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Want to learn about “Injection Techniques in GC” or “Practical Maintenance and Troubleshooting in GC”? Sign up to join our half day course during Pittcon, Chicago.

Please join one of our courses presented next Pittcon in Chicago:

“Injection Techniques in GC”:? Monday,? March 2,? 08:30-12:00, Session: SC1230.

“Practical maintenance and troubleshooting in Gas Chromatography”: Tuesday, March 3, 08:30-12:00, Session: SC1231

For location, visit short course office at S100C.

 

Injection Techniques in Gas Chromatography

In Gas chromatography the most important process is to get the sample into the column. If sample transfer is not optimized, the results will not be reliable. Goal of this course is to understand the different injection techniques used and the process how to obtain a narrow injection band.In this half day course we will discuss the basics of the most popular injection techniques that are used in Gas chromatography. Techniques like split, splitless, direct, on-column and large volume injection will be discussed in detail. Also the selection of liners, retention gaps and columns will be addressed.All techniques will be explained using practical examples. At the end we will zoom into some typical “injection” troubleshooting examples

 

Practical maintenance and troubleshooting in Gas Chromatography

In Gas chromatography 90% of the troubles experienced, is happening in the injection system. In this training we will discuss the purpose and impact of the critical parts (consumables) present in split and splitless injection systems and how this impact in a maintenance schedule. At the end we will discuss a series of practical examples via troubleshooting exercises. In this half day course we will discuss the maintenance and optimization challenges for Split and Splitless injection techniques. We will zoom in carrier gas choice and purity, tubing, connections, septa, ferrules, seals, liners, column-coupling, installation and column maintenance. Also column operation/optimization and extending column life time will be discussed.

 

Chiral separation on a C18 column? Separation of d- and l-amphetamines, Part II

To continue my blog part 1 (Part 1:https://blog.restek.com/?p=67087) , where I have briefly discussed the importance of separating the d and l isomers to accurately identify the illicit isomer using an achiral method on a Raptor C18 column employing a pre-column derivatization technique. Today I’d like to discuss more about the matrix of interest, sample preparation and derivatization, chromatographic and mass spectrometric conditions.

Sample Preparation: 50 ?L of calibration standard or QC sample prepared in analyte free pooled human urine (spiked in the range of 50-5000ng/mL) was aliquoted into a micro centrifuge tube. 10 ?L of a working internal standard (20 ?g/mL (±)-amphetamine-D11 and (±)-methamphetamine-D11 in water) and 20 ?L of 1M NaHCO3 was added and vortexed at 3000 rpm for 10 seconds. After vortexing, 100 ?L of 0.1% (w/v) Marfey’s reagent (1-fluoro-2-4-dinitrophenyl-5-L-alanine amide) prepared in acetone was added, vortexed, and heated at 45 °C for 1 hour in a water bath. Samples were allowed to cool to room temperature before the addition of 40 ?L of 1M HCL in water. The d- and –l amphetamines are now converted to DNPA derivatives. The sample was then vortexed and evaporated to dryness under nitrogen at 45 °C. Samples were reconstituted in 1 mL (20x dilution) of 40:60 water: methanol (v/v) and filtered using Thomson SINGLE StEP standard filter vials (cat# 25893) and then injected for LC-MS/MS analysis.

Matrix…. What Matrix? Currently urine is the sample of choice for pain management or toxicology labs for routine drug testing, because of its ease of sample collection and clean up as well. So we bought 9 lots of drug free human urine lots from Bio IVT, the samples were derivatized and analyzed on LC-MS/MS to check for any presence of endogenous Amphetamines and other significant interference peaks. All the urine lots were free from interferences or presence of amphetamines. Pooled urine was prepared from the analyte free 9 urine lots and was used for method development and validation. Surine was evaluated as well and no matrix peaks were evident and amphetamines were well separated.

Urine dilution factor? How do I decide which dilution factor was right for this analysis. Well, I derivatized both 100uL (10x) and 50uL (20x) pooled urine: 20x dilution gave best results with good sensitivity and less matrix effects compared to 10x and increasing dilution rate more than 20x decreased the signal sensitivity.

