Archive for 2010

Finally! A True Peak Capacity Increase for GCxGC

An ongoing controversy in comprehensive two-dimensional gas chromatography (GCxGC) is whether a true peak capacity increase has ever been achieved. You can read all about it in the article cited below, but in summary, Blumberg et. al state that a properly optimized one-dimensional (1D) GC system produces higher peak capacities than an improperly optimized GCxGC setup with the same primary column. Furthermore, they indicate that essentially every GCxGC report in the literature is on non-optimized systems. You see, we GCxGCers often sabotage our 1D separations to broaden peaks before they enter modulators so we can get at least 3 slices across first dimension peaks for second dimension (2D) separations. To some extent, this is because of speed limitations in modulation and detection technologies when GCxGC is done with typical 30m x 0.25mm 1D columns. That is, peaks are relatively narrow on properly operated 30m columns and that means very fast (and impractical) modulation cycles are necessary for GCxGC.

Comparison of one-dimensional and comprehensive two-dimensional separations by gas chromatography.
L.M. Blumberg, F. David, M.S. Klee, and P.Sandra.
Journal of Chromatography A, 1188 (2008), 2-16.

My colleague Mark Merrick of LECO Corporation and I decided to approach this problem another way: go slow! Perhaps it’s my Oklahoma drawl that gave me this idea…

We collected data on 120, 150, and eventually, 60m, columns that “naturally” produce broader peaks for modulation with today’s technology. We were able to meet (or closely approach) Blumberg’s suggestions for using Speed Optimized Flow and Optimal Heating Rate (see previous blog) such that our 1D separations were optimum, and then we modulated appropriately for the second dimension separation. To some extent, any separations achieved in the 2D can be considered “gravy” for the meat-and-potatoes 1D separation. Put that terminology in your GCxGC lexicon book…

An example of this is shown in the figure below by contrasting 15m and 120m primary column GCxGC separations. The 120m column is operated very efficiently and we maintain primary column separations, important given that the fog oil we analyzed is full of isomers. We’re also using the second dimension very nicely to spread things out, in this case with the orthogonal 2D column, a high thermal-stability, shape-selective stationary phase developed under Frank Dorman’s direction by Mike Wittrig. You can check out more on GCxGC of fog oil via the article below.

Characterization of military fog oil by comprehensive two-dimensional gas chromatography.
A. Kohl, J. Cochran, and D.M. Cropek
Journal of Chromatography A, 1217 (2010) 550-557.

Finally, and you regular readers knew this was coming. I have to tie peak capacity into my ongoing South African trip somehow and this time it’s through a leopard. The leopard’s coat is beautiful, with nicely patterned rosettes that are somewhat evenly spaced throughout. Think of those rosettes as peaks fully occupying a contour plot (the GCxGC chromatogram) and there you have the perfect use of peak capacity. Conversely, imagine the leopard with one diagonal line on its body (non-orthogonal column selection). Or how about lots of run-together spots in both dimensions (inefficient 1D separations)? Probably not near as purty, eh? Same in GCxGC!

Enjoy my Mala Mala leopard photographs by clicking on the thumbnails to expand and then use the browser back button.

Next blog: how to use a GCxGC Calculator to help maximize peak capacity in comprehensive two-dimensional GC.

Speed Optimized Flow and Optimal Heating Rate in Gas Chromatography

Leon Blumberg and Matt Klee have written some excellent articles on optimization of gas chromatography for flow and oven temperature programming that seem to have flown largely under the radar of many (most?) practicing gas chromatographers. I’ve put two significant references below on Speed Optimized Flow (SOF) and Optimal Heating Rate (OHR) that I think you’ll want to check out. SOF and OHR are aimed at maximizing peak capacity under relatively fast analysis conditions. Until you get these articles (you will, won’t you?), let me show you how I used these concepts to improve throughput and sensitivity for polybrominated diphenyl ether (PBDE) analysis using GC.

