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How Dirty Are You? Part 3…Gloves…The Question

Portrait-Photo-boxer-photo

http://mistressmaddie.blogspot.com/2013/03/figaro-figaro-fiiiigarooo.html

This third installment of How Dirty Are You? is all about gloves. Outside of solvent compatibility, there is a lot of room for personal preference regarding the type of lab gloves people can use.

I found 5 different gloves in our lab. The bright green gloves and yellow gloves are the two types most often used by my lab mates. I use the bright green gloves. I work with QuEChERS extracts all the time so I was most interested in background resulting from gloves contacting acetonitrile. I have already investigated the bright green gloves when I was tracking down some peaks in my process blank for a QuEChERS experiment. I was seeing some repeating peaks which were identified as alkanes via GC-TOFMS. Through some further investigation, I was able to confirm that these alkane peaks were from my favorite bright green gloves. That got me thinking about contamination peaks from other gloves…

What We Did

 

We tested the background generated by the 5 glove types found in our lab by soaking a small piece of glove in 1 mL of acetonitrile for 30 minutes. The samples were tested via GC-TOFMS after the 30 minutes. For relative intensity, a dashed line at the peak height of a 2 ppm PAH standard is drawn on the chromatograms. But you will not see the results until my next blog!

 

Questions

Post or email me your answers.

Email me with your answers and look for the data in my next blog.

1. Which gloves (see below) gave the worst background?

 

Bright Green Gloves

Baby Blue Gloves

Yellow latex Gloves

Green Gloves

Royal Blue Gloves

2. What common additive was found in multiple gloves tested?

 

Dyes

Antioxidants

UV Stabilizers

gloves2

Finally, some increased sample loading capacity for PAHs! Part 4: 30m x 0.32mm x 0.50µm Rxi-5ms

This may be the last post in my series on sample loading capacity for PAHs on Rxi-5ms GC columns.

Sample loading capacity for PAHs on a 30m x 0.25mm x 0.25µm Rxi-5ms GC column

Does a thicker film GC column increase sample loading capacity for PAHs? 30m x 0.25mm x 0.50µm Rxi-5ms

Sample loading capacity for PAHs, part 3: 30m x 0.32mm x 0.25µm Rxi-5ms

I finally got an obvious increase in sample loading capacity for PAHs by going to a 30m x 0.32mm x 0.50µm Rxi-5ms:  bigger bore x thicker film.  I say “obvious” because now I can see it by the peak shapes in the chromatograms (essentially answering Jaap’s question – “What do chromatograms tell us?”).  I think in the interest of not being long-winded, I’ll let the chromatograms tell the story and summarize the series below.  As previously, the EZGC™ Method Translator was used to provide the GC conditions when going from the 30m x 0.32mm x 0.25µm Rxi-5ms to the 30m x 0.32mm x 0.25µm Rxi-5ms.

  1. PAHs, especially larger ones like benzo[b]fluoranthene (5 rings) up to benzo[ghi]perylene (6 rings), and higher, easily overload 0.25mm x 0.25µm and even 0.25mm x 0.50µm 5% phenyl-type columns like the Rxi-5ms (and Rxi-5Sil MS) and corrupt overall peak capacity, separation efficiency, qualitative identification (particularly due to coelutions of isobaric species), and quantitative accuracy.
  2. If extra sample loading capacity (above ~10 ng each on column?) for PAHs is very important, a 30m x 0.32mm x 0.50µm column offers a balance between sample loading capacity and efficiency of separation, at the expense of increased run time versus 0.25mm columns.  (Note that the efficient flow rate for the 0.32 x 0.50µm column is going to be around 1.8 ml/min helium, so a mass spectrometer must be capable of pumping this flow.)
  3. Sample loading capacity estimates in ng on column may be lower than is typically stated and needs additional investigation with other compounds.

4 Fig 1

 

4 Fig 2

4 Fig 3

4 Fig 4

 

4 Fig 5

 