Sensitivity issues? One of our customer complained about poor signal intensity of the analytes even after derivatization, a chemists nightmare. I blame the derivatizing reagent here, it is very light sensitive and should be prepared fresh in acetone and stored in dark at 40C. However, the derivatized samples were found to be stable for at least 48 hours in the auto sampler rack, no significant fluctuations in signal intensity was noticed.

Can Cleaning the MS source help? Definitely, especially in this particular case because you are just diluting the urine sample. I clean my MS source once a week with water, Methanol and Acetonitrile after running hundreds of urine samples or any other biological matrix like whole blood, oral fluid etc..

Chromatography: Now that our samples are derivatized and ready for the analysis, I’d like to discuss more about the LC conditions. A Raptor C-18 100 x 2.1 mm, 2.7 ?m column was utilized for the separation of the DNPA derivatives of Amphetamines and their respective deuterated internal standards in 7 minutes (Figure 1 & 2). Using a simple derivatization and dilution procedure along with a Raptor C18 column— good baseline resolution of the target compounds was obtained, allowing easy peak identification and quantitation.

Fig 1: Separation of d- and l- Amphetamine and Methamphetamines enantiomers in fortified Human Urine at 500ng/mL (TIC)

 

Fig 2: Separation of d- and l- Amphetamine and Methamphetamines enantiomers in fortified Human Urine at 500ng/mL (XIC)

Instrument Conditions

Analysis of amphetamines by LC-MS/MS was performed on a Shimadzu Prominence HPLC equipped with a SCIEX API 4000 MS/MS. Instrument conditions were as follows and analyte transitions are provided in Table 1.

Analytical column: Raptor C18 2.7 ?m, 100 mm x 2.1 mm (cat# 9304A12)

Guard column: Raptor C18 EXP guard column cartridge (cat# 9304A0252)

Mobile phase A: 0.1% Formic acid in water

Mobile phase B: 0.1% Formic acid in methanol

Flow rate: ???0.5 mL/min

Injection volume: 10 ?L

Column temp.: ?35 °C

Ion mode: Negative ESI

 

 

 

 

 

 

 

Table 1: Analyte Transitions for the Analysis of Amphetamines by LC-MS/MS.

 

 

 

 

 

 

 

 

 

Hopefully this workflow and some of the tips discussed above on both sample prep and chromatography and can help get you some good baseline separation and resolution of these enantiomers to identify the illicit isomer.

In my next blog I will share some interesting data that mimics the real urine sample concentrations containing very high and low concentrations of the legal and illegal isomers present in urine and optimization of derivatizing procedure. Stay tuned till next one….

References:

  1. Newmeyer, N. M, Concheiro, M and Huestis, A. M. J Chromatogr A. 2014; 1358: 68–74.
  2. Foster, S. B and Gilbert, D. D. J Analytical Toxicology.1998; 22:265-9.

Falling Victim to One of LC’s Classic Blunders: Mismatching Your Diluent and Mobile Phase

An early lesson most of us learn in liquid chromatography is this: Always match your diluent to your mobile phase.? Once the exams are done, if you learned this in a college course, or your manager has walked away, you start flexing this “requirement” a little to see how matched they really need to be.? I get it! You don’t want to have to blow off all your organic, because that takes time.? Or if you’re scouting various columns, you just want to use the same sample you used in HILIC and reversed-phase tests.

In some cases, you can get away with this possibly because your analyte has plenty of affinity for your stationary phase, and that extra bit of strong solvent isn’t enough to ruin your separation.? While in other cases your analytes elute well passed the dead volume of the column, so your peak shape is still good.? In these instances, it can be OK to bend the “rules” a little.? Sometimes though, you have an analyte so sensitive, that even 5% residual acetonitrile in your diluent is enough to ruin your chromatography altogether.? I present to you this case study on acrylamide.

