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3-MCPD blog part 2: How hot we can go with inlet and can we use regular split/splitless inlet?

In my last blog, I (hopefully!) made a case that switching to split injection for analysis of 3-MCPD esters is a viable option. Now I’d like to show that if we stick to split, we can also use much higher temperatures and if we can do that, we can also use regular split/splitless inlet.

To evaluate, I used a similar approach as last time, i.e. set of calibration standards. Standards were analyzed at 120°C and 280°C with MMI inlet and at 280°C using split/splitless inlet (Figure 1).

Figure 1: Comparison of calibration curves of 3-MCPD-d5 obtained using different inlets and inlet temperatures.

Unfortunately, the data were not normalized with an internal standard, therefore, the response of second analysis (split/splitless) was higher than for the analysis with MMI due to evaporation. Nevertheless, the calibration curves from analysis using MMI inlet are almost identical for both temperatures, showing that the temperature of the inlet has little to no effect on the analysis (Fig. 1).

This means that with split injection we can use either MMI (PTV) or regular split/splitless inlet.

Next time I’ll look into how switching extraction solvents affects the analysis!

This Valentine’s Day is all about QUICK connections…

This year, you do not have to be in a long-term, committed relationship in order to embrace Valentine’s Day. In fact, this year is dedicated to “QUICK connections.” You say WHAT???? Check out the following video and you will understand:

Choosing the right dispersive SPE for GC-MS/MS analysis of celery

I’ve recently started experimenting with QuEChERS extractions for pesticide analysis. The available options are overwhelming, especially when it comes to dispersive solid phase extraction (dSPE) for the cleanup. For starters, I’ve been looking at the smaller volume dSPE (2 mL, summarized in Table 1), because I didn’t want to waste the raw material, solvents and salts. Using 15 mL dSPE we’d be using 6-8 mL of extract, while 2 mL dSPE uses only 1 mL, which allows for more replicates per extraction.

Table 1: Summary of available dSPE

Read the rest of this entry »

What are GenX and PFBS? Why are they important in PFAS analysis?

Analysis of PFAS (per- and polyfluorinated alkyl substances) has been a hot topic for envoironmental labs for the last several years. Currently there are two C8 based PFAS compounds (PFOA and PFOS) with a health advisory level of 70 ppt in drinking water announced by the US EPA. In 2018 at the National Leadership Summit, the US EPA announced that they will work with other US federal agencies to establish human health toxicity values for two additional fluorinated compounds, hexafluoropropylene oxide dimer acid (HFPO-DA, a.k.a. GenX), and Perfluorobutanesulfonic acid (PFBS). They have been made to replace PFOA and PFOS, respectively.

(GenX and its precursor acid hydrolyze into HFPO-DA. Credit: Courtesy of Mark Strynar and Laurence Libelo/US EPA)

If your laboratory has a plan to add these compounds to your PFAS analyte list, HERE is what it looks like on our Raptor C18 column (9304552).

If you already have GenX and PFBS in your analyte panel, look what can be done faster on our Raptor C18 column (9304552). Total runtime of 4 min with often early-eluting PFBS well retained at 1.6 min for reliable quantitation.

Are you looking at even more analytes in the PFAS group? Do you want a sub-2µm particle size column solution?

I’m glad you asked. Don’t miss our 34 PFAS compound analysis with a short 8-min runtime solution below.

And did I mention why you need a PFAS Delay Column (27854)?

Stay tuned for more PFAS research updates from us.

Limitations of Alumina: What you should NOT inject on alumina / Al2O3 columns

Alumina (Al2O3) capillary PLOT columns have been available for about 35 years. They are best suited for separation of C1-C10 hydrocarbons and especially the C1-C5 range. The alumina has unique selectivity making it possible to separate all the unsaturated hydrocarbons at temperatures above ambient, see figure 1. While its an excellent choice for unsaturated hydrocarbons, special attention is required for components like pentadiene and 1,2-butadiene which can show reactivity on alumina, see  https://blog.restek.com/?p=8284

WHAT DOES ELUTE FROM ALUMINA?

Fig 1 C1-C4 hydrocarbons separated on Al2O3 PLOT

Compounds that elute are basically all hydrocarbons up to C12 and  aromatics up to the xylenes. Permanent and noble gases like Nitrogen, Oxygen, Hydrogen, Neon, Argon, Krypton, Xenon will elute virtually without retention. Also CO elutes from alumina together with methane and the permanent gases. These gases can be separated, but the oven temperature has to be lowered to -100C or lower.

