Optimizing Mass on Column to Balance Sensitivity Requirements and Calibration Range with Split Injection

This is a continuation of the EPA Method 8270 blog series started in January of 2016. Previous posts: 1, 2, 3, 4, and 5.

We’ve been focusing on the advantages of split injection analysis, while highlighting the weaknesses of splitless injection. This blog is going to revisit the topic of column overload in more detail – focusing on optimizing a split injection method to maximize sensitivity while maintaining and extended calibration range. This is especially critical if you are migrating your method to a 20 m x 0.15 mm x 0.15 µm or 20 m x 0.18 mm x 0.18 µm column for fast analysis with the new GC Acclerator.

Typical semivolatiles calibrations on a 30 m x 0.25 mm x 0.25 µm 5-type column range from 0.5 µg/mL or 1.0 µg/mL to over 100 µg/mL (many analysts target 160 or 200 µg/mL). However, this column dimension (as well as the 0.32 mm ID format) will usually show signs of peak overload with less than 10 ng of any individual component on column. Isobars that elute close together quickly become coelutions as mass on column increases. Figure 1 highlights the most popular example of this, the benzo[b]fluoranthene and benzo[k]fluoranthene isomeric pair. The three highest concentration calibration standards (120, 80, and 40 µg/mL) do not meet the 50% valley resolution criteria under splitless conditions [technically, the resolution criteria are only evaluated at the midpoint used for the CCV evaluation, but they are good indicators of accurate integration potential]. The extreme peak fronting resulting from column overload makes it impossible to accurately integrate and generate a linear calibration including these points. Additionally, the peak apex of benzo[b]fluoranthene shifts more than 0.2 minutes (12 seconds) across the calibration standards, another symptom of severe column overload.

Figure 1 – Benzo Fluoranthene isomer resolution ( B and K ) at increasing mass on column

The concentration-dependent retention time shift requires wide windows in the data processing software, increasing the likelihood of identification errors when the automated integrator processes the data. This results in more time required for manual data review and integration [which can be a headache for those of you manually recording before and after chromatograms in compliance with your manual integration policies] with an elevated risk of error.

Under split conditions, the isomers meet resolution criteria (50% valley) in each of the 9 calibration standards, and the peak apices shift by at most 0.04 minutes (2.4 seconds) from 0.1 µg/mL to 120 µg/mL, indicating only minor peak overload at the high end of the calibration range. Figure 2 shows a comparison of splitless and split benzo fluoranthene isomer chromatography over the same calibration range.

Figure 2 – Comparison of benzo fluoranthene isomer retention time variation as injection concentration increases using splitless (top) and split 10:1 (bottom) injection techniques

The minimal shift of concentration-dependent retention times allows for a much more narrow integration window and a greater confidence in peak IDs. This is important because compound concentration isn’t the only factor that can cause retention time shifts. Complex sample matrices with an excess of co-extracted material can have the same effect on splitless injection by greatly broadening the initial sample band at the head of the column. Split injection minimizes this by transferring a fraction of the injection to the head of the column.

Figure 2 is a good illustration of how column overload is managed by split injection – but it introduces a new problem that needs to be dealt with: detector overload. Initially, when collecting the splitless injection data, we dropped the gain factor to 0.3 (from the default of 1.0), which reduces the voltage applied to the multiplier below the tune optimized level. This reduces instrument sensitivity, preventing detector overload at the high end of the calibration curve (120 ng on column). For the 10:1 split injection, we increased the gain factor to 3.0 (adding almost 250 volts to the tune optimized level) to make sure sensitivity wasn’t an issue at the low end of the calibration because the split injection delivers 1/11th of the sample to the analytical column. This was a gross over-correction, causing compounds with strong molecular ion responses (such as PAHs) to overload the detector at concentrations as low as 40 µg/mL. Through trial and error, we determined that the best balance between low-end sensitivity and high-end overload occurred at a gain factor of 0.8 (see Figure 3). This appears to be instrument dependent, as we operate a similarly configured 5977a with an optimized gain factor of 1.0 and see similar performance.

Figure 3 – Comparison of benzo fluoranthene isomer peak shapes demonstrating the effect of reducing the gain factor on detector overload.

Once you have established your linear mass on column range, you can adjust standard concentrations and the split ratio (or injection volume) to maximize your calibration range while maintaining sensitivity to meet method required LOQs.

Cannabis Concentrates Part II: We’re Heading to Space!

