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As an R&D Chemist charged with developing the perfect packing method for new LC columns, one of the challenges is finding a packing method that results in a column which not only passes QC specifications but also passes our arduous lifetime analysis. During lifetime testing our columns are subjected to 1,000 continuous injections at 12,000 PSI (a pressure far higher than what most LC operators will ever need- it’s the equivalent of being under 27,680 feet of water!).
You may ask yourself, “Why bother actually doing 1,000 injections at high pressure? Couldn’t one just run the column at high pressures or flow rates and see lifetime degradation?” What many people don’t realize is that the principal cause of column degradation over time are variations in pressure that occur with each individual injection. These pressure ‘pulses’ are unique to each make and model of HPLC or UHPLC, and are controlled by the structure and mechanics of the instrument.
Finding a rugged packing method that can withstand this kind of punishment isn’t easy – and proved especially challenging when developing the packing methods for our 1.8 µm Force columns. Once we finally hit on the right method I was exceedingly (embarrassingly) excited and jumped at the chance to pit our new ‘survivors’ against the competition. Utilizing our lifetime test and two replicates of each column, we assessed Restek Force C18 (1.8 µm, 2.1 x 50 mm) column performance in comparison to similar dimension, particle, and stationary phase columns from two well-known competitors:
From the graph above, you can see that Restek and Brand 1 columns showed excellent ruggedness, and maintained ≥94% of their original efficiency over the course of the lifetime test. Brand 2 column performance suffered however, with one column steadily dropping in efficiency by the end of the test, and the other maintaining efficiency for 250 injections before plummeting, after which peak integration became impossible due to peak splitting.
At Restek one of our core values is pride in our work. Seeing the results of our hard work in R&D and knowing that we can offer these excellent LC columns to customers gives me great pride to be a Restek R&D chemist.
Thanks for checking out my first blog post!
p.s. Our other dimensions and phases of Force columns are just as rugged as the ones I’ve shown you above. If you want to see another one of my beautiful graphs and learn more about Force please check out our overview here: Force Performance LC Columns Overview .
Clogging and over pressuring are definitely problematic for Liquid Chromatography columns. Back in 2005 John Dolan’s sage advice to chromatographers to make an LC column last forever was simple, “Never open the box!” As John pointed out, this is not practical and I have to agree. But as an LC column manufacturer I have seen remarks from customers regarding clogging as though it was it was a flaw in the column. Unfortunately, there is not really anything that we do when we manufacture a column that would cause one column clog faster than another. Typically, if a column clogs, it is due to particulate matter that is larger than the pores of the column frit getting stuck in the frit and not allowing the flow to pass through. As obvious and simple as this sounds, it can still be overlooked.
To understand LC column clogging, it is important to know how a modern LC column is built. It is actually pretty straight-forward. A metal tube has an endfitting attached to the bottom. This fitting has two purposes. It provides a means to attach your LC’s tubing to the column and it holds a porous filter or frit in place. Consider the frit as a semipermeable membrane. It has pores that allow the liquid mobile phase to pass through, but are not big enough to allow the column packing particles to pass through. I’ll talk more about the frit later, as it is very important to clogging.
Back to building the column. Once one of the endfittings, the bottom one, is attached to the tube, the packing material, or bonded particles (think of spherical silica of generally uniform diameter, coated with C18 ligands) are mixed into a solvent solution to make a slurry, and pumped into the column tube under pressure. Obviously, the solvent from the slurry passes through the frit that was fixed to the end of the tube with the endfitting and the particles are stopped by the frit. The slurry is pumped into the tube until the tube is filled to the top with particles. You can then remove the tube from the packing pump and affix another frit and endfitting to the open end of the tube. Now the particles that were packed into the tube cannot get out either end and you have an LC column. The particles are packed so tightly into the tube that there is no extra space between the packing material and the frit. Any extra space would be called a void and a void in a column will spoil your day. It can result in problems that will affect the efficiency and peak shape in your separations. For this reason, we recommend that the user never try to take off the endfittings from their column. You will never get a tight fit like a column manufacturer can. So if you take the fittings off the column it is now pretty much scrap. Even though I simplified the packing process here, it is actually a very precise process that takes a great deal of time to perfect. Different particles behave differently as do different phases, column lengths, and different internal diameters. Better to leave it to the professionals.