4 Fig 6

Sample loading capacity for PAHs, part 3: 30m x 0.32mm x 0.25µm Rxi-5ms

My last post, “Does a thicker film GC column increase sample loading capacity for PAHs? 30m x 0.25mm x 0.50µm Rxi-5ms”, generated some interesting internal discussions between me, Jaap de Zeeuw, Chris English, and Chris Rattray, as to what constitutes overload of a GC column.  Jaap really focused on peak shape and contested my assessment that there was not a “substantial” difference between the 0.25mm x 0.25µm and 0.25mm x 0.50µm columns as regards overload, particularly for the 50 and 25 ng each PAH chromatograms.  So in addition to presenting PAH overload results for a 30m x 0.32mm x 0.25µm Rxi-5ms, which is also supposed to have increased sample loading capacity because of its wider bore, I zoomed in on the benzofluoranthene peaks for 50 and 25 ng amounts on column at Jaap’s suggestion for better inspection of the peak shapes.  So check out the first two sets of chromatograms below, which include 3 chromatograms each for 30m x 0.25mm x 0.25µm Rxi-5ms, 30m x 0.25mm x 0.50µm Rxi-5ms, and 30m x 0.32mm x 0.25µm Rxi-5ms columns.  The peak shapes do not look that much different to me, except for what appears to be a slight proportional narrowing of peaks for the 0.32mm x 0.25µm column with 25 ng PAHs, but perhaps readers can offer their opinions.

The next four sets of chromatograms demonstrate to me that there is still no dramatic increase in sample loading capacity for these PAHS with either a thicker film (0.25mm x 0.50µm) or a wider bore (0.32mm x 0.25µm) column. Essentially, these larger ring PAHs are just not “soluble” in the 5% diphenyl stationary phase, leading to overload, except at lower levels.  So up to this level of data collection, I still recommend the 30m x 0.25mm x 0.25µm Rxi-5ms GC column for semivolatiles/EPA PAH work because of faster run times, better separations, and narrower peak widths (which leads to better detectability).

As previously, the 30m x 0.32mm x 0.25µm method was translated from a previous method using the EZGC Method Translator.

Overload 3 Fig 1

Overload 3 Fig 2

Overload 3 Fig 4

Overload 3 Fig 5

Overload 3 Fig 6

Think twice before purchasing a 3/16” OD packed column

So you may be asking yourself, why the cautionary statement? Well, it’s very simple, many of our customers who purchase 3/16” OD (outside diameter) packed columns have a difficult time installing them into their GC oven.  Why?  Sometimes finding the appropriate fitting/ferrule is difficult.

Let’s say you have a packed column instrument. If your injection port was designed for 1/4” OD columns, then installation is usually not an issue.  Most customers can use a 1/4” x 3/16” OD reducing ferrule, like Restek 20258. To center the column properly (which is recommended if you plan to inject your sample into the packed column) and if you have an Agilent GC with a packed column injection port, you can use our kit 21650, which resembles what is shown in the photo below.  The silver thing that looks like a GC injection port liner is called a “centering sleeve”, and as you may guess, its purpose is to center the packed column in the injection port.

21650

 

Now let’s look at a few scenarios where installing a 3/16” OD packed column is not so simple. Many modern GC’s have injection ports designed for 1/8” packed columns.  So how does one install the larger 3/16” OD packed column into this smaller injection port?  You will need a short piece of 1/8” OD tubing and a 3/16” to 1/8” reducing compression (straight) union like Swagelok SS-300-6-2. Unfortunately, we do not sell this fitting.  However, we do sell a 1/4” x 1/8” reducing union, Restek 23170.  Simply replace the 1/4” metal ferrule set with ferrule 20258.

What if you have a GC designed for only capillary columns? Normally we would recommend a “pigtail” set-up, like described in this FAQ.  How do I install a packed column into a capillary GC injection port?  However, you would need a 3/16” x 1/16” reducing (straight) union, like Swagelok SS-300-6-1. Unfortunately, we do not sell this fitting either.  However, we do sell a 1/4” x 1/16” reducing union, Restek 23169.  Simply replace the 1/4” metal ferrule set with ferrule 20258.

In summary, before purchasing a 3/16” OD packed column, make sure you have the proper fittings/ferrules available for installation. I briefly mentioned this in my post titled Things to Consider Before Ordering a Packed Column.  To save yourself frustration, do a little homework first to determine if a 3/16” OD column is really your best choice, and if not, consider an alternative OD based upon your particular instrument.

 

Does a thicker film GC column increase sample loading capacity for PAHs? 30m x 0.25mm x 0.50µm Rxi-5ms

After my post, “Sample loading capacity for PAHs on a 30m x 0.25mm x 0.25µm Rxi-5ms GC column”, a ChromaBLOGraphy reader posed the question about how a 0.50µm column would work.  It’s a great question, because column vendors routinely advertise that thicker films have more sample loading capacity.  But do they really, and if so, how much more?  Let’s test it!

