Use the “Subscribe” link at right. It just takes a few seconds!
The range of reference standards available from Restek is very wide, which is great. But finding a particular formulation among all those options can be a challenge. And, let’s face it, filling out a form to inquire about a custom mix can be a pain in the neck.
That’s why we built our new Reference Standards — Search, Select, and Custom Request tool.
We know it’s all about the compounds, so we’ve done a lot to make specifying them easy. The input mechanism verifies compound names and looks up the CAS numbers for you. Better yet, you can paste in whole lists and enter your compounds in bulk.
Once you’ve told us your compounds, our new tool will suggest catalog products that might meet your needs. The matching components in each suggested mix are highlighted in bold. Easily move products back and forth to pick what works best for you.
If you choose to add one or more of the suggested products to your cart, their compounds are filtered out of the list. Any remaining compounds pass through to be requested as a custom. And you always have total freedom to disregard the product suggestions and send our chemists your entire list.
Save yourself time and effort sourcing reference standards from Restek!
As mentioned in my previous post What is Different about Prep LC?, we ask that you start by developing your method on an analytical scale column. Once you have accomplished this, and wish to start using prep scale HPLC for purification, you will need to determine what size preparative scale (“prep”) column would be most appropriate. Below I have presented a possible scenario for selecting a prep column, based on method development done on an analogous analytical scale column. The following is intended for experienced LC users. Please note that these are my thoughts on how this could be done, based on information that is currently available on the topic. Some laboratories that use prep LC on a regular basis may have other practices that work for their specific application.
Column IDs for our prep columns range from 10 -30 mm, whereas the maximum ID for analytical scale is 4.6 mm. The primary consideration for determining the size of a prep column is the desired capacity for sample loading. Columns have a capacity for sample volume and sample analyte mass, so both should be considered. While some instrument manufacturers address sample load in terms of total sample weight, I will be discussing this in terms of total sample volume, since that translates more directly in practical terms. Theoretical sample volume capacity for our prep columns can be anywhere from 40 µL to about 2 mL, but the practical limit should be determined on a case by case basis for each application.
Load Capacity at Analytical Scale
I would start by experimentally determining a practical limit for injection volume on the analytical scale column, with an analytical scale detector in line. For hints on estimating sample injection volumes, please see our FAQs on the subject here: http://www.restek.com/Pages/faq_lc#pkd2. To do this, make a series of injections of a representative sample with volume increasing in increments. (Please see below for an example.) If you are not pleased with results for these injections, try a lower range of volumes. Make sure your representative sample contains the same impurities that you wish to remove from your production scale sample. It will be important also to use a detector that allows you to see both the compound of interest as well as the impurities you wish to remove. Then, come up with a volume that represents an acceptable maximum load volume. Use that number to calculate your scale-up later.
If your prep/production scale sample is expected to be in higher concentrations (in mass of analyte compound per volume units) than you used for the volume test, or perhaps expected to be variable, you should also determine a limit for the mass load at analytical scale. The same technique of doing successive injections in increments will work for this. Examine the peak shape and separation to see what mass of analyte you would be comfortable with loading to get acceptable results for your application. Please keep in mind that practical limits for one analyte may be different from other analytes, based on its unique properties and peak symmetry.
Example at Analytical Scale
The analyst has a 150 x 3.0 mm column, and makes successive injections at 2, 5, 10, 20 and 30 µL.
In this case, 20 µL is determined to be the largest acceptable volume, using a sample at concentration of 15 µg/20 µL = 0.75 µg/µL = 750 µg/mL.
Suppose that the sample given to process at prep scale will vary from time to time. The analyst performs some test injections to determine the maximum mass for the compound of interest per injection. For the 150mm x 3.0 mm column, a series of injections is made containing 10, 15, 30 and 60 µg of analyte at the maximum injection volume of 20 µL. Test injections contain 500, 750, 1500 and 3000 µg/mL, respectively.
Suppose, for this example, that a maximum mass load of 3000 µg/mL is established for the injected sample (60 µg per 20 µL). This information will be used in the example following the next section to help determine the size for a prep column.
Determining Prep Scale Column Dimensions
The next question to ask is “How much sample do you want to be able to load per injection?” As shown in the FAQ mentioned earlier, optimal injection volumes are directly related to cylinder volume of the column. If you are keeping the same column length, the change in optimal injection volume is proportional to the cross-sectional area, A= πR2, where R is the radius. This would be represented mathematically by this equation:
Please note that some sources suggest overall capacity is equally influenced by column length, but opinions on this vary. For our calculation purposes here, we will assume column length stays the same, so length is not a consideration.