Theoretical and Practical Aspects of Fast GC. M.S. Klee and L.M. Blumberg.
Journal of Chromatographic Science, 400 (May/June), 237-247 (2002)

Optimal Heating Rate in Gas Chromatography. L.M. Blumberg and M.S. Klee.
Journal of Microcolumn Separations, 12(9), 508-514 (2000)

I used a 15m x 0.25mm x 0.10µm Rtx-1614 column with various helium carrier linear velocities (determined at starting oven temperature) or SOF. We think of helium being optimum in the 20-40 cm/sec range. SOF in mL/min for helium, by Blumberg’s definition, is 8 x Column ID (mm), or in this case, 8 x 0.25 to give 2 mL/min SOF. I also used the OHR for GC oven temperature program, no matter what the flow was. And OHR, by definition, is 10 / void time (holdup time in min). Remember that you can either determine the void time by injecting an unretained component, or get it by using the HP Column Pressure/Flow Calculator (free as a download from Agilent). I’ll let you work out the math and get right to the figures to make my points.  By the way, click on the thumbnails to see the figures well and then use the back button of your browser to get back to my wonderfully written text!

A couple of other details: After setting the linear velocity where I wanted it at starting oven temperature in the figures below, I used Constant Flow. And the work was done with electron capture detector (ECD).

Can you tell any differences in the first two figures, the ones where I varied the linear velocity, finally ending with SOF? I’ll give you the answer. There is essentially no difference in the separation efficiency, but the analysis time drops from 25 min to about 9 min. Amazing, huh?! Don’t believe it? Well review the next figure, the one with four chromatograms on it showing resolution between two important BDEs, 49 and 71. The resolution for all practical purposes is the same. Notice anything else? Since those chromatograms are all plotted on the same scale, we are improving our sensitivity as we move towards SOF. That is because we are keeping peaks narrow by having optimized flow and optimal column heating rate. We get a five-fold increase in sensitivity from 20 cm/sec to SOF with no tradeoff in separation!

Finally, if you really want to set a speed record, go for hydrogen. SOF for hydrogen carrier is 10 x Column ID (mm), or in this case 2.5 mL/min. Matching that with OHR, we now have a 6.6 min analysis time for this set of PBDEs. I call this Chromatographic Magic. But it’s really just good science. Thanks Leon and Matt!

Helium carrier at ~ 20 and 30 cm/sec with optimal heating rate conditions for PBDE analysis.

Helium carrier at ~ 40 cm/sec and Speed Optimized Flow with optimal heating rates for GC-ECD of PBDEs.

Resolution between critical BDEs is similar, but sensitivity improves with narrower peaks from Speed Optimized Flow and Optimal Heating Rate GC-ECD conditions.

Opitmized Fast GC with Hydrogen Carrier!

Overview of the development of PLOT columns

I was invited by Ron Majors to submit a historical overview of 2011-jaap-pasfoto4the development of the PLOT column. As there have been many scientific publications around this topic, I tried to write the development of capillary columns from personnel experiences in the laboratory, starting with glass capillary columns, introduction of the fused silica column and implementation of adsorption materials in fused silica columns.

Adsorbents usually are the enemies of polymer chemists as these scientists usually fight adsorption. In Gas-Solid chromatography we let the adsorption work for us. Development of peak tailing in adsorption chromatography is usually limited as activity is used as separation parameter. That’s why adsorption columns usually have long life time.

On the downside, because of the high retention, adsorbents are strong trapping media. When oven is cold, even the most volatile impurities in the carrier gas, are trapped on the adsorbent and may elute as a “ghost” peak or raised base line. Also higher boiling materials will retain on these columns, which can lead to pollution and sometimes pre-columns/back flush is essential.

The overview on PLOT column development has appeared in the October issue of LC/GC, under the “column watch” edited by Ron Majors.  LC/GC is not only available as a printed copy, but is now also digitally available at:

http://digital.findanalytichem.com/nxtbooks/advanstar/lcgc_na1010/index.php#/18

Or

http://chromatographyonline.findanalytichem.com/lcgc/Column%3A+Column+Watch/The-Development-and-Applications-of-PLOT-Columns-i/ArticleStandard/Article/detail/691677?contextCategoryId=48336

Journey to the Center of the Earth: Mponeng

My friend and colleague, Professor Ernst Breet at North-West University in Potchefstroom, South Africa, arranged for a very special trip to the Mponeng Mine for us recently. Mponeng (which means “Look at Me” in the local African language) is the world’s deepest gold mine. After a brief induction, we each donned coveralls, boots, eye and ear protection, a hard hat with a miner’s lamp, and a self-rescue breathing apparatus, and then took an unbelievably fast elevator down through the main shaft almost 4000m (about 2 ½ miles for you metrically challenged people, like me…).

