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TO-15 + PAMS + TO-11A = China’s HJ759 + PAMS + HJ683 Part 3: Formaldehyde Sampling in Air Canisters

In my previous blog (TO-15 + PAMS + TO-11A = China’s HJ759 + PAMS + HJ683 part 2: Deans switching and TO-15/PAMS) I covered the combination of the TO-15 and PAMS (or HJ759 and PAMS) methods into a single run using a Deans Switch and FID/MS detector set up. I said that I’d revisit integrating TO-11A (i.e., HJ683 in China) methods, but first let’s talk about the methods a bit.

TO-11A differs from TO-15 since it involves the capture and derivitization of formaldehyde and other carbonyls on a coated adsorbent tube rather than air canister sampling. The carbonyls react with 2,4-dinitrophenylhydrazine (DNPH) to form a hydrazone derivative in the adsorbent tube. The DNPH derivative is then eluted off of the tube using a solvent and the resulting solution is analyzed by  HPLC/UV. Having two completely different sampling systems and instruments can be a rather large investment in both time and money, so it’s easy to see why there is a push to use canister sampling and GC/MS analysis instead.

So why the separate setup? Formaldehyde is capable of being analyzed directly by GC with no dervitization, although there are some issues with it being low mass and sharing ions with carbon dioxide. See Fig. 1 below illustrating our combined HJ759/PAMS method, using both air and helium as a fill gas for the canister.

Fig. 1 – Formaldehyde in helium (top) and air (bottom), showing interferences from air/CO2 in the bottom chromatogram.

Even if the chromatography can be made acceptable the issue is that formaldehyde is unstable in canisters, creating the risk of false negatives and low response. Canister coating has advanced since the TO methods were originally written though, and one maker of sampling canisters claims they can collect formaldehyde with no losses. If true that would be a game changer for air analysis, so we decided to put it to the test using their canisters. Six canisters were spiked with 100 ppbv of formaldehyde. Three were filled with dry air and three with air at 80% relative humidity to see if water content had any effect. The canisters were then tested every 12 hours for several days and we were not able to replicate their results, with the data showing a quick and drastic drop in formaldehyde response.

 

Fig. 2 – Average formaldehyde loss in dry and humid competitor canisters. N=3 for each data point and error bars show the standard deviation of the replicate canisters.

How bad was the formaldehyde stability? As seen in Fig. 2 in half a day the formaldehyde had dropped to less than 80% of the original amount in the dry canisters, and less than 60% in the humidified ones.  The humidified canisters held steady at near 50% afterwards, but the dry canisters continued to drop until they were less than 40% after 4 days. The water in the humidified canisters likely traps the formaldehyde initially (Day 0 to Day 3) then slowly releases it later (Day 3 on out), causing the initial lower and higher ending results. Given the quick initial drop in what is the simplest scenario of just formaldehyde in air it’s hard to see this as a viable sampling method, even if the samples are rushed to the lab as quick as possible.

While the push for a universal air method is understandable, at this point the sampling techniques don’t support it and it appears that TO-11A will live on as an independent method. And for those of you interested in aldehyde and ketone in air analysis, Restek has you covered with standards and HPLC columns.

https://www.restek.com/chromatogram/view/LC_EV0532

https://www.restek.com/Technical-Resources/Technical-Library/Air-Sampling/env_EVSS2393A-UNV

SPME Fundamentals: Don’t forget the salt for HS VOCs!

Long story short: We were comparing head space (HS)-SPME data with some colleagues, when they asked us “how are we using a 4 minute extraction time on our brewed coffee, when they need 10 minutes to achieve comparable results?” I told you coffee was on the horizon in my last blog. After comparing the 12 or so HS-SPME extraction and desorption parameters from our method and our colleagues’ method, we could not find anything very divergent. Of course, you know where this story goes based on the title of the blog… Our colleagues were not adding salt to their HS samples. In fact, they were surprised to hear that we were doing this.

Why would we add salt to our HS-SPME samples? Long answer: be sure to check out pages 3 and 4 of “A Technical Guide for Static Headspace Analysis Using GC.” Short answer: it does not matter if we are using HS-Syringe or HS-SPME; the addition of salt to the sample matrix (coffee in this example) will often lower the partitioning coefficient (K) for some target analytes, in particular polars. So, for all of the HS-SPME samples we run, we add sodium chloride. How much salt? Several articles have indicated ~20 – 30% wt/wt salt is optimum [1], but of course you should determine the optimum amount for your particular application.