Gases like N2O are well retained on the Alumina column, and show good peak shape, see: https://blog.restek.com/?p=52551

Several chlorofluorocarbons are retained, however the type of alumina and deactivation used will determine the (re)activity, see below.

 

We will discuss here the components we should NOT inject starting with the ones that have the biggest impact: water

WATER

Traces of water will deactivate the alumina and the result is that retention decreases see figure 2.  Water influences overall retention and changes the selectivity of the column.  This can be seen when analyzing polar hydrocarbons, like methyl acetylene.

What can be done?

  • Remove the water from the sample. This is difficult and adds a lot of time.
  • Elute the water after every injection. This can be done by heating the alumina at high temperature (200/250) for 8-10 minutes. The AluminaBOND MAPD can be heated up to 250C, speeding up this process.
  • If water is present at trace level, give the column a conditioning after XX analysis. Usually XX is determined when the target peak runs out of the integration window.
  • Use a Rtx-Wax pre-column (thick film) via a valve in series with the Alumina. Water will be retained on this column, while C1-C6 will elute first. When the C1-C6 have entered the alumina, the water peak is switched to vent or sent to a second detector (if it needs to me monitored)

Fig. 2 Impact of water on retention of Al2O3

POLAR COMPOUNDS (alcohols, amines, acids, amino alcohols, ammonia)

Polar compounds will not elute as a peak from the alumina column. If they elute, they will elute as a “blob” or a raised baseline and once they are baked out of the column the column will continue to perform well.

If polarity of component decreases, the impact on the alumina will also decrease. So, ketones, ethers and aldehydes will still be adsorbed but are easier to elute at higher temperatures.

HALOGENATED COMPOUNDS

Halogenated compounds may elute OK from the alumina column. We can measure several CFCs using AluminaBOND CFC columns, see figure 3. Reactivity can occur with some halogenated components, Figure 4 shows examples for 1,2 dichloroethane and 2-chloropropane, which decompose into propylene and vinylchloride while splitting off a HCl molecule.

Fig. 3 CFC separations on AluminaBOND CFC

Fig. 4 Reactivity of alumina

CARBON DIOXIDE

Carbon dioxide will be adsorbed completely, but it takes significant amounts of CO2 to impact retention times. If the retention times decrease, condition the column for a few hours at its maximum temperature limit.

SULFUR COMPOUNDS

Sulfur compounds will be strongly adsorbed. Hydrocarbon samples containing Sulfur compounds in the sub ppm range (COS/H2S) can be analyzed with minor changes in retention time.

 

HEAVIER HYDROCARBONS

Hydrocarbons C12 and above will not elute as discrete peaks and will manifest themselves as a raised baseline. The alumina column will appear to “bleed”.  If it bleeds, it is an indication that there is something adsorbed on the column. Make sure you periodically condition the column at maximum programmable temperature until the baseline is flat.

SILOXANES

Be aware that siloxanes can also stay on the column and will produce a raised baseline. This is often caused by the septum. For light hydrocarbons, try to use a moderate injection port temperature (80-100°C). You do not need a 250°C injection port temperature for mixes that contain C1-C6 compounds.

 

BEST SOLUTION

In all cases when there are components present that are adsorbed on alumina, and that do impact your retention and separations, the best way is to use a pre-column that can be back-flushed. This way the adsorbed component do not make it onto the analytical alumina column and retention times for hydrocarbons will be reproducible. You can choose to use a short (1-2m) alumina column as a guard, and do the back flushing using valve switching or using a Deans (flow) switching. Today these systems are very easily to setup and operate.

 

 

Trade in Splitless for Split: Faster Speed & More Miles Analyzing 3-MCPD / Glycidyl esters

In my recent work with analysis of 3-MCPD and glycidyl esters, I reviewed the available methods by AOCS and they list splitless or pulsed splitless injection. Splitless injection theoretically transfers all of the sample onto the column, allowing the analyst to achieve trace levels of detection. However, there are limitations. Both splitless hold-time and focusing (or lack thereof) have an effect on peak shape (broadening and tailing) when set incorrectly. Another consideration is the presence of derivatization agents which can damage and decrease column lifetime. The split injection (shoot-and-dilute approach) overcomes these limitations, however, since the majority of the sample is vented there is concern about achieving adequate sensitivity. There is an excellent blog about using this approach for the analysis of used motor oil here! Read the rest of this entry »

SPME Fundamentals: A Look into Incubation and Extraction Temperatures

Being fairly new to anything comes with some good lessons learned. My recent foray into Solid Phase Microextraction (SPME) has been a lot of fun, but I’d be lying if I said it has not been frustrating, complete with some face-palming moments.