The environment in the mine is a hot 31°C with very high humidity, but we only got to experience those “mild” conditions thanks to a sophisticated cooling system. The rock face way down there is 60°C and the “air” temperature would be a deadly 55°C without cooling. Ice is made for the cooling with huge fridge plants that use vacuum conditions to reduce the boiling point of water to 0°C. Water vapor is released, and ice is scraped from the top of the ultra cold water and sent down the mine to a dam for to provide chill for fans.

After a long, long walk, we eventually got to an area where workers were mining by cutting away gold-bearing rock with compressed air hammers. This rock is loaded into trams, taken to the main shaft area, and lifted to the top with elevators, where gold is eventually separated from the bulk rock for further processing.

I was amazed at the working conditions in the mine: the heat and humidity, the noise, and the claustrophobic working spaces in the gold-bearing materials area. This combined with the huge infrastructure and technology to support the effort, as well as the numbers of workers (several thousand), made the fact that the mine only produces 10g gold per ton of rock quite shocking.

As usual, and if you stuck around this long, you might say eventually, there is a link to gas chromatography in this blog. Restek provides gold-plated inlet seals for Agilent split/splitless injectors, and I’m appreciating those much more after my journey to the center of the earth: Mponeng! Mike Goss and I recently invented what we call a “flip seal”, which allows two uses for a gold inlet seal, something that seems very timely now.  Hmmm… I wonder if any of the gold came from Mponeng.

Flip Seal Dual Vespel® Ring Inlet Seals

Flip Seal Dual Vespel® Ring Inlet Seals

Flip Seal Dual Vespel® Ring Inlet Seals Special Offer

Enjoy the pictures of my trip to Mponeng!

Entrance to Mponeng Mine, the World's Deepest Gold Mine (near Potchefstroom).

The Main Shaft for Mponeng Mine. The cables are attached to the elevator that takes you down, down, down...

 

Randall, one of the Chief Mining Engineers for AngloGold Ashanti, operators of Mponeng, tells us how it's done at 4K underground.

Working at the bottom in the gold-bearing area. Can't be claustrophobic and make it a full day here!

The Gold! Not much, huh?

Jack and Ernst in Mponeng

Back up top where they make the ice to cool the mine.

Low-Pressure System: Gas Chromatography, Not Weather… Synthetic Cannabis

We have a very bright guy named Jaap de Zeeuw who works for Restek. Years ago he invented a system for low-pressure gas chromatography by where he used a 0.53mm GC column attached to a mass spectrometer, and a restrictor column (e.g. 0.50m x 0.10mm) press-fitted to that column and installed in a split/splitless GC inlet. This restrictor allowed for a helium head pressure to be maintained on the inlet while the 0.53mm column was operated at sub-ambient pressure. The low column pressure shifts the carrier gas optimum linear velocity higher, which means faster analyses without a loss in efficiency. And, you get the benefit of high capacity from the 0.53mm column.

I recently used low-pressure GC-TOFMS on a LECO Pegasus instrument as part of a project directed by my colleague, Amanda Rigdon, to characterize synthetic cannabis, a blend of herbs that is sprayed with cannabinoid mimics (e.g. the JWH compounds like JWH-018) and marketed as “incense” (e.g. Spice, K2, Voodoo Child, Puff, Tribal Warrior, etc.). This incense is smoked by a user to achieve a high, not completely unlike that from smoking marijuana.

Synthetic Cannabis (Wikipedia)

The GC analysis of these compounds, due to their relative involatility and sometimes active functionalities, is not trivial. In fact, for some GCxGC work we did, the analysis times on a 15m x 0.25mm x 0.25µm Rxi-17Sil MS primary column were about 50 min. Low-pressure GC-TOFMS allowed much quicker run times, and produced peak widths on the order of 2 sec, which helps with sensitivity.

Check out the low-pressure GC-TOFMS analysis of a 12 component mix of synthetic cannabinoids obtained from Cayman Chemical (Ann Arbor, MI, USA). Complete in < 10 min!