Welcome back! It’s been a little while since my last blog, but in that time, we’ve been doing some interesting things regarding cannabis research. Last time, I discussed that we would be analyzing residual solvents in cannabis concentrates and today, I’m going to show one of the methods that we’ve been working on. If you missed the previous blog, be sure to check it out here!

Before we get into the sample preparation techniques that we’ve been focusing on, let’s go over some initial things. First, we need to select a column that has the capability of resolving these light weight, volatile compounds (see Table 1). An easy way to do this is through Restek’s Pro EZGC Chromatogram Modeler. After placing our compound list into EZGC, we saw that the program gave us numerous selections with the first column being the Rtx-502.2. However, since our future work includes terpene analysis, we decided to go with the Rxi-624Sil MS, 30 m x 0.25 mm x 1.40 µm (cat. # 13868). This column has better high temperature stability and it also lines up perfectly with what Amanda chose for her method using Full Evaporation Technique (FET).

Now that we have our column selected, we can move on to the main event! When thinking about how to approach this problem (analyzing residual solvents) we started out by using Jason Herrington’s favorite method: KISS. For those who haven’t heard of KISS, it stands for Keep It Simple, Stupid (sorry, no Gene Simmons here). We wanted to keep everything as simple as possible to make your life in the lab as stress free as possible. To do that, we thought that the easiest approach to this analysis would be through headspace (HS). Headspace samples are prepared by taking a sample and adding it to a dilution solvent in a HS vial (we’re using a 20 mL vial). Depending on the analytes of interest, an inorganic salt (Sodium Chloride, Sodium Sulfate, etc.) can be added to the vial as well to help lower the partitioning coefficient of the more polar compounds. For more information on HS analysis, please check out Restek’s Headspace Technical Guide!

In our current method, which we will call HS-Syringe-GC-MS, we are taking the headspace from a 6 mL sample. All standards (STDs) were prepared as follows: 3 g sodium chloride (NaCl) was measured into a 20 mL amber headspace vial (cat. # 23086) with screw top cap (cat. # 23090). 6 mL of deionized (DI) water was then added to the vial. All STDs then received internal standards. Lastly, STDs were capped and vortexed at 3000 rpm for 10 seconds, inverted, then vortexed again for 10 seconds at 3000 rpm. Luckily for us, we are using the CTC PAL RTC rail system, so the vial equilibration and injection are automated. Once equilibrated, 500 µL of the headspace is drawn into a gastight syringe and injected into the gas chromatograph.

 

*Note: If you are not using a rail system, don’t fret! This method can easily be done with a manual setup.

 

For more detailed specs, please refer to Table 2 & 3, which has our rail and GC-MS parameters.

 

Now that we’ve gone over how we are preparing our samples, let’s take a look at the chromatography (Figures 1 & 2):

 

 

This total ion chromatogram (TIC, Figure 1) shows that we are able to resolve all 19 compounds of interest. Some of the polar compounds that have higher partitioning coefficients are a little more difficult to see. However, in the extracted ion chromatogram (EIC, Figure 2), you can see that we were in-fact able to drive them out of the water and into the headspace. The only compounds that are not baseline resolved are the Toluene-d8 and Toluene. The resolution value for these two compounds is 1.38, which is resolved enough for accurate integration. Since Toluene-d8 is our internal standard, we’ll probably be changing our internal standard in the near future, so that Toluene is completely baseline resolved.

 

 

I know we threw a lot at you here, so I think this is a good place to take a break! We do have more to show you, but we don’t want to give it to you all at once. We want to leave you hungry for more! To recap, we were able to take a very simple approach to this analysis by using the HS-Syringe technique and with the help of EZGC, we were able to resolve all of our compounds with hardly any difficulty. In addition, this method can also be done with a manual HS-Syringe and run on a GC-FID. Like I said, we have much more to cover, so stay tuned for our other HS approach in our next blog!

Alicat Flow Meters for Your TO-11/13/15/17 (Anything Air) Laboratory: Let’s Talk About Errors and Ranges

If you already own an Alicat flow meter, then I will soon be preaching to the choir. However, if you do not have an Alicat flow meter and you work in a toxics organics (TO) air laboratory, whether it be U.S. EPA Method TO-11A or TO-17, etc… then you do not know what you are missing out on. Short story: instantaneous pressure and vacuum flow measurement you can fit in your back pocket. ‘nuff said! However, if you are currently in the market for one or soon to be after that sales pitch I just laid on you, then read on…

We offer 2 different flow ranges for the Alicat: low flow (0-50 mL/min) and high flow (0-500 mL/min). Sometimes we hear the question “can I use only the high flow meter to set the flow on my Passive Air Sampling Kit at 0.5 mL/min and 250 mL/min or do I need to have the low flow meter as well? After all, the high flow meter does have a range of 0 – 500 mL/min?”

