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Cannabis Concentrates Part III: A Second Extraction Approach

And we’re back! Previously, we discussed analyzing residual solvents via HS-GC using a gas-tight syringe. If you missed it, be sure to check it out here! Today, we are talking about a sample extraction technique that is less common, but has great capabilities; known as Solid Phase Microextraction (SPME).

SPME is a sampling technique consisting of a fiber, coated with a sorptive phase. When placed into the headspace of a sample, analytes sorb (adsorb or absorb depending on fiber type) onto the fiber, and are desorbed off the fiber in the GC inlet. We have decided to use the SPME Arrow instead of a traditional SPME fiber for this analysis. The SPME Arrow has a much larger sampling volume, but its best attribute is its durability compared to a traditional fiber (see the figure below).  For more information on SPME, be sure to check out Jason Herrington’s blogs here.

 

 

When moving from the HS-Syringe method to the HS-SPME method, a couple extraction parameters must be changed. The HS-Syringe performs best under long equilibration times at higher temperatures, whereas the HS-SPME performs best under shorter equilibration times at lower temperatures. We have the data to support this, so check out Chromablography in the future for the proof! These main differences between the HS-Syringe and HS-SPME can be seen in the tables below. The reason for this drastic difference in temperature is if the SPME Arrow is placed in a hot vial, the fibers temperature will rise. The SPME Arrow’s thermally conductive metal core magnifies this problem. Because the fiber is at a hotter than the laboratory atmosphere, the analytes on the fiber will desorb off before the fiber reaches the inlet. Some compounds of interest, like propane and butane, show incredibly low responses using elevated temperatures. By keeping the fiber at a cooler temperature, we are able to keep those analytes on the fiber until it is desorbed in the GC inlet.

 

 

The chromatography comparing the HS-Syringe and HS-SPME methods can be seen below.

 

 

Notice that the HS-Syringe gives better response for propane, isobutane, and n-butane. You can see the HS-SPME method quickly has excellent recoveries for higher molecular weight compounds. The peaks tail using HS-SPME, but this is a normal result for this type of extraction technology. Overall, things look good on both the HS-Syringe and the HS-SPME Arrow. Stick around for future blogs where we will go into more depth with both techniques!

 

Are you experiencing helium supply issues and rising costs?

Helium supply issues are nothing new.  At Restek we have been discussing this off and on for years now.  The following articles and information are just a small selection of what is available on our website and our ChromaBLOGraphy, and are there to help you make informed decisions about alternatives to helium, and reduced helium consumption in your lab.

The History of Shortages

http://www.restek.com/Landing-Pages/Content/gen_B007

Donald Duck voices silent and birthday balloons fall everywhere as helium disappears…

Will We Go Over the “Helium Cliff”?

Alternatives to Helium

For years we have been writing and lecturing about using alternatives gases to helium in gas chromatography:

GC Carrier Gases – Alternatives to Helium

GC carrier gases, do you really have a choice?

Hydrogen as carrier gas: Always available, Cost effective and Fast

Reduce Helium Consumption by Using Nitrogen Purge Gas for VOCs in Drinking Water

 

Benefits and Considerations of Converting to Hydrogen Carrier Gas – http://www.restek.com/Technical-Resources/Technical-Library/Petroleum-Petrochemical/petro_PCTJ1729-UNV

Gas Generators

If you choose to move away from helium to hydrogen or nitrogen as a carrier gas, or purge gas, then a gas generator would be a great source for a consistent flow of clean gas.  Gas generators have been used for many years for just this purpose.  Yes they can be a high initial capital expense, but they can pay for themselves in as little as one year when you compare how much you would have been paying for helium in bottles.  The modern gas generators have a series of safety devices built into them so that the concerns over using hydrogen, for example, are diminished to a point that it is no more of a problem than any other gas that is used.

