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- Is the problem with standards or only with samples? If the low response is only with extracted samples, the problem is elsewhere in your method. Please see the blog post Now you see it, now you don’t-Troubleshooting method recovery problems.
- When was the standard ampule opened? If not fresh, open a new one. As mentioned in the blog post Extending the expiration date on a reference standard, the expiration date no longer applies once the container is opened.
- What were the storage conditions for the standard? As discussed in the blog post Handling your analytical reference standards, temperature and light exposure can make a world of difference. Be sure to check your certificate of analysis for proper storage conditions and precautions.
- If the standard is a mix of analytes, are all of them low or high? If you made a dilution, check your math and measurement techniques for dilutions that were made. Repeat them and verify. Make sure the initial volume used for dilution from the vial is made correctly, as discussed in the blog post Chemical reference standards; don’t just snap and pour.
- If you have any ampules from other lot numbers or vendors, prepare parallel dilutions and compare. Particularly if you have previously done this analysis and are not getting what you expect, obtain another standard and prepare it exactly in the same way and on the same day, alongside the one you are having trouble with. A stability issue can only be confirmed if you have data that shows that you are able to get acceptable results with another lot number or standard from another vendor.
- If you usually analyze this standard in combination with other standards, prepare your test dilutions without adding any other standards to the mix. Because the analytes can interact and affect each others’ stability, this is critical to isolate the issue. If you get to this point and have confirmed an issue with one product and not the other, contact the vendor. If this is a Restek product, contact us in Tech Services for assistance in resolving the issue.
- If your test dilutions demonstrate a problem with all of the lots or sources tested, then there is probably something unique to your analytical system that is causing a low response. If you would like assistance with troubleshooting on your HPLC or GC, feel free to contact our Tech Services group for help at Support@restek.com. We will need to see your operating conditions and settings, as well as chromatograms, so please be prepared to send a copy of those by email.
I hope this has been helpful. Thank you for reading.
I have the privilege of working with a number of talented people here at Restek and when I approached Sharon Lupo about a project last year known affectionately as “The Big Pain” I wasn’t sure how successful she could be.
Think about it: Opioids, Barbiturates, Anti-Depressants, Anti-Epileptics, Hallucinogens, etc. all have vastly different chemical make-ups. How else would they be able to treat the variety of ailments they do, or be able to be abused the way they are? The challenge is not only in the chromatography, but in speed of analysis and detection as well.
With lots of resourcefulness, help from her co-workers, and me mostly staying out of the way, a very comprehensive screening method was developed along with class specific analyses as well. 231 compounds, across ten classes, with detection in ESI(+) and ESI(-), were screened in 10 minutes. Optimized conditions for all the classes have also been completed. So whether you are looking for morphine or PCP, MDMA or THC, we have a method for you.
Sharon isn’t able to attend MSACL 2015 this year, but be sure to check out our presentation of her work at our Monday morning workshop, March 30th, which covers all aspects of this challenging project.
If you have been following this blog series, then you already know that we found numerous (upwards of 64) compounds in electronic cigarette solutions. You also know that we found even more (approximately 82) compounds in e-cigarette vapor. More importantly, you know not to waste your time testing the e-juice, because the vaporization process found in e-cigs results in a vapor with a different chemical profile than the liquid; and results in the production of toxic/carcinogenic VOCs like formaldehyde. What you do not know is how we have been testing our e-cigarette vapor. So without further ado I introduce to you our simple sampling device for collecting electronic cigarette vapor:
So what do we have here? We have an electronic cigarette connected to a thermal desorption (TD) tube and the other end of the TD tube connected to a 50 mL gas tight syringe. To collect a vapor sample from the e-cigarette we simply pull on the gas tight syringe, thereby drawing the e-cig vapor across the TD tube for collection. Subsequent to sampling we place the TD tube in our Markes UNITY for thermal extraction. So there it is in short form.
Now for those of you who like details continue reading on. The TD tube we chose for our work consists of the following 3 sorbents: Tenax TA, Carbograph 1TD, and Carboxen 1003. We chose this tube for its ability to look at C2 to C32 compounds, thus giving us our best chance of seeing as many VOCs and SVOCs. We chose the 50 mL gas tight syringe, because we already knew from the peer reviewed literature that a standard e-cig puff was anywhere from 35 to 70 mL (we chose 40 mL for the record). Lastly, it is important to note that the peer reviewed literature suggested that e-cig vaping topography was usually a puff over a ~4 second period. So obviously we drew our puffs over ~4 seconds. One last important point… the LED lit on the e-cigs when we drew on the syringe, indicating that vaporization was taking place. This is a very important point, because… well… you already know now.
