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Inject more fish extracts before GC maintenance is needed using shoot-and-dilute GC

Persistent organic pollutants (POPs) like dioxins, furans, polychlorinated biphenyls (PCBs), and polybrominated diphenyl ether (PBDEs) are lipophilic and therefore may be found in fatty matrices like fish.  In order to remove the high lipid coextractives, lengthy cleanup procedures that require multiple cleanup cartridges are often used.  I recently presented work at the Dioxin meeting where we took a shortcut and analyzed PBDEs and other halogenated flame retardants (HFRs) in fish using a quick QuEChERS type extraction, and a PSA pass through cleanup (Get the poster here!).  While the PSA pass through does a good job at removing a large amount of fat (we reduced the fat by 50-70%), there is still a considerable amount of nonvolatile residue remaining in the final extract.  This is especially evident in the mackerel sample that we analyzed where 38 mg/mL of nonvolatile residue remains after the PSA cleanup.

In order to combat the negative effects of injecting so much nonvolatile material onto the column, such as decreased response of high MW compounds and increased inlet activity, we used a 10:1 split injection.  A split injection (aka shoot-and-dilute GC) takes advantage of the sensitive detectors we have (ECD, MS/MS, HRMS) and keeps the system up at least twice as long when compared to a splitless injection.  The flow through the liner is much faster when compared to a splitless injection and the majority of the sample is swept into the split vent and not onto the analytical column.  Just remember to clean that split vent once in a while (it can get pretty gross)!

The chromatograms below highlight the advantage of using a split injection for fatty samples like fish.  In all of the experiments I used a Sky Precision liner with wool and a 15m x 0.25mm x 0.10µm Rtx-1614 column.  The sequence was the following: split injection of HFR standard, splitless injection of HFR standard, split injection of mackerel sample, split injection of HFR standard, splitless injection of HFR standard, 2 x split injection of mackerel sample, followed by the standards again.  I repeated this sequence with increasing numbers of the mackerel sample injections in between standard injections until the system failed (I could no longer quantify my results).  As you can see the response for the last two eluting peaks, syn and anti-Dechlorane Plus, are dramatically decreased after only three mackerel injections.  The split injection standard did not fail until 15 sample injections.

The increased ruggedness of using shoot-and-dilute GC was also highlighted by Jack Cochran in this blog post where he made repeated injections of used motor oil.  So if you have dirty samples and sensitivity to spare, try the shoot-and-dilute GC approach and think about all those extra things you could be doing instead of GC maintenance!

Halogenated flame retardant standard injected before and after multiple mackerel sample injections.  Response of late eluting flame retardants (syn and anti dechlorane plus) is reduced after only 3 sample injections.

Halogenated flame retardant standard injected before and after multiple mackerel sample injections. Response of late eluting flame retardants (syn and anti dechlorane plus) are reduced after only 3 sample injections.

 

HFR standard injected before and after multiple mackerel sample injections.  A split injection allows more samples to be analyzed before system maintenance (liner change, column trimming) needs to be performed.

HFR standard injected before and after multiple mackerel sample injections. A split injection allows more samples to be analyzed before system maintenance (liner change, column trimming) needs to be performed.

Fast Sample Preparation and Shoot-and-Dilute GC for Flame Retardants in Fish

The shoot-and-dilute GC technique (split injection) is perfectly matched for the fast sample preparation approach of QuEChERS.  The QuEChERS concept provides a fast, multi-residue extraction and “just enough” cleanup.  The technique is quick and minimizes solvent usage, but the resulting extract can contain a large amount of coextracted nonvolatile material.  A split injection is an advantageous injection technique for dirty samples because less nonvolatile material ends up on the column and the flow through the liner is MUCH faster compared to a splitless injection.  Don’t just take my word for it; see these blogs for more information.