In an attempt to show our colleagues the power of salt, we analyzed the headspace of brewed coffee samples with and without NaCl, all other variables being equal. We added 30% wt/wt NaCl to 10 mL to achieve saturation, which would help ensure consistency across samples. In addition, all samples were incubated (2 min) and extracted (various times) with a shaker speed of 250 rpm (with and without salt); but we threw in a wild card of 1000 rpm. The results of all this may be found in the following figure:

As you saw in the previous teaser, we observed numerous VOCs in the HS of brewed coffee; however, more on this in a future blog. For today, we are looking 2-furanmethanol, which has been shown to be an excellent differentiator between coffee bean roasts [2]. Here are the take-away messages:

  1. As you may see by the red trace, using a shaker speed of 250 rpm on samples with no salt correlates to 2-Furanmethanol (and other VOCs not shown) not reaching equilibrium until perhaps 960 seconds. I say perhaps, because it is hard to say when equilibrium was reached, as we did not extract longer.
  2. You will also see by the green trace, using a shaker speed of 1000 rpm on samples with no salt indicates equilibrium was reached at 480 seconds.
  3. Finally, the purple trace, using a shaker speed of 250 rpm on samples with salt clearly shows equilibrium was achieved at 240 seconds and the overall response was higher than the other two scenarios

Hopefully it is clear why our colleagues were extracting for 10 minutes and still not able to achieve what we observed in 4 min extractions (it was clear to them). It was all about the salt! Now, you could say something like “weighing out salt is time consuming, messy, and the juice is not worth the squeeze.” To which I would say “stay tuned for our Life Hack on Weighing Out Salt for HS Samples in an up-coming blog.” Till next time…

References

  1. S. W. Myung, H. K. Min, S. Kim, M. Kim, J. B. Cho and T. J. Kim, “Determination of amphetamine, methamphetamine and dimethamphetamine in hman urine by solid-phase microextraction (SPME)-gas chromatography/mass spectrometry,” J Chromatogr B Biomed Sci Appl, vol. 716, no. 1-2, pp. 359-65, 1998.
  2. C. I. I. Rodrigues, C. M. Hanson and J. M. F. Nogueira, “Coffees and Industrial Blends Aroma Profile Discrimination According to the Chromatic Value,” Coffee Science, pp. 167-176, 2012.

SPME Arrow for Acrylamide in Potato Chips

I love potato chips and I love the SPME Arrow. So, after talking with my colleague Joe Konschnik about taking advantage of the SPME Arrow to analyze acrylamide and other off-flavor compounds in potato chips; I immediately grabbed a bag of chips from my file cabinet (yes, I keep snacks readily at hand and I already confessed my love) and headed to the lab.

We originally set out to see if we could use the SPME Arrow to analyze acrylamide, as this compound has had a recent resurgence in the media. Acrylamide is a by-product of high temperature (i.e., >120 °C) cooking of specific food products [1]. Acrylamide is formed during the frying, baking, and roasting of starch-rich foods, such as potatoes and grains, in addition, acrylamide is often found in tobacco smoke. Therefore, acrylamide exposure is mostly inhalation and ingestion of certain foods. The International Agency for Research on Cancer (IARC) has classified acrylamide as a Group 2A (probably carcinogenic to humans) compound [2]. Therefore, the European Commission published their Recommendation on the monitoring of acrylamide levels in food [3]; and the US Food and Drug Administration (FDA) published their guidance to minimize human exposures to acrylamide [4].

The following work was carried out as a proof of concept experiment to evaluate the efficacy of utilizing SPME for acrylamide in various matrices. In particular, a headspace (HS)-SPME approach was desired, so as to minimize the sample preparation steps and solvents; and minimize the amount of matrix interference. Of course the SPME Arrow was utilized, as Colton and I have already demonstrated increased sensitivity with the SPME Arrow over traditional SPME fibers. The following figure and table provides the results and details (respectively) of analyzing the HS of ground potato chips.

