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If you are an avid chromatographer you likely subscribe to LC/GC magazine or its popular electronic version, E-Separation Solutions. It is definitely the place to go for news and information related to all things Chromatography. Over the years Restek has had many an article published in LC/GC and, full disclosure, we advertise there as well. Who in the business doesn’t? It is the place to be seen.
Back in June a couple of Restek’s LC R&D scientists had an article published entitled, “Effects of Column Inner Diameter and Packed Bed Heterogeneities on Chromatographic Performance.” In case you missed it this summer you can read it here: LC/GC Article
While we got some really good feedback on this article we were thrilled when LC/GC released The Top 16 Articles of 2016 issue and our article came in at #7 on the list. It was not too surprising to us. Anyone that reads the article can see the tremendous amount of research and thought that went into its preparation as well as the relevance to current chromatographic trends. Not unlike the research and thought that goes into the development of Restek’s premium performance LC products. Authors Ed Franklin and Ty Kahler are a part of a team of talented scientists and engineers that brought to life amazing LC products like Raptor™ SPP column, Roc® LC columns, and the new Force™ Premium LC/UHPLC columns. Products which consider the needs of today’s chromatographers, performance, robustness, and innovation.
There is some significant LC firepower in the R&D lab at Restek and it goes into every product that we produce…no matter what the inner diameter is.
They may be called PFP, PFPP, pentafluorophenyl, fluorophenyl or F5 phases. What they all have in common is the structure shown above (the pentafluorophenyl group) attached to the silica particles.
Most modern phases contain a propyl spacer between the above group and the silica, so that the phase looks something like this:
A “PFPP” phase refers to the above structure with the propyl spacer. However, quite often the other names apply as well, depending on the column and vendor. We are quite proud to offer our new PFPP phases, the Raptor Fluorophenyl and the Force Fluorophenyl, which are designed using the latest advances in bonding technology. The following are some questions that may come to mind.
What makes the phase useful?
Due to the presence of the fluorine atoms, a fairly polar phase is created that allows for HILIC separations. The electronegativity of the fluorine atoms, in combination with the delocalized electrons from the aromatic ring, result in increased retention for charged bases (like amine compounds) and other electronegative compounds (those containing halogen atoms, O, or N). Also, due to the presence of the aromatic ring and propyl spacer, the phase still retains some nonpolar characteristics, so it can also be used in reverse phase mode.
I recommend the following if you wish to read more about HILIC separations and characteristics of PFPP phases:
Restek USLC, Ultra Selective Liquid Chromatography
Reliable HILIC LC-MS/MS Analysis of 4-Methylimidazole (4-MEI) on Raptor FluoroPhenyl Columns
How should these phases be used?
For HILIC mode, use a mobile phase that is at least 50% organic solvent. If using a gradient, start with high organic and program up to high aqueous content. Often a mild modifier, such as 0.1% formic acid is used in both mobile phase A and B. Here are some examples:
For reverse phase mode, conditions similar to what is used with C18 are common, except that sometimes the gradient starts with a higher organic content to encourage retention of highly polar compounds, perhaps 30% organic or higher. Here are some examples:
How do I condition this phase before using?
Equilibration times will vary according to application. If using for the first time or after changing solvents in your mobile phase(s), it may take as much as 50 column volumes or more. It is recommended to always start with a new column when developing methods, as the phase is sometimes permanently altered by certain mobile phase modifiers and buffer salts.
How do I prepare my sample to use with this phase?
Samples should be as clean as possible, as the phase is somewhat sensitive to differences in sample matrix. If sample matrix is an issue that does not lend itself well to cleanup, it is best to quantitate using matrix-matched calibration standards. As with any LC column, particulates should be removed by filtration prior to loading on the instrument.
I hope this has helped to answer some of the questions you might have about using PFP phases.
Thank you for reading.