Shoot-and-Dilute GC – Used Motor Oil and PAHs – Rxi-5ms GC Column Performance

PAH Separations – Rxi-5ms GC Column – Shoot-and-Dilute GC

Using the Restek EZGC Method Translator and Flow Calculator to Support Shoot-and-Dilute GC Method Development – Going from GC-ECD to GC-MS

Screening fish and other fatty foods for the presence of halogenated flame retardants is important from a human health perspective.  While the historical PBDEs have been phased out in the US, some of the newer high-production flame retardants such as those found in Firemaster® 550, do not have any available food occurrence data.  In order to develop a screening method for halogenated flame retardants, we paired the fast, multiresidue, sample preparation concept of a modified QuEChERS extraction and a quick extract pass-through of a PSA (primary secondary amine) cleanup cartridge.  The PSA pass through removed large fatty acid interferences and the samples were then analyzed using GC-ECD and GC-MS/MS with a 15m x 0.25mm x 0.10µm Rtx-1614 and a 10:1 split injection.  Even though we employed a split injection, the sensitive detectors allowed us to detect in the low ng/g range.

Analysis conditions for GC-ECD and GC-MS/MS analysis. The oven conditions are listed in the EZGC method translator. In the original column the GC-ECD condtions are listed. The translated conditions are for the vacuum outlet GC-MS/MS.

Analysis conditions for GC-ECD and GC-MS/MS.  The oven conditions are listed in the EZGC method translator. In the original column the GC-ECD condtions are listed. The translated conditions are for the vacuum outlet GC-MS/MS.

QuEChERS_HFRs_FishPSA Pass Through_FishFish_Recovery

The North American Chemical Residue Workshop – Sharing Science and Fun in the Sun

NACRW kicked off again with the Restek Vendor Seminar.  We shared dinner and drinks while Jonathan Keim gave a very informative presentation highlighting some of the uses of the Restek EZGC Method Translator and Flow Calculator.  The translator/flow calculator has many uses including:

  • Translating methods to increase speed of analysis by decreasing column length, decreasing inner diameter,  switching to a faster carrier gas.
  • Updating the oven temperature program through Translation after column trimming for maintenance so peak elution orders do not change.
  • Improving Original methods in separation and/or speed of analysis by solving for Efficiency or Speed in Translation.
  • Translating methods from GC-FID (or other atmospheric outlet detector) to GC-MS (vacuum outlet) or vice versa.

You can download the translator here.

Throughout the rest of the 3 day conference in St. Pete Beach, Florida we heard very interesting presentations about multi-residue, multi-class analytical methods, residues in honey bees and some of the latest and greatest in mass spectrometry.  With a total of 37 oral presentations, over 100 poster presentations and 8 vendor seminars, the meeting was packed full of great information for all attendees.  Not to worry, we were still able to squeeze in some fun into the meeting as well.  An opening reception, a dinner cruise (that unfortunately didn’t actually cruise), beach volleyball and a beach run were all included in the social program.  Good science and good fun can always be had at the North American Chemical Residue Workshop (formerly the Florida Pesticide Residue Workshop).

Next year should be even better, when our own Julie Kowalski takes the helm as the President of the organizing committee.

Jonathan "Munch" Keim takes the stage to introduce the Restek EZGC Method Translator Flow Calculator

Jonathan “Munch” Keim takes the stage to introduce the Restek EZGC Method Translator and Flow Calculator 

The Restek Vendor Seminar draws a crowd at NACRW.

 

Cindy Ross, Michelle Misselwitz, Amanda Rigdon and Mike Chang enjoy the dinner cruise that unfortunately didn't cruise!

Cindy Ross, Michelle Misselwitz, Amanda Rigdon and Mike Chang enjoy the dinner cruise that didn’t cruise!

Flame Retardants on my Mind and on Your Electronics and Your Furniture and….