 

 

In this series, we are going to take you on a journey, looking at different SPME parameters. Not only do we hope that this series will teach you about the fundamentals of SPME, but also that it can assist you during method development.

To kick off this series, we are going to start with how incubation/extraction temperature affects compound response. This will cover compounds ranging from very volatile to semi volatile compounds. This topic originally came up during some method development for a residual solvents in cannabis products application (check out that series here!) where we wanted to move from a headspace syringe (HS-Syringe) method to a headspace SPME (HS-SPME) method. Not having the most experience with SPME, it was thought that we could just take our HS-Syringe parameters and use them for the HS-SPME method. And as you may have guessed, we were wrong.

To understand what temperature is best for doing HS-SPME (keep in mind that we are using the SPME Arrow for this study), we set up an experiment where we tested incubation/extraction temperatures from 30°C to 80°C and then compared each compound’s averaged area.

 

 

This was also compared to the HS-Syringe, where the incubation temperature was set to 80°C, which is a traditional temperature for this type of analysis.

 

 

 

 

These results are very interesting. Just looking at the HS-SPME results, having an incubation and extraction temperature of 30°C gives the best response for the hydrocarbons, especially for the hydrocarbons that have higher boiling points. The more polar compounds respond a little differently though. For example,the response increases for methanol and ethanol as the incubation and extraction temperature increase. These samples are in water, so it makes sense that these compounds need higher temperatures to partition out of the water and into the headspace. However, for the majority of the compounds (17 out of 22) HS-SPME at 30°C gives better response than at 80°C.

When the HS-Syringe method (80°C, 45 min) is compared to the results for HS-SPME (30°C-80°C, 2 min), the HS-Syringe does give better response for some compounds, but when taking in all of the data, the HS-SPME results are very close and for the majority of the compounds, it gives much better response in a fraction of the time.

So, why does HS-SPME at 30°C give better response than at 80°C for the majority of the compounds? I mean come on, more is always better, right?

Well, as Pawliszyn pointed out in 1995, high temperature can have an adverse affect on the absorption of analytes to the fiber coating. This is due to the decrease of partitioning coefficients, meaning that at higher temperatures, while the analytes will release from the matrix, does not mean that they will absorb to the fiber coating. The analytes are more soluble in the fiber coating at lower temperatures versus higher temperatures and this makes a lot of sense knowing that to desorb the analytes off the fiber and into the gas chromatograph requires the inlet to be at higher temperatures rather than lower.¹

If you have encountered this problem before, when switching from a traditional HS-Syringe method to a HS-SPME method, we know how you feel! Luckily, if you simply adjust your incubation temperature, you should see a nice boost in response for most of your compounds of interest!

It is important to note that the incubation time for all of above results was 120 s and the extraction time was 120 s for the SPME Arrow. Perhaps one could incubate and extract for less time at the higher temperatures. However, clearly higher temperatures were detrimental at the current times. Stay tuned in the future when we look at incubation time.

 

 

References:

  1. Quantitative Extraction Using an Internally Cooled Solid Phase Microextraction Device. Zhouyao. Zhang and Janusz. Pawliszyn Analytical Chemistry 1995 67 (1), 34-43

 

 

 

 

Sorry for the delay. Here is your chromatogram with and without the PFAS Delay Column.

Sometimes I hear this from customers: “I don’t need a delay column. I haven’t had any trouble without it so far.”

It might be true. You may not have any issues without the delay column, especially when your samples are, for example, highly PFAS contaminated soil samples because the interference is too small to affect your end result.

I’ll show you the difference between PFAS analysis with and without the delay column.

Don’t be surprised. The chromatogram on the left is a blank sample injection! As you can see, there are peaks at the retention times you would expect the compounds from your sample to come out. Imagine what can possibly happen if you don’t have the delay column and you inject your sample containing low levels of PFAS.

                    

The chromatogram on the right also shows a blank sample injection, but this time the PFAS delay column was installed. You can see that the sharp peaks disappeared, but where are those delayed ”peaks”?   The elevated signals coming AFTER the expected retention times of the compounds of interest are the system-related PFAS that were held up by the delay column.