We already learned how to calibrate our TO-15 system, what our concentration units mean, and how to properly read our vacuum gauge while pressurizing samples. We are clearly going out of order here, because I have never covered how to prepare a working standard (necessary for calibrations) from a stock standard. Therefore, the following multi-part blog series will demonstrate how to use a static dilution to make a working standard.
Okay… first things first. We need to determine our stock standard concentration, canister volume, stock standard injection volume, and final canister pressure. How we go at deriving all of the aforementioned may vary. For example, we often have a 1 ppmv stock standard and a 6 L canister, so those variables are gimmes. More often than not we also know the final canister concentration we desire. For today’s example we will use 2.0 ppbv.
All that is left to determine is the stock standard injection volume and final canister pressure. For the following example we will say that we want a final canister pressure of 30 psig. Now that we have everything we need, but the stock standard injection volume, the math is as follows:
First we determine the final volume of gas in the canister. We know we start with a fully evacuated canister at -14.7 psig and pressurize the canister to 30 psig. So we recognize that we have added 3 atmospheres (~14.7 psig/atmosphere) to the canister. Therefore, our final gas volume is 3 x 6 L = 18 L.
Remembering that we want 2.0 ppbv from 1 ppmv, the rest of the math looks as follows:
Where we are solving for the stock standard injection volume (x), 18 L is our gas volume from above, 0.002 ppmv is our desired concentration, and 1.00 ppmv is our stock standard concentration. The result of this is 0.036 L = 36 mL.
We now know that if we inject 36 mL of our 1 ppmv stock standard into a 6 L canister and pressurize this canister to 30 psig, the final concentration will be ~1.97 ppbv.
We can take the above math and manipulate it any way we want it. And by that I mean, we can move x (what we are solving for) to the top right if we want to solve for the final desired concentration or to the bottom left to solve for the final gas volume, etc… In my experience, we are generally solving for x on the left-hand side, where we are either solving for the injection volume or final gas volume (which ultimately is extrapolated out to final canister pressure).
Now that I have given you some examples, I will give you the shortcut I use when in the lab. Without further ado I introduce you to the:
Stay tuned for next time when I blog about how to introduce a stock standard into a canister…
As of May 31, 2015, 1100 Series HPLC systems were categorized “End of Guaranteed Support” (EGS) by Agilent. Agilent would prefer it if you purchased a new system from them, but 1100 HPLCs are dependable workhorses. Why not keep them running with instrument replacement parts from Restek? Check out our selection, easily sorted by instrument and module: http://www.restek.com/Supplies-Accessories/HPLC-Accessories/Instrument-Accessories-Parts
We also offer PM kits for 1100 HPLCs. The autosampler PM kit (cat.# 25271) can be found here: http://www.restek.com/catalog/view/9668 and the pump PM kit (cat.# 25270) here: http://www.restek.com/catalog/view/9669.
We still keep our older Agilent instruments running with Restek replacement parts. Look at our HP 1050 in our R&D lab. Still going strong!
Let’s see pictures of your older instrumentation. Email a picture to me, and I’ll send you $10 Wizard Dollars (US only): email@example.com
While HPLC may be tempting to use for terpenes analysis, a GC/FID or GC/MS is really the most straightforward and recommended way of analyzing terpenes in cannabis. Terpenes, being relatively volatile and neutral, lend themselves rather nicely to GC in general.
As you can see from Figure 1 below, coelutions of the cannabinoids and terpenes are very likely when analyzing real cannabis samples by HPLC-UV methods. A Shimadzu Prominence 20AD HPLC system and Raptor ARC-18, 2.7um, 150 x 4.6mmID column (catalog# 9314A65) was used in Figure 1.
Figure 1- Standards at equal concentrations
At 205nm, there are many co-elutions of terpenes and the cannabinoids, making identification and quantitation extremely difficult, if not impossible, for either class of compounds.
Conversely, cannabinoids can be analyzed by HPLC-UV, as long as the correct UV wavelength is chosen. At 220nm, terpenes yield a very low signal. Because cannabinoids of interest are present at a much higher concentration than terpenes, in addition to providing a stronger UV signal at 220nm, cannabinoids can be reliably analyzed at a wavelength of 220nm. See Figure 2.