Hydrogen as carrier gas: Always available, Cost effective and Fast

Gas Management in Labs – http://www.restek.com/pdfs/GNSS1758A-UNV

Working Safely with Hydrogen as a Carrier Gas – http://www.restek.com/Technical-Resources/Technical-Library/Editorial/editorial_A016

Using Hydrogen for Gas Chromatography – http://www.restek.com/Landing-Pages/Content/gen_B008

Restek’s Selection of Parker Balston Gas Generator – http://www.restek.com/Supplies-Accessories/GC-Accessories/Gas-Generators

Applications

I still hear your hesitancy and want to have some convincing evidence about using alternative gases in your applications.  Well luckily for you we have done a lot of work on this, and here are a few examples:

Environmental

Organochlorine pesticides – http://www.restek.com/pdfs/EVAR1935-UNV.pdf

Organochlorine Pesticides Analyzed by Gas Chromatography – Electron Capture Detector with Hydrogen Carrier Gas and Concurrent Solvent Recondensation – Large Volume Splitless Injection

VOC Analysis: O.I. Analytical Nitrogen Purge Gas Application Note with Restek Rtx-624 GC Column

Petro

ASTM D2887 – http://www.restek.com/pdfs/PCAR2320-UNV.pdf

ASTM D7213 – http://www.restek.com/Technical-Resources/Technical-Library/Petroleum-Petrochemical/petro_PCAR2269-UNV

DHA – http://www.restek.com/pdfs/PCAR2891-UNV.pdf

Others

Blood Alcohol – https://blog.restek.com/?p=6374

Cannabinoids – https://blog.restek.com/?p=7704

Fragrances – https://blog.restek.com/?p=13850

Pro-EZGC

You may not be able to find an example of your application in our database, but you can model it using Pro-EZGC.  The Pro-EZGC suite allows you to enter your current conditions and then change certain parameters to translate the method.  These parameters include the carrier gas.  So you can model and improve your analysis before you even start performing experiments and method development on the instrument.  The following link will take you to the Pro-EZGC suite and the tutorials to help you:

http://www.restek.com/ezgc

 

 

 

 

 

 

Trans fatty acids analysis part 2: Let’s look at actual samples with incurred TFAs

In my previous blog, I’ve tried and failed to acquire a sample that contained any trans fatty acids (TFAs). While this is a great news for everyone’s health, the scientist in me was somewhat disappointed. That’s why I first decided to cheat a little bit and look at different TFAs – ruminant TFAs. Ruminant TFAs are products of bacterial metabolism of polyunsaturated fatty acids in the rumen of cattle, sheep or goats, contributing up to 6% of total fat.1 They are present in both dairy products and meat. The major difference between artificial and ruminant TFAs is their distribution. Partial hydrogenation produces TFAs with almost Gaussian distribution, with highest abundance for trans-9 C18:1, while ruminant bacteria skews the distribution towards for trans-11 C18:1 (up to 42 wt%, Fig. 1). It’s also noteworthy that trans-C16:1 can contribute up to 20% of ruminant fats but it is not present in partially hydrogenated oils unless they originate in marine oil.1

Figure 1: Distribution of trans-C18:1 isomers. Adapted from Stender et al.1

The obvious choice for analysis was a dairy product with the most milk fat, i.e. butter. Butter contains up to 80% milk fat, therefore, theoretically, 5% of TFAs (about 0.5 g per serving). In many cases, TFA content is low enough that it doesn’t have to be declared on the label. Our analysis shows the trans-vaccenic acid (C18:1 trans-11), albeit in very small abundance (Fig. 2). While both columns perform well in terms of separating trans-vaccenic acid, the Rt-2560 provided better separation of C10:0 from the matrix. A different approach to sample preparation could result in better separation of C10:0 on Rtx-2330.

