Michelle Misselwitz « ChromaBLOGraphy: Restek's Chromatography Blog

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Mining through complex data and non-target analysis is made easier with GCxGC-TOFMS

Friday, March 29th, 2013 by Michelle Misselwitz

I am currently working on a project looking for pesticides in herbal tea. At this point, I am using the contents of one tea bag (~1.5g) and 10mL of freshly boiled water with a QuEChERS extraction. I am still working through the details on the impact of the hot water and what type of cleanup will be necessary for pesticide determination, so that will be a blog for another day. I first decided to do some split injections of the raw extract to see what type of natural products are in some of the herbal teas I picked up at the grocery store.

Using GCxGC-TOFMS with a 30m x 0.25mm x 0.25µm Rxi-5Sil MS in the first dimension and a 1m x 0.25mm x 0.25µm Rtx-200 in the second, I processed the data by doing a “peak find” and library search for anything with a S/N of at least 100. I then get my peak list and sort according to the NIST library similarity number, so that the highest matches are at the top of the list. I start scanning through the list and see what names sound either like a pesticide or something else interesting. For example, I came across the name niacinamide with a similarity of 912, and a spectra that seemed fairly unique. I did a quick google search on the name and (according to Wikipedia) it is a main ingredient in an acne medication and has shown to have anti-anxiety properties among other potential medicinal uses. This type of peak find is made possible by the spectral deconvolution of the time-of-flight mass spectrometer.

I also mine through the data by taking advantage of the ordered chromatograms produced by GCxGC. This means that compounds with a similar structure, or a homologous series, will elute in a band across the chromatogram. I found another compound, lupeol, which is a triterpenoid that has been noted to have several medicinal properties. By looking at peaks that are eluting near lupeol, I found sitosterol which is a phytosterol that may reduce cholesterol.

While I don’t have standards to positively confirm peak identity, a high NIST match similarity with a spectra that contains abundant high m/z ions increases my confidence in the peak assignments. I really enjoy digging through the data and seeing if anything interesting pops up. Using GCxGC-TOFMS can give you A LOT of data to mine through, but with spectral deconvolution and ordered chromatograms, it makes it much easier.

The NIST library spectra, the peak true (spectral deconvoluted) and caliper spectra all show a high match similarity for Niacinamide

Sitosterol and Lupeol have a similar structure and elute in a band in the GCxGC-TOFMS chromatogram

Posted in Food, GCxGC, QuEChERS, Tips & Tricks | Add a Comment »

Half the Column, Same Chromatogram. Trimming your GC Column and Maintaining Resolution.

Friday, March 1st, 2013 by Michelle Misselwitz

Oftentimes, when dealing with complex matrices that have a lot of non-volatile material, you may need to trim the front end of your column to prolong the column lifetime. I was recently working on a project where I wanted to see how short of a column I could use and still maintain resolution between some isobaric pairs of brominated diphenyl ethers (BDE 49 and BDE 71).

I started with a 15m x 0.25mm x 0.10µm Rtx-1614 column and GC instrument conditions that were optimized by Pro ezGC. I began trimming loops off of the front of the column and accurately measured the new column length by injecting air into my GC-MS to determine the holdup time. Then, I used the Agilent GC pressure/flow calculator to decrease the column length until the calculated holdup time matched my experimental holdup time. This new column length was used in the Agilent GC Method Translator software to calculate my new method. By doing this the analytes are eluting from the column at the same temperature, no matter what column length we use. This provides essentially identical chromatograms and maintains resolution (See figure below). I think I could have continued trimming my column, but I ran into two issues. First, my head pressure was reaching 0psi because I was operating under vacuum outlet conditions. Second, the oven ramp rate was getting so fast that my instrument couldn’t properly keep up. Really this just goes to show that you can trim your column in half (and the run time!), while still maintaining resolution, by properly translating the method.

In order to translate your methods remember the following things:

Measure new column length using holdup time
Input new column length and all original conditions into method translator (select translate only)
Make sure to change ALL parameters necessary of new translated method (including your new column length) into GC software
Check your instrument maximum heating rate to make sure your instrument can keep up to new method conditions.

The resolution between BDE 49 and BDE 71 is maintained while trimming the column almost in half. The analysis time is also cut in half!

Posted in Enviro, Faster Analyses, GC/MS, Maintenance, Tips & Tricks | 5 Comments »

GCxGC is the Star at 4th Multidimensional Chromatography Workshop!