In summary, HPLC-UV analysis of terpenes in cannabis is not recommended, and will likely cause more issues than it will provide solutions.
A good solution to the coelutions by HPLC-UV is to choose a GC headspace method. Interferences from the complex sample matrix, as well as the much less volatile cannabinoids can be eliminated then.
You may have seen my last blog post about guard holders and cartridges that we have available for our various LC product lines, “Which guard cartridges and holders go with which LC analytical columns?”
Along those lines of discussion, the following note is available to also show what specific products are currently offered for this: http://www.restek.com/pdfs/GNAR2492-UNV.pdf
Reflected in the above note are some changes in what we are offering for the cartridges that are used with the Trident LC Holders. Starting January 1, 2017, we are discontinuing the 20 mm length cartridges for the Ultra, Pinnacle, Allure and Viva columns. This is a result of declining popularity and lower demand. We will, however, continue to sell the 10 mm length guard cartridges for the Ultra, Pinnacle, Allure and Viva columns.
In conjunction with this change, corresponding Trident guard holders for the 20 mm cartridges will be discontinued as well. This includes some of our older catalog numbers, such as 25085, 25086, 25060, and 25062 (XG-XF fitting for 20 mm).
While we feel confident that, in most cases, the 10 mm cartridge length is sufficient for analytical columns, we apologize for any inconvenience this may cause.
Thank you for your continued interest in using our LC products.
In Europe, the last transition periods named in REGULATION (EU) No 1169/2011 on the provision of food information to consumers are running out on Dec. 13th, 2016. Since then, a nutrition declaration is mandatory for nearly all packed food in Europe, wherein the amounts of fat, saturates, carbohydrate, sugars, protein and salt have to be included. The content of the mandatory nutrition declaration may be supplemented with an indication of the amounts of one or more of the following:
(b) polyunsaturates; …
This was my starting point to investigate some of the Restek solutions in measuring Fat content in Food. The nutrient fat is one of the main energy providers from Food. Fortunately the civilized world is not suffering from a lack of nutrient energy, but from the opposite, which, unluckily, leads to some of the so called lifestyle diseases. This underlines the importance of such nutrition declarations, but also the importance of scientific investigations about different fat classes.
Different fats are all built in the same simple way. The trivalent alcohol Glycerine is esterified by fatty acids. Though simple in its basic structure, fats are varying by hundreds of different fatty acid combinations, some of them identified as healthy, others as unhealthy. For example, mono- and polyunsaturated fatty acids (MUFA and PUFA) are known as more healthy than saturated fatty acids (SFA). Unfortunately we also have to divide between cis- and trans-unsaturated fatty acids, whereas cis-fatty acids are recognized as more healthy than trans-fatty acids. A special role is given to polyunsaturated fatty acids (PUFAs) depending upon the position of the double bond closest to the ester group: n-3 (also notated as ω -3) systems and n-6 (ω -6) systems, wherein the ω -3 systems seem to have a special healthy role in human metabolism.
Sounds confusing? It is, especially for the analyst, who is asked to classify fats in food and other matrices. The huge amount of different, structurally nearly identical compounds, which cannot be easily resolved in an MS, makes it impossible to use an LC approach for this task. Fats itself cannot be transferred into the gas phase to use an easy GC approach. So a lot of techniques are standardized from organizations like AOAS, ISO or DGF to cut the fats into glycerine and its fatty acids, followed by derivatization of the fatty acids into their Fatty Acid Methyl Esters (FAME) for further GC measurement.
The next question is, which GC column would be best to analyze the remaining complex mixture of Fatty Acid Methyl Esters? Restek has developed a wax column, specially designed for such a challenging approach, the FAMEWAX column. So what, if I would recommend this column? I am sure that you are sure that I believe in the quality of Restek products. I am a Restek employee and in the end you would have to trust in my or Restek’s reliability. Or you have to check different columns by your own.