I recently returned from the Brominated Flame Retardant Workshop in Indianapolis, Indiana. Going to a conference that covers analytical, occurrence and fate, biological and toxicology really puts what Restek does and the products that we make into perspective. Being able to analyze flame retardants in biotic and abiotic matrices is only the first challenge. The analytical methodology and data is used to monitor the levels in humans and the environment, and that data is then used for toxicology studies. Finally, and hopefully, that data is used to form policy that will in turn protect the environment and human health. The BFR meeting covers each of these important links surrounding flame retardants and I always come home from that conference and think about the work that I do to hopefully help the analytical scientist, and the changes I can potentially make around my home and office to reduce exposure to flame retardants (and other persistent organic pollutants).

I enjoyed many of the presentations at the BFR workshop, but one in particular stood out because it is a real problem, but not one that I normally think about. Where do you send your old electronics? With the rapid pace of new technology there is now a rapid increase of electronics waste. Electronic waste (e-waste) recycling in concept is a good thing. We really don’t want all of that in our landfills and electronics contain many precious metals that can be re-used. However, as Li Li from The College of Environmental Sciences and Engineering, at Peking University presented, the process of recycling the e-waste is, in many cases, rudimentary at best. Much of our waste is being shipped to developing nations that use children for labor. The e-waste is being burned in open areas, often very close to where the working families are living. Burning the electronics and plastic casings that are coated with flame retardants and other chemicals creates a toxic smoke that contains lighter brominated diphenyl ether congeners (PentaBDE), dioxins and furans, mixed brominated and chlorinated dioxins and furans, polycyclic aromatic hydrocarbons and heavy metals (just to name a few).

 

 

Much of the e-waste recycling is done in or near residential homes and employs children to help sort and disassemble old electronics.

 

Toxic smoke containing heavy metals and many organic pollutants from burning electronics waste located in someone’s “backyard”.

Spring is finally here! Unfortunately so are the ticks…

After a long and cold winter here at the Restek headquarters in Bellefonte, PA, USA it seems that spring is finally here!  It is so nice this time of year to get outside and enjoy the weather and the beautiful outdoors that Pennsylvania has to offer.   Unfortunately after a hike in the woods there is always the required tick check.  Ticks are nasty little arachnids and can carry a number of diseases, including Lyme disease.  I’ve had to pull a few off of myself, but animals seem to be even more susceptible to getting ticks since they are closer to the ground and walk through the grass and brush where ticks like to hide out.  Repellants like DEET (N,N-diethyl-m-toluamide) can be applied on exposed skin and clothing to protect from ticks.   Permethrin containing products can also be used to treat clothing and other outdoor gear that may be exposed.   It is always important to read the application instructions carefully and apply the product only as directed (for you and your animals!).

Another thing to consider is that many of these topical treatments and other personal care products eventually end up in our streams and lakes.  Jack Cochran and I, with the help of Cory Fix, worked on a project where we were using GCxGC-TOFMS to evaluate the Las Vegas Wash, an urban river that flows into Lake Mead.  In order to see very low levels of both targeted and non-targeted analytes we took 4L of wash through a disk extraction and concentrated to a final volume of 1 mL.  We found a lot of interesting things in that water and of course DEET was one of them!

The sample preparation for the Las Vegas Wash sample followed EPA Method 527.  The combination of a large sample volume (4L) and combining and concentrating the extracts to 1 mL, allowed low ppt detection.

The sample preparation for the Las Vegas Wash sample followed EPA Method 527. The combination of a large sample volume (4L) and combining and concentrating the extracts to 1 mL, allowed low ppt detection.

We found hundreds of chemicals in the sample.  These included prescription drugs, illicit drugs, flame retardants and other personal care products.

This is only a very small list of what we found. We actually detected hundreds of chemicals in the sample. These included prescription drugs, illicit drugs, flame retardants and other personal care products.

Are fatty acids overwhelming your QuEChERS dSPE PSA cleanup and causing issues in your GC analysis? Get more cleanup capacity with cartridge SPE cleanup!