Thursday, January 31st, 2013 by Michelle Misselwitz

My colleagues and I made the road trip to the Ontario Ministry of the Environment in Toronto for the 4^th Multidimensional Chromatography Workshop. This workshop organized by Eric Reiner at the MOE is a veritable who’s who in the world of GCxGC. I personally love going to this workshop because of the wide variety of topics covered by not only excellent scientists, but engaging speakers as well (and it’s free). The topics covered fundamentals of multidimensional chromatography, including alternative modulators, GCxGC (or as suggested GC×GC) nomenclature, and retention markers for two-dimensional chromatograms. The program then switched gears towards applications of multidimensional chromatography, with a focus on comprehensive two-dimensional gas chromatography (GCxGC). The first day included fire debris analysis, forensics, chlorinated dioxins and furans, and my personal favorite of GCxGC going to Mars! The second day started with a few talks on metabolomics, characterizing the odor of human decomposition (yum!), and novel halogenated contaminants in the environment. The program is listed below, including links to some of the presentations (note how far many of these speakers traveled to Toronto in January!).

This workshop attended by over 160 people this year just keeps growing. There seems to be a strong desire to continue to grow this workshop, which will require moving to a larger auditorium and possibly adding a third day of presentations. It really shows that GCxGC is gaining familiarity across a broad range of possible applications. If you are interested in learning more about comprehensive two-dimensional gas chromatography, or are just getting started, check out our GCxGC brochure (GCxGC Columns, Your One Source for 2D Chromatography). The brochure has a handy column combination guide and some of the applications that we have done here in the lab. We also have numerous posts about GCxGC here on ChromaBLOGraphy that you can view by either searching or clicking the GCxGC link under Categories on the sidebar.

The 4^th Multidimensional Chromatography Workshop Jan 8 & 9 2013

Tuesday January 8, 2012:

Multidimensional Chromatography – past, present, future Philip Marriott, Monash University, Victoria Australia

Optimization Aspects of Comprehensive Two-Dimensional Gas Chromatography Tadeusz Gorecki, University of Waterloo, Waterloo, ON

Study of complex VOC mixtures by GCxGC-TOFMS Jef Focant, University of Liege, Liege Belgium

The Use of GCxGC TOFMS for Forensic Applications Frank L. Dorman, K Organtini, J. Westland, E. Ly, A. Brockman, J. Stubleski, Penn State, State College PA, USA

Bidimensional Retention Normalization in GCxGC: Advances and Perspectives. Jean-Marie Dimandja, Spelman College, Atlanta GA, USA

High Speed Deans Switch for GCxGC Separations: Principles and Applications John Seeley, A, Ghosh, S. Seeley Oakland University, Rochester MI, USA

How soft ionization, high mass resolution and a comprehensive data analysis could further enhance the significance of GCxGC-TOFMS. Thomas Gröger, M. Schäffer, M.Saraji, M. Sklorz, M. Eschner, J. Wendt, R. Zimmermann, Helmholtz Zentrum, Munich Germany

GCxGC-TOFMS for Chlorinated Dioxin and Furan Analysis in South Africa, Jack Cochran, EJ Reiner, T. Kolic, K. MacPherson, L. Quinn, M. Brits, C. Swiegelaar, J. de Vos, P. Gorst-Allman, Restek, Bellefonte PA, USA

Time controlled Cryogenic Zone Compression (t-CZC) GC-HRMS – can we detect attogram amounts of TCDD? Dirk Krumweide, Thermo Fisher, Bremen Germany

Identification of New Markers of Wood Smoke Exposures in Firefighters using GCxGC-TOF-MS- Sujan Fernando and Brian McCarry, McMaster University, Hamilton ON

Analysis of blubber extracts of Peponocephala Electra by GC x GC-high resolution TOF-MS. Teruyo Ieda, Gerstel Japan

GCxGC for Space Science – Opportunities and Challenges for Deep Space Organic Analysis. Mark Libardoni, Southwest Research Institute, San Antonio TX, USA

Wednesday January 9, 2012:

Using GCxGC-TOF/MS to elucidate metabolome differences in complex biological matrices. Catherine Rawlinson, Joel P. A. Gummer, Robert D Trengove, Murdoch University, Perth Australia

Identifying biomarkers of P. aeruginosa antibiotic susceptibility using GCxGC-TOF and Statistical Compare. Heather Bean, JM Dimandja, J.E. Hill University of Vermont, Burlington, VT, USA

Investigating GCxGC separations using selective column chemistry and compound derivatization pairings for common metabolomics chemical compounds Julie Kowalski, Michelle Misselwitz, Jack Cochran, Restek, Bellefonte PA, USA