But what, if others would have done this before? Think about the most demanding ones? Instrument suppliers. If they have to proof their complex systems in front of a customer, they do well to know everything about the performance of a strategic compound like a GC column. And so they do. I recently found an application note from Shimadzu Europe, comparing different types of GC columns for this approach, including Restek’s FAMEWAX column, which I mentioned before.
The finding of the Shimadzu scientists was that under six different types of columns the FAMEWAX column with a 30m length performed as good or better as five others with 60 m length. The complete application note can be found here:
Another interesting publication using our FAMEWAX column was published by young scientist Annika Ostermann from the research group of PD Dr. Schebb at the University of Veterinary Medicine in Hannover, Germany.
This research group investigates the role of ω -3 fatty acids in human and veterinary metabolisms. For this work, Annika Ostermann compared different derivatization and extraction procedures suitable for the determination of the fatty acid composition in plasma and tissues as fatty acid methyl esters using gas chromatography. Sample preparation and derivatization methods for the analysis of small amounts of tissue and low plasma volumes is more challenging than determining FAME in food, where sample amounts may not play such a big role.
Annika Ostermann provided the following chromatogram, showing this nice separation for the interesting compounds in her work. The complete work was published in PLEFA (Prostaglandins, Leukotrienes and Essential Fatty Acids) and can be found here.
Being a GC chemist for almost 38 years I always wondered why most LC separations are performed on a C18. A C18 is an extremely good non-polar stationary phase and is the foundation of reversed-phase chromatography, where polar mobiles phases are used with non-polar stationary phases. But there are so many C18 phases, how do you choose the correct one? There are also a lot of separations where analytes are polar, one of the big advantages of LC is that you can analyze much more polar analytes than GC without derivatization. For these types of analytes why do you choose a C18 at all?
A basic rule in chromatography is that you choose a stationary phase that shows interaction with your analytes. In a GC hydrocarbon separation, for example, a polydimethyl siloxane like Rtx-1 is used. For alcohols and glycols, a polyethylene glycol like Rtx-Wax is used, or for optical isomers a chiral stationary phase is used that shows specific affinity to one type of isomer.
In LC, multi-ringed structures, substituted ring structures, and small polar analytes are frequently analyzed. These analytes will interact with C18 through dispersive forces, but they may also interact with the silica substrate through hydrogen bonding or cation-exchange. These interactions are often referred to as “silanol” interactions and they are often thought of as undesirable. In fact, with many separations these silanol interactions contribute significantly to the retention of the analytes. This means silanols can be both beneficial, by creating retention; and detrimental, if they are not controlled or if the silica surface is not consistent. Differing silica substrates is one of the main reasons all C18’s are not the same.
So the question that came to my mind is how do you take advantage of additional retention mechanisms for polar analytes in reversed-phase? One way is with the Biphenyl stationary phase which was originally developed at Restek.
The Biphenyl stationary phase retains compounds through the same dispersive forces as a C18 but it also allows for more polarizable substances to be retained. Basically, the pi electrons available on the Biphenyl phase create retention with analytes which are electron deficient. Because there are so many pi electrons in conjugation you get much better retention for small and polar analytes than on a phenyl-hexyl or diphenyl type phase.
I would argue this actually make the Biphenyl an even BETTER choice than a C18 when starting your method development. An example I saw at a recent food meeting showed >600 pesticides in one run using LC/MS/MS on Restek’s Raptor Biphenyl phase.
As with any phase chemistry there is always some type of downfall and in the case of the Biphenyl it is UV bleed. A dirty little industry secret is that ALL phases bleed whether you know it or not, it just so happens that with C18 you don’t see the bleed with UV or MS detectors. In the case of the Biphenyl you can see phase bleed in certain instances by UV. If this is the case contact Restek Technical Support and they can coach you through how to minimize or eliminate it.
