SPME Arrow Robustness = Confidence

When you see an analytical chemist confidently swagger through the laboratory, you cannot help but ask yourself “is he/she using SPME Arrows?” You say this now, because you know that the SPME Arrow bolsters copious amounts of confidence, which all stems from the increased mechanical robustness and phase volumes, over traditional SPME fibers that is. But let us get a little more detailed about the SPME Arrow size. The following Figure and accompanying Table provides you with everything you need to know:

I will not bore you with discussing the nitty-gritty details, so let us talk about what this all really means. For me it means two things. The first is that I can have a lapse in judgement and decide to close my SPME Arrow as if it were a retractable (clicker) ball-point pen by pushing the Arrow tip down on the laboratory bench. It looks like so after:

But that is okay, because I always carry my Victorinox SwissTool for moments like this. So, I simply bent the Arrow back like this:

Ultimately my Arrow looked like this when I was done:

You will notice I circled the one part of the Arrow. This is the back side of the bend, and during the bend and repair the carbon was clearly discolored in this section. But I still took this very Arrow and racked it up and ran it just like I had done before the bend. And I still had close to 100% collection efficiency. Try that with your Traditional SPME… I dare you!!! When you have this kind of SPME mechanical robustness, you cannot help but strut across the lab. Stay tuned for next time where I talk more about SPME Arrow dimensions and how this corresponds to an increase in sensitivity and even more swagger.

Extractable Petroleum Hydrocarbons (EPH) Method – Why it is important?

Spills and releases of petroleum fuels are the largest source of environmental contamination in the United States. Massachusetts published a method that addresses the need to account for semi-volatile aliphatic and aromatic fractions of gasoline using gas chromatography (GC) analysis and a flame ionization detector (FID). This method was designed to measure the collective concentrations of extractable aliphatic and aromatic petroleum hydrocarbons in water and soil/sediment matrices. Extractable aliphatic hydrocarbons are collectively quantitated within two ranges, C9 – C18 and C19 – C36. Extractable aromatic hydrocarbons are collectively quantitated within the C11 – C22 range.

Aromatic and aliphatic compounds are separated from each other by processing the sample through silica gel cartridges. The extracts are then analyzed using two separate calibrations. The reason for this stems from data suggesting that aromatic compounds are more toxic than aliphatic compounds. This approach, known as Extractable Petroleum Hydrocarbons (EPH) Method, characterizes sites according to their toxicity. Because this method is unique in its ability to determine human health hazards, and yet can still be used to calculate diesel range organics; it has been adopted by other states in the US and in Canadian provinces.

Resprep EPH Fractionation SPE Cartridges (cat # 25859) provide method-specific performance for EPH analysis of soil and water samples through complete separation of aliphatic and aromatic compounds into distinct fractions, while providing extractable background levels guaranteed to fall under the strict reporting limits of Massachusetts and New Jersey EPH methods. Restek’s newly optimized silica gel cartridges have superior lot-to-lot reproducibility and storage stability ensured by rigorous QC testing and moisture-resistant packaging. Resprep EPH Fractionation SPE Cartridges from Restek are manufactured in a strictly controlled environment with the simple goal of giving you consistent results – every day, every time. Resprep cartridges provide easy, reproducible, and guaranteed EPH analysis.

The fractionation process is very critical to accurately determine the amount of aliphatic versus aromatic compounds. As shown in Figure 1, the fractionation of aliphatic compounds includes C9 – C36 and the surrogate 1-Chlorooctadecane (COD). COD is a good marker for the end of the aliphatic range. The beginning of the aromatic range starts a couple milliliters later with the first compound, 2-Methylnaphthalene, starting to elute.

The importance of a reproducible and background free silica gel cartridge will be shown in the next segments of this EPH blog series.

Does that SPME Arrow come with a free lunch?

Here at Restek our company has a core value referred to as “In the Light.” For me, I like to think of it as no skeletons in the closet. So, time for me to shine a little light. I already introduced you to the SPME Arrow. I also addressed the SPME Arrow’s larger diameter and if there are any associated issues of septa coring. Today I reveal how much lunch costs.