Here is my answer: Both meters will work, but there will be a lot more error associated with the high flow meter when measuring down to 0.5 mL/min. I say this, because the flow meter accuracy is defined as: ± (0.8% of Reading + 0.2% of Full Scale). The following table shows you 3 possible scenarios that could play out with the two different flow meters when trying to measure flow.


For scenario 1 we see how using a low flow (0 – 50 mL/min) meter to measure an actual flow of 1 mL/min could result in a potential reading ranging from 0.892 – 1.108 mL/min, which is perfectly acceptable for most people. However, for scenario 2 we see how the errors add up on the high flow (0 – 500 mL/min) meter when trying to measure 1 mL/min. In fact, you could be reading 2 mL/min when in reality it is 1 mL/min. That is not to say you will, as these errors/ranges are the maximum (i.e., worst case scenarios). You could also read 1 mL/min for 1 mL/min. The point is, you will not have the confidence in the low flow measurements (e.g., 1 mL/min) when using the high flow meter. However, you can be confident with the low flow meter. Scenario 3 is here to demonstrate: 1) that you may not use a low flow meter to measure 100 mL/min, as it is above the meter’s operational range and 2) to show you that the high flow meter is accurate when applied appropriately.

Sorry for the long-winded answer. Since I love to work in analogies, my shortcut/analogy response is often as follows: would you make a 1 µL injection on your 500 µL syringe or do you own several syringes? Again, ‘nuff said! So, the short answer is that most of us have to use both flow meters.

A Simple Tip for Quicker Searching

One of the questions that Restek’s Technical Service Group is asked frequently is “how do I find ____ on your website?”

The Restek website contains tens of thousands of products and supporting resources. In the past, my colleagues have written posts on using our online search function and how to use our searchable chromatogram database. In this post, I wanted to share a simple tip for finding information on the website more quickly and easily.

Let’s say you’re searching for plug valve. Go to our web site and enter plug valve into the search box in the upper right corner of the page.

This search will return 82 “Product” and 149 “Resource” hits containing the words plug and/or valve. Wow — that’s quite a few results to sift through…

But – what if you were to surround that search with quotes?

Doing this will narrow the search results to hits containing the term plug valve, rather than just the individual words plug and/or valve. Now, only three “Product” and three “Resource” results show up when you hit “Enter”! Keep in mind, however, that using this method will keep a lot of results from showing up. If your search is too specific or has a spelling error, then the page you want could get filtered out.

This tip also applies to most major search engines. Perhaps you have already used trick on Google? In this case, plug valve vs “plug valve” on Google takes the search from 6,990,000 down to 584,000 hits.

There you have it – a better and faster way to search http://www.restek.com/ for the product(s) or information you need.

I hope this little tip is helpful to you. A special thanks to my colleagues Bradley Pike, Brenda Cogan-Seprish, Chris Nelson, Travis Raup, and Kent Rauch for their valuable help with this post.

 

Can I use a 100% aqueous mobile phase with my LC column?

You might be asking this because you have read that not all columns are OK to use with highly aqueous content (>95%) in the mobile phase.  Or maybe the caption caught your eye because you have recently encountered some difficulty with using highly aqueous mobile phase.

There is a phenomenon called “phase dewetting” that can occur with LC phases that are composed of the longer alkyl chains such as C18 and C8.  Phase dewetting results in a sudden and dramatic loss of retention, as well as poor peak shape. If you would like to read more about phase dewetting, I suggest reading the following:

A C18 is a C18, right?