First I used the EZGC Method Translator to get the GC oven program for the 30m x 0.25mm x 0.50µm Rxi-5ms.  Ultimately, the translation results in a slower oven program rate to try and keep the elution temperatures about the same for the compounds of interest so that no compounds suddenly coelute, or worse, switch elution order.  As you can see from the chromatograms below, the 0.25mm x 0.50µm Rxi-5ms does not show a substantial sample loading capacity increase versus the 0.25mm x 0.25µm for the PAHs of interest.  Given that the run times are longer for the thicker film column, and the peak widths are broader (leading to reduced sensitivity), there seems to be no dramatic advantage to using the thicker film column in this case.

25_50 PAH Xlate

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

25_50_PAH 50

 

25_50_PAH 25

 

25_50_PAH 12_5

 

25_50_PAH 6_25

 

25_50_PAH 3_13

Pesticides/Fungicide cocktails responsible for Death of Bees: analysis via GC-MS

 

2011-jaap-pasfoto4-smallResearchers at the University of Maryland and the US Department of agriculture may have uncovered the mystery of high mortality in honeybees. These insects are responsible for pollinating over 130 different crops in the US alone. Loss of this pollinator species could cost the US $15 Billion annually. The EU (European Union) issued a temporary ban against the neonicotinoid pesticides. Honeybee deaths may be caused by  a ‘witches brew’ of pesticides and antifungals.

 

The scientists are narrowing down which pesticides and antifungals (fungicides) impairs the bees by investigating pollen of different crops (almonds, apple, cranberry, watermelon, pumpkin, cucumber and blueberries). Since a different cocktail of pesticides is used on varying crops researchers have the ability to determine which pesticides have the greatest effect on bee mortality.

On average crops were sprayed with nine different pesticides and one crop tested had 21 different pesticides present. The combination of these chemicals reduces the bees’ resistance to the parasite Nosema ceranae. Bees with compromised resistance die. Some of the most notable pesticides were chlorothalonil, pyraclostrobin (fungicides) and fluvalinate, amitraz (insecticides). This research was published in the scientific journal PLOS One.

Chlorothalonil is analyzed by GC and can be performed on a non-polar Rxi-5ms type column or a stabilized Rxi-5Sil MS. For a list of applications see:
http://www.restek.com/chromatogram/search?s=type:GC::chlorothalonil

Insecticide Fluvalinate and pyraclostrobin fungicides, are a bit more challenging:
http://www.restek.com/chromatogram/search?s=type:GC::chlorothalonil::fluvalinate

We have done this one only using comprehensive (GCxGC-TOF). Here Chlorothanil is also determined. Fig 1 shows a typical chromatogram. In the second dimension the Rtx-200 was chosen showing very good separation of peaks of interest over the whole separation space.

fig. 1 pesticides GCxGC

Fig 1: separation of pesticides using GCxGC-TOF, 30m x 0.25mm Rxi-5SilMS in first dimension and Rtx-200 in second dimension

Benzo[b]fluoranthene as part of the EFSA PAH4

I’ve already had one reader of my last post, Is separation of benzo[b]fluoranthene and benzo[k]fluoranthene on 5% phenyl-type GC columns really that important for environmental analyses? ask why I had benzo[b]fluoranthene in red text on the chromatograms.  I had planned to include something about it being on the EFSA PAH4 list in that post, but I’m an old man, so I forgot.  Here goes, in more detail…

The European Food Safety Authority (EFSA) in their Polycyclic Aromatic Hydrocarbons in Food – Scientific Opinion of the Panel on Contaminants in the Food Chain (Adopted on 9 June 2008), issued the following statement:

“The CONTAM Panel concluded that benzo[a]pyrene is not a suitable indicator for the occurrence of PAHs in food. Based on the currently available data relating to occurrence and toxicity, the CONTAM Panel concluded that PAH4 and PAH8 are the most suitable indicators of PAHs in food, with PAH8 not providing much added value compared to PAH4.”

The PAH4 are:

Benz[a]anthracene, Chrysene, Benzo[b]fluoranthene, Benzo[a]pyrene

The PAH8 are:

Benz[a]anthracene, Chrysene, Benzo[b]fluoranthene, Benzo[k]fluoranthene, Benzo[a]pyrene, Indeno[1,2,3-cd]pyrene, Dibenz[a,h]anthracene, Benzo[ghi]perylene

As you can see, Benzo[b]fluoranthene is a member of the PAH4, possibly the most significant group in estimating carcinogenic potential of food due to PAH content. Importantly, this doesn’t mean that benzo[j]fluoranthene and benzo[k]fluoranthenes are NOT present in food; they are, anytime benzo[b]fluoranthene is present.

