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The title says it all… we’re taking our e-cig method and jumping on the EZGC Method Translator and Flow Calculator bandwagon. Why… well because all the cool kids are doing it. No seriously… to simply reiterate how easy it is to use the Method Translator. In fact, the EZGC Method Translator makes life so easy you can kick back, put your feet up, and vape away on an e-cig. Okay, so maybe we do not endorse such behavior, but you get the point!
So we (Amanda, Colton, and me) took our e-cig solution analysis method and used the method translator to switch from helium to hydrogen carrier gas. It was as simple as plugging in our current method parameters (which you may find in part II of this blog series) and then letting the translator do the work. As you will see below, the flow bumped up to 2.5 mL/min and the oven ramping jumped to 54 °C/min. All of this correlates to our 4.84 min run dropping down to a mere 3.11 min. But most important… we no longer use helium!
Now let’s take a look at how this… well… looks!
Pretty nice, but let’s compare this to our previous method. The following overlay will show you the difference between the “old” helium (blue trace) and “new” hydrogen (red trace) carrier gas methods:
Now let’s look at this from a different angle. Here the overlay has taken into account the off-set due to hydrogen being faster:
Once again, looking good! Now I know some of you may be saying… “what the heck is this guy thinking; I can’t ramp at 54 °C/min… explicative!!!”… Which is certainly a fair statement, minus the explicative. We just want to demonstrate what you may achieve with a fast-ramping oven like we have. So for the rest of you… our original 35 °C/min ramping method should work well. So let’s just capitalize on the hydrogen like so:
Even with the slower oven rate, the hydrogen method gets our compounds eluted a minute faster. The problem is that we left an extra minute hanging out there in no-mans-land. So we can recoup that minute by simply dropping the final oven temperature from 260 to 240 °C like so:
So there you have it… several hydrogen-based methods for the analysis of electronic cigarette solution. Regardless of which method you chose… the EZGC Method Translator and Flow Calculator made for a simple transition between helium and hydrogen carrier gas. I literally ran all 4 chromatograms (yes, I know there are technically 5, but there was a duplicate in there) back-to-back as you see above… with no “trial and error” or “guess and check”. Now we have a shorter run, with super crisp peaks, and no helium consumption. The problem is… I now have to figure out what to do with all this extra time!?!?
It is well known by people who analyze environmental and food safety matrices for semivolatile organic compounds like pesticides and PAHs that you occasionally have to trim the GC column to restore peak shapes degraded by nonvolatile matrix material that builds up on the inlet side of the column. (As an aside, change that liner and seal, too!) What gets a bit murky is how you change the GC conditions after trimming to maintain the same elution orders for peaks of interest. Consider using our new EZGCTM Method Translator and Flow Calculator (MTFC).
In the example below, I’ve made three (exaggerated) trims on a GC column that had an original nominal length of 30m, our 30m x 0.25mm x 0.25µm Rxi-5ms, which resulted in a column length of 23.7m. Using the MTFC, enter the Original column dimensions (determine the original length accurately using MTFC, too!), and the original GC conditions. Keeping the flow rate the same, enter the Translation column length, and simply watch MTFC spit out the updated oven program rate for you, which it will do when you have “Translate” selected under Results. Note that the 30.7m and 23.7m chromatograms look almost identical, except that the 23.7m run is faster due to the increased oven program rate necessary to keep compounds eluting at the same temperature (the thing they need to do to avoid elution order flip-flops).
That faster run creates a bit of a problem, the need to update any Selected Ion Monitoring / Selected Reaction Monitoring windows and/or Calibration Table Expected Retention Times. Again, MTFC to the rescue! Just use the “Speed” factor in Results to calculate the expected retention times, as follows:
Predicted retention times for Translation = Actual Original retention times divided by “Speed”.
The table below indicates the calculation above works very well. Now, just cut and paste the new retention times into the method and calibration areas and keep using that same column and save money.
If you want to follow along with this example, the Original Method and the Translation Method are:
30.7m x 0.25mm x 0.25µm Rxi-5ms, 1.4 mL/min constant flow He, GC-MS, split injection
Oven: 90°C (0.1 min), 8.5°C/min to 330°C
23.7m x 0.25mm x 0.25µm Rxi-5ms, 1.4 mL/min constant flow He, GC-MS, split injection
Oven: 90°C (0.1 min), 12.5°C/min to 330°C
In the last e-cigarette blog we (Amanda, Colton, and me) showed you a quick proof of concept for analyzing the major constituents of e-juice. Working from that starting point, we realized that some labs may not have access to a GC-MS and/or some may only be interested in a very quick screening method for the nicotine content in e-juice. So… that is exactly what we’re showing you today.