Remember the PFAS delay column is installed BEFORE the injector in the LC-MS/MS workflow and system-related interference will enter the delay column in a continuum from the mobile phase bottle and make their way through the analytical column to the detector. Unlike the injections of your sample which focus analytes at the head of the analytical column before the gradient starts, the interferences are continuously fed and they will be retained as they go through the delay column, diluted as they go through the injector, then retained again on analytical column not necessarily being focused much at any stage; hence resulting in broad and elevated signals.

Yes, the delayed interferences are broad because they enter the system continuously, and the signal is only elevated instead of looking like a sharp peak because the interferences exist from the very beginning of your analytical workflow (mobile phase) and are never focused on the analytical column like your sample. The magnitude of the delayed signal elevation depends on how long you equilibrate your LC column (length of column equilibration is related to how much your LC-MS/MS system components leach out the interferences onto the delay column) and what LC-MS/MS system you use (different LC-MS/MS systems may have different levels of system-related interferences depending on the materials used in various component parts).

Another way of minimizing system-related interferences is to replace all plastic parts in the workflow which may have PFAS leachates with PFAS-free parts such as PEEK. Still, the possibility of having background interferences from the workflow is endless since different LC-MS/MS systems may need different parts to be replaced. This approach might be too time-consuming to be practical, and you can still struggle with finding unknown sources of the interferences.

Sometimes you may analyze PFAS samples with high concentration for remediation projects,  e.g. contaminated soil from military bases or fire department practice sites, and sometimes your samples can be drinking water samples, which usually require low level detection (parts per trillion or even below).

Regardless of the LC-MS/MS systems you use for PFAS analysis and the level of concentrations you work on, having the PFAS delay column installed in your workflow BEFORE the injector will make you headache-free in your method development, method validation, and your day-to-day PFAS analysis.

Cannabis Residual Solvent Method Development Made Easy with Free Modeling Software

In a typical cannabis testing laboratory, finding time and resources to improve or develop methods can be scarce.  Even in our own applications group, finding time to “quickly” generate data can be a challenge with multiple projects taking place at the same time.  However, there’s a tool that can help make this process more efficient and alleviate some of the burdens that you might encounter during this process.

Recently, I received an external inquiry regarding our Cannabis Residual Solvent Mix #1 (cat.# 34105).  During the email conversation, I shared a chromatogram and method which Colton developed last year.

After sharing the method, I was asked if we offered a standard for ethylene oxide and had any supporting chromatograms using the same conditions above to show where the compound eluted since our customer wanted to develop a method for California’s residual solvents list.  Unfortunately, we didn’t have this chromatogram.  To make things worse the instrument needed to generate the chromatogram was running samples for another project.  Yet, within minutes of Colton informing me of this news, he generated a model chromatogram containing ethylene oxide using his run conditions.

 

How was this possible?!?!  We have Pro EZGC® Chromatogram Modeler to thank for this!  This free program allows users to spend a little time in the office to save a lot of time in the lab.  To find out more about EZGC, check out Chris English’s post “I Can’t Drive 55” – The Pure Power of EZGC.

https://blog.restek.com/?p=8195

And thanks to Colton’s quick thinking we were able to answer our customer’s questions in less than a day.  So the next time you’re planning to modify a method or develop a new one, use the Restek Pro EZGC® Chromatogram Modeler to help you get started!  https://www.restek.com/proezgc

Does your cannabis lab need a Residual Solvents standard? Look no further, friend!

So, we got the news. Cannabis labs and application chemists are sick and tired of residual solvent standards in DMSO. Instead of continuing to offer you something that can be difficult to work with, we put our heads together at Restek to offer you a new RSA standard in N,N-Dimethylacetamide (DMA)!

Not only is this standard offered in the preferred solvent across the industry, but it also includes 20 of your favorite compounds at 3000 ppm! The Residual Solvents #1 (cat# 34105) CRM pairs very nicely with some Topaz Liners (cat# 23280) and the Rxi-624Sil MS (cat# 13868). Finally, when we combine all three of these products, we are left with the delicious solution shown below.

 

You can also check this out on the web here!

 

So, if you’ve been trying to get your hands on a residual solvent standard, look no further! Restek has you covered!

 

Be on the look out for more Cannabis reference standard solutions. We’ve got some good stuff brewing up!

Photo Credit: DavidCardinez