Figure 2: Separation of butter FAMEs using GC-FID

After analyzing butter, I decided to go back to a grocery store and take a good look at nutrition labels of more suspected products. Shelf-stable or frozen pastries were all in clear, but I had luck in the frosting aisle. I selected two types of chocolate frosting which declared 1g of TFAs per serving. The first frosting tested proved to be mislabeled since no TFAs were detected and the pattern of C12:0, C14:0 and C16:0 suggested replacement of partially hydrogenated oil by palm or palm kernel oil.

Fortunately for my analysis (not so fortunate for consumers), the second frosting showed a whole slew of TFAs. Figure 3 shows a close-up of the C18:1 region of chromatogram overlaid with cis/trans FAME standard mixture (#35079, in blue). Interestingly, Rtx-2330 was able to separate the standard mixture reasonably well but failed to separate the same isomers (namely C18:1 cis-6 and cis-9) in the real sample (Fig 3A). The second column (Rt-2560) separated those two peaks fairly well, moreover, it showed an additional peak directly following methyl oleate (C18:1 cis-9, Fig 3.B).

Figure 3: Separation of C18:1 isomers in frosting using GC-FID. The red trace is the frosting sample, blue trace is the cis/trans standard.

 

To conclude, while Rtx-2330 provides fairly good separation, it is crucial to use Rt-2560 to separate as much of cis/trans isomers as we can.

More about TFAs and Rt-2560 here and here!

 

PS: Have you ever tried to use Rt-2560 on GC-MS? Coming up in my next blog post!

References:

  1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2596737/

What’s in a name? A C18 by any other name would not be the ARC-18

You have probably read or been told numerous times and in numerous ways that not all C18 columns are the same. And that is very true. Particle morphology, bonding chemistry, and add-ons like end capping all influence the retention and selectivity of this workhorse LC phase. At Restek we have an interesting C18 phase that we call the ARC-18 that I would like extol upon if I may.

The name ARC-18 is descriptive of one of its most useful characteristics. I actually had the pleasure of coming up with this name (a love/hate task for any product manager). The “AR” in ARC-18 stands for “Acid Resistant” which is very descriptive of this C18 phase. Through steric hindrance or the add on of bulky side groups near the ligand connection to the silica the bond is protected from the attack of acidic mobile phases that can cleave off the C18 and cause your columns to lose retention.

 

The acid resistance of a sterically protected C18 is well documented. We often describe this in comparison to non-protected C18s. And its advantages are highly sought after. Especially when you want a long lasting and stable column for LC-MS/MS where the mobile phases are typically acidic. An area where that is particularly important is in the analysis of peptides. My colleagues here at Restek did some fantastic research to demonstrate how the acid resistance of the ARC-18 allows for the flexibility of acidic adjustments to optimize peptide analysis.   http://www.restek.com/pdfs/PHAN2615-UNV.pdf

While there are other sterically protected C18s on the market you may find that these phases are also end capped. Encapping is typically done to prevent analyte interaction with the silica surface which can cause secondary retention exhibited as peak tailing. We found that C18s that were both sterically hindered and end capped (e.g. TMS) could take longer to equilibrate and therefore increase your run time. The endcapping can also cause the peak shape of some basic compounds to suffer. We wanted a C18 with a well-balanced retention profile for many different types of compounds like pesticide panels.

Another area where this phase shines is in cannabis potency analysis. We have demonstrated excellent LC-UV separation for 16 Cannabinoids on both our Raptor 2.7 (shown below) and 1.8µm columns utilizing the unique selectivity of the ARC-18 phase.

16 Cannabinoids on Raptor 2.7µm ARC-18

So if you need a robust C18 column for LC-MS/MS, a selective column for cannabis potency, a well-balanced C18 for a wide array of pesticides, or a versatile column for peptides, have a look at the ARC-18. It is truly ahead of the curve.

How often do I need to get my electronic flowmeter recalibrated?

The answer is it depends. It depends on how often you use it. It depends on the environment it has been exposed to. It depends on what your company’s, industry’s or government’s regulations on recalibration are. It depends on whether you have accidentally dropped or banged the device. It depends on how confident you are of the results your are getting.