Benefits of GCxGC-TOFMS approach for chemical characterization of environmental risk factors in the Sudden Infant Death Syndrome (SIDS) and Sudden Intrauterine Unexplained Death (SIUD) Daniela Cavagnino, A. Siviero, A. Mantegazza,V.Termopoli, P. Palma, G. Famiglini, A. Cappiello, Dani Instruments, Italy

Multidimensional LC for shotgun proteomicsor Proteomics. Herman Lam, Ivan Chu, Ricky Ng, Ricky Kong, S.O. Siu, Maggie Lam, Yuen Zhao and Edward Lau. CVG, Toronto, ON

Planar Microfluidics in Multidimensional Chromatography, H.J. Cortes, R. Shellie, J.Luong, K. Parlevliet. H. J. Cortes Consulting, LLC., Midland, MI. USA

Further exploration in the quest of orthogonally PH Stefanuto, JM Dimandja, JF Focant, University of Liege, Liege Belgium

Characterizing human decomposition odour: an application of Thermal Desorption-GCxGC-TOFMS, Sonja Stadler, PH Stefanuto, M Brokl, JF Focant, S Forbes University of Ontario Institute of Technology, Oshawa, ON

Making sense of the fume: GCxGC TOFMS analysis of mainstream tobacco smoke, M Brokl, L Bishop, C Wright, C Liu, K McAdam, JF Focant, : University of Liege, Liege Belgium

GCxGC Analyses of Naphthenic Acid Esters in Oil Sands Composite Tailings, Dave Bowman, B. E. McCarry, McMaster University, Hamilton ON

The Analysis of Mixed Bromo/Chloro Planar Compounds Produced as Combustion Byproducts Kari Organtini, Frank L. Dorman and Mark Merrick, Penn State, State College PA, USA

Open Forum with Manufacturers

Tags: Chlorinated Dioxins, Chlorinated Furans, environmental forensics, fire debris, GCxGC, GCxGC-TOFMS, Metabolomics, Multidimensional Chromatography
Posted in Conferences, GCxGC | Add a Comment »

EZGC Made Even Easier – Free web version now available!

Monday, December 17th, 2012 by Michelle Misselwitz

A few of my colleagues, Julie Kowalski and Chris English, have written blogs about the original CD based Pro ezGC modeling software (ezGC really IS cool! and Pro ezGC Software: Manna from Heaven or a Clunky DOS-Based Program). The basics of this software is to add a library of compounds by entering the retention times of a slow analysis and a fast analysis, add column and instrument conditions and then it will model separations for you. I have used this tool many times, and sometimes I don’t like having to enter in all of that information, but it does reduce method development time if you have a tricky isobaric interference. The software is admittedly a little clunky and has some bugs to it, but once you know the work around it can be a very powerful tool.

Well lucky for you we have taken the basics of the original Pro ezGC software and made this powerful tool even easier by updating the user interface, adding a huge library of compounds so you don’t have to, and made it available for FREE on the website (link). We currently have thousands of the most common compounds on a variety of column phases, and we will be adding more as time goes on, based on feedback from customers. Now, all that you need to do is enter in a list of compounds that you would like to be separated and voila! The EZGC Chromatogram Modeler will then give you a modeled chromatogram separating all (hopefully) of your compounds of interest. If you think that the results are strange or would like to change some of the values (oven temperatures, maybe column choice or dimensions) just contact the Restek Technical Service using a very handy link in the big yellow box and they can help you out with more specifics.

Hopefully this simplified approach to the EZGC software will help with some initial method development. It has saved me a lot of time in the lab, and now that you don’t have to worry about outdated software and inputting libraries, it should save you even more time! Let us know what you think, and if there are analytes missing from the library let us know, and maybe we can add them to the library in the future.

Example of modeled chromatogram using new web based EZGC Chromatogram Modeler

Tags: Chromatogram Modeling, EZGC, Method Development
Posted in Optimizing Applications | Add a Comment »

House Dust and Milk, Getting fired up about BFRs

Thursday, May 31st, 2012 by Michelle Misselwitz

Do a Google search on “flame retardant” and you are bound to be hit with a lot of scary news stories about flame retardants being found in a variety of places (food, dust, hands, breast milk…). It seems pretty hard to avoid exposure to something like flame retardants since it can be found in your furniture, computers, carpet, and of course your food. One story that has been picked up by many news outlets is about a recently published scientific paper on children’s exposure to polybrominated diphenyl ethers (PBDEs). Researchers from Duke University, Centers for Disease Control and Prevention (CDC) and Boston University determined blood, hand wipe, and house dust PBDEs levels. These levels were related to sex, age and race as well as breastfeeding practices and parents’ education of study participants. So why all of the attention from the general news media? The study indicates that there is a potential relationship between socioeconomic status and exposure levels. The study also reports trends with age, breastfeeding and hand/mouth activity. This paper is published in Environmental Health Perspectives, and the paper can be found here.

