passed through the disk and captured in a collection flask.  Both approaches will prevent you from being able to extract your spiked analytes, but the analytical data will reflect that differently.

If you’re moving from LLE to SPE and you’re seeing QC recoveries drop from near-perfect to near-disaster, take another look at the dilution solvents you’re using to prepare your stock standards and surrogates.  Just as flow rates are important when processing samples by SPE, so too are the solvents you use for preparing standards.

Share your extraction challenges in the comments below!

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