 

 

 

 

 

 

 

 

 

All instruments degrade with time
Most measuring devices drift out of tolerance, and some devices need more frequent calibration than others.  The reasons depend on the technologies used in the device and where the device is being used.  When the device is primarily electronic in nature, the resistors, capacitors, and solid-state components that comprise it will deteriorate with time and exposure to heat, cold, and radiation.  As a result, the accuracy of the measurements made by the device also degrade with time until its specifications are exceeded.  Usually, the calibration process can compensate for this degradation through electrical adjustments to the device.  When calibration cannot bring the instrument back into specification, some repairs or part replacements may be needed.

Restek spent a lot of time developing and testing our Proflow 6000 flowmeter.  Under the controlled lab conditions we subjected our devices to we found that our devices stayed within acceptable calibration ranges for at least a year.  However, not all customers use or store the devices in the same way in which we tested them.  Therefore it is up to customers to determine there own frequency of testing.

When customers return devices for recalibration the collection of as-found data gives a good snapshot of how out of calibration a device is (this data is given in the recalibration certificate).  In most cases one or two calibration points are slightly out of range.  This is a good indication that the device was still providing accurate data up to that date.  If, however, the as found data shows significant drift from the calibration then it is an indication that there is damage to the flow sensor manifold.  In our experience this has occurred from exposure to too high a flow rate, even it if is a short burst (always establish flow before applying the flowmeter to measure the flow), or that the device has been dropped or banged.

To always get the most accurate flow measurements, contact Customer Service to send in your flowmeter for recalibration (cat.# 22656-R).

The Chromatography Picture-Show

If you’re caught up on all your favorite shows, might I suggest the Restek Video Library for your next binge. Most of the episodes only run about two minutes, so there are few extraneous subplots. (And, so far, no car chases. Not a single one in over 50 videos! But new episodes are added all the time, so who knows?)

screen shot showing the library and its menu location

The cast is superb, and delivers performances packed with practical wisdom. Their costumes probably won’t dominate the awards, but the concise and helpful info they’re providing outshines such considerations. Also of note is the use of exotic filming locations such as laboratories and even the abstract realm of cyberspace. You feel like you’re right there. The cinematographer’s boldness is most apparent in the unflinchingly informative depictions of the guts of LC and GC instrumentation.

Diverse plotlines offer something for nearly everyone. From the heartbreak of tailing peaks to the triumph of a clean, square cut on a fused silica column, from the thrill of proper LC column storage to the drama of detector lamp replacement, it’s all there. There’s even a very special episode about finding the reference standards that match your requirements.

Although these gems have yet to show up on any of the major streaming services, they’re easy to track down. You can even use your phone and take them into the lab with you. Here’s the hookup:

http://www.restek.com/Technical-Resources/Technical-Library/Video-Library

https://www.youtube.com/user/RestekCorp

Have there been any new applications developed for Diquat and Paraquat?

I am happy to answer “yes” to this question. Those of you who have been doing this analysis might have been using our Ultra Quat column that I talked about in an earlier blog post here, “I have an Ultra Quat column and I know how to use it…”

 

This updated method uses the Raptor HILIC-Si, which is a newer product we are excited to have. Here is what the application looks like:

http://www.restek.com/images/cgram/lc_fs0512.pdf

 

We are pleased with this application, particularly since it does not require any ion pairing agents or chaotropic salts. The mobile phase modifiers are very common ones that most labs will have on hand and extended equilibration times are not needed (as compared to NaPF6 or Heptafluorobutyric acid). The run time is short and sweet.  :-)

Here is a link to the product information for the column we used:

http://www.restek.com/catalog/view/50802/9310A52

 

I hope you find this post helpful and thanks for reading.

Which Headspace Vial Cap Is Right For Me?