Back to frits. They are so important to clogging because just as they are the right size to let liquid through but not the particles in the column, they allow your HPLC mobile phase pass though when you connect the column to your HPLC. Now keep in mind the particles inside an LC column are very small. A typical HPLC column has 3 to 5 micron particles. If you look on the internet you will find that an average human red blood cell is about 5 µm in diameter. Now that is similar to a relatively large HPLC column particle. UHPLC columns typically have particles that are less than 2 µm in diameter. Remember the pores in the column frit need to be smaller than the particles inside the column. And when a column manufacturer says that a column has 3 or 5um particles know that that is an average. Silica is a natural product so there is a distribution of particle sizes inside that column. A 3um column could have, for instance, particles ranging from 2 to 4um in it. It is very typical for a column manufacturer to use a 2um average pore size frit for 5um particle columns and a 0.5um average frit for 3um or smaller particle columns. I say average for the frit pore sizes because of how they are made. Frits are typically sintered metal. Sintering is process of heating up small uniform metal bits to near melting point and then pressing them so that they fuse together. The spaces that are left between the fused metal bits are the pores. By being layered they create channels or pores in the material. Some may be larger or smaller than others but the process is controlled and repeatable to make a more or less uniform material. This porous metal material is cut into small discs that HPLC column manufacturers use as frits.
So when you are injecting your sample into the column consider what is in your syringe. Was your sample soil or water, blood or urine, or maybe a plant extract? Do you know what size particles are in your sample? Will they pass through the column frit or will they get stuck in the pores? There are some very easy ways to be sure that you won’t clog the frit on your column and give it a nice long operating lifetime. The easiest thing to do is prefilter your sample. If you know that your column has a 0.5um frit, you should prefilter it through a membrane that is smaller than 0.5um, perhaps a 0.45um or 0.2um syringe filter or filter vial. The better you can filter your sample (translation the smallest filter you can use) the better. Maybe you think that your sample is free of particles. Try centrifuging it. If you can see anything at the bottom of the tube, you have a dirty sample. But let’s face it, the human eye could never see a 1um or even a 10um particle in a solution. Of course there are other ways to protect your column’s precious frit. A precolumn disposable frit for example. Again choose one that is, ideally, smaller than the frit in your column. Or a guard column. A good quality guard column will have 2 frits on it just like your column. If the precolumn frit or guard column get clogged, just throw them away and get a new one that is what they are for. Your column is protected.
Your support team at Restek can help you with several different options to help you keep your column from clogging. Remember, if you want your column to last forever, keep it in the box. But if you want your column to maintain its operating pressure under use for a long time, you now know how it works so think before you inject.
Restek manufactures several different residual solvent standards. These can be used for testing according to USP <467>, the EP monograph, or other residual solvent assays, such as medical cannabis testing. The calculations involved for the use of these standards has led to questions for our technical team. This post should help provide guidance for their usage. I’ll use USP <467> in my example calculations, but they can be applied to other methodology.
Since USP <467> is a limit test, each residual solvent has a concentration limit based upon its potential risk to human health. For that reason, not all compounds are at the same concentration in our reference standards. As an example, Benzene has a limit of 2 ppm for the Class 1 Residual Solvents. Catalog #36279 contains benzene at 10 mg/mL. This solution would be what USP refers to as Residual Solvent Mixture RS.