So, my potato chips did not have a whole lot going on in the HS. In fact, ultimately, we had to fortify them with acrylamide at 0.5 µg/g. As you may have also seen and as expected, the SPME Arrow continues to give us more information over the traditional SPME fiber (no surprise there). Yes, a lot more work has to be done, but we were happy to see we could recover the spiked acrylamide from the chips as a proof of concept. Additionally, we were able to find 2,5-dimethylpyrazine, which is known for it’s off-flavor characteristics.

This story does not end here, as I happen to really love coffee and espresso even more than potato chips (yes, I have this in my office too. It really is quite nice). We have also started to look at ground coffee and brewed coffee headspace with the SPME Arrow as well. I will leave you with the following teaser c-gram generated with our new triple-phase SPME Arrow and brewed coffee HS, which is loaded with compounds. In particular, next time we will look at the furans we found. Till next time…

 

References

  1. WHO, Consultations and Workshops: Health Implications of Acrylamide in Food. 2002.
  2. IARC, IARC Monographs on the Evaluation of the Carcinogenic Risks to Humans, Vol. 60, Some Industrial Chemicals 1994.
  3. EU, COMMISSION RECOMMENDATION on the monitoring of acrylamide levels in food. Official Journal of the European Union. 3.6.2010.
  4. FDA, Guidance for Industry Acrylamide in Foods. 2016.

 

 

GC Inlet Liner Selection, Part I: Splitless Liner Selection

Splitless injections are used when detection of trace amounts of analytes is necessary and the goal is to recover close to 100% of all analytes that are injected into the instrument.  During a splitless injection, the split vent is closed for a predetermined amount of time, directing all inlet flow onto the column (with the exception of the septum purge).  Because of the slow flow rates, splitless injections can be tricky.  These slow flow rates can contribute to band broadening (wider peaks), as well as longer residence times in the liner, leading to increased interactions with any active sites.

I conducted an experiment to compare various liner configurations for use with splitless analyses of liquid injections.  I wanted to compare liners based on recovery across a wide molecular weight range, as well as reproducibility from injection to injection.  To do this, I injected a series of hydrocarbons ranging from C10 up to C40.  As these compounds are all in the test mix at equal mass, ideally, peak area responses for all compounds should be the same.  A common phenomenon, known as molecular weight discrimination, occurs in the GC inlet when there is incomplete vaporization and therefore incomplete transfer of heavier compounds, with the heaviest compounds showing much less recovery compared to lighter compounds.

The following liner configurations were compared using the splitless conditions listed in Table 1 below.

Table 1: Instrument conditions for liner comparisons.

Figure 1 shows how some common liner configurations compare for peak area response across the molecular weight range when performing splitless injections:

Figure 1: Comparison of peak area response across a wide molecular weight range for various liner configurations used in splitless mode.

As previously mentioned, an ideal liner will minimize molecular weight discrimination, leading to equal responses across all compounds.  You can see that the single taper liner with wool and the double taper cyclo liner achieve this.  Both the wool and the cyclo corkscrew provide extra surface area, which enhances vaporization by increasing the heat capacity of the liners.  The low pressure drop liner had similar performance for C20 and higher; however, there is some loss of more volatile compounds, perhaps from the wool being located higher up in the liner, leading to losses out of the septum purge vent. The presence of a taper helps to direct the sample to the column, as well as minimize interactions with the gold seal, which can otherwise be detrimental to performance with the slow carrier gas flows used in splitless injections.

When it comes to reproducibility, the liners with wool and the cyclo corkscrew also performed best (See Figure 2).  These features create a turbulent zone, allowing for reproducible mixing with the carrier gas upon injection.  They also serve to “catch” the sample, preventing analytes from hitting the bottom of the inlet where they can condense and get lost.

Figure 2: Liner reproducibility comparison across wide molecular weight range.

Conclusions

Overall, I would recommend the use of a single taper liner with wool or a cyclo liner for use with splitless injections of liquids.  The single taper liner with wool is the more cost effective solution; however, for those that do not want to use wool, the double taper cyclo is a viable second choice if you’re looking for the best splitless performance.  As you can see from the data above, if your analytes are on the more volatile side of the spectrum, the use of wool or a cyclo may not always be necessary for recovery and reproducibility.  Depending on matrix, though, these features can help to catch involatile material, as well as septa particles, preventing column contamination.