Cayman Chemical Synthetic Cannabinoids

Many thanks to Paul Kennedy at Cayman Chemical for help with this project and to Scott Grossman of Restek for providing the drawing of a low-pressure GC system.

Low-Pressure GC-TOFMS of Synthetic Cannabinoid Standard from Cayman Chemical

Zoom of Low-Pressure GC-TOFMS Chromatogram of Synthetic Cannabinoids

QuEChERS Workshops in South Africa

One of the best things about working at Restek is their commitment to continuing education for chromatographers, both inside and outside of their doors. This takes many forms, including the seminars produced by Rick Parmely, our Training Director, and through other efforts like the QuEChERS workshops I’m currently doing in South Africa. In the last two weeks, I’ve had the pleasure of watching many “students” extract their own samples using the EN QuEChERS approach, and we’ve even analyzed those extracts by fast GC-TOFMS (more on that in another blog…), all in about 3 hours, not bad considering the time involved in more classical extractions.

Two workshops were held at the University of Pretoria, one at the Forensic Chemistry Laboratory in Cape Town, and two were held at the University of KwaZulu-Natal (Pietermaritzburg and Westville campuses). We extracted fruits, vegetables, spices, wine, sediments, flavorings, herbs, freeze-dried mussel tissue, and other matrices for pesticides, drugs, medicinally active compounds, PAHs, PCBs, and other compounds. We did it all!

Even with a method as simple as QuEChERS, these workshops could not happen without the dedicated efforts of many people. My colleague Julie Kowalski produced the excellent training binder for the workshops. LECO Africa sponsored my attendance, and provided the able assistance of Peter Gorst-Allman and Alexander Whaley to keep things running smoothly. Thulani Webster Shubangu at the Forensics Chemistry Laboratory in Cape Town was a gracious host. The South African Chemical Institute, thanks to Martin Dovey at FlavourCraft, sponsored the workshops at KZN campuses, with Ross Robinson at PM going above and beyond the call of duty to retrieve our lunch in a hail storm! In addition, lab activities were greatly facilitated by Yvette, Alan, Neil, Carol, Anita, and others whose names I have forgotten, but whose helpfulness I haven’t. A good time was had by all.

Enjoy these photographs of the activities at KZN PM…

Martin Dovey of FlavourCraft weighs his flavor samples for QuEChERS extraction.

Students at the University of KwaZulu-Natal weigh samples for the QuEChERS workshop.

Checking out the QuEChERS extractions at KZN PM!

Centrifuging the extracts. This is easy...

We're almost done and we can analyze the extracts...quick.

The hail storm that almost ruined our pizza lunch (and did ruin Ross Robinson's clothes!).

Why pay for Technical Support if you can have at least the same as a Free Service from Chromatography Specialists?

Many laboratories experience the same challenge:  They have many chromatographic applications running with their GC and LC systems, and as long as there is no problem, everything is fine. Trouble starts when a problem is observed and data looks “different”.. At that point troubleshooting will start and an experienced user has to look at the system.

Chromatography applications need maintenance. For a system that is used with the same samples and similar matrices, maintenance can be turned into preventive maintenance.  This means BEFORE trouble starts the maintenance is already performed. This can be changing liners, seals, septa,, cutting a meter of the column inlet or replacing a complete guard column.

Practically sample load is different and so are the maintenance intervals.

This is also valid for troubles that may evolve during operation.  Challenge of many laboratories is that the GC systems sold are often presented as a “simple”, self diagnostics, operate with minimal trouble, so you  need a less educated person to operate them.

People should be very careful about this. Does the operator realize how important the correctness is of the data that was generated?  Or what decisions are made based on the produced data?  And: what can happen if the data was not correct? Legal impact can be huge.

To operate GC-systems one must also take responsibility for the data and this can only be present if you know it is correct. This means: knowing what is happening inside the instrument.  Here comes the challenge and opportunity: The level of knowledge on chromatography in the labs is decreasing which is a risk, so a new market is developing:  The market of Chromatography expertise.

You see all kinds of commercial services offered where you can sign up for: Chromedia,  ChromAcademy, etc. but you have to pay a significant membership fee depending on degree of service.

I like to get your awareness for a free service that is always out there, and that is the added value that a few specialized chromatography companies can deliver. They do not want to sell a GC or lC systems, but are pure specialized in the chromatography part, which is the heart of every chromatographic system.