Restek will be attending the 13th Workshop on Brominated and other Flame Retardants in Winnipeg, MB, Canada next week, June 4 and 5^th. Interestingly, three of the authors from the paper discussed above will be presenting work at the conference; Heather M. Stapleton, Andreas Sjödin, and Thomas F. Webster. I will be presenting some of my work about BFRs in human milk and a poster about some of the newer phosphate flame retardants found in house dust.

If you are attending, please stop by our booth and meet the Restek crew; Dominique Lavigne, Julie Kowalski and me, Michelle Misselwitz.

Posted in Conferences, Enviro | Add a Comment »

Free EPA Meeting! New technologies, techniques and quality assurance

Friday, October 28th, 2011 by Michelle Misselwitz

Last week I attended the 21st Annual Quality Assurance Conference hosted by EPA region 6. I really love attending this meeting because of the relaxed atmosphere, the quality of the talks, and no registration fees. Talks are in one hour time slots, so it really lends itself nicely to discussions, instead of just lecture style.

This year had some fantastic talks on a broad range of topics. Steven Moser from the Oklahoma Department of Agriculture gave a nice presentation about developing a quick method for Aminocyclopyrachlor (Imprelis) in vegetation and soils by LC-MS/MS that increased sample throughput by 10x from the original method (wow!). Imprelis is an herbicide that is used to kill broad leaf weeds, but not trees. However it was quickly discovered that it was affecting the surrounding trees resulting in Dupont pulling the product. You may remember that Chris English blogged about this topic when the EPA first revoked the registration.

Laura Autry from the US EPA gave a presentation about collecting information about all of the EPA’s monitoring programs, compiling a database and figuring out what some of the gaps and needs are to fulfill the EPAs monitoring goals . One of the common themes she talked about is the need for development and utilization of state of the art technology and allowing for more flexible approaches to environmental monitoring.

Doug Stevens from Waters gave an interesting talk about using Atmospheric Pressure Chemical Ionization for GC/MS. I was familiar with the technique for LC/MS, but this was the first time I have heard about APCI for GC/MS. Since everything is at atmospheric pressure (no vacuum) you are not limited by flow, column dimensions and can use a wider range of CI reagents.

These were just a few of the talks I attended last week. Most of the presentations will be available on the EPA Region 6 website in a few weeks, including the ones I gave about analyzing tarballs and seawater, using a regular split/splitless injection port for large volume injections, and increasing throughput for method 8270 using a split injection.

Posted in Conferences | Add a Comment »

Milk Matters – Using QuEChERS extraction approach with GCxGC determination

Tuesday, September 6th, 2011 by Michelle Misselwitz

Last week I had the honor to present my work at the 31^st International Symposium on Halogenated Persistent Organic Pollutants (Dioxin 2011). While it is still a work in progress, Jack Cochran and I evaluated the QuEChERS extraction approach with a silica SPE cleanup for PCBs and BFRs in cow milk and human breast milk. The QuEChERS extraction used hexane:acetone (1:1), as an alternative extraction solvent to acetonitrile, and the QuEChERS Unbuffered packets . The hexane:acetone provided a good extraction of the fat in the cow milk samples, which is important since both PCBs and BFRs are lipophilic. To remove the coextracted fat, part of the QuECheRS extract was loaded on a 500mg Silica SPE cartridge and eluted with 5 mL hexane, which gave good recoveries of the PCBs and BFRs in cow milk (see table). The samples were analyzed by comprehensive two-dimensional gas chromatography (GC x GC) with electron capture detection (ECD). The use of a 30m x 0.25mm x 0.25µm Rxi-XLB in the first dimension and a 1m x 0.15mm x 0.15µm Rxi-17Sil MS in the second dimension allowed simultaneous determination of both the PCB and BFR compounds of interest. (View more GC x GC products here) The ECD was used for two reasons: first it allowed low pg detection of the multiple halogenated compounds, and second, it keeps the Cheap in the QuEChERS approach.