As Hailey pointed out last time, the SPME Arrow has a larger sheath. With only one exception, all of the SPME Arrows have an outside diameter of 1.1 mm. We also have a 1.5 mm Arrow which sports 250 µm of PDMS. To put things in perspective, a traditional SPME fiber has an outside diameter of ~0.63 mm. So, in order to take advantage of the SPME Arrow’s mechanical ruggedness and increased phase volumes, you will have to install a GC Inlet Conversion Kit. Obviously, we have these available for you. In addition, your traditional SPME fiber holder will not work with the SPME Arrow. So you will have to use an appropriate SPME holder. And finally, if you are using a “rail” system (e.g., Gerstel MPS) on your GC, you will have to contact your rail manufacturer to secure an appropriate set-up for the SPME Arrow.

So there you have it… that is the catch! Relatively painless in my opinion. Especially when I know you have a drawer full of these:

It is important to keep in mind that these conversion kits do not render your inlet SPME Arrow only. You can continue to use the same inlet as usual for everything else. The problem is that the original inlet on your GC can not accommodate the increased SPME Arrow diameter.

Excellent SPME question Hailey!!!

Last time, I told you I hunt for organic compounds with SPME Arrows, as opposed to the traditional SPME fibers, because I greatly appreciate the mechanical robustness and increased phase volumes afforded by the SPME Arrow. That blog has since received the following excellent comment/question from Hailey: The “sheath” part of the Arrow looks to be much thicker than the traditional setup; is septum coring an issue when injecting manually into a GC?

I thought this warranted the current blog. So first off, let me start by saying Hailey is spot on with her observation of the increased sheath. I plan to talk more about dimensions in a future blog, but for now you should know that everything but the hub (i.e., the screw end) on a SPME Arrow is bigger than a traditional SPME fiber. As my one colleague would say, the SPME Arrow is “heavy duty!” But if we take a look at the following picture, you will notice the SPME Arrow (left side) has an arrow-like tip, from which it garners its name.

This arrow tip accomplishes the following 2 things:

  1. Facilitates the smooth (i.e., core-free) penetration of vial and gc septa.
  2. Acts as a cap when the Arrow is retracted, thereby protecting the phase from any contamination.

You will also notice from the above picture that the traditional SPME fiber (right side) has an open-ended syringe-like tip, which is just begging to grab a hold of some septum and do some coring. So, truth is Hailey, we have more coring issues with traditional SPME fibers than the SPME Arrow.

BUT… like my father always said: a picture is worth a thousand words. So, let us take a look at the following septum, which has received over 150 injections from a SPME Arrow.

Here is the top side:

Here is the bottom side:

Do you see any coring? I do not! But let us take a closer look. The following picture is the same septum, but with me applying pressure:

When I apply pressure to the septum what you see is what I describe as a slit. The SPME Arrow’s tip appears to cut (not core) the septum and make its own “duckbill-like” lips, something you might be familiar with on a Merlin Microseal.

So Hailey… I greatly appreciate your question, as you touched on an excellent topic that I am sure other potential end users will be asking as well. Oh… the short answer to your question is “NO.” And yes, my fiancée says I tend to get a little long-winded.

 

 

I hunt with arrows… SPME that is!

When you say “I hunt with arrows” in central PA, you better be prepared for a long conversation about whitetail deer. So, I better clarify that I am talking about hunting for organic compounds with a SPME Arrow. What is all this I am talking about?

Time for a brief history lesson: Janusz Pawliszyn (University of Waterloo) developed solid phase microextraction (SPME) and published the first article on the technology back in 1990. After filing a patent on SPME fibers, the technology was later licensed to Supelco. The 17-year patent meant the following two things: 1. No one else could offer traditional SPME fibers and 2. No one could make any improvements to the technology. However, now that the patent has expired, Restek is able to offer traditional SPME fibers. In case you have not been paying attention to Restek’s product portfolio (not sure why not), we have been offering traditional Solid Phase Microextraction (SPME) fibers since the summer of 2016. A nice addition to our product offering; however, nothing incredibly revolutionary.