Today, I’d like to cover a question that I have been receiving from cannabis labs who are doing residual solvents analysis. This blog may not only help cannabis labs, but also help labs analyzing other matrices looking for residual solvents or very volatile compounds. The question is, “Which type of vial and vial cap combination should we use to do our analysis?”

This is referring to the screw-thread caps (18 mm) and crimp caps (20 mm) for 20 mL headspace (HS) vials. So instead of just recommending one or the other based on “feel” or price, we decided to run a little experiment to compare the performance of the two styles. The experiment was designed to analyze a few very volatile compounds and gather data on how well these two types of caps prevent the compounds from escaping the HS vial over the course of a specific period of time. To familiarize yourself with the products that we were working with, please reference Figures 1 & 2.

 

Figure 1. Screw Cap HS Vial: PN#23082 and Screw Cap: PN#23092

 

Figure 2. Crimp Cap HS Vial: PN#21162 and Crimp Cap: PN#21761

 

Sample preparation included adding propane, butane, and isobutane standards in N,N-Dimethylacetamide (DMA) along with α,α,α-Trifluorotoluene as the ISTD to a 20 mL HS vial. To model the vial cap’s ability to keep these volatile compounds from escaping the vial, we designed a time study. In this study, 14 samples were prepared for each vial type and the samples were split into two sets of 7. Set one (T0) was run immediately after the samples were prepared and set two (T6) was run 6 hours later. It should also be noted that the crimp cap pressure was set to model the tightness of a screw cap. This meaning that the amount of torque needed to remove a screw cap was similar to that of the torque needed to spin a crimped, crimp cap.

The average compound area count from T0 to T6 were compared by calculating the relative percent loss over the 6 hour period of time. The relative percent difference for each HS vial type can be seen in Figure 3.

 

Figure 3. HS Vial Cap Comparison

 

From the data shown in Figure 3, we can see that the screw caps (blue) did not seal as well from T0 to T6 compared to the crimp caps (red). It is also worth noting that we see very little change in our ISTD over the 6 hour time period for both the screw cap and the crimp cap.

After looking at the data and taking everything in, I think it is suffice to say that if you are working in a lab doing HS analysis of very volatile compounds (gases), you should be using crimp caps. However, even if crimp caps are being used, one still needs to be cautious of the fact that the lighter, more volatile compounds, like the compounds shown here, are still capable of escaping the HS vial. Now, with all this being said, I don’t want to give the impression that screw cap HS vials are worthless. They can definitely be used in HS analysis, especially if the compounds being analyzed are on the less volatile side or your time from sample preparation to analysis is kept to a minimum. As an example take a look at the minimal differences of α,α,α-Trifluorotoluene, so for many customers analyzing the standard list of solvents and volatiles a screw cap may be appropriate.

Tailing solvent peak during split injection? Check your column installation distance!

When performing splitless injections, it is not uncommon to witness a tailing solvent peak, given the large amount of solvent introduced onto the column over a relatively long period of time.  For split injections, however, only a small portion of the solvent loads onto the column and it occurs very quickly. This results in a relatively sharp solvent peak.

Recently, I set up a method with a 20:1 split and upon my first injection I noticed a badly tailing solvent peak, which I wasn’t expecting (Figure 1 red trace).  My gut feeling told me something was occurring within the inlet and the solvent wasn’t split properly.  I removed my column from the inlet to find that the installation distance was too low. The recommended installation distance for an Agilent split/splitless inlet is 4-6mm above the ferrule and my column was only 1-2mm above it.  This would lead to the column being below the inlet seal surface, creating an area of dead volume, where residual solvent would not get swept away by the higher split flow.  After correcting the installation distance, my problem disappeared (Figure 1 blue trace)!

Figure 1: Improper installation distance of column can create excess dead volume, leading to tailing.

Figure 2: Capillary installation gauge

 

Generally, I pre-swage my ferrule to the column at the proper distance using a capillary installation gauge, but I was in a rush and didn’t do that this time. I probably bumped the column while installing, inadvertently pushing it down.  I highly recommend using a capillary installation gauge, which allows you to pre-swage your ferrule to the column at the exact specified distance for installation (Figure 2).  It only takes a few seconds and greatly improves consistency of installation.  The table below shows the capillary installation gauges that Restek carries for various instrument makes.