The point I was going to make is, that in a pinch, an Rxi-5ms might be a better choice for screening of the EFSA PAH4 in food, because it would only have the absolute coelution of triphenylene and chrysene (at m/z 228). But a better choice is an Rxi-PAH GC column that will separate:

Triphenylene and Chrysene (m/z 228)

Benzo[b]fluoranthene, Benzo[k]fluoranthene, Benzo[j]fluoranthene, and Benzo[a]fluoranthene (m/z 252)

Benzo[e]pyrene and Benzo[a]pyrene (m/z 252)

Dibenz[a,j]anthracene, Dibenz[a,c]anthracene, and Dibenz[a,h]anthracene (m/z 278)

Is the Rxi-PAH only suitable for food PAH analyses? Absolutely not.  It will work very well for environmental PAH analysis where more comprehensive profiling is desired (e.g., environmental forensics, such as oil spill and other fingerprinting work), including as a primary column in GCxGC-TOFMS.  But keep in mind since it was designed with a thin film to more easily elute involatile PAHs like coronene and dibenzopyrenes, you need to inject less material on it or trim the front of the column more frequently for maintenance purposes.

Now where did I put those car keys?

Is separation of benzo[b]fluoranthene and benzo[k]fluoranthene on 5% phenyl-type GC columns really that important for environmental analyses?

Some gas chromatographers might say so because of the following statement in US EPA Method 8270D (Semivolatile Organic Compounds by Gas Chromatography – Mass Spectrometry (GC-MS):

“11.6.1.4 Structural isomers that produce very similar mass spectra should be identified as individual isomers if they have sufficiently different GC retention times.  Sufficient GC resolution is achieved if the height of the valley between two isomer peaks is less than 50% of the average of the two peak heights.  Otherwise, structural isomers are identified as isomeric pairs.  The resolution should be verified on the mid-point concentration of the initial calibration as well as the laboratory designated continuing calibration verification level if closely eluting isomers are to be reported (e.g., benzo[b]fluoranthene and benzo[k]fluoranthene).”

The language above ignores the fact that benzo[j]fluoranthene is a polycyclic aromatic hydrocarbon (PAH) isomer consistently found at significant levels in environmental (and food) samples, and more importantly, that it coelutes with either, or both, benzo[b]fluoranthene and benzo[k]fluoranthene on 5% phenyl-type GC columns. In this blog post, I will define the extent of the coelution on 30m x 0.25mm x 0.25µm Rxi-5Sil MS (5% phenyl as silphenylene, similar to DB-5MS) and 30m x 0.25mm x 0.25µm Rxi-5ms (5% phenyl as diphenyl, similar to DB-5).

PAHs were first analyzed using hydrogen efficiency-optimized flow (EOF), and optimal heating rate (OHR), with an Agilent 6890 GC-FID. Injections of PAH standards (prepared from SV Calibration Mix #5 / 610 PAH Mix and a custom benzo[j]fluoranthene standard) at a split ratio of 100:1 into a 4mm Precision split liner with wool served to minimize injection band widths to keep overall system efficiency high.  In addition to OHR, chromatograms for each column were generated at 1.5 x OHR and 0.5 x OHR to force different elution temperatures for the benzofluoranthenes to see what effect that had on their separation.

As you can see in Figure 1, the Rxi-5Sil MS shows an intractable coelution for benzo[b]fluoranthene and benzo[j]fluoranthene under all experimental conditions, with elution temperatures around 278, 293, and 257°C for OHR, 1.5 x OHR, and 0.5 x OHR, respectively.  The Rxi-5ms has a different coelution, that of benzo[j]fluoranthene and benzo[k]fluoranthene for OHR and 1.5 x OHR, but interestingly is able to partially resolve the benzofluoranthenes under 0.5 x OHR conditions (Figure 2).  Elution temperatures in this case were approximately 280, 295, and 258°C (OHR, 1.5 x OHR, 0.5 x OHR).

What does this coelution situation mean for environmental analysts? First, all quantitative values reported for benzo[b]fluoranthene and benzo[k]fluoranthene are likely inaccurate for at least one of the isomers depending on the GC column choice if using a “5-type” stationary phase.  Second, technically speaking, analysts should not be reporting the benzofluoranthenes as “individual isomers” according to the language of EPA Method 8270D when using a “5-type” GC column.

