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Cannabidiolic Acid (CBDA) Reference Standard now Available!

Today is a momentous day! Restek’s new CBDA standard is live and for sale. There has been a ton of interest in this compound in the cannabis industry, but it has historically been pretty much impossible to source. Restek now has this ISO Guide 34 and 17025 – certified reference standard available at 1mg/mL in acetonitrile. Acetonitrile is an LC-friendly solvent that also helps to enhance the storage stability of this compound. Including this standard in your LC potency analysis will allow for accurate quantification of CBDA in cannabis products. For pricing and ordering information, contact one of our friendly customer service representative at csreps@restek.com or call 800-356-1688, ext. 3.

While this standard is definitely a little pricey, a little bit of it can go a long way. In solution under refrigeration, this standard is stable for months at a time. During analyses, only a small amount is injected, so to stretch the standard you have, you can use limited volume inserts (LVIs) in your autosampler vials. Without an insert, a minimum of ~0.5mL is required in the bottom of the autosampler vial in order for the autosampler needle to get to the sample. These inserts allow as little as 50µL to be used in the vial while still being accessible to the autosampler needle. Using these low-cost inserts can really save on standard consumption, which can translate to significant cost savings for standards. Just be sure not to fill the LVIs all the way to the top – some room needs to be left for the volume of the syringe during sampling.

My preferred vial/cap/LVI combination consists of the 2mL, 9mm Short-Cap, Amber Screw-Thread Vials, Short-Cap, Preassembled Screw-Vial Closures with PTFE/Silicone Septa, and 50µL Glass Big-Mouth Inserts with Bottom Spring. This combination works on both Agilent and Shimadzu GC and LC autosamplers, so they can be used throughout your lab, and the screw caps allow for easy re-capping of your standard vials, reducing evaporation, which allows your standards to last even longer. If you have a different instrument configuration, check out our handy instrument reference for these vials.

I’ll stop the autosampler and melt the LVI with you…

Another interesting story involving Jack and limited volume inserts (LVI)….

see the first…

 

melted LVI single_v3

The Raptor Biphenyl blows away a conventional C18 in this gunpowder assay

Structures Apr2014 BlogBiphenyl propellants cgram Apr2014

A very enjoyable aspect of my position within Restek is the opportunity to work with an experienced and interesting network of chromatographers throughout Europe. This includes the talented scientists at ALSAC in Uppsala, Sweden, who recently evaluated a Raptor Biphenyl column in a customer assay of gunpowder materials. ALSAC (Addtech Life Science Applications Center) has a strong focus on applications/method development and education for their network of internal and external customers.

The target compounds, ethyl centralite (1,3-diethyl-1,3-diphenylurea, or EC) and 2N-Diphenylamine (DPA) are used as stabilizers in standard propellant. Quantitative determination of these analytes is routinely performed by QC departments after the production of gun powder. DPA can also be used as a pre- and postharvest scald inhibitor for apples. Both analytes are rather polar and insoluble in water. In conventional LC, they elute very early on a standard C18 column and are not well resolved, even with a complex mobile phase gradient. This makes accurate quantification very difficult.

Fortunately, we have LC column choices beyond a C18 for compounds of this type! Raptor (speed and efficiency with a superficially porous particle) and Restek’s own biphenyl phase (excellent aromatic selectivity) result in significantly better retention and selectivity. The end result is a fast assay with great resolution, using a simple mobile phase (0.1% TFA in water : acetonitrile) and short gradient program.

Many thanks to Ilya Zelikman, Ph.D., from ALSAC in Uppsala, Sweden for sharing this interesting project and data with us.

The International Network of Environmental Forensics 2014 Conference

800px-St_johns_rear_buildingsThe International Network of Environmental Forensics (INEF) 2014 Conference will be held at St John’s College in Cambridge, UK from August 4-6.  Environmental forensics involves the use of a broad array of scientific techniques to not only identify a pollutant in the environment, but also apportion its introduction source and timing, often in support of finding a responsible party for a legal claim.  The INEF 2014 Conference will bring together scientists and other environmental forensics experts for lectures, posters, and discussion.  For you analytical chemists, there will be GC and GCxGC and LC and more.  I am on the Programme Committee (notice the extra “me” on Program in honor of our generous hosts in the UK!), and invite you to attend this meeting, or even contribute, as the abstract deadline has been extended to April 21.  See the brochure for additional details.

I hope to see you in Cambridge.

Jack Cochran

TO-15 dilution factors, sample volumes, and the all new Mega Can Calculator – V1.02

So I was recently asked “how do I calculate the dilution factor on a canister?” Now ya’ll get this blog as a direct result. So here we go with the following example…

First we start with a clean and fully evacuated canister. Regardless of size a fully evacuated canister will have a pressure of -14.696 psig (note the g for gauge). Also note that -14.696 psig = -29.92” Hg.

Next we ship the canister out to the field and a sample is collected. Typically a canister is sampled until -2.5 psig (-5.09” Hg). Yes, not all of the canister is consumed. This has to do with the capabilities of maintaining “constant” flow with a passive flow controller, but that is for another blog.