More Technical Service “Red Flags” – LC

http://www.chromatographyonline.com/lcgc-blog-chromatography-technical-tips-polar-analyte-retention-and-phase-collapse

http://files.pharmtech.com/alfresco_images/pharma/2014/08/22/55290841-3e3e-433b-a5d4-adb7f6495a06/article-32973.pdf

http://www.waters.com/webassets/cms/promotion/docs/Polar-Retention-Reversed-Phase-LC-Webinar-%20Pres-Kevin-Jenkins-Oct2016.pdf

 

As mentioned in one of the above listed blog posts, “A C18 is a C18, right?, our “Aqueous C18” phases are quite different from conventional C18 phases.  This includes our Ultra Aqueous C18 and Pinnace DB Aqueous C18 phases.  We also have a few other phases that can be used with up to 100% aqueous mobile phase. You might notice from reading the referenced articles that pore size and bonding density has some impact also. Here is an all- inclusive list of Restek LC column phases that will tolerate 100% aqueous conditions:

Ultra Aqueous C18

Ultra IBD

Ultra Biphenyl

Ultra PFP Propyl

Ultra Aromax

Ultra Cyano

Ultra C1

Pinnacle DB Aqueous C18

Pinnacle DB IBD

Pinnacle DB Biphenyl

Pinnacle DB PFP Propyl

Pinnacle DB Cyano

Viva C4

Viva Biphenyl

Viva PFP Propyl

ROC Phenyl-Hexyl

Roc Cyano

Raptor Biphenyl

Raptor Fluorophenyl

Force Fluorophenyl

Force Biphenyl

Please note that although the above phases are known to be aqueous tolerant, we may not have generated data (chromatograms or application notes) at >95% water content in the mobile phase. This is simply because we have not performed the application and, most likely, there has not been much demand for using a high aqueous mobile phase for applications that are commonly run on that column phase.  If you do have any questions about whether or not your column is suitable for an application, please feel free to contact Tech Service at support@restek.com.

I hope you found this post helpful.

GC compounds – poor peak shapes and missing peaks

One of the most common questions I am asked by customers is “Why is my peak shape so poor?” Another is “I do not see a peak for my compound, what should I do?”

Sometimes they are developing a new method and have no experience with the compound(s) of interest; other times things worked well in the past and now they don’t. Depending upon the exact circumstances my questions/suggestions may vary somewhat, but the first thing I usually do is to ask a few questions (Q) to help me narrow down what may be the issue.

Q1.  Has this analysis worked well on this specific instrument in the past?  If so, when was the last time?  What has changed from when it worked well until now?  Has the instrument recently been serviced or had extremely “dirty” samples analyzed on it?

Q2. Has this analysis worked well on any instrument in the lab in the past?  If so, when and on which instrument make/model?  Is the current GC similarly equipped to the previous model?

Q3. Is this a new analysis for this lab?  If so, what method is being followed?  Is it a common, well-publicized method like those from EPA, ASTM, USP, NIOSH, etc., or is it an obscure method that was found searching the web?

Q4. How does one know for certain that this particular analysis is going to work well on a specific instrument?  In other words, have you found any data/chromatogram/method showing that this analysis should work well (or is even possible) on your instrument?

Simplify GC column selection and optimize GC separations

Some of the questions listed above may seem somewhat repetitive, but they really are not. Depending upon the answers to the questions above, possible suggestions (S) are listed below.

S1. If a specific instrument has provided good peak shape(s) in the past, we would suspect that it should be able to do so again.  I suggest changing the column, cleaning the injection port, replacing the injection port liner, cleaning and/or replacing the detector and associated consumables, verifying all gas flows, etc.

S2. If this analysis has not been performed successfully on this particular instrument, but it has on other similarly-equipped instruments, I suggest trying to perform this analysis on the instrument where it last worked.  Assuming this instrument is working well, if the same issues occur, there likely is something else going wrong that is not related to a single instrument.  Verify your chemical reference standard is OK.  Verify the instrument gases are clean.  Look at whatever these two instruments have in common and investigate further.

S3. If this analysis has never been performed in your lab but you are following a method, I suggest contacting the author(s) or agency that developed the method.  It’s possible they may have heard of similar issues and be able to quickly provide you a solution.

S4. If you are not certain that your instrument is capable of performing a particular analysis, I suggest searching for chromatograms/methods showing the analysis using a similarly-equipped instrument. You need to make sure your peak of interest isn’t eluting under the solvent peak (if a solvent is involved), co-eluting with other peaks, and that you have allowed enough time for your compound to elute from the column.

In summary, by reviewing the answers to these questions, it usually becomes easier to determine a logical approach to finding a solution. For example, if you know the analysis has worked in the past, you know it should be able to work in the future, and a methodical step-by-step troubleshooting approach will usually provide the solution.  But if you are uncertain if a particular analysis will work using your column/instrument/detector, your time may be better spent researching to make certain you have chosen the proper column & instrument & detector.