Acrylamide is a small polar molecule (show image).? It is akin to Iocane powder.? ?It’s odorless, tasteless, dissolves instantly in liquid, and is [not] among the more deadly poisons known to man.? Though as a side note it is thought to be a probable carcinogen with repeated prolonged exposure. ?In reality, acrylamide is soluble in water and methanol and is very soluble in acetonitrile. Additionally, it is difficult to retain on most LC columns.? Earlier this year, we developed a product specifically for acrylamide analyses and have developed applications in various matrices.

Early on in method development, we noticed that acrylamide was very sensitive to residual organic solvent leftover from the sample preparation procedure.? In the published EN 16618 method, the final extract is in a water/methanol eluate.? The step before analysis is a lengthy blow down; a step many, including myself, are tempted to short change.? In the QuEChERS method, the final sample diluent is acetonitrile (MeCN), which is even more detrimental to chromatographic analysis and requires complete solvent replacement.

To better understand the sensitivity of acrylamide to organic diluent, we deliberately performed an extraction of acrylamide from potato chips/crisps and added organic solvent in known volumes to see how this changed the chromatography from an ideal sample prep to one that might be more “lazy.”? In our method, we use a 100% aqueous mobile phase with 0.001% formic acid, meaning that our diluent should also be 100% aqueous.? ?As more organic was added to the diluent, retention time decreased and peak width broadened.? While 10% methanol in the diluent still gave decent retention and chromatography, 20% or more methanol resulted in poor peak shape and retention.? In the case where there is even 5% residual MeCN, massive peak broadening occurred and when more MeCN was present, the acrylamide peak split and then eventually eluted with the matrix contaminant peak.

While acrylamide qualifies as an extreme example, this data shows the necessity of ensuring your diluent matches your starting mobile phase.? We also noticed that having strong organic in your autosampler rinsing routine could result in a similar effect on your chromatography.? So in 2020, and beyond, next time you see poor chromatography with early eluting or poorly retained peaks, consider checking your diluent composition.? Your solution might be as simple as making sure your diluent better matches your mobile phase.

Happy New Year from all of us here at Restek!

 

What impurities are hiding in your monocyclic aromatic solvents?

As we approach the end of the year we are indulging in a bit of nostalgia and most experts agree that reminiscing about the past is a good thing1. It makes us more optimistic and generous. Researchers also noticed that in cold rooms we are more inclined to nostalgize than if we are in a warm environment. And, since my boss, Chris, is a fan of cold office spaces, I will spend a few minutes on what we did last year.

Past year, we devoted some time to validate the products for the ASTM Petrochemical Method Chromatography Product Guide. The ASTM guide contained mostly D02 GC methods – methods for the analysis of petroleum products. Now, we have expanded it to include D16 ASTM chromatographic methods. D16 Committee is responsible to standardize testing of the aromatic, industrial, specialty, and related Chemicals.

One of the more popular methods in the D16 section is ASTM D75042, a method for the analysis of trace impurities in monocyclic aromatic solvents by GC and the effective carbon number. This method uses one analytical procedure and column for an analysis of a variety of industrial solvents from different sources. This includes synthesized or refined solvents such as benzene, toluene, p,m,o-xylene, ethylbenzene, and styrene. Check out our ASTM guide for chromatograms of a few commercially available solvents.

The analysis is quite long, and if we simplify, the only resolution requirement in the method is to separate any impurity from the solvent being analyzed. The impurities in the solvent are reported as a sum of total non-polar hydrocarbons, a few individual mono-aromatic compounds, and later eluting aromatic compounds are summarized and reported as a group. Reported analytes can be easily separated on a long column which led to the use of shorter columns cited in the literature.

I evaluated the 20mm x 0.18mm x 0.36?m Stabilwax column and found it to have sufficiently separated the method resolution mixture in p-xylene.

Figure 1: Chromatogram of a method resolution mixture in p-xylene overlaid with a synthetic blend of compounds most commonly found as an impurity in monocyclic aromatic solvents on a 20m column. The analysis conditions were optimized using the Restek EZGC method development suite (method translator and chromatogram modeler)

Read the rest of this entry »

Two for the Price of One?Using CPP to Simultaneously Clean Pesticide and PCB Extracts

In a recently published blog, we introduced the advantages of cleaning EPA Method 8081 extracts using CarboPrep Plus (CPP) as a replacement.? The sample preparation for PCBs by EPA Method 8082 is commonly combined with the pesticides EPA Method 8081 for the extraction step then split into two portions for sample cleanup.? The logical question is: can I run the pesticide/PCB extract through CPP prior to splitting for separate analysis, thereby avoiding separate clean up procedures?

