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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
Which includes dilution factors and sample volumes (before and post dilution).*
*Model not included with calculator.
Last 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”.
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.
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.
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.
If you are determined save a column that you’ve just pulled out of storage, try the following:
- 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.
- 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.
- Once your pressure is in normal range, increase the flow rate gradually to your normal operating flow rate or close to it.
- 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.
- 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.
- 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.
- 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.
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
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.
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…
During recent discussions with colleagues, it became apparent that my interpretation of the internal standards (ISTDs) used for the U.S. Environmental Protection Agency’s (EPA’s) Method TO-15 may not mean the same thing to everyone. So before I kick off this discussion, let me first breakdown the terminology (as defined by me) to establish some common ground:
- Tuning Standard: Utilized to “tune” or optimize the MS detector parameters to ensure that mass assignments and abundances are correct. TO-15 utilizes 4-bromofluorobenzene (BFB) as a tuning standard.
- Internal Standard: Utilized for calibration and quantification by accounting for instrument variability in response and/or retention time from run to run. TO-15 utilizes bromochloromethane, 1,4-difluorobenzene, and chlorobenzene-d5.
- Surrogate Standard: Utilized to monitor for bias/variability throughout the entire sample extraction process. TO-15 makes no mention of surrogate standards; however, they are generally compounds similar to the target analytes in chemical composition and behavior in the analytical process, but which are not normally found in environmental samples.
- We only need the aforementioned definitions, so I will not be covering any of the following: matrix spikes, blank spikes, field duplicates, laboratory control spikes, matrix spike duplicates, method blanks, and the list goes on infinitum… depending the analyst, laboratory, and/or method.
Now that we have our terminology squared away, let us use EPA Method 625 as a benchmark for our discussion. With 625 you add surrogate standards to the sample pre-extraction, which allows you to evaluate your extraction efficiency… and then you add internal standards post-extraction, but pre-analysis, which are utilized for quantification. Of course all of this is injected on an instrument, which has been tuned.
Now with that paradigm at the forefront of our minds, it is important to note that most of the commercially available TO-15 preconcentrators add the “ISTDs” (quotation on purpose… hint hint) from a separate, designated canister onto the preconcentrator traps for each sample analysis, prior to the loading of the sample. So therefore the TO-15 “ISTDs” go through almost (I will explain in the next sentence) the entire “sample extraction” process. To be more in line with the 625 paradigm the standard(s) would have to be added to each canister prior to extraction, which is feasible, but just not done because the TO-15 preconcentrators allow end user to cheat this step.
In any event…any variability associated with the extraction process (e.g., breakthrough, relative humidity effects, desorption efficiency, etc…) would be represented by a change in response in the ISTDs. This… to me… sounds more like a surrogate standard/internal standard hybrid. You get the best of both worlds in my opinion and you do not have to do anything but let the software work for you.
But I already know my opinion (and now you do)… so I want to know what others are thinking. So if you care about air (I know… a little lame) please feel free to chime in here? Do you consider the TO-15 ISTDs to truly represent just ISTDs or are they more like surrogate standards… and why?
After a long and cold winter here at the Restek headquarters in Bellefonte, PA, USA it seems that spring is finally here! It is so nice this time of year to get outside and enjoy the weather and the beautiful outdoors that Pennsylvania has to offer. Unfortunately after a hike in the woods there is always the required tick check. Ticks are nasty little arachnids and can carry a number of diseases, including Lyme disease. I’ve had to pull a few off of myself, but animals seem to be even more susceptible to getting ticks since they are closer to the ground and walk through the grass and brush where ticks like to hide out. Repellants like DEET (N,N-diethyl-m-toluamide) can be applied on exposed skin and clothing to protect from ticks. Permethrin containing products can also be used to treat clothing and other outdoor gear that may be exposed. It is always important to read the application instructions carefully and apply the product only as directed (for you and your animals!).
Another thing to consider is that many of these topical treatments and other personal care products eventually end up in our streams and lakes. Jack Cochran and I, with the help of Cory Fix, worked on a project where we were using GCxGC-TOFMS to evaluate the Las Vegas Wash, an urban river that flows into Lake Mead. In order to see very low levels of both targeted and non-targeted analytes we took 4L of wash through a disk extraction and concentrated to a final volume of 1 mL. We found a lot of interesting things in that water and of course DEET was one of them!
Patterns in your HPLC chromatography can exhibit telltale signs that point toward probable sources of error. In this brief series of posts, we have looked at possible scenarios that you may encounter in the laboratory and how you might approach resolving those difficulties.
Any time you have difficulty with poor peak shape, it is also likely you’re having an issue with loss in response. For this reason, you may also find the previous two posts in this series helpful:
As in the previous posts for this series, these suggestions assume that you are working with an established method and had successful results in the past. As always, it is critical to note when the change occurred and how it might correlate to changes in the system. Equally important is to change one thing at a time to identify the source(s) of difficulty. The following are examples of things that could contribute to fronting. I suggest investigating these in this order:
1. Column phase collapse- This is easy to rule out because it is always accompanied by an obvious shift to shorter retention times. This is something that can occur in reverse phase LC when using mobile phases that are more than 95% aqueous. Usually this can be resolved by pumping 100% acetonitrile for several column volumes. It is best to avoid risking phase collapse by using an appropriate column phase, such as our Ultra Aqueous C18 or Pinnacle DB Aqueous C18 columns, when highly aqueous mobile phases are used. For further reading on the topic, please see the following:
2. Incompatible solvent composition of sample- As mentioned in the earlier trouble shooting post on Loss in Response for Some, but Not All Analytes, this can impact the shape and sensitivity. This is often more pronounced for the early- eluting peaks.