Next time we can cover more on our vapor sampling methodology, compounds of interest, concentrations, and implications.
Need help finding a ferrule to install your column? Part 3: Perkin Elmer GCs
You’re ready to install a column into your GC and realize you do not have ferrules to do the job. Which ferrule do you choose? The parameters to consider when choosing the correct ferrule are instrument make and model, nut size and type, ferrule material, and column ID. A visit to our ferrules home page will provide a general overview of various ferrules offered by Restek. By navigating the page, you can narrow the selection by choosing these parameters. This post will focus on Perkin Elmer GCs.
Perkin Elmer inlets use 1/16 inch fittings to install capillary columns. Some inlets require reducing adaptors as the body of these inlets and large fitting mounted in the GC oven is 1/4 inch. Restek and Perkin Elmer sell 1/4 to 1/16 inch reducing adaptors.
Perkin Elmer FID, ECD, NPD and FPD detectors come with 1/8 inch fittings. To install capillary columns in these detectors you can opt for a 1/8 inch nut and reducing ferrule. Or you can purchase a detector reducing adaptor just like the inlet. Restek and Perkin Elmer sell 1/4 to 1/16 inch reducing adaptors.
Detector Adaptors for Perkin Elmer FID, ECD, NPD and FPD: Ours are supplied with a 1/8 inch nut required to attach.
To install a capillary column into the TCD, make-up gas may be required for low capillary flow rates. Restek does not sell the fittings or adaptors for the TCD.
Our Vespel®/graphite ferrules will work with Perkin Elmer style fittings and nuts. Our 100% graphite ferrules will not.
- Restek does not sell Perkin Elmer style nuts.
- Restek sells a compression style reducing adaptors that will accept 1/16 inch nut. These will accept graphite and Vespel®/graphite capillary ferrules.
- Once you’ve determined which fitting is on your GC you can go to the table in part 1 and match the ferrule ID to capillary column ID and choose the appropriate ferrule.
Perkin Elmer packed column inlets are not on-column. They are equipped with liners (glass or quartz); Restek does not sell this type of packed column inlet liner. The fitting size on both the packed column injector and detectors are 1/8 inch diameter. To connect a 1/8 inch packed column all that is required is a 1/8 inch nut and ferrule.
If you have a 3/16 or 1/4 inch packed column, you will need to purchase from Perkin Elmer the 1/4 inch adaptors for the injector and detector. Then use a 1/4 inch nut and either a 1/4- 3/16 inch reducing ferrule for a 3/16 OD column or a 1/4 ferrule for 1/4 inch OD column
My next post, Part 4 of the series, will discuss Thermo Scientific GC’s ferrules, nuts, and helpful hints. Thank you for reading.
Despite what is sometimes advertised, setting up optimized methods for multiresidue pesticide analysis is typically not “easy peasy” and can be quite time consuming. Luckily, some colleagues are willing to share their method with us.
Thank you Kelli, James, and Jon!*
The downloadable spreadsheet contains retention times, transitions, ionization and collision parameters for our LC Multiresidue Pesticide Kit. The work used a Waters Acquity LC and an AB SCIEX QTRAP® 5500 LC/MS/MS system. Analysis conditions are listed on the last tab. The column used was an Ultra Aqueous C18 (cat# 9178312).
Although conditions may vary on different instruments, this information will dramatically shortcut your own method development. A similar spreadsheet using an LC-QQQ can be found here (see 5th bullet point).
*Kelli Sikorski, PhD, James Wittenberg, PhD, Jon Wong, PhD
You’re ready to install a column into your GC and realize you do not have ferrules to do the job. Which ferrule do you choose? The parameters to consider when choosing the correct ferrule are instrument make and model, nut size and type, ferrule material, and column ID. A visit to our ferrules home page will provide a general overview of various ferrules offered by Restek. By navigating the page, you can narrow the selection by choosing these parameters. This post will cover Bruker/Varian systems.
