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Book Review: Echoes of Life: What Fossil Molecules Reveal about Earth History.

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.

The striking similarities between cholesterol and sterane.

The striking similarities between cholesterol and sterane.

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.

Sterane and Hopane patterns (shaded areas) by GC-MS SIM using an Rxi-5Sil MS 30m x 0.25mm x 1.0µm film column.

Sterane and Hopane patterns in MC252 Crude (shaded areas) by GC-MS SIM using an Rxi-5Sil MS 30m x 0.25mm x 0.25µm film column.

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

Petroleum Biomarkers and Tarballs at the 36th International Symposium on Capillary Chromatography and the 9th GCxGC Symposium

Gulf Oil Spill Blogs

http://blog.restek.com/?cat=3

 

Injection Techniques used in GC: Names and What are we actually doing with each Technique?

2011-jaap-pasfoto4-small

 

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.

 

 

 

Split injection

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

Splitless injection

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)

Figure 1 With splitless injection, the solvent must look like the trunk of a tree.. Note that the peaks that elute immediate after the solvent are already very sharp, indicating the focusing

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. Liners with wool 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).

fig2

Figure 2: position of column in a Uniliner. Note there is a seal formed by the polyimide outer coating

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, 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

fig3

Fig. 3 Inside the PTV the syringe needle is guided by the tapered liner, to realize an on-column injection

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.

http://www.restek.com/catalog/view/36032

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

Fast Sample Preparation and Shoot-and-Dilute GC for Flame Retardants in Fish

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.

Shoot-and-Dilute GC – Used Motor Oil and PAHs – Rxi-5ms GC Column Performance

PAH Separations – Rxi-5ms GC Column – Shoot-and-Dilute GC

Using the Restek EZGC Method Translator and Flow Calculator to Support Shoot-and-Dilute GC Method Development – Going from GC-ECD to GC-MS

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.

Analysis conditions for GC-ECD and GC-MS/MS analysis. The oven conditions are listed in the EZGC method translator. In the original column the GC-ECD condtions are listed. The translated conditions are for the vacuum outlet GC-MS/MS.

Analysis conditions for GC-ECD and GC-MS/MS.  The oven conditions are listed in the EZGC method translator. In the original column the GC-ECD condtions are listed. The translated conditions are for the vacuum outlet GC-MS/MS.

QuEChERS_HFRs_FishPSA Pass Through_FishFish_Recovery

The North American Chemical Residue Workshop – Sharing Science and Fun in the Sun

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.

Jonathan "Munch" Keim takes the stage to introduce the Restek EZGC Method Translator Flow Calculator

Jonathan “Munch” Keim takes the stage to introduce the Restek EZGC Method Translator and Flow Calculator 

The Restek Vendor Seminar draws a crowd at NACRW.

 

Cindy Ross, Michelle Misselwitz, Amanda Rigdon and Mike Chang enjoy the dinner cruise that unfortunately didn't cruise!

Cindy Ross, Michelle Misselwitz, Amanda Rigdon and Mike Chang enjoy the dinner cruise that didn’t cruise!

Which LC column should I use for Method 8330B explosives analysis?

cropped 526

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.:

 

cropped lc 526cropped lc 527

 

 

If you have a system that has a pressure limit of at least 600 Bar, we suggest our Raptor™ Biphenyl as the primary column and Raptor™ ARC-18 as the secondary column.

Here are chromatograms. Again, for best resolution, please click on the chromatogram:

cropped lc 530LC531

 

 

 

 

I hope this clarifies some things and gets you started in the right direction. Thank you for reading.

 

[26] What do Chromatograms tell us? Base line is Rising with a Near Constant Slope..

2011-jaap-pasfoto4-smallChromatograms 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”.

Fig.1  Ghost peaks appearing every analysis

Fig.1 Ghost peaks appearing every analysis

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.

Possible solutions:

  • 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.J.J_Thomson

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.

Cavendish Lab Bldg (Large)JJ Thomson Plaque (Large)Physical Chemistry (Large)Cavendish Lab 03 (Large)

Be sure to check out the Restek presentations at NEMC!

DCThe National Environmental Monitoring Conference (NEMC) will be held this Monday (August 4th) through Thursday (August 7th) in Washington, DC. Here is the technical program for you to peruse. As you may see, Restek will be providing the following talks:

Jason Herrington @ 3:30 Monday

Christopher Rattray @ 4:30 Monday

Christopher Rattray @ 3:30 Tuesday

For more information on the conference check out the NEMC homepage.