Fatty acids are important molecules in the human body because they are used as a source of fuel.  There are many food sources of both “healthy” and “unhealthy” fatty acids.  Many sources of dietary fatty acids come from fruits, vegetables, seeds, nuts, and animal fats.  The QuEChERS methodology was developed to analyze pesticides in fruits and vegetables, however many scientists (including Restek) have adopted this approach to analyze pesticides in other commodities (tobacco, dietary supplements) or a further deviation of looking at other residues (PAHs, PCBs, PBDEs) in other food types (milk, tea, seafood).  My colleague, Julie Kowalski, wrote a really nice 3 part series on QuEChERS for Separation Science that starts with the basics and finishes with using QuEChERS as a concept, or tool, instead of a direct method (A Primer, Beyond the Basics, The Concept).

Let’s get back to fatty acids.  They are in foods, we want to test those foods, and they don’t play nicely with the GC injection port, and can completely overwhelm your target analytes. In the QuEChERS method, PSA (primary secondary amine) sorbent is used in the dispersive solid phase extraction (dSPE) cleanup to remove fatty acids.  However, sometimes the capacity of the dSPE format is just not enough to provide an effective cleanup of the extract for analysis.  I found this out first hand when I was trying to develop a method for determining PCBs and PBDEs in human milk using the QuEChERS concept.  In my initial method development I was using GCxGC-ECD, but found that my target analytes were shifting retention times by almost 30 seconds!  A quick look on the TOFMS and we confirmed that large amounts of fatty acids were still present in the extract.

We then moved to a 500 mg PSA cartridge SPE (cSPE) cleanup step to better remove the fatty acids present in human milk.  The process was actually pretty simple.  After a quick conditioning step with acetone, I loaded 2.5 mL of my extract on the cartridge, for this project my extract was in 1:1 hexane:acetone, and simply pulled it through the cartridge.  After pulling vacuum for 2 min to dry the cartridge, I eluted with 5 mL hexane.  Since human milk also has a significant amount of fat, I needed to further clean up the extract with a 500 mg silica cartridge.  The cSPE PSA did a really great job of removing all of those fatty acids as you can see in the GCxGC contour plots of the NIST SRM below.

The top contour plot shows the NIST SRM with a silica cSPE cleanup only.  It is clear that large amounts of interferences (fatty acids) are present in the extract.  The bottom contour plot shows that the addition of a PSA cSPE pass through then the silica SPE cleanup provides a much cleaner extract!

The top contour plot shows the NIST SRM with a silica cSPE cleanup only. It is clear that large amounts of interferences (fatty acids) are present in the extract. The bottom contour plot shows that the addition of a PSA cSPE pass through then the silica SPE cleanup provides a much cleaner extract! Both GCxGC chromatograms are on the same scale.

The Rtx-1614 separates MORE halogenated flame retardants than just Polybrominated Diphenyl Ethers (PBDEs)!

Halogenated flame retardants (HFRs) are used in numerous household and office products, including electronics, carpeting and furniture.  Polybrominated diphenyl ethers (PBDEs), a subset of HFRs, have been found to be persistent and bioaccumulative in the environment.  While the main technical mixtures of PBDEs have mostly been phased out of production and use, the concentrations in the environment have not been declining and are currently still widely monitored.  Other halogenated flame retardants are now being used to replace the PBDEs that have been phased out.  There is still debate whether these replacements will be more environmentally friendly than their PBDE counterparts and monitoring the levels and occurrences of these HFRs are important to understand potential environmental and biological implications.  A recent PBS episode of “To The Contrary” highlighted some of these issues.

Achieving GC separations for such a large group of compounds can be difficult. In addition to shared quantification ions making chromatographic resolution necessary, the thermal stability of the compounds must also be carefully addressed.  Decabromodiphenyl ether (BDE 209) is a notably difficult analyte since it can thermally degrade in the GC inlet and on the GC column. A 15 m x 0.25 mm x 0.10 µm Rtx-1614 GC column with a fast elution profile limits thermal degradation of BDE 209 while maintaining resolution of BDE 49 and BDE 71.