Example of Conversion to Prep Scale
Suppose you wish to load a sample volume that is 100 times greater than the maximum volume you determined at analytical scale and your analytical column was 150 mm x 3 mm, then your calculations would look like this:
According to the calculation, you should select a column with radius of 15 mm or larger. A prep column with inner diameter of 30 mm should work for this.
Applying this calculation to the previous example where the maximum volume was 20 µL for a 3 mm ID column, scaling up to a 30 mm ID column would allow you to inject up to 2 mL on the prep column. Suppose that the maximum mass load was 60 µg, or a concentration of 3000 µg/mL for the injected sample. In this case, scaling up to a 30 mm ID column would allow you to inject up to 6000 µg or 6 mg for your compound of interest.
Determining Flow Rate
Another significant difference with prep LC is the flow rate. In order to get the best efficiency for a larger ID column, the flow rate must be increased. As was the case for injection volume, the flow rate also is directly proportional to the cross sectional area of the column, so if you’re converting from analytical scale to prep size, you would multiply the initial (analytical scale) flow rate by the ratio of the radius for the prep column squared over the radius of the analytical column squared. (It is equivalent and sometimes easier to use the diameters rather than the radii.) See formula below:
Where F = flow rate, R = radius and D = diameter. A subscript of “1” indicates analytical scale, while subscript “2” indicates prep scale.
As an example, if your flow rate with a 3 mm ID analytical column was 0.4 mL/minute and you are scaling up to a 30 mm ID column, an equivalent flow rate would be calculated this way:
If you are not certain whether your LC system can accommodate this increased flow rate, please consult your operating manual or contact the manufacturer to ensure it can handle the desired flow rate.
I hope you have found the suggestions in this post useful. Thank you for reading.
Dan Li, Chris English, Jack Cochran, Jason Thomas, and Rebecca Stevens
The alkyl and aryl esters of 1,2-benzenedicarboxylic acid, better known as phthalates, are widely used as plasticizers. Mainly added to polyvinyl chloride (PVC), phthalates make plastic products more durable and flexible. Phthalates are everywhere: plastic toys, PVC water pipes, wallpaper, artificial leather, electrical wire insulation, glue, nail polish, lipsticks, hair spray, plastic water bottles, paints and printing ink are all formulated using phthalate additives. These seemingly endless applications create a big marketplace. Health concerns bubbled up more than a decade ago over their potential to disrupt endocrine signaling .
Aside from health concerns, their ubiquity can cause frustration for chromatographers. Phthalates leached from laboratory consumables, such as rubber tubing, plastic syringes, pipette tips, plastic filters, plastic beakers, plastic stir bars, plastic vials, 96-well plates or other plastic labware can interfere with a lot of chromatographic analysis (both GC and LC). Common lab interference phthalates include diethylhexyl phthalate, diisononyl phthalate, di-n-butyl phthalate (from polytetrafluoroethylene), butyl benzyl phthalate, di-n-octyl phthalate and dimethyl phthalate (from cellulose acetate and Parafilm) . In addition, lab gloves used in sample handling processes are another source for phthalates contamination.
The Rtx-CLPesticides/Rtx-CLPesticides2 GC column set provides unique selectivity and rapid determination of pesticides by GC- μECD (micro Electron Capture Detector) in the low pg range. The advantages of the μECD are clear; low detection of halogenated compounds with minimal interference from hydrocarbons and a variety of other sample matrix. The conjugate electrophore gives phthalate molecule good sensitivity on ECD detector as well. EPA methods 8061A and 606 also recommended ECD for phthalate analysis. Therefore, phthalates can interfere with target compound identification and quantification. This study was designed to determine where the phthalates elute on these pesticide columns relative to the US EPA 8081 chlorinated pesticides.
A significant number of phthalates were observed interfering with pesticides analysis (see the chromatogram and the table). Among contaminants, di-n-butyl phthalate and diethylhexyl phthalate are the most notorious in envirionment due to their low molecular weight, easy partition from polymer matrix, solubility in water, and high usage in polyvinyl chloride production.
Need suggestions to avoid phthalate contaminants? Sometimes avoiding the problem is better than solving the problem. Plastic is the most common source of phthalates. Use glass labware instead of plastic. Although micropipette tips and centrifuge tubes are made from polypropylene, which should be phthalate-free, phthalates can still leach from plastic pipette box, plastic pakage or plastic caps. When using glass, baking-out is a simple way to remove contaminants. In addition, rinsing glassware with redistilled solvents was found to be effective in eliminating phthalates contamination during sample preparation.