Calculating Splitless Valve Time for Splitless Injection GC with the New EZGC Method Translator and Flow Calculator – Pesticides

Properly setting the splitless valve time (SVT) for splitless injection in gas chromatography (GC) is probably one of the most overlooked optimization parameters for pesticide analysis today.  Having too short of a SVT leads to poor transfer of active and involatile pesticides, especially as the liner becomes contaminated with “dirt” from injected sample extracts.  This leads to poor quantitative results and possibly the premature need to recalibrate the GC.  The reason a proper SVT is so important has to do with the long sample transfer time from the inlet to the GC column inherent in splitless injection.  In general, the flow through the inlet is in the same order-of-magnitude as the capillary GC column outlet flow, which means it’s slow.

A rule-of-thumb for setting the SVT (sometimes known too as purge valve time) is to sweep the inlet liner completely with carrier gas 1.5 to 2 times before opening the split valve.  This range provides a buffer for quantitative transfer as the liner/seal gets dirty and analytes have to “chromatograph” out of the dirt to get to the column, while keeping the transfer time short enough to avoid a long solvent tail that might obscure earlier eluting analytes.  We provide an easy way to calculate splitless valve time through our new EZGC™ Method Translator and Flow Calculator (MTFC).

Using the Flow Calculator:

  • Enter your Column length, inner diameter, film thickness, and starting oven temperature.
  • Enter your Column outlet flow, and click “Atm” or “Vacuum” depending on whether you’re using an ambient pressure (Atm) detector like ECD, or a vacuum-outlet detector like a mass spectrometer (MS).
  • Enter your inlet temperature and your inlet liner volume (this can be calculated or if you’re using an HP or Agilent GC inlet liner, you can find liner volumes in the MTFC Glossary).
  • The Splitless Valve Time range that should be used is calculated automatically.

I’ve outlined the steps graphically below.  For additional reading, try these ChromaBLOGraphy posts:

Setting the splitless injection purge valve time in gas chromatography

Splitless injections of dirty samples on a single taper with wool liner result in quick degradation of its performance, especially for involatile polycyclic aromatic hydrocarbons

SV 01zz

SVT 02

SVT 03x

SVT 04

[25] What do Chromatograms tell us? My peaks broaden and look worse after every analysis: Impact of the Transfer Line in GC-MS

2011-jaap-pasfoto4-small

 

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.

 

This was an interesting case.  The customer makes a sequence of 8 analysis and every next analysis the chromatogram looked different. Not a little different but the last peaks completely broadened and disappeared, see fig 1 for the first 4 runs out of the series of 8.  The early eluting peaks look similar.

Figure 1. First 4 runs of series of 8 Semivolaatiles. Note with every run more compounds broaden and disappear.

Figure 1. First 4 runs of series of 8 Semivolaatiles. Note with every run more compounds broaden and disappear.

It was an analysis of semi-volatiles using GC/MS, using splitless injection on a 30m x 0.25mm Rxi-5Sil MS, film 0.25 micron, a column used a lot for this type of work because it offers inertness and low bleed.

 

As the change happened with every new injection, it could have been contamination of liner caused by highly dirty sample.  In my life I have never seen such big differences just between a few injections.

An other way to get such extreme “smeared” peaks, is when there is a lot of solvent condensation on the column. This can happen if the oven temperature is much below the boiling point of the solvent. Because of excessive condensation, peaks can look very broad. As methylene chloride was used here, this could also not be the case.

The last option must be a cold spot or a “zone” that changes in temperature after each analysis.

 

After checking that, it became clear: it was the temperature of the transfer line which was “OFF”.  After turning it “ON” the results were as in figure 2 and peak width/response was back to normal.

 

Fig. 2 After transfer line temperature was turned on, peaks were again narrow and sharp

Fig. 2 After transfer line temperature was turned on, peaks were again narrow and sharp

Probably at the first analysis the transfer line was still hot, but every next run, it became colder. When components pass through a hot section, nothing will usually happen, but passing a colder section, the retention increases a factor 2 every 15 C lower temperature.

That means that a sharp peak can easily be converted into a “blob” as we see  happening in fig 1.

As the temperature of transfer line was decreasing in time, the effect became more visible also at more volatile compounds.

It’s very important to eliminate cold spots, so make sure the transfer line temperature is at least similar as the final oven temperature. This is also a reason why detector temperature must be above final column temperature.