Before I answer, I should explain that the reason the single extract is sometimes split for post extraction processing is the sulfuric acid procedure cannot be applied to the pesticide portion, it will degrade the acid sensitive analytes.? Sulfuric acid cleanup has been used with PCB extracts since it can be effective at removing much of the colored material that Florisil leaves behind, so it is possible that CPP may be used instead of splitting the sample which results in additional extraction steps.

?Why clean the PCBs extracts at all?? If pesticide extracts are run on the same instrument regular maintenance would be required to meet the stringent inlet inertness requirements for endrin and DDT.? PCB methods typically do not have the same stringent requirements, however samples typically have high concentrations of non-volatile matrix compounds that build up in the inlet effecting chromatography, which effects quantitation.

An effective cleanup is also important since chromatographic interferences make it difficult to perform Aroclor pattern identifications. ?Other challenges include weathering which can significantly change the Aroclor pattern combined with different Aroclor mixtures.

One characteristic to the CPP cartridge based upon experience with GCB, lies in its strong affinity for certain compounds.? In this case, the non-ortho PCB congeners have varying levels of interaction to GCB.? The logical question is how the pattern of various Aroclors will be affected by this preferential adsorption of some congeners relative to others when using CPP. Jason Thomas set off on a crusade to answer these questions. First, he started by running Aroclors through the cartridge using the standard elution method of 10 mL hexane/acetone 90:10. ?Second, he ran the individual congeners through in a series of mixes, collecting fractions in 5 mL increments and identified congeners that required an additional amount of solvent for elution.? Finally, Jason compared our concentrations with the various Aroclor ratios of congeners reported in a paper by Frame & Cochran1.

First, all PCB congeners eluted in the first 5 mL elution volume except for the following PCBs in Table 1.? The congeners that exhibited retention beyond the first 5 mL of elution were those possessing no chlorine substitution at the otho positions (known as non-ortho PCB congeners) as illustrated in the chart below:

Figure 1: Recoveries of selected PCBs with different chlorine substitutions measured against amount of elution solvent necessary to remove the analyte from the carbon.

The non-ortho PCB congeners have a longer retention on the carbon, but for quantitation of Aroclors, these congeners may not be relevant as it is recognized that not all 209 are actually present in the Aroclor mixtures, which is where the Frame-Cochran paper comes in handy.? Ratios of these congeners in each specific Aroclor were reported.? Adding this information to the above table, it becomes apparent that only five of these non-ortho congeners (highlighted in orange) are expected to appear in any meaningful quantity as defined here with a cutoff of 0.3%.

As an example, let us compare the effect of CPP on the elution of Aroclor 1221 using the standard pesticide Florisil methodology, 10 mL 10% acetone/hexane elution solvent.? We will specifically focus on PCB-15 in Aroclor 1221 as it represents the congener with the highest % representation in a given Aroclor, (4.2% in 1221).

Chromatogram 1: Demonstration of the elution of Aroclor 1221 using standard pesticide Florisil methodology. Control standard is shown in blue and the extraction standard is shown in red.

There is a notable response missing from the CPP processed standard (red trace) versus the control standard (blue trace), roughly half, as expected with a 10 mL elution-based data in Table 1 above.? As indicated, PCB-15 is a minor component of the Aroclor 1221 pattern, the absence of which will not affect quantitation.

This does not constitute a significant change in the overall appearance of 1221.? In the event of quantitation however, choosing this as one of the quant peaks will provide some degree of error, but there are clearly several more prominent and representative peaks to choose from in order to establish the 3 to 5 peak calibration required by EPA 8082.

None of the heavily retained congeners are present in the heavier Aroclor mixtures, 1254 – 1268.? This may also be corroborated by comparing a 1254 standard processed with CPP and one without.? No discernable difference exists between the two suggesting that none of the components of this Aroclor is retained on the SPE cartridge.