Columns: Rtx®-CLPesticides 30 m, 0.32 mm ID, 0.32 μm (cat.# 11141) and Rtx®-CLPesticides2 30 m, 0.32 mm ID, 0.25 μm (cat.# 11324) using Rxi® guard column 5 m, 0.32 mm ID (cat.# 10039) with deactivated universal “Y” Press-Tight® connector (cat.# 20405-261); Sample: Organochlorine pesticide mix AB #2 (cat.# 32292), Pesticide surrogate mix, EPA 8080, 8081 (cat.# 32000); Injection: Inj. Vol.: 2 μL splitless (hold 0.3 min), Liner: Splitless taper (4 mm) (cat.# 20799), Inj. Temp.: 250 °C; Oven: Oven Temp: 120 °C to 200 °C at 45 °C/min to 230 °C at 15 °C/min to 330 °C at 30 °C/min (hold 2 min); Carrier Gas: He; Detector: μ-ECD @ 330 °C; Notes: Instrument was operated in constant flow mode. Linear velocity: 60 cm/sec @ 120 °C. This chromatogram was obtained using an Agilent μ-ECD. To obtain comparable results, you will need to employ a μ-ECD in addition to dual columns connected to a 5-meter guard column using a “Y” Press-Tight® connector. Concentrations are 8-80 ppb for pesticides and 10 ppm for phthalates.
 J. Annamalai, V. Namasivayam. Endocrine disrupting chemicals in the atmosphere: their effects on humans and wildlife. Environ. Int. 76 (2015),78-97.
 A. Reid, C. Brougham, A. Fogarty and J. Roche. An investigation into possible sources of phthalate contamination in the environmental analytical laboratory. Intern. J. Environ. Anal. Chem. 87 (2007) 125-133.
The goal of prep LC is not to produce the best looking chromatogram.
Prep LC can be used to purify a material for manufacturing or research purposes, or it can also be used for sample cleanup prior to analytical measurement, such as in gel permeation chromatography. Most Restek customers are more involved with using it for purification processes, so I will stick to that for this discussion. Products purified by prep LC might include pharmaceutical products, test materials for research or industrial chemicals, to name a few. What surprises some analysts is that the separation criteria are often not as stringent for prep LC as compared to its analytical counterpart. Perfectly symmetric peak shape and baseline resolution are often not required. The goal is not to produce an aesthetically pleasing graph, but to produce a purified product in a way that is overall the most economical and most expedient.
Column eluent is at least partially recovered and used, not sent to waste.
A very big difference in prep LC is that usually the column eluent is separated into timed intervals to make “cuts” of the sample for purification. This is typically done by attaching a fraction collector immediately following the detector and a non-destructive detector, such as UV. Often a switching valve is used to direct flow either to a waste container or to the fraction collector, or the fraction collector itself may contain the switching valve. The system or its components are programmed to begin collecting fractions as soon as analyte peaks become visible on the detector. The collected fractions are usually concentrated/ solvent exchanged to produce a final product or to perhaps to allow analysis by HPLC, GC, or other techniques for an intermediate product. Often multiple aliquots of the same sample are injected repeatedly to purify more of the starting material. Under this scenario, the desired fractions from each chromatographic run would likely be combined to produce the final product. It would be quite common to analyze aliquots from intermediate products or perhaps from individual fractions to monitor progress prior to combining them at the end.
Requires larger column IDs.
The size of the column in prep LC is obviously much larger than analytical LC. For these applications, this is needed to accommodate a larger sample size and also to make using it most cost effective. Column IDs for our prep columns are from 10 -30 mm, whereas the maximum ID for analytical scale is 4.6 mm. Our selection of prep columns can be found here on our website:
Requires method development on analytical scale first.
Since prep HPLC columns are more expensive and have limited availability, we ask that you first develop your method on an analytical column of the same phase. It makes sense to confirm that your compounds of interest can be separated sufficiently before you “graduate” to prep scale. Often analysts will start with a 4.6 mm ID column first. Please do make sure you do this with the exact same column phase to make it worthwhile. In other words, if you plan to use an Ultra C18 prep column, perform your method development on an Ultra C18 analytical column with an ID of 4.6 mm or smaller.
Requires specialized hardware.
Using a prep LC column brings with it certain requirements in terms of LC system hardware. For that reason, many analysts will use a system specifically designed for prep chromatography. Here are some examples of components that would be different:
- Solvent pumps capable of higher flow rates, up to 150 mL/min
- Larger sample loops
- Higher pressure rating valves and components
- Detectors with prep size flow cells
For example, here are links to information on systems available from Shimadzu and Waters:
I hope this post has helped to give you a better idea of what is involved with prep LC and how it is different. Please stay tuned for the next post, where we will discuss scaling up from analytical scale to prep scale LC. Thank you for reading!
My third installment of Technical Service Red Flags contain six more situations that cause us to pay attention and ask questions. I hope you find these helpful.