If you have any questions, feel free to send me an email.

You may find the links below helpful.

Thank you for your time.

GC compound responses lower than expected? Maybe this will help.

General (very general) guidelines to help meet GC detection limits.

Broken fused-silica columns – it’s rare, but it does happen.

Troubleshooting GC Syringe Issues – Part 2

Should I use LC or GC for my analysis?

What happened to my GC column?

 

Restek Proudly Supports Young Scientists at Germany’s 28th Annual Separation Science Working Group’s PhD Student Conference

Helping young scientists grow into the chromatographic industry’s next generation of innovators and leaders is an opportunity we relish at Restek.

For a company dedicated to the science of separation, it is invigorating to see the work of aspiring chromatographers, and there is no better place to see that than the annual PhD Student Conference hosted by the Separation Science Working Group, a part of the German Chemical Society (GDCh).

This year’s conference, located in Hohenroda, Germany was no exception.

Organized and led by students, the conference is a great opportunity for the attendees to network and exchange ideas. It’s also a valuable chance for companies to be introduced to the next generation of chromatographic talent! Recruiters from several international chemical companies like Dow Chemicals and BASF, as well as instrument suppliers like Agilent and Thermo used the opportunity to present open positions for young scientists.

This year’s conference featured sections on Food and the Environment, Bioanalyses, and the development of New Analytical Methods.

Drivers for new developments were the coupling of LC to Ion Mobility Spectrometry to enhance peak capacity for complex samples like biological materials or those found in petro chemistry, LC/ICP/MS methods for speciation analysis in biological matrices, and the miniaturization to Nano-LC, or a Lab-on-a-Chip approach.

For the first time, the organizers asked for two tutorials out of the industry. Restek was chosen to give one of them. The talk about “pro ezGC – Chromatogram modeling as a powerful tool for method development and in education” found a lot of interest, especially from University members involved in educating young students in separation science.

This was also Restek’s 12th year presenting an award for the top three presentations delivered by PhD students, as voted on by the audience. Next to BGB and Springer Publishing we granted the young scientists with vouchers for Restek products, worth 1.500 €, 1.000 € and 500 € to support their woek is proud to announce this year’s award winners:

  1. Sebastian Pallmann from Ludwig-Maximilian University, Munich with his presentation about “Realization of Hadamar Transform Capillary Zone Electrophoresis on a standard and unmodified CE Instrument”
  2. Kevin Eckey from the University of Münster with his presentation about “Studies about degradation Kinetics of Sulfamethoxazole under UV irradiation conditions”
  3. Renata Gerhardt from University of Leipzig with her presentation about “A droplet-based interface for fractionation of on-chip separations and surface enhanced Raman detection”

We congratulate the winners and all of the participants of this year’s conference for their outstanding contributions to the science of chromatography.

We look forward to seeing familiar and new faces alike at next year’s PhD Student Conference!

Sampling and analysis of landfill gas / biogas, an overview

Lately it seems that the sampling and analysis of landfill gas / biogas is a popular topic among our customers.  When initially ask about the topic, I had an idea of what customers were trying to do, but didn’t really have a firm grasp on the topic.  As I dug deeper, I began to realize just how broad and complicated this topic actually was and no longer was I surprised customers were asking us for some guidance.

I was aware that gas analysis via GC was a primary aspect of monitoring landfills.  GC analysis of most or all fixed/permanent gases including CH4, CO2, N2, O2, CO and H2 was often mentioned by most customers.  I also knew that H2S and NH3 were typically monitored because of their negative health effects above certain concentrations and their unpleasant odors.

After a brief discussion of these gases, the next topic usually discussed was trace VOC’s (Volatile Organic Compounds) collection/sampling and analysis. Unlike the fixed gases which are commonly collected in air cans and/or sample bags, trace VOC’s are routinely collected onto thermal desorption tubes to concentrate the sample in order for the instrument/detector to be able to detect these trace levels.

Those two sampling/analysis were about the extent of what I knew. Only after talking with customers and reading more on the topic did I realize there were multiple other sampling/analysis performed associated with landfill gas / biogas.  These included mercaptans, siloxanes, non-methane hydrocarbons/organics, aromatics, alcohols, etc.  It seemed like the list of compounds could be quite extensive.

