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Just like in my original post, Technical Service “Red Flags” – GC, this post contains several additional Red Flags we look (email) and listen (phone) for in technical service.
Trying to set the carrier gas flow rate for a packed column without using a flow meter. You may want to review this previous post.
When you have poor or no compound response for only the early eluting or later eluting compounds, but response for the other compounds are normal.
Purchasing a compressed gas cylinder/tank regulator without knowing which CGA fitting is needed. Need some help in choosing the correct gas regulator?
Trying to cut capillary fused-silica or metal (MXT) tubing with scissors or wire cutters (or some similar household cutting tool).
Using a splitless liner for split injections. While you may obtain acceptable results for sensitivity and reproducibility, obtaining a linear calibration curve may be difficult.
Trying to analyze Polywax® standards without using an on-column injection port, the proper simulated distillation GC column, and FID.
I hope you have found this second installment of “Red Flags” interesting and informative. Thanks for reading.
A dip tube (also known as an outage tube) provides vapor space above liquefied gases in a sampling cylinder so that if expansion occurs with an increase in temperature, the pressure is not significantly increased. Basically, the length of the dip tube is used to determine the filling capacity of the cylinder.
Outage is expressed as a percent of the total cylinder volume, based on the ratio of the vapor space to the total length of the cylinder with a maximum available outage of 50%. The dip tube is welded directly to the male inlet of the valve and is cut to a length ranging from one inch to 5 ¼ inches (available in ~1/8 inch increments).
• Dip Tube Length = Cylinder Length x Percent Outage (as a decimal)
Typically, the ideal dip tube length will provide a ~20% void within the cylinder based on the cylinder length and the physical (expansion) properties of the sample. To calculate for this length, determine the overall length of the cylinder and multiply by 0.20 to obtain the desired dip tube length in inches. Note that this is an approximate calculation (+/-20%) since several cylinder design variables will influence the actual volume.
Restek offers 10, 20, 30, 40, and 50% outage options. A 10% outage would translate to a 90% fill capacity, 20% would equate to 80% fill, 30% would be a 70% fill, etc.
The following chart provides suggested dip tube lengths for a ~20% outage (~80% fill) based on Restek supplied sample cylinder dimensions.
To calculate a ~10% outage, multiply the cylinder length by 0.10. For example, Restek catalog #22925 (1000cc High Pressure Cylinder) has a length of 10.9 inches. 10.9 inches x 0.10 = 1.09 inches (or about 1 1/8 inch). Multiply by 0.30, 0.40, or 0.50 respectively for 30, 40, or 50% outage values. Again, keep in mind that all these calculations and the associated dip tube lengths are approximate.
We offer two types of dip tube materials: stainless steel and Sulfinert-treated stainless steel. Stainless steel is recommended for general purpose applications, while Sulfinert is superior for the collection and analysis of low-level sulfurs in the ppb range. Make sure to match the dip tube (stainless steel or Sulfinert) to the sample cylinder material.
Placing an order for a Restek supplied dip tube is easy. Simply contact our US Customer Service Team at firstname.lastname@example.org or your local Restek representative. Specify dip tube length in inches or percent outage when ordering. Note that when ordering a treated dip tube, the end of the part will not be Sulfinert treated after we cut the tube to length.
To learn more about dip tubes, refer to ASTM D3700 (Standard Practice for Obtaining LPG Samples Using a Floating Piston) and ASTM 1265-05 (Standard Practice for Sampling Liquefied Petroleum (LP) Gases Manual Method) as well as the link below.
In summary, appropriate dip tube selection is a critical part of a successful liquefied petroleum gas sampling. Review your method specifications, select the dip tube length based on cylinder volume and sample composition, and determine the best material based on sample inertness requirements to ensure successful sampling.
I am happy to share that Restek’s own Jack Cochran has recently been awarded the North American Chemical Residue Workshop (NACRW) George and Wilma Fong Award. This award is given to individuals in recognition of their service to the workshop. This service award is named after the founders and organizers of the meeting, George and Wilma Fong, who were also the inaugural award recipients. This award was introduced in 2011 when George and Wilma officially retired from organizing the meeting…after 48 years! Jack first attended the workshop in 2000 and has been volunteering and helping to shape the meeting ever since…even happily participating in the annual “wave” photo…taken by George. Jack has also served as the President of the meeting in 2010 and was chosen to serve on the Board of Directors for NACRW starting in 2015.
I know Jack was excited to see George and Wilma attending the workshop this year as were many long time attendees.
To find out more about the history of NACRW, read the interview of George written by Jack in 2011.
Join the NACRW LinkedIn page too!
Photo credit George Fong.