If an analyst needs to report benzofluoranthenes as “individual isomers”, a 30m x 0.25mm x 0.25µm Rxi-17Sil MS (or even a 15m version) will work well.  If the challenge is to separate the benzofluoranthenes and, triphenylene and chrysene (228 m/z coelution on 5-type columns), consider the Rxi-PAH GC column.

BF Fig 1

 

BF Fig 2

Sample loading capacity for PAHs on a 30m x 0.25mm x 0.25µm Rxi-5ms GC column

Sample loading capacity for a GC column (also known as “column capacity” and “sample capacity”) is essentially the amount of non-active compound that can be put on a GC column and chromatographed at some set of conditions where the peak shape is symmetrical. Conversely, if a GC column is overloaded with a component amount, the peak will exhibit the classic “shark fin” shape.  Most vendors estimate the sample loading capacity for a 0.25mm x 0.25µm GC column to be around 50-100 ng per analyte.  But is this even close to accurate?  Perhaps, but it depends on the compound of interest.

Environmental analysts already know that polycyclic aromatic hydrocarbons (PAHs) are prone to overload on GC columns, especially the typical “five” type columns used for semivolatile organic compound analysis in EPA methods, e.g., 8270 and CLP. Although the CLP semivolatiles method states that a GC column should be able to “accept up to 160 ng of each compound listed in Exhibit C (Semivolatiles), without becoming overloaded”, I could find no quantitative data indicating when overload occurs for Exhibit C PAHs on the commonly used 0.25mm x 0.25µm GC columns.  That’s my lead-in to say, “I’ll test it myself!”  With the follow-up… “And post about it on ChromaBLOGraphy”.

All work was done with a 30m x 0.25mm x 0.25µm Rxi-5ms utilizing hydrogen efficiency-optimized flow, and optimal heating rate, with an Agilent 6890 GC-FID.  Injections of PAH standards (prepared from SV Calibration Mix #5 / 610 PAH Mix) at a split ratio of 10:1 into a 4mm Precision split liner with wool served to minimize injection band widths to keep any peak deformations associated with the column, and not the inlet.  As you can see in Figure 1 by the “shark fin” shape of the peaks and the unacceptable resolution between benzo[b]fluoranthene and benzo[k]fluoranthene, and indeno[123-cd]pyrene and dibenzo[ah]anthracene, 200 ng of each of these PAHs greatly exceeds the sample loading capacity of this column. Figure 2, at 50 ng each compound, also shows gross overloading of PAHs, and although the separation starts to improve between critical pairs, the retention times are still shifted later based on peak deformation.  Finally, at 12.5 ng (Figure 3), the chromatogram looks good in separations and retention times, comparing relatively nicely to Figure 4 at 3.13 ng, where we would not expect overload.

Even at 25 ng of later eluting PAHs (Figure 5), we have overload, which puts the maximum sample loading capacity of the 30m x 0.25mm x 0.25µm Rxi-5ms GC column under these operational conditions between 6.25 and 25 ng each PAH, an order-of-magnitude less than what is suggested to be possible by the CLP method.  Can we tolerate overload and still get our work done?  That depends on your data quality objectives, which might include:

Separation between critical, often isobaric, pairs.

Overall required peak capacity for the chromatogram (the 200 ng overload cut the peak capacity by almost 3).

Capacity of the detector to handle overload (MS systems are very sensitive these days).

I prefer the peaks look more like those seen in Figure 7, and given the sensitivity of today’s MS systems, we can inject less (maybe via “Shoot-and-Dilute”), see almost as much, and probably keep our systems up longer due to less involatile “dirt” put on the GC column.

Figure 1 OL

 

Figure 2 OL x

 

 

Figure 3 OL

 

Figure 4 OL

 

Figure 5 OL

 

Figure 6 OL

 

Figure 7 OL

 

 

 

What are these O-rings for that I received with my baseplate trap?

Some of you have noticed that included with several of our baseplate traps are a package of two small O-rings. Technical Service has been informed that these are the O-rings which fit onto the check valves on your baseplate (see red arrows below).  Does this mean that you need to replace these O-rings?  Not necessarily.  Carefully inspect the O-rings on the check valves, and if you do not see any signs of wear (cracks, deformation, etc…), you probably don’t need to replace them.  However, if they do exhibit signs of wear, go ahead and replace them.

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 Baseplate 4

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If you also need the larger O-rings that are located on the baseplate, at the base of the check valves (as identified by the green arrows), you can obtain those by purchasing Restek catalog number 22023.

22023