According to the monograph, you are to do the following:
Class 1 Standard Stock Solution— [Note—When transferring solutions, place the tip of the pipet just below the surface of the liquid, and mix.] Transfer 1.0 mL of USP Class 1 Residual Solvents Mixture RS to a 100-mL volumetric flask, previously filled with about 9 mL of dimethyl sulfoxide, dilute with water to volume, and mix.
Using Catalog #36279
10,000 µg/mL x 1.0 mL / 100 mL = 100 µg/mL
Transfer 1.0 mL of this solution to a 100-mL volumetric flask, previously filled with about 50 mL of water, dilute with water to volume, and mix.
100 µg/mL x 1.0 mL / 100 mL = 1.0 µg/mL
Transfer 10 mL of this solution to a 100-mL volumetric flask, previously filled with about 50 mL of water, dilute with water to volume, and mix.
1.0 µg/mL x 10 mL / 100 mL = 0.1 µg/mL Class 1 Standard Stock Solution
Class 1 Standard Solution— Transfer 1.0 mL of Class 1 Standard Stock Solution to an appropriate headspace vial containing 5.0 mL of water (place the tip of the pipet just below the surface of the liquid for dispensing), apply the stopper, cap, and mix.
0.1 µg/mL x 1.0 mL / 6.0 mL = 0.0167 µg/mL
Note: Final volume = 6 mL (1 mL of Class 1 Stock Solution + 5 mL Water)
This solution is the one analyzed by headspace GC/FID. It is less important to consider the 0.0167 µg/mL concentration of benzene than it is to know that there is 0.1 µg of benzene in the vial. This 0.1 µg of benzene is the amount partitioned into the gaseous headspace and injected into the GC
1 mL of 0.1 µg/mL = 0.1 µg (benzene)
With your drug substance or excipient, you are to do the following according to USP:
Test Stock Solution— Transfer about 250 mg of the article under test, accurately weighed, to a 25-mL volumetric flask, dissolve in and dilute with water to volume, and mix.
250 mg / 25 mL = 10 mg/mL
Test Solution— Transfer 5.0 mL of Test Stock Solution to an appropriate headspace vial, add 1.0 mL of water, apply the stopper, cap, and mix.
Since this is the solution analyzed by headspace GC/FID, it is more important to know how much article is present and not the concentration.
10 mg/mL x 5 mL = 50 mg of article tested
The limit for benzene is 2 ppm (0.002 µg/mg), which happens to be the concentration you would find by having 0.1 µg of benzene in your 50 mg of article.
0.1 µg (benzene) / 50 mg (article) = 0.002 µg/mg (2 ppm)
You can do the same math for any of the other compounds in the Class 1 Mix.
The dilution scheme for Class 2 Solvents is similar to the Class 1 Solvents. Since Class 2 Solvents are considered not as toxic as the Class 1, the limits are much higher. Since the allowable limit is so much greater, Restek has accounted for the first dilution with the Class 2 Solvents. This is in Catalog #36012. For the Class 2 Solvents, I will use acetonitrile as an example.
Following USP and the calculations outlined above, the procedure would be:
2050 µg/mL x 1.0 mL / 100 mL = 20.5 µg/mL
20.5 µg/mL x 1.0 mL/ 6.0 mL = 3.417 µg/mL or 20.5 µg of acetonitrile
20.5 µg (acetonitrile) / 50 mg (article) = 0.41 µg/mg (410 ppm) = limit for acetonitrile
I hope you find this helpful.
I recently posted a Blog regarding some notes about Fats and Oils, FAMES and our Famewax column, which can be found here. In Food Industry the challenge may be a bit heavier, if the amount of Trans Fatty Acids is requested.
Therefore some European Countries have taken action and have introduced a legal limit of IFTAS in Food, starting with Denmark in 2003, followed by some other countries like Austria, Hungary and Latvia. Voluntary self-regulation industrial measures have been implemented in Belgium, Germany, the Netherlands, Poland, the UK and Greece. Legal measures limiting in this area exist also in Switzerland, Iceland, Norway and the US.