Thermodynamic modeling software like Pro ezGC, can aid method development for closely eluting compounds.  This modeling program was used to determine the best separation of all target analytes, keeping in mind that increased residence time in the column would reduce response to thermally labile compounds. Check out the chromatograms below for the actual separations achieved from the modeled program.  What’s even better now is that all of these compounds are available here on the Web EZGC Chromatogram Modeler.  That means that if you only want to model a subset of these compounds, you can see what the best instrument conditions are for your specific separation!!

Halogenated Flame Retardants on Rtx-1614

Figure 1: Instrument conditions from Pro eZGC modeling maximizes resolution while keeping the analysis time to 25 min. Separation is increased between BB 153 and BDE 154, however EHTBP and DP syn are now coeluting.
Oven: 75°C (1 min) to 210°C at 18°C/min, to 310°C (4 min) at 8°C/min
Flow: 1.6 mL/min

    Figure 2: Alternative instrument conditions based on best efficiency flow and optimal heating rate. This results in a fast analysis time, but compromises resolution between BB 153 and BDE 154.     Oven: 75°C (1 min) to 330°C (2.8 min) at 25°C/min     Flow: 1.4 mL/min

Figure 2: Alternative instrument conditions based on best efficiency flow and optimal heating rate. This results in a fast analysis time, but compromises resolution between BB 153 and BDE 154.
Oven: 75°C (1 min) to 330°C (2.8 min) at 25°C/min
Flow: 1.4 mL/min

If you are planning on using a subset of this list for your own modeling in the Web EZGC Chromatogram Modeler you can copy and paste from the original Excel file for this table (Halogenated Flame Retardants Excel Table).HFR Table

 

Analyzing pesticides in herbal tea using QuEChERS and GCxGC-TOFMS of course!

Herbal tea, a non-caffeinated drink made from plants, herbs, or spices has been used throughout history for its potential medicinal benefit.   Various herbal material is mixed depending on the desired medicinal or flavor properties for the tea.   As with any plant based commodity, there is the potential for pesticide residues to remain in the final product.   Herbal tea also falls into a gray area of pesticide regulation because it can also be viewed as a dietary supplement.

It can be very challenging to detect trace levels of pesticide residues in dried plant material found in herbal tea.  The extract, even after cleanup can contain a large amount of coextractive material that can completely overwhelm the target pesticides, making trace detection very difficult. Furthermore, nonvolatile material not removed during extract cleanup deposit onto the inlet and column requiring more frequent maintenance to be performed (Figure 1). Luckily we have been down this road before and came out the other side successfully (See Dietary Supplements and Tobacco application notes).  When encountered with trace level pesticides and difficult food based commodities we employ the QuEChERS methodology and evaluate and quantitate the samples using GCxGC-TOFMS.

The tea samples were first ground to a powder and then we weighed 1 g of tea and added 10 mL of water to hydrate the sample.  After letting that sit for 30 min, we went through the EN QuEChERS extraction procedure.  The extracts were cleaned up using dSPE tubes containing 150 mg MgSO4, 50 mg PSA, 50 mg C18, 7.5 mg GCB.

The primary column was a 1 m x 0.25 mm Rxi Guard column connected to a 30m x 0.25mm x 0.25µm Rxi-5ms.  The guard column (aka retention gap) allowed for better solvent focusing and improved the peak shape of the early eluting pesticides while protecting the analytical column (Figure 2). The secondary column was a 1 m x 0.25 mm x 0.25 µm Rtx-200.  All of the columns were connected using the SGE SilTite µ-Union.  For quantification, I used our new QuEChERS Performance mixes which cover a range of pesticide volatility and overlapped with many of the GC amenable pesticides commonly found in tea or the other ingredients included in the herbal teas.  The herbal tea “Wildberry Zinger” had the most incurred pesticides of the teas that we evaluated (Figure 3 and Table I).