As I already told you, SPME technology has seen no improvements in the last two decades. BUT (bold and capitalized, so time to pay attention)… now Restek can finally offer something new and exciting. Cue the Restek PAL SPME Arrow! The SPME Arrow was designed to overcome the following two most significant disadvantages associated with traditional SPME fibers: 1. Limited mechanical robustness and 2. Small phase volumes. If you have been using traditional SPME fibers, then you know exactly what I am speaking of. In fact, I know you feel the following pain:

You sneeze on a traditional SPME fiber and the thing is bent. Now let us take a look at a SPME Arrow:

Here is a better image to give you a direct comparison between traditional and Arrow SPMEs:

 

Right off the bat you will notice the increased size of the SPME Arrow, which results in increased mechanical stability and phase volumes. All of which we can dive into next time. So stay tuned to this multi-part blog series, where I break down in detail what a SPME Arrow is and what kind of performance  you can expect from a SPME Arrow.

Making a TO-15 Working Standard: Part 2 How-To Video

Last time we learned that in order to make a TO-15 working standard from our stock standard we need to determine our stock standard concentration, canister volume, stock standard injection volume, and final canister pressure. We covered how to go about deriving all of the aforementioned, the associated math, and I even provided you with the Super Standard Calculator – V2.01. Today I am providing you with the following video to show you exactly how to make a working standard via a static dilution:

Troubleshooting Injection Volume Variation in the Concurrent Solvent Recondensation – Large Volume Sample Injection Technique

I’ve started receiving a number of questions about the large volume injection technique called Concurrent Solvent Recondensation – Large Volume Sample Injection. Most recently, a question came through the Tech Service group about injection volume variation. I had encountered a similar problem when working on the 50 µL injection for the combined 1,4-Dioxane and Nitrosamines (EPA methods 522 and 521) application note (pdf). I had written up an examination of the problem, but it didn’t make it into the final draft. Fortunately, this blog is an ideal platform to share the work. (Tip – there is shortcut on the right of the blog’s home page to all the CSR-LVSI related blogs)

The default speed for a fast autosampler injection using a 10 µL syringe is 6,000 µL/min. When you increase the syringe volume to 100 µL and leave the ALS configured for a fast injection, the injection speed increases to 60,000 µL/min. During method development, we found that a standard single taper liner with wool did not provide enough packing material to arrest the 60,000 µL/min, 50 µL injection of dichloromethane. The high inlet flow pushes the large volume of liquid sample through channels in the wool, causing some of sample to enter the head of the column as a liquid. This causes peak fronting and splitting; two defects which are not amenable to reproducible integration and quantification. We addressed this problem by slowing the injection speed to 4,000 µL/min and adding more wool to the single taper liner, bringing the total mass of in situ deactivated quartz wool to approximately 15 mg.

One of the major challenges of combining the highly volatile compounds from EPA Method 522 with the less volatile nitrosamines from EPA 521 was minimizing the retention time variability of the early eluting compounds (THF, 1,4-dioxane, and NDMA). Early runs collected under the final GC analytical conditions showed great retention time variation for these compounds. Figure 1 shows the retention time variation of 1,4-dioxane-d8 over the course of nine sequential injections to be approximately 0.5 minutes. This is unacceptable for any analysis, but especially so for the trace-level SIM analysis.

Figure 1 – 1,4-Dioxane-D8 retention time variation over the course of nine 50 µL injections

By watching the injection sequence, it was determined that the retention time variation was largely due to variations in the injected volume, indicated by bubbles of various sizes present in the syringe from injection to injection. Adding a viscosity delay to the ALS program slightly improved retention time reproducibility, but the root cause of the problem was determined to be linked to the large volume syringe plunger design. Figure 2 shows the problem syringe (bottom) and the solution (top).

Figure 2: Switching to the more rugged, hermetically sealed fixed-needle ALS syringe (top) greatly increased retention time reproducibility for the more volatile components by more precisely controlling the injected volume. Top 100 µL gas-tight syringe with fixed 26/23 gauge needle (cat.# 005668); bottom: 100 µL syringe with removable 23 gauge needle (cat.# 005665);

Keeping the short viscosity delay and switching to a fixed needle gas-tight syringe with a PTFE-tipped plunger significantly improved the retention time stability of the volatile components. In addition to the PTFE tip, the gas-tight syringe plunger maintains its diameter down the entire length of the syringe barrel. Still, internal standard and surrogate compounds eluting close to SIM window borders were occasionally lost due to retention time drift. Figure 3 illustrates the reduced retention time variation shown by tetrahydrofuran-d8 following the change in syringe design.