 

Are My Shut-off Valves Contaminated?

Figure 1: Experimental set-up for testing valve contamination (plug valve shown). Gas flow was set at 20mL/min for 1 hour (1200mL total extraction volume) with oven held at 35⁰C and the split vent closed. After a one hour hold, oven was ramped to 330⁰C @ 20⁰C/min. An Agilent 7890A was used with an Rxi-5MS (30m x 0.53mm x 0.25µm) (cat. #: 13425) to allow for high flows. Inlet liner was a Topaz 4mm single taper liner with wool (cat #: 23303).

Shut-off valves provide a convenient means to control gas flows and are often used in conjunction with gas chromatography systems.  There are several types of shut-off valves including ball valves, plug valves, and diaphragm-sealed valves.  Some of these valves, ball and plug valves included, use a silicone-based lubricant to make opening and closing the valve easier and to extend lifetime by reducing wear on moving parts.  Unfortunately, this lubricant can off-gas into the flow path and lead to system contamination, causing baseline interferences.

Use a gas filter containing a hydrocarbon trap between lubricated valves and the GC to prevent any off-gassed components from entering your system.

I performed an experiment where I attached ball valves (cat. #: 23144) and plug valves (cat. #: 23146) directly behind the GC EPC and set a high flow of carrier gas for one hour (1200mL of Helium total volume), while leaving the split vent closed (Figure 1).  The GC oven was left at 35⁰C to trap off-gassed components on the head of the column.  Following this, I ran a series of “no-injection” blanks, with a temperature program, to qualitatively see if anything was off-gassing from the valves.  This experiment was also performed with a Super Clean Triple Gas filter (cat. #: 22019) installed between each valve and the instrument.

I found that the plug valves introduced a significant amount of contamination to the system (Figure 2).  Ball valves produced some contamination, as well, but at a much lower level than the plug valves (Figure 3).  This is likely due to the different opening and closing mechanisms of each valve and the much larger surface area of the lubricated plug.  Based on this, I would never recommend using a plug valve without a filter for carrier gas lines and would encourage the use of filters even with a ball valve.  Testing of multiple valves of each type showed similar results.

When these experiments were repeated using a filter in between the valve and the instrument, this contamination was effectively captured, as can be seen in Figure 2 and Figure 3, for each respective valve.  Note that I used a triple filter (traps hydrocarbons, moisture, and oxygen); however, a hydrocarbon filter is most critical for trapping this specific type of contamination.  Nonetheless, gas lines should have moisture and oxygen traps prior to the instrument, as well, hence my choice of the triple filter.

Figure 2: Contamination introduced from plug valve (red trace), measured with a GC-FID. By contrast, using a filter between the valve and the instrument (blue trace) produced a clean baseline.

Figure 3: Contamination introduced from ball valve (red trace), measured with a GC-FID. Note that this is at a much lower level than the plug valve, but could still interfere with trace analyses. Using a filter between the valve and the instrument (blue trace) produced a clean baseline.

 

I was surprised at the amount of contamination witnessed, especially from the plug valves.  Many GC gas plumbing guides show shut-off valves downstream from the filters, placed directly before the instrument.  Keep in mind that with the large volume of gas passed over the filter with the split vent closed, this was designed to be somewhat of a worst-case scenario; however, similar results could be witnessed simply from letting an instrument sit idle for a period of time.

For these experiments I used an oven temperature of 35⁰C to trap off-gassed components on the column.  There are undoubtedly additional volatile components that are not effectively captured using this method.  Off-gassing of these volatile components could potentially create major contamination issues with sampling techniques where valves are placed before analytical traps for volatiles, such as thermal desorption units, purge and trap, etc.