From there we receive the canister in the lab and then very often the canister is pressured to 5 psig before analysis (this makes for faster loading of the sample onto the preconcentrator).

So how much has my original sample been diluted by going from -2.5 psig to 5.0 psig. To calculate the dilution factor for this canister we use the following equation:

Dilution Factor = [Pressure (after dilution) + Pressure (atmospheric)] / [Pressure (atmospheric) + Pressure (before dilution)]

Dilution Factor = (5 psig + 14.696 psig) / (14.696 psig + (-2.5 psig) = 1.61

So the concentration we get out of this canister we multiply it by 1.61 to get the dilution corrected concentration.

Now very often during the same conversation I get asked “what is my sample volume?”

Keeping with the same example, we calculate the volume of sample in the canister by using the following equation:

Sample Volume = Pressure Change / Initial Pressure x Canister Volume

Sample Volume = -14.696 psig – (-2.5 psig) / -14.696 x 6000 mL

Sample Volume = 4979 mL

For those of you who do not want to reinvent the wheel… I now offer you the

Mega Can Calculator – V1.02

Which includes dilution factors and sample volumes (before and post dilution).*

 

 

 

 

 

 

 

 

*Model not included with calculator.

Do I smell Cannabis in the Lab?

2011-jaap-pasfoto4-smallLast week I was traveling in Europe to present seminars on practical topics like trace analysis, faster analysis and troubleshooting.

During such seminars you visit companies and you always learn something.  One of the companies we visited was a company that did forensic analysis and were specialized in cannabis measurement.  My colleague already explained to me, that when entering the company, I probably would smell a familiar smell.

He was referring to the typical Dutch image because of the tolerance towards growing and using Cannabis, which you can purchase everywhere in the Netherlands at the so-called “coffee shops”.

coffee shop

When we presented the seminar we also discussed about the possibility of clogging the split lines as was published some time ago, see:  http://blog.restek.com/?p=5454.  Split lines do not only “clog”, the splitted sample components will also get into the lab.

 

When we discussed about “where the sample went”,  when it was splitted off, an interesting discussion started. The sample components that are splitted off, are normally trapped in an in-line split filter. (or trap).   Such filters are filled with charcoal type  adsorbent and are standard installed when the GC is setup. However, they need to be replaced very regularly as this “trap” will behave like a normal “packed” column, and components will break through once the filter is saturated.

Split line filters are available, see: http://www.restek.com/Supplies-Accessories/GC-Accessories/Gas-Purification?s=type:spl_vent

In this lab, It seemed that the methods used for cannabis measurement, were split methods, meaning a significant part of the sample was split-off. As the split-vent traps were not regularly replaced, it would explain why there was a clear “smell” of cannabis hanging around these labs.  It may also explain why the analysts usually were in pretty good mood. fly

Dimensions of NORM-JECT® syringes

We often get asked for dimensions of our NORM-JECT® syringes.  As a result, I decided to take the information tech service has been provided and put it in a convenient place for our customers.  Below is the information for Restek part #’s 22766 through 22778.  For those of you who need it, I hope you find this information useful.

NJf2

Resurrecting an old reverse phase LC column- should I, could I, how would I???

Well, if we had an Easter egg hunt and you found an egg that was from last year or the year before, you could probably tell the difference and you would know pretty quickly if it had gone bad.  Let’s just say that is not so easy with HPLC columns. If you’ve dealt with this much, you’re aware that it is difficult to fully know the history of column usage for one that you find in a drawer somewhere. That is the biggest problem.  Maybe it was in good shape when placed in storage or maybe not. Maybe it was stored properly or maybe it wasn’t.  If there is any question about the proper way to store, please see the LC column usage and care instructions.

If it is a rugged column phase such as a C18 and it truly has not been used, its condition will depend on how well it was sealed on the ends, the climate/temperature and what solvent it contained. Theoretically it should be good as new if sealed well, stored properly and no extreme high temperatures are encountered. If it has been used, then its future is not so full of promise.

The worst case scenario would be if it was stored in mobile phase containing buffer salts and then it dried out. A dead giveaway for something like this would be extreme pressure issues if you were to start pumping solvent through it. Things almost never end well when this happens. Other minor issues could occur relating to leakage of solvent over time and drying of the packing material. The result of this could be pressure issues and/or some things that look like phase collapse.