The table below contains all the specifics of interest for a rapid GC-FID screening method for nicotine in e-juice.
We continue to use our thick film volatiles column, because the ultimate goal with e-smokes is to see what is in the vapor. Also, it is already doing an excellent job on the current application. So we simply bumped up our starting temp to 100 and now we have a 5 minute run (see below).
Okay… so there may be a little bit more. As we mentioned last time, we purchased some raw e-juice and analyzed it as is (i.e., straight from the refill bottle). Well since the last blog we have learned that the e-juice is extremely viscous. If you plan to analyze the e-juice raw (i.e., without any dilutions) you will have to set up your injector appropriately (see below). We suggest you include at least one sample wash; lower the sample wash speed down to ~30 µL/min; and you may also want to add in a viscosity delay. Close observation of your autosampler syringe coupled with a little experimentation will get you to where you need.
Alternatively, you could just simply dilute your e-juice, which is ultimately what we suggest. If you want to have quantitative nicotine results, clearly you will need to calibrate your instrument. So when you use our nicotine standard at 1,000 µg/mL you will realize that most of e-juice nicotine concentrations are well above this. So… a nice 100 fold dilution with methylene chloride (yes, methylene chloride and not methanol if you want to look at ethanol) will kill the following two birds with one stone: 1. you no longer have viscosity issues. 2. your concentrations fall nicely into your calibration curve.
Speaking of which… the aforementioned method calibrates fairly well [i.e., the correlation coefficient (r) (not the coefficient of determination (r²)) = 0.99562] from 1.6 x 10-2 to 1.0 mg/mL (see below). FYI – most e-juice nicotine concentrations are ~10 to 20 mg/mL for a 1.8% nicotine solution. Remember… we diluted by 100 fold, so we typically injected 0.1 to 0.2 mg/mL. Well within the aforementioned calibration curve.
The year 1936 marks the beginning of organic geochemistry. It started with Alfred Treibs’ discovery of porphyrins in petroleum; compounds that closely resemble chlorophylls in plant matter. Another 25 years would pass before scientists recognized that these compounds, known as biomarkers, could reveal insights into the evolution of plants and animals spanning a time frame measured in billions of years.
Echoes of Life weaves a complex fabric of stories, peppered with personal details, that describe the emergence of analytical techniques; mainly GC-MS. The authors are a mix of organic geochemist, founding father of biomarker research and a marine chemist/novelist that draw on a variety of perspectives and experiences. Echoes of Life is a well written, digestible story rather than a textbook. One of its enduring facets is the ability to eloquently describe the required transition of geochemist to analytical chemist; a necessity to crack the origins of oil. The book starts as a disjointed collection of stories from finding “life” on the moon to botanists studying leaf waxes. Using mass spectral interpretation it was evident that cholesterol could be found in oil in the modified form of steranes and hopanes. They were “stripped of their double bonds and oxygen containing functional groups reduced to their bare carbon skeletons.” The book follows researchers from around the world arriving at the same conclusion from various fields using different techniques. Described as, “a tribe of scattered chemists using the new technique of ‘coupled GC-MS’ and coming to the same conclusion.”
The Deep Sea Drilling Project (DSDP) revealed chemical traces of algae, zooplankton and microbes that proved to be a chemical chronicle of the last 150 million years. The keys to understanding the history of earth, its climate and life were locked in these biomarkers. For instance the degree of unsaturation in algae’s lipids increased systematically with an increase in temperature. Dialkyl ketones containing 37 to 39 carbon atoms were analyzed to determine the number of double bonds remaining. Combining age of sediment and dialkyl ketone data, scientists were able to estimate regional global temperatures.
The story is a combination of thousands of scientific papers, hundreds of interviews and many anecdotes as a mechanism for moving the story forward. Reflected in these pages is an insatiable curiosity that defines science. This book is a journey of discovery, the human spirit and the quest to understand our surroundings. It is a trip worth taking.