So where did the Biphenyl phase come from? A number of really intelligent people were involved in making it happen at Restek and it included GC polymer chemists, LC R&D chemists, and Applications personnel. It’s a great story about experimentation, fundamental chemistry, and teamwork making a really great and unique product. If you want to learn more about the biphenyl story go to www.restek.com/biphenyl.
Special thanks to Ty Kahler and Paul Connolly for their input in this blog
Dan Li, Katarina Oden, Chris English, and Jason Herrington
Sulfur compounds are reactive, corrosive to pipes, and destructive to catalysts in petroleum refineries. Sulfur emission are strictly regulated globally. When released into the atmosphere sulfur dioxide converts to sulfuric acid resulting in adverse effects on human health and the environment. Sulfur detection is found useful in many other industries; therefore, detection of sulfur compounds in matrices serves a vital role in many application areas.
Sulfur analysis is typically done by gas chromatography (GC). The frequently used sulfur detectors are sulfur chemiluminescence detector (SCD), flame photometric detector (FPD), and mass spectrometry detector (MSD). As sulfur selective detectors, SCD and PFPD have the advantages of measuring components of interest and providing an equimolar response for sulfurs. Compared to SCD, FPD is more robust, less expensive and less complicated for maintenance. One challenge of FPD or PFPD is the hydrocarbon interference, especially those from the chromatographic coelutions, which can cause quenching or signal suppression. In order to minimize the quenching effect, one can either use high split ratio to reduce the amount of hydrocarbon injected or resolve the sulfur species from the hydrocarbons chromatographically. In most cases, small injection volumes or high split ratios are unsuitable for trace-level detection. If cryogenic cooling cannot be used, it is difficult to avoid coelutions using a single detector.
Mass spectrometry is a universal detector widely used in many applications. It provides structural information of the analytes in full scan mode and enhanced selectivity / sensitivity in selected ion mode (SIM). For some volatile sulfur compounds, unique qualifier ions are not available in the presence of impurities; however, selected ion monitoring can reduce the coelution problems in many applications.
Using the MS in tandem with the FPD mitigates the disadvantages of both detectors. FPD provides accurate sulfur amount due to the equimolarity characteristics while MS gives a total profile to include matrix not seen by the FPD. This paper describes the design of a parallel GC FPD-MS and demonstrates its applications.
Analysis of sulfur samples was performed on a Shimadzu GC-MS QP 2010 Plus system equipped with an FPD. Sample introduction was done by manual injection through a split/splitless injector at 200 °C. The injection volume was 1 mL. The GC analytical column was a 15 m × 0.25 mm × 0.25 µm Rtx-1 (Cat # 10120). A three-port SilFlow device (SGE Analytical Science) was employed to split the flow at the end of analytical column to both MS and FPD. Two deactivated, uncoated fused-silica transfer lines (restrictors) were employed to couple the splitter device with MS and FPD, one with a dimension of 84 cm × 100 µm I.D (connected to MS) and the other 75 cm × 250 µm I.D (connected to FPD). Figure 1 shows a schematic drawing of the GC with parallel FPD and MS. Details of instrumentation are listed in Table 1. Both FPD and MS chromatograms for the gas samples were acquired simultaneously.
Figure 1. A schematic diagram of GC-MS-FPD setup.
Table 1. Instrument Conditions
Sulfur standards were purchased from DCG Partnership 1, LTD. (Pearland, TX). They were prepared in two blends due to the stability issue. The components and concentrations are listed in Table 2.
Table 2. Sulfur Standards
Peak shapes are greatly impacted by the inertness of the column. Rtx-1 column offers great inertness as well as sufficient resolution for heavy matrix sulfur analysis, as seen in Figures 2-4.
By using the dual detectors system, a sulfur chromatogram and a simultaneous hydrocarbon chromatogram can be generated from a single injection. For the following examples, the top chromatogram displays the FPD signals and the bottom window displays the corresponding MS profiles. The retention time from both chromatograms matched well. This hardware configuration can be applied to other sulfur application areas.