So how does one determine which analysis is needed? Usually the customer determines the list.  How does a customer determine the list?  If I understood correctly from the references I reviewed, it is determined by local, state and federal agencies based upon their regulations.  For landfills, it appears sampling/analysis requirements can be dictated by the size (acreage) of the site, the age of the landfill, and previous collected data.  For biogas, the starting material and the process (fermentation, digestion or other) usually dictates the compound list and/or method/analysis requirements.

For additional information, I found the following interesting and informative.  I hope you do too.

EPA landfill gas

Collection of Methods for Biogas

Guidance for Monitoring Trace Components in Landfill Gas

General (very general) guidelines to help meet GC detection limits.

Sometimes we (tech service) receive requests from customers who cannot meet detection limits when developing a new method or when trying to follow a current method. They ask for advice on how to meet these limits.  As a result, I decided to provide some (very) general guidelines that I would follow back-in-the-day when I would develop GC methods.

In the context of this blog post, the topic of detection limits is limited to being able to achieve a certain signal/noise (S/N) ratio* for a particular compound, usually ranging from S/N of at least 2.5 to around S/N of 10. Some refer to this as Instrument Detection Limit (IDL).  The topic of detection limits can be very complex, so when reviewing the following, don’t think of it in terms of Quality Assurance, but in terms of optimizing your instrument’s sensitivity.

* Some consider the signal/noise ratio an outdated industry standard, especially when using a mass spectrometer (MS) or MS/MS for detection.

 

As with most procedures performed in a laboratory, there are usually multiple ways to accomplish the same task. These were the steps I followed to optimize my GC’s sensitivity.

 

  1. Make sure all gas flows are measured and adjusted according to the instrument manufacturer’s recommendations. You may need all the sensitivity your instrument is capable of to meet the required limits.

Keep in mind that older instrumentation may not be as sensitive as newer instrumentation, so if you are having difficulty meeting detection limits, ask the instrument manufacturer and/or detector manufacturer if your particular instrument has the necessary sensitivity.

Injection technique is important. You will get the most sensitivity by injecting in splitless mode, or cool-on-column. Large volume injections may be possible with certain types of injection ports, but do not simply inject more to overcome the steps needed to optimize your instrument.

Liners Every Lab Should Own (in my opinion)

Which GC injection port liner to use for gas samples

For maximum sensitivity, make sure that your detector is clean and if needed, replace any worn or malfunctioning parts. If developing a method, make sure the most appropriate detector is chosen (see detector selection section in the link below).

How to choose the correct GC column – Part 3

 

  1. When following pre-existing methods, make sure every step listed in the method is followed. Do not skip steps, do not modify steps, and do not rush through the steps. They are there for a reason.  If developing a method, someone with experience is an invaluable asset.  Make sure the appropriate person is assigned the task.

 

  1. If the method already exists, one or more column choices should be listed within the method. If you are developing a new method, have you performed your due diligence to choose the best column, or at least one capable of performing the necessary separations?

GC column selection and optimize GC separations

EZGC Calculators and Chromatogram Modeler for GC Method Development

If you only are looking for one or just a few compounds, a 15-meter (or shorter) GC column may be all that is needed. However, as the number of compounds increase, or the matrix becomes more complex, a longer column is usually preferred.  Keep in mind that (generally speaking) the shortest GC column with the smallest ID (internal diameter) and thinnest liquid film/phase will usually provide the best signal/noise ratio for each peak, and therefore the most sensitivity, because (in theory) this column should provide the lowest bleed and sharpest peak shape.   But, sometimes a longer column with a thicker film is necessary such as to aid in separation, especially when there are early eluting compounds and/or when extra column capacity is needed.

 

  1. Once the instrument has been optimized and an appropriate column has been chosen, begin your preliminary testing. I would first prepare a (relatively) highly-concentrated chemical reference standard to make sure the instrument would detect the peak(s) so I could determine the retention time(s).  Injecting a high concentration should also help “prime” the surfaces (column, injection port liner, etc.) the compound will contact, minimizing activity for future injections and helping to stabilize the system. Just do not forget to inject a blank (usually pure solvent) to make sure carry-over is not an issue.

As a general rule, if possible, you would want the compound(s) to elute where the baseline is the lowest in order to achieve the best signal/noise. This area is usually after the solvent peak has completely eluted from the column and while the GC oven is at a relatively low temperature (which minimizes column bleed).