Echoes of Life: What Fossil Molecules Reveal about Earth History. Susan M. Gaines, Geoffrey Eglinton and Jurgen Rullkotter. Oxford University Press, 2008. 376 pp. (ISBN 9780195176193 cloth).
Check out Michelle’s work on oil identification: Fingerprinting Crude Oils and Tarballs using Biomarkers and Comprehensive Two-Dimensional Gas Chromatography
Gulf Oil Spill Blogs
When traveling around I experience a lot of confusion on the naming of Injection techniques in Gas Chromatography. The challenge can be the “mode” settings of the GC. Often the GC does not use the same name as what the actual technique is what we are using.
The sample is introduced in a hot liner where only a percentage of the sample enters the column(the sample amount is split). The rest goes out via the split vent. Amount entering the column depend on the actual volumetric flow that passes the split-point (=column inlet). Typical split ratios of 1:5 up to 1:500 are used which allows concentrations that can range from 2ppm to percent levels of sample. Sample transfer from the injection port to the column occurs quickly and the best way to assure even vaporization is by using wool. Often precision liners are used with wool to aid in sample evaporation; for example see: http://www.restek.com/catalog/view/11047
GC setup done in split-mode
Is used in trace analysis and the majority of the sample is transferred onto the column. Transfer times are slower and peaks are broader when compared to split injection. Solvent focusing or analyte focusing are used to get a narrow band at the head of the column. In splitless injection focusing is essential. It focuses (read: concentrates) the components at the inlet of the capillary. This focusing can be realized by using retention (for higher boiling components or using thicker film stationary phases), or by using the solvent effect. This last technique is very powerful and allows focusing of components that elute just a little later then the solvent itself, as a sharp band. (see fig.1)
General –rule of thumb-setting is that during the splitless injection time, the oven is set at a temperature 20°C below the (atmospheric)boiling point of the solvent. The injection time is the time needed to empty the liner volume, which usually is between 60 and 90 seconds. After the injection time has passed, the split-vent (or purge valve) is opened and the liner is flushed (this will take out the last molecules of solvent, generating a very sharp solvent peak, see fig 1. At the same time the oven is programmed and the separation starts. For details on injection time, you can also use the EZ-GC Flow calculator, see http://www.restek.com/ezgc-mtfc
Splitless injection only works for components that elute later then the solvent. Singel gooseneck/single tapered (Sky)-liners with wool on the bottom are recommended.
The GC setup done in splitless mode
Direct injection using a split/splitless inlet system
In a direct injection, all the sample is transferred into the column. There is no splitting done. Special liners are developed for the direct injection, we call the “uniliner”, see: http://www.restek.com/catalog/view/11053
The uniliner have the tapered part in the bottom, allowing to make a “Press-Tight” type connection, (see fig. 2).
Uniliners for Agilent GC also have a “side hole”, which is required to make the EFC work correctly. As most GC’s do not have a separate “direct injection mode” to choose from, the GC is setup in “splitless” mode. This assures that all of the sample enters the column. Here is where the confusion starts, as we are really performing a “Direct” injection.
Uniliners are mostly recommended for low level analysis and we cannot use the splitless technique. For instance if the analytes of interest elute before the solvent peak.
As all sample in the liner is transferred into the column, and the chromatographic separation starts immediate after injection, often the solvent peak will show some broadening and tailing and early eluting peaks after the solvent may elute on a skimmed baseline. The best results are obtained by injection fast and use of 0.53mm ID columns.
Direct injection with a PTV inlet system
One can also do a Direct injection using the PTV (programmed Temperature Vaporizer). This technique is often used for High temp. simdist. The sample is introduced in a cold liner using the taper at the bottom, which is rapidly heated to high temperature. As all sample transfers, it’s direct injection. In a PTV configuration the software allows for the injection port to track the oven. Generally the injection port should always be kept 10°C above the oven temperature.
GC software is configured for PTV, but be aware, it goes by many names…
Direct injection using Valves
In petrochemical methods, often valves are used for injection of the sample. The sample size is determined by the sample loop or by the internal volume of the rotor. This is also a direct injection as all the sample is transferred into the column. To make transfer quantitative, often the valves are heated or are positioned in the oven or a heating-box. The GC software allows for valve times for sample transfer.