Get in touch with such companies specialized in Chromatography. They have been talking with customers for many years and have built up a huge knowledge on all aspects of practical chromatography.

For instance at Restek, we have specialized  people for petrochemical, environmental, forensics, food, fragrance,  pharma, but also for troubleshooting LC columns, Sample prep. , GC Columns, Reference materials, Air sampling etc.  These people do method development and know the practical side of the real world applications, as they also work direct with customers. There is very little that these people have not seen. This resource is available via:  http://www.restek.com/Contact-Us/Technical-Service

The ChromaBLOGraphy will also give you an idea about the technical capabilities of our team.

Alternatively you can join chromatography forum’s and feed your question there. Regretfully forums seem to have a relative short life. If someone knows an interesting one, please share.  One of the latest is via Linkedin

http://www.linkedin.com/groups?mostPopular=&gid=93188

Other than that: In case I have a challenge, I want to be helped fast. I contact people I know can help me, instead of passive waiting via the forum.

Good night, Sleep tight; don’t let the Bed Bugs Bite!

When my parents tucked me in at night with this little saying, I never thought bed bugs actually existed.  Now it seems news articles about the resurgence of bed bugs in the United States are being published at an alarming frequency.   Although bed bugs have actually been around since the 1700’s most had been wiped out by the use of pesticides like DDT in the World War II era.  Because of this heavy reliance on pesticides to mitigate the bed bug problem, most bed bugs today have developed a resistance to commonly used pesticides.   I recently attended a talk at the EPA Region 6, 20th Annual Quality Assurance Conference by Weste Osbrink from the U.S. Department of Agriculture on bed bugs.  He noted that there are a few non-chemical means of helping to eliminate a bed bug infestation.  One way is to treat the area with extremely cold or hot temperatures.  It is important to make sure that all possible infected areas are treated.  This means disassembling furniture, taking pictures off of the wall, and even removing baseboards.  One can prevent bed bug infestation by using caulk around any possible openings around piping, use a plastic mattress cover, and fill any cracks or voids. After attending this very informing, yet kind of disturbing talk I went back to my hotel room and thoroughly inspected the mattress for any signs of bed bugs.  The first sign of a bed bug problem is little blood spots on the sheets and mattresses.  If you find that you have a bed bug problem and non-chemical means to treating a bed bug infestation are not feasible, the EPA has just released a Pesticide search tool to help consumers choose a safe product to use for bed bug infestations.  I guess one thing that may help you sleep better at night is there have been no reports of diseases being carried by bed bugs.  So good night, sleep tight; and seriously don’t let those bed bugs bite!

When your data goes BOING! An electrifying tale of FID troubleshooting!

Recently my colleagues were performing dual inlet/column/detector analyses of 2,4-dinitrophenol (2,4-DNP) using flame ionization detectors (FID) in an Agilent 6890 gas chromatograph.  They noticed something odd.  The results from the front side were considerably lower and less reproducible than those observed from the back, even though they were essentially set up the same way.  Bah!  That has to be an injection port issue, right?  Perhaps not.

Being excellent detectives, they isolated the cause to the detector by simply switching the columns between detectors back and forth while keeping the inlet sides the same and unperturbed.  In doing so, they observed that the results always stayed with the front detector regardless of which inlet was used.  Curious.  Even more curious was the maintenance that seemed to solve the problem.

Having determined the detector was the likely source of the problem, the first thing they did was replace the FID jet.  That did not improve the results.  Crud.  Next they began to wonder if the spring that makes the connection between the collector body and the FID signal board was the problem.  A seasoned vet examined the spring and felt that it looked like it was extending too far into the collector housing and postulated that an electrical connection may be the problem.  Other seasoned vets felt that this spring should not be considered adjustable.  Uh oh.  They decided to try the spring idea and adjusted the length the spring, pushing it farther in the metal tube (away from the collector) a few millimeters.  See Figure 1 for some pictures that illustrate this.  And the result?