Although Bruker/Varian GCs can come equipped with many injector options, selection of nuts and ferrules is simpler than my previous post on Agilent ferrules. These injectors all use Bruker/Varian style Capillary nuts and standard 1/16 inch compression ferrules to install capillary columns. The detectors on Bruker/Varian GCs that are designed for capillary columns use the same nuts and ferrules as the injectors.
For chart on ferrule size in relation to column ID see part 1
For table on ferrule material description see part 1
A word on ferrule material choice: I found using Bruker/Varian nuts with 100% graphite ferrules, upon tightening the nut the graphite material will start to extrude out the nut hole.
Bruker/Varian GCs can come equipped with two different packed column ports.
The 1040/1041- On-column Isothermal injector for 0.53mmID capillaries and packed columns. You will need to verify your current installation hardware before you begin. The 1041 comes equipped with 0.53mmID installation hardware. Adaptor kits for 1/8 inch packed columns and 1/4 inch packed columns are sold separately. These kits include adaptors for both Injector and detector sides. Restek does not sell these adaptor kits. Packed columns for this injector should have a void in the inlet side to allow the needle to inject directly into the column.
The 1060/1061 Flash Vaporization Isothermal for 0.53mmID capillaries and packed columns. This inlet has an adaptor with a deactivated glass insert. Restek does not sell this adaptor or inserts. You will need to verify your current installation hardware before you begin. The 1061 comes standard with 0.53mmID installation hardware. Adaptor kits for 1/8 inch packed columns and 1/4 inch packed columns are sold separately. These kits include adaptors for both Injector and detector sides. Restek does not sell these adaptor kits. Packed columns for these inlets can be packed full with no inlet void needed.
- The nuts and ferrules required to install the packed columns would be standard compression ferrules with compression style nuts.
- To install a packed column into a capillary injection port, you would use a “pigtail” setup.
Please refer to part 1 for suggestions on on-column centering adaptor kits.
In summary, the key points to remember are:
- Use Bruker/Varian nut and standard compression ferrules to install capillary columns.
- Know the ID of your column to match with the correct ferrule size.
- For packed columns: know which packed column adaptor you have in your GC.
- Match the correct packed column OD with the ID of the ferrule.
My next post Part 3 of the series, I will discuss Perkin Elmer ferrules, nuts, and helpful hints. Thank you for reading.
Often we get asked for a column recommendation for a complex GC analysis. This is especially true for gas analysis. Probably the most common request is for separation of fixed (permanent) gases (like O2, N2, CO, CO2, N2O, etc…) plus separation of larger (higher-boiling point) compounds (light hydrocarbons, moisture, sulfur compounds, etc.). Unfortunately, there is seldom a single column solution for this type of analysis. So what is the solution? A two (or more) column set-up with valve switching may be needed to separate all the compounds.
Just to be clear, the two-column set-up/solution mentioned above is not dual-column analysis, where two different columns are used in parallel (most of the time these columns are referred to as the primary analytical column and the secondary, or confirmation column). Nor is it two different columns connected in series using a union-type fitting.
Instead, it is (generally speaking) two different columns where the outlet of the first column is connected to some type of valve or other switching device, and the inlet of the second column is also connected to this same switching device. For example, the first column (let’s call it Column #1, or a pre-column, or a stripper column) has the sample injected into it. The outlet of Column #1 is connected to a switching device. A second column (let’s call it Column #2) is connected to a different port of this same device. When the last component/compound of interest elutes from Column #1 onto Column #2, the carrier gas flow is redirected by this switching device so that no other compounds/components are sent onto Column #2, but directed elsewhere (a detector, another column, back-flushed to vent, etc.).
An example of (rotary) valve switching can be seen on the instruction sheet for our D3606 column set (which separates benzene from ethanol in spark ignition fuels). D3606 Application Column Instruction Sheet (PDF) Column #1 holds up the fuel matrix allowing only the compounds of interest to pass to Column #2.
Additional valve applications can be found here. Valve Applications – Valco Two Position Injectors and Valves
Note that these aren’t the only types of valves used; there are also other options available for both packed and capillary columns. Several common terms you may hear include Multiport Valves, Capillary Flow Technology, Deans Switching System, Multidimensional Switching Systems, Multi Column Switching, and Fluidic Switching Devices (among others). To read more about these terms, I suggest reviewing the links below, or typing them into Google.