After 28 injections of tea samples, nonvolatile residue was present on the liner.  After changing the liner GC performance was restored.

After 28 injections of tea samples, nonvolatile residue was present on the liner. After changing the liner, GC performance was restored.

The use of the 1 m retention gap allows for better solvent focusing and improves the peak shape of the early eluting pesticides while protecting the analytical column.

The use of the 1 m retention gap allows for better solvent focusing and improves the peak shape of the early eluting pesticides while protecting the analytical column.

Although the herbal tea is very complex, the GCxGC-TOFMS helps to separate the matrix from the pesticides of interest.

Although the herbal tea is very complex, the GCxGC-TOFMS helps to separate the matrix from the pesticides of interest.

Percent recoveries for a subset of the pesticides evaluated.  We achieved good recoveries at both spike levels and found a few incurred pesticides too.

Percent recoveries for a subset of the pesticides evaluated. We achieved good recoveries at both spike levels and found a few incurred pesticides too.

From Glass to Fused Silica in Corning, NY, USA

I almost hate to admit it, but oftentimes when I am enjoying a vacation, I can’t help but think about chromatography and how it touches so many aspects of life.   Last week I was in Corning, NY and went to the Corning Museum of Glass which has a fantastic display of all facets of glass.  The museum started with artistic sculptures, then a historical perspective of glass making, including a display on borosilicate glass used to make scientific glassware (including the Kuderna-Danish Concentrator and the glassware used for soxhlet extractions).  I’m sure my family loved hearing how all of the pieces of glassware are used! At Restek, we acquired Glastron, a manufacturer of specialized glassware, to expand our glassware products.

The final area in the museum is the Innovation Center which is an interactive science and technology exhibit that houses the optics gallery.  I couldn’t ignore the connection to chromatography when the exhibit started explaining how Corning chemist J. Franklin Hyde made fused silica in 1934 from pure liquid chemicals instead of melting dry mineral ingredients like other glass products. Fused silica therefore, has a much higher melting temperature than other traditional glasses. It took another 45 years until fused silica was used for capillary GC columns by adapting a process for the manufacturing of fiber optics. The development of fused silica capillary GC columns changed the entire field of gas chromatography and continues to be the chosen platform for efficient, fast separations.  It makes me wonder, what is the NEXT invention that will change the face of chromatography like fused silica capillary columns did decades ago?

We attended a glass blowing demonstration at the museum that was really great!

Watching the glass blowing demonstration at the museum was really neat!

The 33rd International Symposium on Halogenated Persistent Organic Pollutants, Dioxin 2013

The last week of August I attended the POP symposium in the steamy Daegu, Korea.  With over 500 attendees representing 42 countries the technical content was both informative and interesting.  The conference mainly focuses on persistent organic pollutants named in the Stockholm convention.  These include several organochlorine pesticides, polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF), polychlorinated biphenyls (PCB), along with some of the newer additions of perfluorooctane sulfonic acid (PFOS), polybrominated diphenyl ethers (PBDEs) and the proposed chemicals, hexabromocyclododecane (HBCD), short-chained chlorinated paraffins, chlorinated naphthalenes, hexachlorobutadiene and pentachlorophenol.  While those POPs alone can fill a week full of material ranging from analytical, toxicological, fate, transport and levels in the environment, other organic pollutants were added to this year’s program.  Several presentations focused on an emerging issue of pharmaceuticals and personal care products (PPCPs) in water and also on the analysis of polycyclic aromatic hydrocarbons (PAHs).  Speakers from Brazil, Vietnam, Korea, Australia, Canada, Belgium and the USA, just to name a few all discussed the issues of organic pollutants in our environment and the levels that are found in humans and animals.  It really highlights the importance of persistent organic pollutants and that environmental and food safety are truly global issues!

We were serenated by some very talented traditional Korean musicians during the opening reception!

We were serenaded by some very talented traditional Korean musicians during the opening reception!