Figure 3: Tetrahydrofuran-d8 retention time variation following the change in autosampler syringe design used in the CSR-LVSI experiments.

Eluting more than a minute earlier than 1,4-dioxane-d8, tetrahydrofuran-d8 showed even more retention time variation than 1,4-dioxane. The peaks are still eluting in a 6 – 8 second window, but this is a large improvement over the 30+ second spread we were seeing in our initial results. This indicates there is still some volume variation in the injections, but the internal standard correction should be sufficient to account for this.

The less volatile analytes, such as N-nitroso-di-n-propylamine-d14 did not display the same retention time variation, indicating analyte focusing was occurring rather than solvent focusing (Figure 4).

Figure 4: N-nitroso-di-n-propylamine-D14 retention time variation over the course of nine 50 µL injections. Being much less volatile, the thick film of the analytical column traps this compound until the oven reaches a much higher temperature than 1,4-dioxane or THF. This makes its retention time dependent on the oven program, not the injected solvent volume. The peak area is still injection volume dependent, and its use as the internal standard for the nitrosamines normalized the calculated recoveries for the small variations in injection volume.

 

What can I use to Qualify my HPLC instrumentation?

If you are setting up LC instrumentation in a new lab, you may find yourself looking for ways to perform Operational Qualification (OQ) tests. The requirements for qualifying a new instrument vary, depending on regulatory needs and SOP requirements, as well as the specific instrumentation. They might involve tests to confirm parameters such as solvent pump flow rate, gradient composition, linearity, reproducibility and wavelength settings (for UV, fluorescence and PDA detectors). For other detectors such as the ELSD or Mass Spec, this may also involve parameters like the measurement of the gas flow rate through the nebulizer, temperature settings and mass tuning.

Here are the products that Restek has to offer:

  • LC OQ Gradient Standard (acetone), catalog #30012
  • LC OQ Wavelength Accuracy (erbium perchlorate), catalog #31053
  • LC OQ Linearity Test Mix Kit (caffeine at 5, 25, 125, 250 and 500 ug/mL), catalog #31805
  • LC OQ Standards Kit, catalog #31069, contains catalog numbers 30012 and 31053 shown above, as well as catalog #31068, LC OQ Linearity Kit, containing ethyl paraben and propyl paraben at 5, 10, 15, 20 and 25 ug/mL each. Catalog #31068 is available separately, although not currently shown on our website.

We also have other test solutions that are intended to monitor column performance, not the instrument. You can find the full list of test mixes for LC here. http://www.restek.com/Reference-Standards/Test-Mixes?s=type:lc   For mass spectrometry qualification (LCMSMS), test solutions are often sold by the instrument manufacturer, so please contact them in this case.

Some of the above products may also be used for Performance Qualification (PQ) testing, which would be performed more frequently than the OQ test and applied to across the different vendor brands that the analyst may have in the laboratory.  This is also sometimes referred to as Performance Validation (PV) and exact requirements are determined by the laboratory. Instrument manufacturers usually provide their own guidelines, at least for OQ testing, which we recommend that you also consider.  Here is what we found that the various LC manufacturers suggest:

 

From Agilent:

https://www.agilent.com/cs/library/service/public/HPLC%20Hardware_Operational%20Qual-Col2.pdf

For gradient composition test in the above, acetone is used as mobile phase B, with UV detection. Please note that acetone is not normally used in mobile phase, due to its high UV absorbance. Agilent suggests using a liquid flowmeter for testing pump performance. We do not sell this type of flowmeter, so instead we suggest using a volumetric flask and stopwatch to manually do this. Mixing chamber performance is usually monitored by the gradient composition test, since it affects this directly.

 

From Waters:

https://www.waters.com/webassets/cms/library/docs/720000498en.pdf (see pages 349-350)

For the gradient composition test, Waters takes a different approach. They use more typical mobile phases, such as acetonitrile/water, and monitor by injecting a caffeine standard at various mobile phase percentages. Then they plot the retention time versus the ratio of water/organic solvent. Their test for flow rate is more complicated because they are also determining the linearity of various flow rate settings.