For those of you not familiar with the term “red flag”, or phrase “It raised a red flag”, it basically means that something does not sound correct, or is very unusual or uncommon. In tech service, we listen for these “red flags” and if we hear one, we will either offer a cautionary statement if we consider something is dangerous or offer unsolicited professional advice if we believe it is needed. In many cases, it may simply be asking a customer to repeat a statement which is questionable.
So what are examples of “Red Flags” in technical service? A few of the more common ones are below.
GC capillary columns which are connected in series (using a simple union-type connector) rather than to a switching device. If one needs to connect two capillary columns in series (not including a guard column), I usually ask why this needs to be done. This is especially true if the columns contain dissimilar phases. If you are connecting multiple columns in series, you may want to review the following:
Requests for packed/micropacked columns which are longer than 5-meters. Although packed columns longer than 5-meters are used, columns less than 4-meters are much more common. Carrier gas head-pressures needed for long packed/micropacked columns tend to be very high, and unless a valve/sample loop (or other switching device) is used, loss of sample through the GC injection port septa via blow-back is common if using a (Gas-Tight) syringe injection. To read more about packed column basics, see Packed Column information for the beginner
If interested in ordering a packed/micropacked column, or to obtain a quote, you may want to review:
Using a Uniliner in split-only mode. I even commented on it several years ago in this post:
Liners Every Lab Should Own (in my opinion) Just remember, these liners are designed for splitless injections only (if you experience carryover, try turning on the split flow at 5mL/min after the last compound elutes).
Injecting 1µL (or more) of an aqueous (water) sample in splitless mode. Sample back-flash can be a real concern. Capillary GC Column Killers – Part 4
Splitting a sample after the GC column and sending part of the sample to an atmospheric detector (like a FID) and the other part to a mass spectrometer (mass spec). Remember that the mass spec is under vacuum, so it will want to pull more of the sample/carrier gas into it. Because different mass specs have different pumping capacities, there is no kit or product that we sell which will guarantee equal flow to each detector.
Using air as a carrier gas. Please remember that with almost all GC columns, oxygen will cause irreparable damage to the column’s phase (through oxidation). While it is true that at room temperature very little damage/oxidation will occur, at higher temperatures damage will occur almost immediately.
So the next time one of us in technical service asks you to repeat yourself, it may be because we thought we heard a “Red Flag“. Thanks for understanding.
I recently spoke to a customer who said she was having a difficult time finding the catalog number of the column she was trying to order. I asked for the catalog number, and she provided 70112-6850. I instantly knew what the problem was, she paired a MXT column catalog number (70112) with a fused-silica column suffix number (-6850). I began to wonder how many of our other customers experience the same confusion. As a result, I decided to write this post to help clarify this topic.
As you may know, especially if you have a GC without a “full size” oven, not all GC capillary columns will fit into your GC oven. For example, several of the most common requests we receive are for columns which fit into the oven of most SRI GC’s (and Buck Scientific) and an Agilent 6850 GC oven. In addition, with the increase in number of compact mobile GC’s, smaller OD (outside diameter) columns are more popular than ever.
Fused-Silica capillary columns
So what do you need to know when purchasing a new capillary column for your instrument? Let’s start with fused-silica columns. The majority of our fused-silica columns listed in the catalog and on the website (five-digit catalog numbers which begin with a “1” or “4”) are wound onto cages (which we call the 11-Pin cage) which are approximately 7.25-inches outside diameter (and approximately 1.5-inches wide).
If you need a smaller cage, we also offer a 5-inch OD cage (and approximately 3-inches wide). To obtain a column on this smaller OD cage, simply take the fused-silica column five-digit catalog number and add a -6850 suffix to this number.
Cage (with string)
For example, the catalog number of a Rxi-1ms, 30m x 0,32mmID x 4µmdf column wound onto a 7.25-inch OD cage is 13396. To have this column wound onto a 5-inch OD cage, add the suffix -6850, so the new catalog number becomes 13396-6850. Please note that many of the -6850 columns are not listed on the website. If you do not see the catalog number you need on the website, contact customer service.
MXT (Metal) capillary columns
Our metal MXT columns are the most popular choice for capillary columns which need coiled (wound) to outside diameters (OD) less than 5-inches. Unlike fused-silica tubing which can become stressed the smaller you wind it, metal tubing does not have this problem (within limits). However, once the metal tubing has been wound (coiled), do not try and coil it to a different diameter because kinks in the tubing may result.
There are three popular sizes (OD) with MXT columns. The product web pages for MXT columns normally list the OD for that particular part number, so be sure to take a look before ordering.
The first is similar to fused-silica capillary columns; 7.25-inch OD. These columns include our MXT-PLOT (Porous-Layer-Open-Tubular), our MXT-Biodiesel TG, and our MXT-1HT SimDist columns (Simulated Distillation).