 

GC Inlet Liner Selection: An Introduction

GC inlet liners play an important role in GC sample introduction. The sample’s first contact is with the liner and from there it is transferred to the analytical column. In the case of liquid injections, the sample must be vaporized inside of the liner prior to transfer.  Choosing a proper inlet liner for your analysis is critical, as it can affect both analyte recovery, as well as injection to injection reproducibility.  Inertness must also be considered, especially when analyzing active compounds, such as pesticides, acids, or bases.

Liner selection can sometimes seem like a daunting task, because there are so many configurations available, including different shapes, sizes, packings, such as wool, etc.  The best liner is going to depend on your sample type, nature of analytes, and injection type.

In the following blog series, I would like to discuss selection of liners for split and splitless liquid injections, based on optimizing performance.  I will also blog about liners for use with gas analyses and PTV inlets, as well as direct injection uniliners.  I will post a final blog discussing the importance of liner inertness.  I hope this upcoming series will be useful to anyone who is confused by the myriad of liners available and wonders if they are actually using the best liner for their analysis.

A Hoppy Little Story

Now that we’re getting into the warmer months of the year, you’re probably starting to see pop-up beer gardens or people outside relaxing with a nice cold one. Since the beer industry has been evolving and people are enjoying craft beers more and more, there are tons of unique brews on the market. If you are not a beer connoisseur, then you may not know what gives your favorite India pale ale or summer lager its intriguing characteristics. The ingredient responsible for the bitterness and much of the aroma are the hops. There are an incredible number of different hop varieties and every single variety has its own personal profile! Some common U.S. hop varieties include Cascade, Centennial, Citra, and Nugget. But, you may be asking yourself, “what makes these hop varieties so different?” TERPENES! Terpenes are a class of organic compounds that are comprised of isoprene units and they are what give hops their unique aroma and flavor profiles. There are many different classes of terpenes, but the classes that we are interested in are the mono- and sesquiterpenes, which include limonene, humulene, pinene, myrcene, to name a few.

http://www.chemspider.com/Chemical-Structure.20939.html

Limonene – http://www.chemspider.com/Chemical-Structure.20939.html

Since I have an interest in quality craft beers, I wanted to dive into a variety and look for some terpenes. Luckily for me, a friend of mine in Restek’s reference standard department grows his own hops. Thanks, Joe! When these hops were harvested last year, Joe vacuum packed me his finest Hallertau variety.

Ground Hops

The hops were added to a Blixer processor with dry ice and then ground into a very fine powder. 0.5 g of the hop powder was measured into a 50 mL QuEChERS tube (cat# 25846), followed by 10 mL of isopropanol. The mixture was vortexed for 5 seconds, then sonicated for 5 minutes, repeated three times. 1 mL of the supernatant was filtered using a 13 mm, 0.22 µm, PTFE syringe filter (cat# 26142) and added to a 20 mL headspace (HS) vial (cat# 24685). 19 mL of RO water was then added to the 20 mL HS vial, which was then capped and ready for analysis. Further sample preparation included the CTC PAL RTC rail system, where samples were analyzed via solid phase microextraction (SPME), using direct immersion (DI), with gas chromatography-mass spectrometry. A divinylbenzene (DVB) SPME-Arrow (cat# 27486) was used for this SPME Arrow-DI-GC-MS method.

 

 

 

 

Total Ion Chromatogram of Terpenes in Hops

 

Using Restek’s Terpene Mix 1 & 2 (cat# 34095 & 34096), we were setup to identify 23 different terpenes. After normalizing the responses for terpenes to 100% we were able to gather a nice breakdown of the terpene profile for Joe’s Hallertau. Using the NIST mass spectral database, we were also able to identify several other terpenes (i.e. beta-Phellandrene), but these were not added to the pie chart.

 

 

So, the next time you’re enjoying a cold one on a hot summer day, give it a good sniff to get an idea of what terpenes may be in there. Every beer has its own unique hoppy little story to tell! Cheers!