Figure 2 shows a set of FPD and MS Total Ion Chromatograms (TIC) of sulfurs and hydrocarbons. In this case, a series of hydrocarbons coeluted with sulfur components. Recognizing specific sulfur compounds using FPD makes it possible to accurately pinpoint the retention time in complex TIC. Quenching, which is caused by the coelution of hydrocarbons, is illustrated in Figure 2. Sulfur signals can be significantly suppressed by the matrices, leading to inaccurate quantification. With the assistance of cryogenic devices or longer columns, sulfurs with limited number of hydrocarbons may be resolved. It is impossible to avoid quenching or signal suppression in gasoline samples containing hundreds of different hydrocarbon compounds with a wide range of concentrations. The use of the MSD in SIM mode can reduce this problem in many cases, while operating in scan mode assists in initial method development, unknown matrices identification, and finding the retention time of interferences.
Figure 2. GC-FPD-MS (full scan mode) detection of sulfur standards (Blend 1) in hydrocarbon matrices.
Figure 3 is a display of sulfur analysis with FPD and MS under SIM conditions. The SIM ions are listed below the chromatograms. The ions are carefully chosen to avoid interferences from hydrocarbons; however, the confirmation of peaks 1, 3, and 4 may be questionable because the qualifier ions were also shared by matrice ions. When a unique ion is not available, different chromatographic column / conditions should be tried to resolve the analytes. Fortunately, we have the ability to use Restek’s free ProEZGC library tool which will allow us to model the elution times of both sulfurs and hydrocarbon interferences under a specific set of conditions, in this case, using the Rtx-1. Conditions can be optimized for specific conditions and allow the use of SIM and the specific retention time as a reliable means of compound identification.
Figure 3. GC-FPD-MS (SIM) detection of sulfur components (Blend 1) in hydrocarbon matrices.
In Figure 4, both FPD and extracted ion chromatograms were collected for all 20 sulfur compounds (Blends 1 and 2). The SIM ions used for each sulfur compound are listed. The relative abundances are different in FPD and SIM responses, which results from the different detection mechanisms. The SIM chromatogram showed improved resolution on hydrogen sulfide and carbonyl sulfide.
Figure 4. GC-FPD-MS (SIM) detection of sulfur components (Blends 1 and 2).
The GC-FPD-MS coupling allows positive identification of sulfurs in complex matrixes and eliminates the need for multiple injections using different columns and detectors.
Dimethyl polysiloxane stationary phases (Rtx-1) provide good retention and resolution for sulfurs. Historically, thick film columns are used since they provide excellent inertness and peak shapes since analytes spend less time in contact with the deactivated fused silica surface. Columns with thinner films demand excellent surface inertness, for example, a thin-film short column (15 m × 0.25 mm ×1µm) was employed, resulting in a 10-minute analysis time. Conditions were optimized using ProEZGC resolving 16 out of 20 compounds (Figure 4) to include low-molecular-weight volatile sulfurs.
The FPD-MS combination is a powerful tool for unknown compounds, especially in the presence of complex matrices. Using tandem FPD/MS detectors provides an additional measure of confirmation not available from using either detector alone.
This paper demonstrates the capabilities of this hardware configuration for sulfur analysis. Further optimizations can be done on different column dimensions, oven temperatures, flow rates, and other parameters by using EZGC programs.
The authors would like to thank Shimadzu Corporation for their consultation with the operation of the QP2010 Plus GC-MS instrument and the FPD. http://www.ssi.shimadzu.com/
As our family of LC products has grown, so have our choices for columns and accompanying guard cartridges. Here is a handy chart to show which guard holder and guard cartridges were intended for use with each type of analytical column. Please feel free to click the links to access each group of products on our website.
*Guard cartridges not available for this column. Options for column pre-filters are listed.
Please note that the Trident LC Holders shown above are also available for use with filters only, as catalog numbers 27470/27471. However, this is less common, as most analyses benefit from including the guard cartridge for full column protection.
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Thank you reading and for using our LC products.