The second popular size (OD) is what we refer to our standard size column which is not actually wound onto a cage, but the coiled column layers are what is referred to in our literature as “Banded” (I call it bracketed). Depending upon the column’s length, these columns may range from an approximate OD of 4.5-inches to 5.5-inches.
Standard MXT Cage
One of the most popular sizes of MXT columns is our 3.5-inch OD, which is the choice for most SRI (and Buck Scientific) and mobile GC’s. Like our “standard” MXT columns, these columns are not wound onto a cage, but unlike the standard columns, these columns simply have a few metal bands which keeps the coils together and are not in layers. This is referred to in our literature as “Bundled” (see photo below). To obtain a column with this smaller OD, simply take the MXT column five-digit catalog number (a five-digit number which begins with a “7”) and add a -273 suffix to this number.
For example, 70112 is a 5-meter x 0.53mmID x 0.1µmdf MXT-1HT SimDist columns. To obtain this column with a 3.5-inch outside diameter (no column cage), the catalog number you will want to order is 70112-273.
If you are wondering about packed columns while reading this, none of these suffixes will work; packed columns have their own list of suffixes. Please review Things to Consider Before Ordering a Packed Column Thank you.
I think by now we’ve all heard that GC potency testing for cannabis or hemp has some drawbacks. That being said, GC is a popular, rugged, and cost-effective laboratory workhorse and is still employed in many cannabis laboratories. The major drawback of GC versus HPLC cannabinoid testing is the fact that the acidic cannabinoids convert to their neutral form in the GC inlet and cannot be separately quantified. This means that the amount of acidic cannabinoids in a sample cannot be reported by GC, and this result is becoming more and more important with the growing popularity of non-smoked cannabis products.
Cannabinoid Acids Convert to Their Neutral Forms in a Hot GC Inlet
While HPLC would be the more straightforward choice for cannabis or hemp potency analysis, some labs only have GC instruments. Joan Serdar at Tetra Analytics is one such analyst. Her lab is equipped with GC instruments, but she wants to accurately quantify both the neutral and acidic forms of cannabinoids in hemp and cannabis – good for you, Joan! In working with Joan, we were able to take a page from the toxicologists’ handbook and derivatize our cannabinoids. What is derivatization, you ask? In a nutshell, derivatization prior to GC analysis involves ‘sticking’ functional groups onto a molecule in order to make it more volatile, more stable, or to improve detectability.
In the case of our cannabinoids, we want to make them more stable so the acidic forms don’t convert to their neutral forms in our GC inlet (see figure above). The derivatization technique we decided to try for this analysis involves N,O-Bis(trimethylsilyl)trifluoracetamide + 1% trimethylchlorosilane (BSTFA + 1% TMCS). This derivatization reagent targets –OH groups and replaces the hydrogen in that group with a trimethylsilyl group, creating an ether that is easier to gas chromatograph than an acid or alcohol.
Hydroxyl (-OH) Groups on Cannabinoids are Derivatized Using BSTFA + 1% TMCS, Making Them Stable in the GC Injection Port
So did it work? The preliminary work performed by Tetra Analytics and here at Restek indicates that this derivatization technique works for all of the cannabinoids of interest for most scientists, resulting in stable, chromatographically-resolved derivatization products. The chromatogram below is a derivatized high-level standard analyzed by my colleague Jack Cochran via GC-FID using the same 15m x 0.25mm x 0.25µm Rxi-35Sil MS column we recommend for underivatized cannabis potency work. The Rxi-35Sil MS had the selectivity needed to separate all of the derivatized cannabinoids. Jack also ran the sample by GC-TOFMS to verify peak IDs and ensure we were getting the derivatization products we hoped to get.
GC-FID Chromatogram of Derivatized Cannabinoid Acids and Neutrals – All Compounds are Resolved on Rxi-35Sil MS
So these results look pretty promising! While HPLC is a more straightforward way to measure cannabinoids, the derivatization used here was very simple and shows really good preliminary results. Stay tuned for more discussion of this method – does it work in the presence of matrix? Find out in our next installment (spoiler alert: it does).
With the last installment of this blog series garnering some comments/questions, I thought it would be appropriate to post the following link to our application note, which has been featured in a lot of these blogs: http://www.restek.com/Technical-Resources/Technical-Library/Foods-Flavors-Fragrances/fff_FFAN2127-UNV
As Applications Chemists in the LC lab, one of the most exciting parts of our jobs is the variety of analyses we are exposed to. One day you are developing a method for potency analysis in cannabis samples, the next you are looking at anti-epileptic drugs in urine. We’re regularly challenged to think outside the box to provide the best solutions for our customers.