 

Photo courtesy of Wil Stewart

Are You Interested in Ultrashort-Chain PFAS Analysis? Be Sure to Screen Your Solvents/Solutions for Contamination

Trifluoroacetic acid (TFA) is the perfluorinated analogue of acetic acid with the shortest possible chain length (C2) among per- and polyfluoroalkyl substances (PFAS). Together with perfluorinated C3 compounds, they are defined as ultrashort-chain PFAS. C2 and C3 PFAS are ubiquitous and very mobile in global aquatic environment including rain, snow, river, and even ocean. TFA, especially, can occur at very high concentration in both drinking and non-potable water sources. As we are developing a dilute-and-shoot LC-MS/MS method for ultrashort-chain PFAS analysis, we realized that the HPLC grade water and methanol could have TFA contamination!  By applying a HILIC/ion exchange column for ultrashort-chain PFAS analysis, a detectable TFA peak was observed upon blank diluent (50:50 LC/MS grade water:HPLC methanol) injection. Further analysis showed that the LC/MS grade water contains relatively higher trace level of TFA (~5 ppt) compared to the HPLC grade methanol we regularly used for LC-MS/MS analysis. In a search for TFA-clean reagent solvents, we tested a variety of water and methanol from different vendors. Using a 10 ppt standard solution (in 50:50 DI water:EMSURE® methanol) as the reference, it was shown that EMSURE® methanol (with plastic container) and JT Baker methanol are much cleaner compared to other brands with either detectable or significant higher level of TFA (Figure 1). As for water reagents, the reverse osmosis (RO) and deionized (DI) waters generated in our facility are much cleaner than the LC/MS grade water.

Figure 1. TFA in Water and Methanol Reagents

So watch out for what kinds of reagent solvents you are using if you want to include TFA in your PFAS analysis. For the trace level of TFA detection, it is necessary to use cleaner solvents for both sample preparation and LC analysis. And one more note, we also experienced that the use of glass HPLC vials could produce TFA contamination. We recommend to use polypropylene vials for ultrashort-chain PFAS or TFA LC-MS/MS analysis to avoid trace level contamination.

Are you analyzing TFA? We welcome you to share your experience in terms of what reagent solvents and labware you are using in the lab.

A Tale of Two Columns (CLPesticides and CLPesticides2)—Part IV: Fast 8081 Method Using GC Accelerator Kit

The moment has finally come to see how we can use the GC Accelerator to get the most horsepower for your 8081 analysis.  If you’ve been following this blog series, you will remember that in Part II, I talked about ways to make your runs faster.  I also showed you our previous fast “7 minute” method, using some of those tricks.  One of the limiting factors for that method was oven ramping ability; the ramps at the beginning and end were as high as a 120V Agilent GC would be able to maintain.  Fortunately, we have enough resolution at those points in the run to actually increase the ramp rate without causing any coelutions.

So how do we do this?  You’ve probably figured out by now that the answer is the GC Accelerator Oven Insert Kit.  With this kit, simply increasing the initial ramp rate and final ramp rate will allow you to elute all analytes in around 5 minutes, with a total oven cycle time close to 10 minutes.  In addition, flow rates can be increased to further capitalize on this benefit.  If, for example, your current total cycle time for 8081 is close to 20 minutes, this will effectively allow you to double the amount of samples you can process in a given amount of time.

The examples below demonstrate this fast method on both 0.32mm ID CLPesticides column as well as 0.25mm ID CLPesticides columns. Table 1 details the products you will need to try either of these methods.  Note that the columns are custom products and are not wound on cages.  This is necessary to fit both analytical columns in the oven with the entire GC Accelerator kit.  Refer to Part III of this series for detailed instructions on how to set this up.

 

Table 1: List of products needed to perform the methods below.  Both the 0.32mm ID columns and 0.25mm columns are listed for you to choose which pair works best for you.  All columns were custom products that are not on cages.