3. Volume overloading-Injecting too large of a volume can result in fronting, since it broadens the peak. You can eliminate this possibility by injecting a smaller volume. Some general guidelines as far as suggested volumes can be found in the FAQ section on our website.
4. Mass overloading- Injecting a solution that is too concentrated in terms of sample material (including matrix) can result in fronting. Often a slight shift to earlier retention times is also observed. Try diluting the solution by a factor of 10 or so and injecting it again. If this is the problem, remember that quantitation standards and samples will need to be injected at a lower concentration to avoid this on an ongoing basis. In some cases, an improved procedure for extract cleanup, prior to HPLC may be needed.
5. Interfering coelution- As is the case with tailing, an interfering contaminant that coelutes can make a peak look like it is fronting. If that is the case, you might see the peak shape change from time to time, or there may be a difference between your unknown samples and your quantitation standards. If you think this might be happening, try using a slower gradient or changing your organic/water ratio and see if the peak separates into two or develops a shoulder.
6. Increase in dead volume- In particular, dead volume at the point of sample introduction onto the column can create a fronting peak shape. This will be most obvious for columns with smaller ID and might be more evident with early eluting peaks, although you may see an effect for all analytes. Make sure the tube fitting at the inlet of the column is fully seated and that you are using the proper fitting and ferrule. If you are using a guard holder that accommodates a cap frit, such as catalog # 25084, make sure that a cap frit is installed; otherwise, dead volume would result.
7. Other physical damage to column- This phenomenon is described in the earlier post Troubleshooting HPLC- Tailing Peaks. A physical change like this is more likely to cause fronting if the anomaly occurs at or near the column inlet. This would most likely affect peak shape for all analytes and may or may not be accompanied by a change in column pressure. A column with this type of damage should be replaced.
Thank you for reading. This concludes this series of posts.
Release of dangerous substances from Construction products – The Difficulty of harmonizing Methods in Europe
Indoor air quality is as important for human health as quality of food or drinking water. Most of us are spending a high percentage of our living time in closed rooms. If, during work time, we are exposed to concentrations of dangerous substances, released from the working process, measurements are mandatory which ensures that these concentrations do not exceed given MAC-values (maximum allowable concentration). At home we may be exposed to a large number of substances known as carcinogenic or toxic, which are released from different construction products like paints, glue, sealing materials, or carpets and other floor coverings. Despite the large amount of potentially dangerous substances around us in our homes, no regulation requires regular measurements. A suitable way out of this problem is to regulate the construction products prior to their introduction to the market.
In Europe the CE-mark of conformity is mandatory for every manufacturer or importer of a certain product, if this product is to be introduced into the European market. Herein the manufacturer or importer claims the product is developed and manufactured in accordance with the product related European Norms (EN).
The basic description in how construction products should be handled is part of the European Legislative Process. The European Community has governed the certification of construction products by the “Construction Products Regulation” (CPR – former CPD – Construction Products Directive).
CPR defines basic requirements for construction works (BWR3) as follows:
“…the construction works must be designed and built in such a way that they will, throughout their life cycle, not be a threat to the hygiene or health and safety of their workers, occupants or neighbours, nor have an exceedingly high impact, over their entire life cycle, on the environmental quality or on the climate, during their construction, use and demolition, in particular as a result of any of the following
a) the giving-off of toxic gas;
b) the emission of dangerous substances, VOC, greenhouse gases or dangerous particles into indoor or outdoor air;
c) the emission of dangerous radiation;
d) the release of dangerous substances into ground water, marine waters, surface waters, or soil;
e) the release of dangerous substances into drinking water or substances which have an otherwise negative impact on drinking water;
f) faulty discharge of waste water, emission of flue gases or faulty disposal of solid or liquid waste;
g) dampness in parts of the construction works or on surfaces within the construction works.”
Based on this CPR regulation European Norms are developed by Technical Commitees (TC) related to a specific construction product, e.g. CEN/TC 134 “Resilient, textile and laminate floor coverings”, responsible for “Standardization of definitions, requirements, classification and test methods and provision of guidance documents and reports for resilient and textile floor coverings and for laminated floor coverings”.
It is the task of these TC’s to harmonize existing National Normings and develop a harmonized European Norm (hEN) which specifies the named construction products. The CPR/BWR3 is one of the great challenges to the TCs which also have to harmonize national ideas of construction products with these basic requirements.
Being part of such a complicated process, TCs often develop their own methods and standards, even if similar behaviours are surveyed, e.g. the release of Volatile Organic Compounds from different construction products. Since indoor air quality is determined by the behaviour of all different construction products, one of the big challenges for the European Community is to standardize tests and analysis methods between different TCs. This process is called “Horizontal Standardization”.