Everything seemed to be going pretty smoothly (or so we thought), so we tried this methodology on a NIST Certified Reference Material of fortified human breast milk (NIST SRM 1954). The fat content was equivalent to the fat content in the cow milk, so no adjustments were made in the cleanup. Analyzing these samples on the GCxGC-ECD gave results that were about 25% off from the certified values (removing two outliers that were biased high from coelutions). We wanted to see if we could get even closer results by using GCxGC-TOFMS and all I can say is wow! Sometimes they say ignorance is bliss, using the selective ECD we thought all was well, but when using a more universal detector, the fatty acids reared their ugly heads (see image below)! Later I found out that even though the fat content is essentially the same for cow and human milk, the fatty acid content is not. There is a much higher concentration of conjugated linoleic acids in human milk that aid in the brain development of the infant. So now we are trying to add a quick PSA cartridge SPE cleanup in addition to the silica cleanup to remove the fatty acids in human milk. Sometimes when you think you are at the end of the road, there is a bend and you see you have much farther to go! So I will continue this journey and hopefully we will get to the end with a method that is environmentally friendly, multi-residue, quick, easy and cheap! (One can dream, right?)

If you are new to GC x GC check out the Restek GC x GC Column Combination Guide!

Separation of BFRs and Aroclors using Rxi-XLB and Rxi-17Sil MS

Average (n=3) % recoveries of spiked cow milk

Fortified Human Milk SRM 1954 on GCxGC ECD and GCxGC TOFMS

Posted in Conferences, GCxGC, QuEChERS, Sample Cleanup | 2 Comments »

Split Injections… The Good, The Bad and The Ugly

Friday, June 24th, 2011 by Michelle Misselwitz

While I’m not necessarily a fan of the old spaghetti western film with Clint Eastwood, I thought it was an appropriate title for what I have seen with my work using a split injection for EPA Method 8270 extracts. You may recall an earlier blog that I wrote about the benefits of a split injection for these dirty extracts. So let’s start with the good. Using a split injection can allow for an increased initial oven temperature (80°C) versus a splitless injection (45°C) because of the faster transfer of analytes through the injection port onto the head of the column. This gives faster analysis times and much faster oven cool-down times, meaning more samples analyzed in a 12hr period. I also noticed increased injection-to-injection repeatability and held a continuing calibration longer before instrument maintenance was needed (see Pittcon poster for more info).

This brings me to the bad… Of course with a 10:1 split there is a compromise in sensitivity. With an Agilent 5975 MSD I was able to get a nice linear calibration curve from 5 – 160 µg/mL (0.5 to 16 ng on-column). Going down to 1 µg/mL was possible for most compounds with a few exceptions for those troublemaker phenols (2,4-dinitrophenol – I am talking about you!). On a positive note, by decreasing the sample amount injected onto a 0.25mm ID x 0.25µm column, sample overloading is minimized. So maybe bad is a little harsh, but it goes with the theme.

Finally, let’s finish with the ugly, and I do mean ugly! After injecting over 300 soil extracts from an underground storage tank site I performed maintenance on my split gas line. When I took off the line that goes from the injection port to the EPC I couldn’t believe my eyes. Not sure ugly cuts it here, I think my words were more like disgusting! A simple rinse with some methylene chloride shined it right back up to looking new. Anyways, lesson learned, there are great benefits to using a split injection for Semivolatiles analysis if you can afford to lose some sensitivity, but make sure to do some routine maintenance on your split lines.

Click for more details on “The Good” side of split injections for Semivolatile analysis

Posted in Enviro, GC Injection Techniques, GC/MS, Maintenance | 3 Comments »

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

Tuesday, October 26th, 2010 by Michelle Misselwitz

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, 20^th 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!

Posted in Conferences, Miscellaneous | Add a Comment »

Split Injections Increase your Throughput for Semivolatiles Analysis by EPA Method 8270

Friday, September 17th, 2010 by Michelle Misselwitz

It seems strange to think that using a split injection verses a splitless injection has anything to do with analysis time, but it does! When you are using a split injection the initial oven temperature can be much higher compared to a splitless injection. Since the transfer of analytes onto the head of the column is much faster for a split injection you don’t have to worry about solvent focusing, or cold trapping which requires the oven temperature to be 20° C lower than the solvent boiling point. In fact, by using a 10:1 split, we started the oven at 80 °C and were able to get good peak shape for early eluters 1,4-dioxane, N-nitrosodimethylamine and pyridine. Now we save on analysis time and the oven cool down time!

For more info, check out the poster I presented at the National Environmental Measurement Symposium (NEMC) a few weeks ago. Benefits of Split Injection 8270-Poster

Posted in GC Injection Techniques, Optimizing Applications | Add a Comment »