This said, there is obviously a lack of homogeneity in the whole EU in relation to limitation of IFTAs in food, what is causing problems in an effective functioning of the internal EU market (e.g. protection of consumers’ health). Therefore SANTE (EU Directorate General of Health and Food Safety) has started a new “Initiative to limit industrial trans fats intakes in the EU”, which may lead to an increasing challenge in Food characterization. The final SANTE statement is supposed to be given during 3rd quarter of 2017.
Measuring the Fatty Acid composition of Food, including IFTAs, is challenging and normally follows the EN ISO 12966-2015, Part 1 to 4 (Animal and vegetable fats and oils –Gas chromatography of fatty acid methyl esters), which is also ground base of some national method developments as given in DGF C-VI 10a/11a in Germany
For us, as one of the leading GC separation column producer, in particular the ISO 129664:2015 “Animal and vegetable fats and oils – Gas chromatography of fatty acid methyl esters – Part 4: Determination by capillary gas chromatography” is the most important part, giving some recommendations of suitable columns to be used.
Chapter 5 (Apparatus) indicates as a suitable Capillary column “fused silica capillary 100 m and 0,25 mm i.d. coated with 100 % cyanopropylsilicone stationary phase to a thickness of 0,20 µm.”
Due to its high polarity this column is not easy to be manufactured, as many users may have recognized in the past. This column cannot be manufactured as a bonded version. Until now, this results in some draw back, like change of polarity during lifetime (having changes in retention times) and in some production inconsistencies, recognized as differing batch to batch reliability. This is due to phase chemistry and could be seen all around commercial available products.
Restek scientists therefore have started an initiative to overcome most of the mentioned issues. Having all production processes under control, starting with the manufacturing of raw fused silica and the in-house production of high end polymers to achieve the best possible stationary phase, we have improved a lot of the manufacturing processes, which gave us the opportunity to implement tighter controls on specifications to guarantee low bleed and a more stable baseline. This initiative leads to more consistency in the product and much more reliability for your measurements.
This sounds like a huge step forward in characterizing the fat composition of Food, especially regarding the analysis of the mentioned ITFAs. Some of the impressive results can be seen here.
But as always, the best proof is to check these results by yourself. Ask your regional Restek Sales Representative about this column and make your own decision.
If you are an avid chromatographer you likely subscribe to LC/GC magazine or its popular electronic version, E-Separation Solutions. It is definitely the place to go for news and information related to all things Chromatography. Over the years Restek has had many an article published in LC/GC and, full disclosure, we advertise there as well. Who in the business doesn’t? It is the place to be seen.
Back in June a couple of Restek’s LC R&D scientists had an article published entitled, “Effects of Column Inner Diameter and Packed Bed Heterogeneities on Chromatographic Performance.” In case you missed it this summer you can read it here: LC/GC Article
While we got some really good feedback on this article we were thrilled when LC/GC released The Top 16 Articles of 2016 issue and our article came in at #7 on the list. It was not too surprising to us. Anyone that reads the article can see the tremendous amount of research and thought that went into its preparation as well as the relevance to current chromatographic trends. Not unlike the research and thought that goes into the development of Restek’s premium performance LC products. Authors Ed Franklin and Ty Kahler are a part of a team of talented scientists and engineers that brought to life amazing LC products like Raptor™ SPP column, Roc® LC columns, and the new Force™ Premium LC/UHPLC columns. Products which consider the needs of today’s chromatographers, performance, robustness, and innovation.
There is some significant LC firepower in the R&D lab at Restek and it goes into every product that we produce…no matter what the inner diameter is.
They may be called PFP, PFPP, pentafluorophenyl, fluorophenyl or F5 phases. What they all have in common is the structure shown above (the pentafluorophenyl group) attached to the silica particles.