GC setup is done by the valve settings in the software…
Sometimes a valve is used before a split inlet. In this case we use a splitted injection setup.
Cold On-Column injection
Here the sample is injected into the column as a liquid. The needle actually gets inside the column and introduces the sample. Injection temperature must be low, to prevent flash, usually 20C below the BP of solvent. For good on column injection, retention gaps are required to correct for the injection error, see: http://www.restek.com/Technical-Resources/Technical-Library/Editorial/editorial_A008. Mostly a 0.53mm ID retention gap is used, which allows most easy entrance with needle.
To make on column work possible, we have to choose the on-column mode of the GC settings; Total flows are very low, and as everything is transported to the column, one must be assure there are no leaks.
Cold on-Column using a PTV inlet system
When using a PTV, one can also use this system in an “on column mode”. Using a tapered liner, but positioning the taper on top, allows the needle to be inserted inside the column, see fig 3.
Also here retention gaps are recommended and before starting the oven program, first the PTV must be programmed to transfer the analytes from the first 5 – 6 cm column inside the PTV;
GC must be setup in on-column mode.
With special thanks to Chris English for practical recommendations on GC settings
The shoot-and-dilute GC technique (split injection) is perfectly matched for the fast sample preparation approach of QuEChERS. The QuEChERS concept provides a fast, multi-residue extraction and “just enough” cleanup. The technique is quick and minimizes solvent usage, but the resulting extract can contain a large amount of coextracted nonvolatile material. A split injection is an advantageous injection technique for dirty samples because less nonvolatile material ends up on the column and the flow through the liner is MUCH faster compared to a splitless injection. Don’t just take my word for it; see these blogs for more information.
Screening fish and other fatty foods for the presence of halogenated flame retardants is important from a human health perspective. While the historical PBDEs have been phased out in the US, some of the newer high-production flame retardants such as those found in Firemaster® 550, do not have any available food occurrence data. In order to develop a screening method for halogenated flame retardants, we paired the fast, multiresidue, sample preparation concept of a modified QuEChERS extraction and a quick extract pass-through of a PSA (primary secondary amine) cleanup cartridge. The PSA pass through removed large fatty acid interferences and the samples were then analyzed using GC-ECD and GC-MS/MS with a 15m x 0.25mm x 0.10µm Rtx-1614 and a 10:1 split injection. Even though we employed a split injection, the sensitive detectors allowed us to detect in the low ng/g range.
NACRW kicked off again with the Restek Vendor Seminar. We shared dinner and drinks while Jonathan Keim gave a very informative presentation highlighting some of the uses of the Restek EZGC Method Translator and Flow Calculator. The translator/flow calculator has many uses including:
- Translating methods to increase speed of analysis by decreasing column length, decreasing inner diameter, switching to a faster carrier gas.
- Updating the oven temperature program through Translation after column trimming for maintenance so peak elution orders do not change.
- Improving Original methods in separation and/or speed of analysis by solving for Efficiency or Speed in Translation.
- Translating methods from GC-FID (or other atmospheric outlet detector) to GC-MS (vacuum outlet) or vice versa.
You can download the translator here.
Throughout the rest of the 3 day conference in St. Pete Beach, Florida we heard very interesting presentations about multi-residue, multi-class analytical methods, residues in honey bees and some of the latest and greatest in mass spectrometry. With a total of 37 oral presentations, over 100 poster presentations and 8 vendor seminars, the meeting was packed full of great information for all attendees. Not to worry, we were still able to squeeze in some fun into the meeting as well. An opening reception, a dinner cruise (that unfortunately didn’t actually cruise), beach volleyball and a beach run were all included in the social program. Good science and good fun can always be had at the North American Chemical Residue Workshop (formerly the Florida Pesticide Residue Workshop).
Next year should be even better, when our own Julie Kowalski takes the helm as the President of the organizing committee.
Over the years, Restek has run applications on a long list of columns for this analysis. It does require a primary and secondary column for analysis, since there is not one column that perfectly separates all compounds on the list simultaneously. Keep in mind that EPA Method 8330B allows for alternate columns versus the ones listed if you can demonstrate proficiency by presenting valid data that meets QC acceptance criteria as described in Method 8000. Our Innovations chemists have worked hard to make column selection easier for this and get you started in the right direction for optimization.