 

From Dionex/Thermo:

http://www.dionex.com/en-us/webdocs/81038-HPLC_OQ_PQ_E_Manual_6.8_Oct2008.pdf

It appears that their approach for gradient composition testing is similar to Agilent’s, using acetone. They monitor the flow rate/precision by collecting solvent for a specified period of time, weighing it. This can be done by weight or volume, but I have found volume is much easier.

 

From Shimadzu:

Shimadzu does not have published procedures for their OQ testing. However, their manuals offer guidance on how their instruments have some automatic functions that assist in these procedures.  As an example, please see pages 8-9 of the following manual for the LC2010HT:
http://www.ssi.shimadzu.com/products/literature/HPLC/C196-E056C-LC2010HT.pdf

Their manual also states that OQ testing can be performed by their representatives at the time of installation (if requested), in addition to the Installation Qualification (IQ) checks.  Any subsequent OQ and PQ tests would likely be performed by regular lab personnel.

 

Here are a couple more links that discuss instrument qualifications in general. As you can see, this is mostly a procedure driven by regulatory requirements:

http://www.forumsci.co.il/HPLC/OQ_Krull.pdf

http://www.chromatographyonline.com/performance-qualification-hplc-instrumentation-regulated-laboratories-0

 

I hope that the information provided here has been useful. Thank you for reading.

New Custom UCMR4 Standard for EPA Method 530 on the Rxi-5Sil MS

We recently stocked custom EPA Method 530 standards made specifically for labs participating in Unregulated Contaminant Monitoring Rule 4 (UCMR4). Quantitative Certificates of Analysis with Data Packs are available for each item. Email csreps@restek.com or phone 800-356-1688 ext. 3 to place your order (items are not available at Restek.com).

EPA Method 530, the Determination of Select Semivolatile Organic Chemicals in Drinking Water by Solid Phase Extraction and Gas Chromatography / Mass Spectrometry was developed on the Rtx-1701, but section 6.12.1 of the method states that “any capillary column that provides adequate capacity, resolution, accuracy, and precision may be used.” 3 of the 4 Method 530 target analytes, butylated hydroxyanisole, o-toluidine, and quinoline have been included in UCMR4; the 4th target analyte, dimethipin, was included in UCMR4 as part of EPA 525.3. It isn’t really clear to me why a new method was made for these compounds instead of relying on EPA Method 525.3. Method 530 uses a 6mL divinyl benzene or polystyrene/divinyl benzene cartridge for extracting 1L where 525.3 has multiple cartridge and disk SPE options. The analysis is a couple minutes faster on the Rxi-5Sil MS under the 525.3 conditions. The selectivity of the two columns are similar, the primary difference being that butylated hydroxyanisole (BHA) is less retained relative to acenaphthene-D10 on the Rxi-5Sil MS. If your lab has limited resources, you should be able to perform both method 530 and 525.3 on one instrument set up for method 525.3.

Cat# 572262 – Method 530 UCMR4 Standard (in Methanol)

Contaminant CAS Registry Number Minimum Reporting Level Standard Concentration
butylated hydroxyanisole (BHA) 25013-16-5 0.03 µg/L 150 µg/mL
o-Toluidine 95-53-4 0.007 µg/L 35 µg/mL
Quinoline 91-22-5 0.02 µg/L 100 µg/mL

 

Cat# 572265 – Method 530 UCMR4 Surrogate Standard (in Methanol)

Contaminant CAS Registry Number Standard Concentration
Quinoline-D7 34071-94-8 500 µg/mL
o-Toluidine-D9 194423-47-7 500 µg/mL

 

Cat# 572266 – Method 530 UCMR4 Internal Standard (in Acetone)

Contaminant CAS Registry Number Standard Concentration
Acenaphthene-D10 15067-26-2 500 µg/mL
Phenanthrene-D10 1517-22-2 500 µg/mL

 

I’ve collected example chromatograms on the Rxi-5sil MS using the same GC conditions that were used for the EPA Method 525.3 UCMR4 standard (Section 10.2.3.1), but with a new SIM program generated using the AutoSIM feature of MSD Chemstation. This was mainly to demonstrate the the two methods can be run on the same GC-MS with the same analytical column.