During this step, no changes to the carrier gas flow rate should be made as this parameter should have already been optimized. However, you may need to optimize the GC oven temperature ramp for compound separation, or other GC temperatures to prevent issues such as compound/sample matrix condensation (which may lead to ghost peaks) or to minimize the break-down of thermally liable compounds.  Sometimes this will not be known until actual samples are analyzed.

 

  1. Once you have decided on the (presumed) best GC temperatures and ramp rate, decrease the concentration of the chemical reference standard with each injection until you have reached the minimum signal/noise ratio allowed for each compound. This will likely be the minimum limit possible with your particular instrument. You may even want to consider using only higher concentration standards which provide a S/N of at least 10 because if the instrument loses sensitivity after analyzing samples, you don’t want to have backed yourself into a corner.  In other words, leave some wiggle room.

 

  1. After performing Steps #1 through #5, it’s time to determine if you can achieve the limits listed in a pre-existing method. Keep in mind, these limits usually include sample size and sample preparation/concentration and not simply the limit of the chemical reference standard injected as described in Step #5. For example, let’s say you were able to detect 1ppm (parts-per-million) on-column of compound xyz.  Through sample preparation, whether it is via extraction and/or concentration or some other means, you are able to concentrate the sample 100x, your new limit would be 0.01ppm, or 10ppb (parts-per-billion).

 

  1. Perform any changes/modifications to optimize the instrument and/or sample preparation (prep) and/or analysis once sample matrix has been injected. Hopefully this won’t be necessary, but with dirty matrices, usually some sort of clean-up step needs to be added. In extreme situations, the sample prep may need to be changed completely, such as going from extraction/concentration to SPME or headspace (compound boiling point will be an important determining factor).

A Technical Guide for Static Headspace Analysis Using GC

Restek PAL SPME Arrow

To elaborate a little more on this topic, consider the three things that may happen to a compound’s response once sample matrix comes into play:

a. No change in compound response. This is the best-case scenario.

b. Compound response decreases and/or the compound may disappear altogether. This is the worst-case scenario. Unless corrected through sample clean-up or changing the sample prep, analysis is going to be difficult or impossible.

c. Compound response increases. Not an ideal situation, but usually not an insurmountable issue. Some may refer to this as “matrix enhancement” or “matrix effect”. I always thought this effect was caused either by the matrix covering compound active sites, or possibly adding to the baseline which may improve a compound’s peak area, but I really don’t know for certain.  To deal with this, I would keep injecting samples (containing matrix) until compound responses stabilized.

 

  1. The information described above is to help you get started developing a method, or to assist you achieve the limits in a pre-existing method, but by no means is this the end of the story. As a matter of fact, sometimes it is only the beginning, and may actually have been the easiest part. To read more on the topic, I found this link very informative.

ANALYTICAL DETECTION LIMIT GUIDANCE & Laboratory Guide for Determining Method Detection Limits

 

I hope you all have found this helpful. Let me know if you have any questions.  Thank you.

Cannabis Concentrates Part I: Introduction to Residual Solvents

Greetings, everyone! My name is Colton Myers and I’m the new kid on the block at Restek…kinda. You may have seen my name before in Jason Herrington’s E-Cig blogs, but that’s from when I was an intern at Restek. Now, I have the opportunity to work in our Innovations lab at Restek and do some research on a hot topic: CANNABIS!

Image Credit: Cannabis Now (cannabisnow.com)

In this blog series, I’m going to discuss analyzing residual solvents in cannabis concentrates. Some of this was previously discussed by my predecessor, Amanda Rigdon, in her blog. Cannabis concentrates are products that are made by extracting the chemical compounds, such as Delt-9-Tetrahydrocannabinol (THC), Cannabidiol (CBD), and other cannabinoids from the cannabis plant. After the extraction, you’re left with an oil, which can then be used to make a variety of different products.

Image Credit: The Travel Joint (thetraveljoint.com)

So, why do you care? Well, many of the cannabis concentrate producers are using different types of solvents to extract the compounds of interest from cannabis. Because of this, methods for testing residual solvents are needed to analyze these products to ensure that consumers are not exposing their body to harmful levels of chemicals. Therefore, many states that have legalized medical or recreational use of cannabis require residual solvent testing.

I know that this may be old news to many of you, but don’t walk away yet! I’m excited to share with you some of the work I’ve been doing on new testing methods, so stay tuned to this new and exciting blog series!