 

Medic istock

 

If you are determined save a column that you’ve just pulled out of storage, try the following:

  1. At a reduced flow rate, pump several column volumes of 40:60 ACN/water (in case any buffer salts are inside). Observe the pressure rating throughout this process.
  2. If you do experience any difficulties with high backpressure at this point, stop the flow and let the pressure dissipate.  Try it again at a lower flow rate and give it some more time.  If the pressure is still excessively high, skip to #5.
  3. Once your pressure is in normal range, increase the flow rate gradually to your normal operating flow rate or close to it.
  4. Pump several column volumes of ACN (For rewetting in case the phase has started to collapse or the packing is dried out). Continue to observe pressure. You should see it decrease as more of the water is replaced by organic solvent.
  5. If you continue to have trouble with pressure, for some columns, you can try flushing in a backward direction. To do this, please see our  LC Cleaning Recommendations (Note: Pumping in a backward direction is not recommended for UHPLC -1.9 µm Pinnacle DB and Raptor™ Columns, although you can still pump through a series of solvents as described in a forward direction).  For these purposes, you can use ACN and MeOH interchangeably.
  6. If you do not (or no longer) experience high pressures, proceed to pump the mobile phase that you plan to use. Equilibrate with at least 7 column volumes before attempting any injections.
  7. If it does not seem that you are making any progress, contact us before proceeding. It may or may not be worth continuing.

If you have tried these suggestions and still have issues with backpressure or poor resolution, then the column has exceeded its lifetime and not likely to be revived. It’s time to replace the column and go home.

Fatty Acid Methyl Esters (FAMES) – converting % by weight to µg/mL

The first time I was asked by a customer about how to convert % by weight of one of our FAME reference standards to µg/mL, I needed to ask for some help.  Because we (tech service) occasionally get asked this question, I thought I would show the calculation in a post.

 

Let’s take Restek catalog number 35077 as an example.  The overall concentration of this Food Industry FAME Mix is 30mg/mL.   Individual compound concentrations range from 2 to 6% by weight.   So what are the individual compound concentrations in µg/mL?   I’m not going to list them all, but rather show you how to perform this calculation.

We list the first compound as C4:0 and at 4% by weight.  Since the total concentration of 35077 is 30mg/mL, to determine the concentration of C4:0 in µg/mL:

4/100 x 30mg/mL = 0.04 x 30mg/mL = 1.2mg/mL

To convert to µg/mL:  1.2mg/mL x 1000µg/mg = 1200µg/mL

 

          ampules

 

If you purchase a neat standard like 35066, then there will be one extra step to obtaining the final answer.   This catalog number contains approximately 100mg.  For the sake of simplicity, let’s say you remove exactly 100mg and dissolve this material into 10mL of methylene chloride.  This will produce a solution with a concentration of 10mg/mL.   Once again, I will use the first listed compound (C14:0 in this case) for an example calculation.

C14:0 is in the neat material at 6% by weight.  Since the total concentration of the solution you prepared is at 10mg/mL:

6/100 x 10mg/mL = 0.06 x 10mg/mL = 0.6mg/mL

To convert to µg/mL:  0.6mg/mL x 1000µg/mg = 600µg/mL

 

I hope you have found these examples helpful the next time you need to perform similar calculations.  Thanks for reading.

World Record Set for Longest Gas Chromatography Retention Time!

My colleague Jaap de Zeeuw holds a Guinness World Record certificate for making and applying the longest GC column ever, 1300m.  That’s quite a feat and I’ve often wondered what the retention times were for that column when you consider the holdup time was probably on the order of hours instead of minutes.  No matter how long the retention times were though on that 1300m GC column, I may have exceeded them on a simple 30m x 0.25mm x 0.25µm Stabilwax, which I’ve been using for a selectivity study conducted by colleagues James Harynuk and Teague McGinitie at University of Alberta.  This work will be presented at the 11th GCxGC Symposium (and 38th International Symposium on Capillary Chromatography) in Riva del Garda, Italy.

The study includes, Rxi-1ms, Rxi-17Sil MS, Rtx-200, and Stabilwax GC columns, which represent a variety of stationary phase polarities and selectivities.  The first 3 have temperature stabilities of 350, 360, and 340°C, respectively, but the Stabilwax only goes to 260°C.  I can chromatograph all of the molecules chosen for the study on the first 3 phases, but when I get to the polycyclic aromatic hydrocarbons (PAHs), some of which are notoriously involatile, I struggle on the Stabilwax.

The first figure below shows the Acquired Sample list for the Stabilwax work, and I purposely started with SV Calibration Mix #5 / 610 PAH Mix because I knew some of the compounds would be hard to elute from the wax GC column.  Well, how about almost impossible to elute?  As you can see in the first chromatogram, even though I had a 40 min final oven temperature hold time at 250°C, none of the PAHs with molecular weights of 252, 276, and 278 eluted.  In chromatogram 2, which is run 3 from the queue, I see carryover peaks for 252 PAHs (benzofluoranthenes, benzo[a]pyrene), but still didn’t get complete elution of the PAHs even though I pushed the final hold time up to 90 min.  OK, this isn’t going so well, this part of the selectivity study…

Eventually, after uninstalling the Stabilwax column, putting it in my office for a few days, and then reinstalling it to run different standards, I finally saw the first 276 and 278 PAHs eluting as massively broad peaks, almost 5 min wide at base.  When I calculate retention time (yeah, sure, I included the time in my office!) for indeno[1,2,3-cd]pyrene, it’s 7.25 days!

Your move, Jaap…

Sample Queue

 

Chro SV-5 01

 

Chro SV-5 03

 

Chro 8270-2 PAH carryover