 

Figure 1: Fast GC Accelerator method on 0.32 mm ID Rtx-CLPesticides and Rtx-CLPesticides2 columns.  All analytes elute near 5 minutes with a total GC cycle time of around 10 minutes.  Link to chromatogram and conditions found here: https://www.restek.com/images/cgram/gc_ev1498.pdf

 

Figure 2: Fast GC Accelerator method on 0.25 mm ID Rtx-CLPesticides and Rtx-CLPesticides2 columns.  All analytes elute near 5 minutes with a total GC cycle time of around 10 minutes.  Link to chromatogram and conditions found here: https://www.restek.com/images/cgram/gc_ev1496.pdf

 

So there you have it!  The GC Accelerator kit can be used to increase sample throughput for dual column methods, in addition to its original intent to be used for GC-MS.  While the above example uses method 8081, similar changes could be made to other methods.  I encourage you to use Restek’s Pro EZGC Chromatogram Modeler to explore speeding up methods using increased ramp rates attainable with the GC Accelerator kit.  Any column can be ordered without a cage by attaching the suffix “-051” to the column catalog number.

ASTM D 3606 17

Keep your benzene in check with ASTM D3606

 

While benzene historically has been used as an additive in gasoline and aviation fuel, it is a known toxic air pollutant and is regulated by both the EPA and European Union. While the ASTM has published both capillary and packed column methods this blog will focus purely on the more robust packed approach. With the addition of ethanol to gasoline modifications have been made to the method to prevent the coelution of benzene / ethanol which allows for the quantification of benzene at concentrations ranging from 0.1% to 5% by volume. Toluene, while less toxic, can be quantified at between 2% and 20% by volume.

 

Since benzene and ethanol are poorly resolved on some column configurations, we have found a solution using a two column set. Column #1 is a 6’ x 1/8” OD (1.8m x 2mm id) nonpolar Rtx-1 polymer, which separates components in boiling point order. After the elution of n-octane (C8), column #1 is back flushed to prevent the heavier compounds from entering column #2, the main analytical column.  Column #2 a 16’ x 1/8” OD (4.9m x 2mm id) column packed with a proprietary polymer that allows complete resolution of ethanol/benzene, R = > 1.50. One thing to remember; if the column set is housed in an auxiliary oven, manually check the oven temperature to be sure it is actually at the method temperature of 135°C.  An incorrect oven temperature can result in poor resolution. If you suspect your oven is not at the correct temperature check the retention time of toluene. One of the most important, yet commonly overlooked problems is oxygen and moisture in the chromatographic system. Regardless of what GC system you’re using always have oxygen and moisture filters installed on the carrier gas line as close to the GC as possible to assure a trouble-free analysis.

 

Stay tuned for more blogs on this topic

Take Care of Your LC System Investment and Minimize Downtime with Routine Maintenance!

For cannabis QA laboratories and producers, developing methods for HPLC analysis of cannabinoids can be time consuming and resource heavy.  While a lot of focus has been given to sample preparation and optimization of method conditions to maximize sample throughput, one VERY critical factor is often  overlooked: routine LC system maintenance.

Keeping a log book to document LC system maintenance and replacing consumable parts on a regular basis can help minimize system downtime.  It also provides a “known good” system baseline to reference for troubleshooting if problems occur.  It’s worth the small investment in record-keeping and routine maintenance time to ensure your expensive system stays up and running to maximize productivity.

We often use “the car example” to help people relate: would you purchase a vehicle for tens of thousands of dollars and not take it in every 5,000 miles for a $30 oil change as recommended by the manufacturer?  Probably not unless you want to risk catastrophic engine damage and a much bigger price tag than $30 somewhere down the road.  Not to mention your customer’s frustrations with longer turnaround times while your system is down.  Regular preventative maintenance is critical for optimum LC performance and prolonging the lifetime of your investment.  Restek offers replacement parts and kits for a variety of LC systems.  Start here to search by instrument manufacturer or part type, or input the catalog number of the vendor part you need to replace in the search box at the top of the page.

If you have an Agilent 1100, you know those are not supported anymore by the manufacturer, but we’ve got you covered with these critical parts:

Autosampler PM Kit

Pump PM kit

Lamps

In future posts, we’ll go into more details about what parts should be changed routinely and what issues they might help prevent for your workflow.  So stay tuned for a closer look at replacement parts that you should keep on hand and be ready to routinely change to minimize the risk of leaks, flow rate and gradient percent inconsistencies, peak area/height inconsistencies, and carryover.

If you can’t wait and need to do some maintenance right away, check out our video library for help with changing a lamp, choosing the proper tubing and fittings, and more!