Chromatogram 2: Demonstration of 1254 recoveries using CPP where blue is the standard and red is the extract. Little to no differences were observed.

In summary, CarboPrep Plus may be used to clean EPA 8082 extracts for Aroclor quantitation, provided caution is exercised in choosing the quant peaks, setting up the possibility of processing a combined pesticide/PCB extract without the sulfuric acid clean up step.

  1. Frame, G. M., Cochran, J. W., Bowadt, S. S. (1996), Taming Complete PCB Congener Distributions for 17 Aroclor Mixtures Determined by 3 HRGC Systems Optimized for Comprehensive, Quantitative, Congener-Specific Analysis. Journal of High Resolution Chromatography, 19: 657-668.

 

Determining Optimal LC-MS/MS MRMs: Sensitivity is NOT Everything!

LC-MS/MS using electrospray ionization (ESI) is a widely used platform for routine target analysis and quantitation. However, one big challenge faced through the use of ESI is the presence of matrix effects. So, what are matrix effects and why are they important? Simply put, when matrix components coelute with the target analytes, they have the potential to enhance or suppress analyte ionization efficiencies which may cause a loss of method precision and accuracy and even result in false positives or negatives. How can we solve this problem? Generally, the approaches can be categorized into 4 main strategies: 1. Sample cleanup to remove the matrix interference; 2. The use of chromatography to resolve the matrix from the analytes; 3. Calibration techniques to compensate for the matrix effects and 4. Modifying detector parameters to reduce interferences.

Sometimes interferences from the sample extracts have similar ions in the MS/MS experiment compared to your target analytes. In this case, choosing a non-interfering MRM (multiple reaction monitoring) transition with less impact is the most effective way to minimize matrix effects without additional sample clean-up or preparation. To demonstrate the effectiveness of this technique, I provided an example of where this strategy was used recently for the analysis of mycotoxins in peanut powder.

Mycotoxins are toxic secondary fungal metabolites and are very harmful to both humans and animals. Peanuts are one of the major food commodities affected by mycotoxins. The following chromatogram shows an example of matrix related interference for HT-2 toxin in peanut powder.? Difficulties with integration were observed by using the primary MRM transition 447.3->285.3 due to an isobaric matrix interference. This was effectively resolved by using a less abundant MRM transition (447.3->345.3) for quantification. In doing so, excellent accuracy and precision were observed. In addition, it saved extra time and cost that would have been needed to improve the sample cleanup and chromatographic separation. However, due to the lower intensity of the second transition (447.3->345.3), sensitivity was reduced and the lower limit of quantitation (LLOQ) had to be adjusted. Applying this approach is an acceptable practice as long as the reduced sensitivity doesn’t affect the ability to reach an LLOQ necessary to meet a regulatory requirement, or data quality objective.

Overall, matrix interferences vary widely in nature, so there is no single approach to cover every scenario. While no method can completely eliminate matrix effects, applying all the tools and resources available to us will aid in finding the best solutions that fit your application to alleviate or minimize the impact of matrix interferences in challenging samples.

GC Inlet Liner Selection, Part III: Inertness

The inlet liner is the first surface analytes will interact with after introduction into a GC.? It is critical that liners are deactivated, as a number of adverse interactions can occur between analytes and the glass surface.? Deactivations typically involve some type of silanization of the surface to cover active sites inherent in glass, such as silanols.? Most liners are made of borosilicate glass, which in addition to active groups like silanols, can also contain metallic impurities (Figure 1).

Many liners are also packed with glass wool; which serves to aid in vaporization, helps to mix the sample, and protects the column from non-volatile material.? Wool can be either borosilicate or quartz, with quartz being preferable, as it contains less impurities.? The high surface area of wool presents challenges in deactivation, where providing comprehensive coverage is essential.

Figure 1: Examples of active sites found on glass surfaces, requiring the need for deactivation. The steric variations of silanols found on the surface require a comprehensive deactivation procedure.