Most modern phases contain a propyl spacer between the above group and the silica, so that the phase looks something like this:
A “PFPP” phase refers to the above structure with the propyl spacer. However, quite often the other names apply as well, depending on the column and vendor. We are quite proud to offer our new PFPP phases, the Raptor Fluorophenyl and the Force Fluorophenyl, which are designed using the latest advances in bonding technology. The following are some questions that may come to mind.
What makes the phase useful?
Due to the presence of the fluorine atoms, a fairly polar phase is created that allows for HILIC separations. The electronegativity of the fluorine atoms, in combination with the delocalized electrons from the aromatic ring, result in increased retention for charged bases (like amine compounds) and other electronegative compounds (those containing halogen atoms, O, or N). Also, due to the presence of the aromatic ring and propyl spacer, the phase still retains some nonpolar characteristics, so it can also be used in reverse phase mode.
I recommend the following if you wish to read more about HILIC separations and characteristics of PFPP phases:
Restek USLC, Ultra Selective Liquid Chromatography
Reliable HILIC LC-MS/MS Analysis of 4-Methylimidazole (4-MEI) on Raptor FluoroPhenyl Columns
How should these phases be used?
For HILIC mode, use a mobile phase that is at least 50% organic solvent. If using a gradient, start with high organic and program up to high aqueous content. Often a mild modifier, such as 0.1% formic acid is used in both mobile phase A and B. Here are some examples:
For reverse phase mode, conditions similar to what is used with C18 are common, except that sometimes the gradient starts with a higher organic content to encourage retention of highly polar compounds, perhaps 30% organic or higher. Here are some examples:
How do I condition this phase before using?
Equilibration times will vary according to application. If using for the first time or after changing solvents in your mobile phase(s), it may take as much as 50 column volumes or more. It is recommended to always start with a new column when developing methods, as the phase is sometimes permanently altered by certain mobile phase modifiers and buffer salts.
How do I prepare my sample to use with this phase?
Samples should be as clean as possible, as the phase is somewhat sensitive to differences in sample matrix. If sample matrix is an issue that does not lend itself well to cleanup, it is best to quantitate using matrix-matched calibration standards. As with any LC column, particulates should be removed by filtration prior to loading on the instrument.
I hope this has helped to answer some of the questions you might have about using PFP phases.
Thank you for reading.
We already learned how to calibrate our TO-15 system, what our concentration units mean, and how to properly read our vacuum gauge while pressurizing samples. We are clearly going out of order here, because I have never covered how to prepare a working standard (necessary for calibrations) from a stock standard. Therefore, the following multi-part blog series will demonstrate how to use a static dilution to make a working standard.
Okay… first things first. We need to determine our stock standard concentration, canister volume, stock standard injection volume, and final canister pressure. How we go at deriving all of the aforementioned may vary. For example, we often have a 1 ppmv stock standard and a 6 L canister, so those variables are gimmes. More often than not we also know the final canister concentration we desire. For today’s example we will use 2.0 ppbv.
All that is left to determine is the stock standard injection volume and final canister pressure. For the following example we will say that we want a final canister pressure of 30 psig. Now that we have everything we need, but the stock standard injection volume, the math is as follows:
First we determine the final volume of gas in the canister. We know we start with a fully evacuated canister at -14.7 psig and pressurize the canister to 30 psig. So we recognize that we have added 3 atmospheres (~14.7 psig/atmosphere) to the canister. Therefore, our final gas volume is 3 x 6 L = 18 L.
Remembering that we want 2.0 ppbv from 1 ppmv, the rest of the math looks as follows:
Where we are solving for the stock standard injection volume (x), 18 L is our gas volume from above, 0.002 ppmv is our desired concentration, and 1.00 ppmv is our stock standard concentration. The result of this is 0.036 L = 36 mL.
We now know that if we inject 36 mL of our 1 ppmv stock standard into a 6 L canister and pressurize this canister to 30 psig, the final concentration will be ~1.97 ppbv.