In summary, when there is no single GC column solution available, ask yourself “Are there two (or more) columns which can perform the necessary separations?” If so, this application may be a candidate for multiple-columns and a switching system. I suggest contacting your instrument manufacturer to see what hardware and/or software upgrades will be needed and contacting us for your GC column needs. Thank you.
Occasionally, we will get a Technical Service call about peak retention times moving during a customer analysis. Early eluting compounds are especially prone to this shifting, but the issue can often be attributed to matrix interferences that are also not well retained. Pregabalin and Gabapentin are two antiepileptic drugs typically analyzed by LC-MS/MS. Because of their small, polar nature, they may be difficult to retain using reversed phase chromatography.
In our example of an antiepileptic drug panel, Pregabalin and Gabapentin are well retained on the Raptor™ Biphenyl column.
See http://www.restek.com/chromatogram/view/LC_CF0616 for full method details.
These two analytes can be accurately quantitated and results are reproducible when looking across different column lots:
However, when analyzing urine samples using a simple dilute & shoot method, quantitation becomes a bit more difficult. Matrix interferences may give the illusion of peaks moving within the chromatogram. In the example below, a sample chromatogram with 100 ng/mL of antiepileptic drugs in human urine was diluted 5x with 0.1% formic acid in water. The retention time of the analytes is remaining the same, but there are clear interferences from the matrix components.
If we take closer look, and extract the MRM transitions for pregabalin, we can clearly see matrix interferences that could make quantitation difficult.
The next time you see your analyte “shifting” retention time, I would recommend running a solvent standard to rule out the possibility of matrix interferences. For more information on this and other challenging analyses, be sure to come to our Monday morning workshop at MSACL 2015 US in San Diego, CA (MSACL Workshop).
In just about all of my pesticide residue work, I use a convenient multicomponent internal standard mix that contains several PCBs as well as popular internal standard compound triphenylphosphate. The mix contains the six compounds in the chromatogram below. I have been working on a method for our GC Multiresidue Pesticides Kit which has about 200 compounds. We will be publishing the method…retention times and SRMs soon!
I am just about to put the kit into action with some citrus and celery samples. Luckily, I realized I had forgotten to add my internal standards to my method. I have provided a downloadable spreadsheet with run conditions and optimized SRM transitions for the six internal standard compounds. I hope this helps shortcut your own method development.
In the last electronic cigarette blog we revealed that e-cig vapor has many more constituents than the propylene glycol, glycerin, nicotine, and flavorings on the manufacturer’s solution list. In fact, we found 82 compounds [i.e., unidentified and identified (some only tentatively)] in the vapor of one e-cigarette vendor. Now if you recall from e-cig blog cuatro, we found 45 compounds in the e-juice. Well since that blog we went over those e-cigarette liquid results with a fine tooth comb and actually found 64 compounds. So… that means our vapor analysis (of the exact same juice) exposed an additional 18 compounds.
Of particular interest was the presence of formaldehyde, acetaldehyde, acrolein, xylenes, and several siloxanes. The observation of these compounds in the vapor is significant for the two following reasons: 1) all three of the aforementioned carbonyls are acutely toxic; in addition, formaldehyde is a known human carcinogen and acetaldehyde is a probable human carcinogen. 2) None of these compounds were present in the e-juice, which indicates they were generated during the vaporization process and/or from the e-cigarette materials. This is exactly what we were hypothesizing about in part I of this blog series. If you recall, this is also why we chose to rock out the thick film volatiles column. Our observations are consistent with the fact that pyrolysis of both propylene glycol and glycerin (something taking place in e-cigarettes) results in the formation of formaldehyde, acetaldehyde, and acrolein. Our observations are also consistent with the fact that polysiloxanes are often used as plastic additives and the majority of the 1st generation e-cigarettes, like those evaluated in this study, are made with plastic bodies. All of the abovementioned have profound implications for how e-cigarettes should be evaluated, especially when considering the fact that the e-cigarette vapor is ultimately what end users are exposed to. So have we made our point by now? I hope so, but just in case… test the vapor not the juice. Stay tuned for the next blogs to see just how we tested the e-cig vapor, what concentrations were observed, and all things e-cig related.