The process of Horizontal Standardization starts with Expert Groups of the Member States of the European Union, organized in one of the Commission’s “Directorate-General (DG)”. For the regulation of construction products the most involved Directorate-General would be the DG Enterprise and Industry. However, since we are talking about risks stemming from the release of dangerous substances into the environment, the DG Environment and possibly also the DG Energy have to be involved.
These DGs will decide to give a mandate to the European Norming Commission (CEN), and then to a leading TC, to horizontally standardize the norms describing the different construction products. The mandate for Horizontal Standardization and Harmonization of the considered European Norms (EN) for construction products is named M/366 and it was assigned to the CEN/TC 351, responsible for construction products.
The full name of this mandate is: “Horizontal complement to the mandates to CEN/CENELEC concerning the execution of standardization work for the development of horizontally standardized assessment methods for harmonised approaches relating to dangerous substances under the Construction Products Directive (CPD)”
During this process of Horizontal Standardization and Harmonization the people in charge had to handle:
- Over 65 product TCs and environmental TCs
- European Commission: SCC, DG Enterprise, DG Environment
- 27 Member States / 30 CEN Members
- Industry, industrial sectors
- CEN bodies: CMC, CSN, CSNPE, SABE
- Regulators, experts from various fields
- Liaisons with interest groups
To keep workload of the acting people manageable, TC 351 has decided to divide the work into 5 Working Groups (WG).
|CEN/TC 351/WG 1||Release from construction products into soil, ground water and surface water|
|CEN/TC 351/WG 2||Emissions from construction products into indoor air|
|CEN/TC 351/WG 3||Radiation from construction products|
|CEN/TC 351/WG 4||Terminology|
|CEN/TC 351/WG 5||Content and eluate analysis in construction products|
Table 1: Technical Bodies of CEN/TC 351 – taken from the homepage of CEN – European Committee for Standardization. HTTP://www.standards.cen.eu
As a company of chromatographers, Restek is mostly interested in the results of the Working Group 2 (WG 2) “Emission into Indoor Air” and how the classification of construction products are realized for this approach.
During the last years WG 2 has developed a horizontal testing standard for the emission of volatile compounds from construction products. This CEN/TS 16516 now has to be adopted into the product standardizations by the TCs of the specific construction products. Until now the CEN/TS 16516 is not a European Norm, but a Technical Specification (TS), seen as a preliminary stage of a European Norm. The complete norming process is promised to be finished during 2015/16.
The CEN/TS 16516 is described as follows:
“This Technical Specification specifies a horizontal reference method for the determination of emissions of regulated dangerous substances from construction products into indoor air. This method is applicable to volatile organic compounds, semi-volatile organic compounds, and volatile aldehydes. It is based on the use of a test chamber and subsequent analysis of the organic compounds by GC-MS or HPLC.” The quoted text was cited from the CEN Homepage.
One of the big International Laboratory Associations, the Eurofins Group, announced in January 2014: “CEN/TS 16516 will become the “mother of all VOC emissions testing standards” for construction products in Europe within the next months and years. It will form the basis of VOC emissions testing for CE marking as soon as the criteria for CE marking have been specified in near future.” (Eurofins Homepage).
Unfortunately, in Europe the situation for the regulation of products is a bit more complicated than in other economic areas. Responsiblity for the risk assessment of dangerous substances lie with the National Authorities (e.g. for Germany the National Authority is the Federal Institute for Risk Assessment (BfR) in Berlin).
National Authorities may choose different ways of assessing the risk of the release of dangerous substances from construction products. Up to now several quality rating systems are established in Europe. The following parameters are included for testing:
Formaldehyde and acetaldehyde are mandatory for all EU countries. In France a “Compulsory VOC emissions labelling” is installed with 10 VOCs and TVOC (Total Volatile Organic compounds) as basic testing values to run into an ABCD label, classifying the construction products. Germany bases his classifying system on a List of more than 200 compounds, published by the Committee for Health-Related Evaluation of Building Products (AgBB), latest version in 2012. This LCI-list (Lowest Concentration of Interest) includes VOC and SVOC compounds as well as a maximum TVOC value. The Belgium classifying system, at the moment, is referring to another list, the EU-LCI-list, including 177 VOC and SVOC compounds of interest. All other EU countries, at the moment, do not have any classification system.
In a country without regulation on VOC emissions, a CE marked product can be sold with the NPD option (“no performance declared”), in a country with existing regulation on VOC emissions this product only can be marked with a CE label if the declaration of performance includes VOC emissions test data, in conformity with the national regulation.
Regarding the production of reference materials, the situation is not easy to overcome. A certified Multi Parameter Reference Material Kit, as Restek has done for VOCs in water or Pesticides in Food (MegaMix) could be possible but a clear European strategy about which components should be targeted and how many countries will participate on a common European LCI list is needed.
Separating and determining VOC’s and SVOC’s out of air is a well-known and well used application at Restek. If someone needs assistance, everybody is welcome to discuss their challenges with our Technical Service group.