We can take the above math and manipulate it any way we want it. And by that I mean, we can move x (what we are solving for) to the top right if we want to solve for the final desired concentration or to the bottom left to solve for the final gas volume, etc… In my experience, we are generally solving for x on the left-hand side, where we are either solving for the injection volume or final gas volume (which ultimately is extrapolated out to final canister pressure).
Now that I have given you some examples, I will give you the shortcut I use when in the lab. Without further ado I introduce you to the:
Stay tuned for next time when I blog about how to introduce a stock standard into a canister…
As of May 31, 2015, 1100 Series HPLC systems were categorized “End of Guaranteed Support” (EGS) by Agilent. Agilent would prefer it if you purchased a new system from them, but 1100 HPLCs are dependable workhorses. Why not keep them running with instrument replacement parts from Restek? Check out our selection, easily sorted by instrument and module: http://www.restek.com/Supplies-Accessories/HPLC-Accessories/Instrument-Accessories-Parts
We also offer PM kits for 1100 HPLCs. The autosampler PM kit (cat.# 25271) can be found here: http://www.restek.com/catalog/view/9668 and the pump PM kit (cat.# 25270) here: http://www.restek.com/catalog/view/9669.
We still keep our older Agilent instruments running with Restek replacement parts. Look at our HP 1050 in our R&D lab. Still going strong!
Let’s see pictures of your older instrumentation. Email a picture to me, and I’ll send you $10 Wizard Dollars (US only): firstname.lastname@example.org
While HPLC may be tempting to use for terpenes analysis, a GC/FID or GC/MS is really the most straightforward and recommended way of analyzing terpenes in cannabis. Terpenes, being relatively volatile and neutral, lend themselves rather nicely to GC in general.
As you can see from Figure 1 below, coelutions of the cannabinoids and terpenes are very likely when analyzing real cannabis samples by HPLC-UV methods. A Shimadzu Prominence 20AD HPLC system and Raptor ARC-18, 2.7um, 150 x 4.6mmID column (catalog# 9314A65) was used in Figure 1.
Figure 1- Standards at equal concentrations
At 205nm, there are many co-elutions of terpenes and the cannabinoids, making identification and quantitation extremely difficult, if not impossible, for either class of compounds.
Conversely, cannabinoids can be analyzed by HPLC-UV, as long as the correct UV wavelength is chosen. At 220nm, terpenes yield a very low signal. Because cannabinoids of interest are present at a much higher concentration than terpenes, in addition to providing a stronger UV signal at 220nm, cannabinoids can be reliably analyzed at a wavelength of 220nm. See Figure 2.
In summary, HPLC-UV analysis of terpenes in cannabis is not recommended, and will likely cause more issues than it will provide solutions.
A good solution to the coelutions by HPLC-UV is to choose a GC headspace method. Interferences from the complex sample matrix, as well as the much less volatile cannabinoids can be eliminated then.
You may have seen my last blog post about guard holders and cartridges that we have available for our various LC product lines, “Which guard cartridges and holders go with which LC analytical columns?”
Along those lines of discussion, the following note is available to also show what specific products are currently offered for this: http://www.restek.com/pdfs/GNAR2492A-UNV.pdf
Reflected in the above note are some changes in what we are offering for the cartridges that are used with the Trident LC Holders. Starting January 1, 2017, we are discontinuing the 20 mm length cartridges for the Ultra, Pinnacle, Allure and Viva columns. This is a result of declining popularity and lower demand. We will, however, continue to sell the 10 mm length guard cartridges for the Ultra, Pinnacle, Allure and Viva columns.
In conjunction with this change, corresponding Trident guard holders for the 20 mm cartridges will be discontinued as well. This includes some of our older catalog numbers, such as 25085, 25086, 25060, and 25062 (XG-XF fitting for 20 mm).
While we feel confident that, in most cases, the 10 mm cartridge length is sufficient for analytical columns, we apologize for any inconvenience this may cause.
Thank you for your continued interest in using our LC products.