They observed a significant improvement in both the response and the reproducibility!  One thing to keep in mind is that they were observing a calculated value called a response factor, which is essentially a normalized measure of the relative response of an analyte to an internal standard.  It was the response factor that got better.  Weird, huh?  If the signal had increased independent of species, the absolute areas for the analyte and the internal standard might have risen proportionally, which would not have resulted in a difference in the relative measurement.  This didn’t happen, though.  They observed a greater increase in response for the analyte, 2,4-DNP, than they observed for the internal standard, which resulted in a higher response factor.  This leads to interesting thoughts about the relative sensitivity of compounds when there are varying voltages applied to the collector.  Table 1 has some representative data illustrating this observation.

What are the conclusions that I took away from this experience?  The first and biggest lesson is that a problem that seems clearly associated with one part of the instrument may actually be caused by something entirely different, which is why methodical troubleshooting helps isolate the source of trouble.  Second, if the problem really did have to do with the spring it taught me to pay more attention to that part of the detector and it made me want to learn more about the fundamentals of the FID.  Finally, I’m not convinced the spring was actually the thing that made the difference, so like lots of investigations, this experience raised at least as many questions as it answered.  But hey, that’s why we do science, right?  Right?  :)

The Disappearance of the Honeybee: Can we Solve Colony Collapse Disorder (CCD)?

Admire 2F (Imidacloprid) is highly toxic to honey bees and therefore can only be used after the removal of the apiary from the cranberry bog.

Adult worker bees leave the hive and never return. Since honeybees cannot live without the colony they inevitably die. The hive has plenty of honey stored, the queen is in good health, and there appears to be no invasion from robbing bees, beetles or moths. Yet the colony is nearly empty. Mysterious bee die-offs are not new, the first reported case of a similar disorder occurred in 1869 (1,2) and was thought to be related to poisonous honey, lack of pollen or a hot summer (1). Since then there have been dozens of CCD-like outbreaks where worker bees have disappeared from the hive and theories abound from; fungus (Aspergillus flavus), mites (Acarapis woodi, Varroa destructor), neonicotinoid pesticides (Imidacloprid, Clothainidin) to sources of pollen (Eucalyptus leucoxylon). Honeybees are delicate organisms with half as many genes present to detoxify foreign substances compared with other insects (3). They are also sensitive to moisture, temperature, diet and relocation. Studies have indicated that CCD hives are under stress and there is either a contagious condition or exposure to a common risk factor (3). Immunodefense genes that are activated in bees can give clues to exposure and current studies are underway to determine what factors are triggering these responses.

            One theory is the use of the neonicotinoid pesticides such as Imidacloprid is contributing to CCD. One mechanism for mortality in termites exposed to Imidacloprid, for example, is the termites leave the nest and cannot remember how to get home. Although studies have shown the acute toxicity for Imidacloprid is 20ppb (3), chronic toxicity is difficult to determine given the limitations on techniques for detection in the single-digit ppb or sub-ppb concentration range in matrix. Another facet of this investigation is the metabolites of this compound are believed to be orders of magnitude higher in chronic toxicity compared to the parent compound (4).

            Alaa Kamel with the US EPA Office of Pesticide Programs examined Imidacloprid and its known metabolites and examined different procedures for the extraction, cleanup and analysis of the bee, bee pollen, and honey. The most effective extraction and cleanup required a 1gm C18 SPE cartridge with the addition of sodium acetate and magnesium sulfate to the sample prior to extraction. Analysis was performed using positive ESI-LC/MS/MS. One of the LC columns used was a Restek Aromax for its superior retention characteristics. Metabolites were better retained on this phase, although some peak tailing was observed. A faster gradient will help to overcome this problem. Imidacloprid was detected at 0.2ppb and metabolites ranged from 3.6ppb for the olefin moiety to 0.2ppb for desnitro olefin imidacloprid (4).  

            The behavior, social and genetic characteristics of these insects make it exceedingly difficult to determine the cause of these latest bee die-offs. Many of the CCD-like events in the past 150 years were never solved. With recent advances in genetic testing, viral identification, and chemical detection, CCD is a problem that will expand the frontiers of science and will eventually be solved.

Admire 2F (Imidacloprid): Can be used to control rootworms, white grubs and a variety of other insects on Cranberries. Aerial application is prohibited.

** Both photographs were taken on Cranberry Road in Eastern Massachusetts on July 3rd, 2010.  Cranberry bogs line the roads. The sign was located on one side of the road and the apiary was on the opposite side.