So far, we have determined optimal conditions for two pairs of columns that we recommend above all other combinations. For traditional HPLC systems with a 400 Bar pressure limit, we suggest using an Ultra C8 as the primary column and an Ultra Aromax as the secondary (confirmatory) column.
Here are chromatograms. For best resolution, please click on the chromatogram.:
Chromatograms are like fingerprints. If you can “read” chromatograms well, you often can find a plausible cause. In this series, we will show a series of GC-chromatograms that are obtained from users and discuss some potential causes for the phenomena. Then we can move into some solutions for improvement.
When using GC sometimes the baseline increases with every analysis. Also sometimes a huge “hump” elutes after all the components of interest have eluted, see fig.1. The “hump” typically shows as a broad peak, indicating “something” is eluting form the column. Normally the base line should be stable after the elution of the peak marked “X”.
Typical causes for such “humps” are:
Built up of later eluting peaks in the column. If there are heavy components in the sample, they will elute eventually. This will result in unstable base line and also in the “hump” from figure 1.
- Heat the column to a higher temperature each analysis, so the heavier components will elute.
- If the column cannot be heated at higher temperature, consider to use a flow – or pressure program. This will also make the late eluting compounds elute faster.
- Use a back flush system. You can back-flush the whole column after elution of the last peak of interest; You can also use a pre-column of the same phase and only back-flush the pre-column. Back-flush systems are used a lot in process analyzers. They will offer the most reliable chromatography. Challenge is, that valves or Deans switching has to be used, which does require a little more understanding of the separation process.
- One can also choose to do more sample prep and remove heavies before the analysis is done.If the “hump” or ghost-peak is not coming from the sample, it may be a contaminant somewhere in the system, that is also accumulating. Check this by doing a blank run (no injection) first; Then also do a solvent injection and see if the “hump” is increasing. GC split/splitless systems often get contaminated at the split-vent line. Because this location is cold, often heavy components can accumulate. See also Blog: http://blog.restek.com/?p=5454That split-vent line need to be cleaned regularly, as well as the charcoal trap that should be connected downstream your split-vent line. Be aware of that. Many of us do not realize this, but it can impact the base line significantly.v Lastly, also check your detector temperature.. If it’s too low, peaks will also broaden..
- Using a solvent, especially in splitless mode, sometimes extracts these contaminants and they get into the column. Ideally this should not happen as there should be a positive flow outside the injection port. Practically, we do see sometimes, that such contaminants can make it back into the system and they show up as ghost / broadened peaks.
International Network of Environmental Forensics Conference – Cambridge, UK – The Invention of Mass Spectrometry and J.J. Thomson
I’m writing this post from St. John’s College at the University of Cambridge in the United Kingdom where I will be speaking at the International Network of Environmental Forensics Conference tomorrow on polychlorinated biphenyl (PCB) analysis with parallel dual-column comprehensive two-dimensional gas chromatography – time-of-flight mass spectrometry (2GCxGC-TOFMS). I could wax on and on about famous colleges and students and teachers here, including Isaac Newton, but what I was most thrilled about was being in the place where the mass spectrometer was invented by J.J. Thomson, who is also credited with discovering the electron.
I strolled down to the site of the old Cavendish Laboratory this morning, where Thomson (and others like F.W. Aston) made historic discoveries, and took a few photographs, which I share below. Thomson foresaw the future of mass spectrometry very well, as you can tell from this quote from his 1913 book, Rays of Positive Electricity and Their Application to Chemical Analysis:
“I have described at some length the application of Positive Rays to chemical analysis; one of the main reasons for writing this book was the hope that it might induce others, and especially chemists, to try this method of analysis. I feel sure that there are many problems in chemistry, which could be solved with far greater ease by this than any other method. The method is surprisingly sensitive — more so than even that of spectrum analysis, requires an infinitesimal amount of material, and does not require this to be specially purified; the technique is not difficult if appliances for producing high vacua are available.”
You don’t even have to read between the lines to see his mentions of sensitivity, selectivity, and universality, things we take for granted in MS today. I like his use of the word “appliances” also. When I think of appliances today, it’s my refrigerator, my clothes washing machine and dryer, my coffee pot… But come to think of it, maybe one day we’ll all have MS appliances in our homes, for personalized medicine, water analysis, indoor air analysis, etc. I think even Thomson would be amazed at the possibilities for the future in mass spectrometry.