In some recent work, my colleague, Shun-Hsin Liang, was tasked to develop a method for nicotine and related compounds in urine using LC-MS/MS. The challenge was finding suitable analytical conditions for a rapid, accurate, and reproducible method geared toward high-throughput testing laboratories. Typically, this analysis uses high-pH chromatography with relatively high concentrations of additives to increase retention, improve peak shape, and reduce peak tailing. These conditions may lead to some additional non-routine maintenance (like changing pump seals and replacing tubing), and if you’re in a high-throughput laboratory, you know non-routine maintenance events always pop up at the most inopportune time.
Shun-Hsin was able to use the Raptor™ Biphenyl column and standard, low pH, reversed-phase LC-MS mobile phases that are compatible with a variety of LC-MS instrumentation. The method provided excellent performance for the simultaneous analysis of nicotine, two major metabolites (cotinine and trans-3’-hydroxycotinine), two minor metabolites (nornicotine and norcotinine), and a minor tobacco alkaloid, anabasine, in human urine. Accurate and reproducible analysis was achieved in less than 5 minutes of chromatographic analysis time, making the column and method well suited to low-cost, high-throughput analysis of nicotine-related compounds.
Please click on the link for more complete details of Shun-Hsin’s most recent work on the Analysis of Nicotine & Related Compounds in Urine Using Raptor Biphenyl.
I get quite a few customer questions concerning peak tailing during LC analysis, and how to best troubleshoot this issue. Peak tailing may be attributed to a variety of different causes including secondary interactions, contamination, column loading, etc. This list goes on and on. I usually ask a few key questions, and generally can give some good advice based on the responses.
- Is this a new problem?
- What are your analytical conditions?
- Mobile phases?
- Which analytes are troublesome?
During some applications work I was doing in the lab, I came across a very familiar issue: All of my peaks are tailing…
- Is this a new problem? Yes, I just started method development.
- What are your analytical conditions? Flow rate – 0.5 mL/min, Temp – 30 °C
- Mobile phases? MPA: 0.1% formic acid in H2O, MPB: 0.1% formic acid in MeOH
- Column? Raptor Biphenyl, 2.7 µm 50 mm x 3.0 mm I.D.
- Gradient? 10-75%B in 5 minutes, re-equilibrate to 10%B for 2 minutes
- Which analytes are troublesome? All of them. A mixture of 6 beta-blockers.
Nothing struck me as particularly odd with the analytical conditions, all analytes were well retained and eluting during the gradient, and there was sufficient re-equilibration time. As I looked at the analyte structures, I noticed that these were all very active compounds.
Based on this information, I believed the peak tailing was attributed to secondary interactions caused by silanol activity. The cartoon below depicts how the secondary amine in the pindolol molecule could be potentially interacting with residual silanols on the surface of the silica.
With the addition of a buffer to my mobile phase, I should be able to mitigate this issue. Since I’m already using formic acid, choosing a complimentary salt, Ammonium formate, will give the best buffering capacity at the desired pH.
By changing the composition of my mobile phase, I was able to reduce the secondary interactions, resulting in much more symmetrical peaks, and even got the bonus of some increased resolution between labetalol and oxeprenolol. With a flow rate of 0.5 mL/min, the total volume of mobile phase used per injection is 1.4 mL of MPA and 1.1 mL of MPB – for all intents and purposes these are relatively equal. The addition of buffer to both aqueous and organic mobile phases ensures that the secondary interactions will be mitigated throughout the entire gradient – solving the peak tailing issues for both early and late eluting compounds.
See http://www.restek.com/chromatogram/view/LC_GN0550 for the final optimized method using 0.1% Formic acid and 5mM Ammonium formate modified mobile phases.
Because the widespread use of preventative veterinary antibiotics has resulted in increased antimicrobial resistance in humans, the US Food and Drug Administration (FDA) released guidelines to address the issue in food animals (December 11, 2013). Similar constraints are also prevalent in the European Union. Recently, European Researchers1 developed a validated method for the determination of veterinary antibiotics in bovine urine by LC-MS/MS. The column chosen was a Raptor Biphenyl column (150 x 2.1 mm, 2.7 um), because of superior resolution and sensitivity relative to other columns tested. While Restek scientists claim no affiliation with these researchers, their universities, their methods or results, we respect their efforts and are proud to provide chromatographic products that make their analyses possible. The paper can be found on the Elsevier’s Science Direct website for purchase. View the abstract here: http://www.sciencedirect.com/science/article/pii/S0308814615004707
1 Department of Veterinary Science and Public Health and Department of Health, Animal Science, and Food Safety, University of Milan, Italy, the Department of Health Sciences, V. le Europa, Campus S. Venuta, Germaneto, and the Department of Chemistry at the University of Nis, Bulevar in Serbia