Given the potential activity shown in Figure 1, there are two main types of adverse interactions that can occur.? One is chemical reactivity and the other is adsorption, explained in further detail below.? It is important to note that not all liner deactivations are created equal.? As seen in Figure 1, several steric variations of silanols are possible, making thorough deactivation difficult. ?Depending upon the specific deactivation reagents, as well as the process with which they are applied, some deactivations may have better performance for active analytes than others.? Restek offers Topaz liners, as our premium deactivated liner.? Topaz performs well for a variety of active analytes, including various pesticides, acids, and bases.

Chemical Reactivity

Chemical reactivity occurs when an analyte reacts within the liner to form new products.? The high temperatures in the inlet combined with active sites can lead to chemical reactions for active analytes.? This can affect the accuracy of the GC analysis, since analytes not originally found in the sample will be produced upon injection.? One example of this is the “breakdown” of endrin and DDT, two chlorinated pesticides (More info: https://blog.restek.com/?p=21873).? Upon introduction into a GC, endrin can react to form endrin aldehyde and endrin ketone and DDT can dechlorinate or dehydrochlorinate to form DDD or DDE, respectively. Figure 2 demonstrates how liner deactivation can affect compound performance, showing different levels of endrin and DDT reactivity.

Figure 2: Comparison of endrin and DDT breakdown on three different liner deactivations. Breakdown percentage is calculated as the relative percentage of reaction products vs total amount of parent analytes introduced into the system. Only endrin and 4,4’-DDT were injected into the system at 50 ppb and 100 ppb, respectively. Liners were single taper with wool, analyzed in splitless injection mode.

Adsorption

Analytes can adsorb to the liner surface through interactions such as hydrogen bonding and Van der Waals forces.? Adsorption can be reversible or irreversible.? With reversible adsorption, analytes may temporarily interact with the liner surface and then slowly load onto the column, potentially leading to peak tailing.? Irreversible adsorption, on the other hand, results in total loss of the analyte, with the analyte “sticking” in the liner.? Figures 3 and 4 show some examples of adsorption for acidic and basic compounds.

Figure 3: Example of 2,4-dinitrophenol response on two different liner deactivations, one showing low recovery due to adsorption within the inlet liner. Liners were single taper with wool, analyzed in splitless mode.

Figure 4: Example of benzidine response on two different liner deactivations, one showing lower recovery due to adsorption within the inlet liner. Liners were single taper with wool, analyzed in splitless mode.

A Word on Injection Mode

While these interactions can occur when using either split or splitless injections, liner inertness is especially critical for splitless injections.? Splitless injections have lower total inlet flows, leading to longer liner residence times for analytes and therefore more time for adverse interactions to occur. Longer residence times subject compounds to higher temperatures in the inlet. In addition, splitless injections are generally used for trace analyses, where activity can have a much larger impact due to the larger ratio of active sites to analyte molecules.? Because of this, using split injections, if possible, will greatly reduce the impact of inlet/liner activity on your analysis.

Conclusions

It is important to be aware of the adverse interactions that can occur within liners in order to optimize analyte recoveries and preserve the integrity of your analyses.? If you are working with active analytes, such as pesticides, acids, and bases, it is essential to choose a liner with a good deactivation as a starting point.? Keep in mind that with use, liner performance may degrade as a result of build-up of non-volatile matrix material.? This will result in the same types of adverse interactions discussed above.? Monitor analyte performance for any signs of chemical reactivity or adsorption, which signals that it’s time to change out your inlet liner.

Also be aware that if you’re using a splitless injection, switching to a split injection can significantly reduce the impact of inlet/liner activity on your analysis.? The catch is that you must still be able to meet method detection limit requirements in order to make this switch.

Links to blogs in this series:

GC Inlet Liner Selection: An Introduction

GC Inlet Liner Selection, Part I: Splitless Liner Selection

GC Inlet Liner Selection, Part II: Split Liners

GC Inlet Liner Selection, Part IIB: Split Liners Continued

GC Inlet Liner Selection, Part III: Inertness

Terpene Analysis Approaches – Part II

Welcome back my fragrance fanatics! Today, we continue exploring approaches for analyzing terpenes. If you missed part one, you can get ca