 

SIM Analysis of EPA 530 UCMR4 Standard Run at Method Reporting Limit (MRL) on the Rxi-5Sil MS

SIM Analysis of EPA 530 UCMR4 Standard Run at 10x the Method Reporting Limit (MRL) on the Rxi-5Sil MS

 

Peaks tR (min) MRL Conc. (µg/mL) 10x MRL Conc. (µg/mL)
1. o-Toluidine-D9 (SUR) 5.64 1.0 1.0
2. o-Toluidine 5.70 0.070 0.070
3. Quinoline-D7 (SUR) 8.22 1.0 1.0
4. Quinoline 8.26 0.20 0.20
5. Butylated hydroxyanisole (BHA) 11.50 0.30 0.30
6. Acenaphthene-D10 (IS) 11.54 1.0 1.0
7. Phenanthrene-D10 (IS) 15.05 1.0 1.0

 

Column Rxi-5Sil MS, 30 m, 0.25 mm ID, 0.25 µm (cat.# 13623)
Sample Method 530 UCMR4 Standard (cat.# 572262)
Method 530 UCMR4 Surrogate Standard (cat.# 572265)
Method 530 UCMR4 Internal Standard (cat.# 572266)
Diluent: Dichloromethane
Injection
Inj. Vol.: 1 µL pulsed splitless (hold 1.0 min)
Liner: 4 mm Single Taper w/Wool (cat.# 23303)
Inj. Temp.: 275 °C
Pulse Pressure: 30 psi (206.8kPa)
Pulse Time: 1.05 min
Purge Flow: 60 mL/min
Oven
Oven Temp.: 70 °C (hold 1.5 min) to 200 °C at 10 °C/min (hold 0 min) to 320 °C at 7 °C/min (hold 3 min)
Carrier Gas He, constant flow
Flow Rate: 1.2 mL/min
Detector MS
Mode: SIM
SIM Program:
Group Start Time (min) Ion(s) (m/z) Dwell (ms)
1 1.543 106, 107, 112, 114 25
2 6.917 102, 108, 129, 136 25
3 9.881 137, 162, 164, 180 25
4 13.297 160, 188 25
Transfer Line Temp.: 280 °C
Analyzer Type: Quadrupole
Source Type: Stainless Steel
Drawout Plate: 6 mm ID
Source Temp.: 280 °C
Quad Temp.: 180 °C
Solvent Delay Time: 1.45 min
Tune Type: DFTPP
Ionization Mode: EI
Instrument HP6890 GC & 5973 MSD

EPA Method 541 on the Stabilwax Column

EPA Method 541 UCMR4 Standard Chromatogram at 10x the Method Reporting Limit on a 30m x 0.25 mm ID x 0.50 µm Stabilwax (cat# 12039).

I recently posted some chromatograms for our new EPA Method 541 UCMR4 Standards, and wanted to focus on the column in a separate post. EPA Method 541 was developed on a 30m x 0.25 mm ID x 0.50 µm wax column from another manufacturer. I used the Stabilwax because it is a relatively low bleed wax column and has similar selectivity to the column used by the EPA. My chromatogram is shown above, while a calibration standard chromatogram taken directly from the method documentation is shown below.

EPA METHOD 541 FIGURE 2 – Reconstructed ion chromatogram (RIC), SIM mode, for calibration standard on a 30m x 0.25 mm ID x 0.50 µm wax column.

There are 2 major differences I wanted to point out:

  1. The Stabilwax column has the same elution profile but it is less retentive at the same phase ratio. The last target compound (chlorobenzene-D5) elutes on the Stabilwax at 8.66 minutes, which is BEFORE the 1st target compound (1,4-Dioxane) elutes on the column used in the EPA Method (10.3 minutes). UCMR4 does not include 1,4-dioxane, so it is not included in the Stabilwax chromatogram. The GC acquisition parameters were the same for both columns, though the detector settings were different because of the SIM window start times. All peaks had eluted on the Stabilwax before the method recommended solvent delay ended.
  2. The bleed is significantly lower on the Stabilwax compared to the calibration standard chromatogram from the EPA method.