Using a liquid leak detector to locate gas leaks instead of using an electronic leak detector. Or worse yet, using both at the same time. We have, unfortunately, communicated with many customers who have accidently “sucked” liquids into their electronic leak detector. If this happens, turn off the leak detector immediately. We suggest allowing it to “dry” in a desiccator or bag of rice for several days. If it no longer works (or it does not work well), contact Customer Service or your distributor and order 22655-R (Leak Detector Repair Service).
Leaving your GC oven temperature at or just above ambient temperature overnight with carrier gas flowing. Moisture condensation can become an issue.
Trying to analyze light gases and/or water vapor using a GC/MS. When NOT to use (GC) Mass Spec
Using our products for applications other than those they were designed for. While I am sure many of our products have multiple uses, we are a chromatography company and our products were designed for our chromatography customers. We will unlikely know if our product will work for your non-chromatography application, and we cannot be held responsible if it does not work. In summary, use at your own risk.
Using a TCD with helium as the carrier gas to quantitate hydrogen. Experiments have shown that obtaining a linear calibration curve can be difficult, if not impossible.
Trying to use our Restek ProFLOW flow meter (# 22656) to measure negative (vacuum) gas flows. This item is only designed to measure flows of clean, dry, non-corrosive gases under positive pressure. If you need to use a flow meter for vacuum gas flows, select one of our Alicat M-Series Flow Calibrators
To read my previous Red Flags posts, the links are below. Thanks.
Derivatization is a widely-used technique for GC sample preparation across many industries and in widely varied matrices from soil to plastics to blood that is used to make polar and active compounds more amenable to good GC analysis. If you’re careful about testing your derivatization procedure during method development, you can be confident that you’ll have a reproducible method that can vastly improve the quality of your GC results. While derivatization does require some extra sample handling, the procedure I developed for cannabis plant matrix is very straightforward and easy to perform:
Derivatization Procedure for Cannabis and Hemp Plant Matrices:
- Place 100µL of plant extract into a 1mL Micro-Vial
- Evaporate to dryness at 50°C under a gentle stream of nitrogen
- Add 50µL ethyl acetate and 50µL BSTFA + 1% TMCS
- Incubate at 70°C for 30 minutes
- Cool and dilute with ethyl acetate if desired
In my last blog, I introduced the concept of derivatization for use in cannabis or hemp cannabinoid testing. Once acidic cannabinoids are derivatized, they no longer break down in the GC inlet and can be quantified separately from the neutral cannabinoids. I demonstrated this through derivatization of high-level solvent standards, but work with solvent standards is a far cry from matrix work, which means the procedure needed to be tested in matrix. To kick off the matrix test, I spiked an extract with the most common cannabinoids of interest, derivatized it using the procedure listed above, and my colleague, Jack Cochran, analyzed it via GC-FID with our Rxi-35Sil MS GC column. We can see that we have a beautiful chromatogram with all of the derivatized cannabinoids separated, and very little matrix interference.
In addition to confirming that all derivatization sites are indeed derivatized by analyzing the standards with GC-MS (this is shown in my last blog), we also tested derivatization efficiency using a cannabis extract previously generated at Penn State University with the help of Professor Frank Dorman and a Police Officer Specialist. Because derivatization is a chemical reaction, the derivatization reagent gets used up during the derivatization reaction. Because plant matrix contains many other derivatizable compounds like sugars and sterols, these other compounds may compete for the derivatizing reagent, possibly resulting in the reagent getting used up before all of our analytes of interest can be derivatized.
So how can we be sure our derivatization is going to completion in the presence of matrix? There are a couple things we can do, the first of which is really simple. We can see in our procedure that we use a hefty 50µL of derivatizing reagent per 100µL of cannabis extract. We know that our extract contains a lot less than 50mg of plant matrix, not all of which is derivatizable. This means that by adding 50mg of BSTFA per 100µL of sample, we can be confident that we have a significant excess of derivatizing reagent as compared to derivatizable groups in our sample. Excess derivatizing reagent means that it will never be completely used up, ensuring the reaction will go to completion no matter what.
A more quantitative way to test derivatization efficiency in a matrix where you can’t get blanks is to evaluate analyte linearity with differing amounts of matrix. For example, if you derivatize four THCA-containing samples prepared using 10, 20, 50, and 100µL of cannabis extract and plot the area of THCA versus sample amount, you should end up with a straight line if your derivatization is going to completion. If it’s not, then you’ll likely see THCA area fall off for the samples containing more matrix since the derivatization reagent is being used up before all the analyte in the higher matrix level sample is derivatized. To test our procedure, we did just that. We can see that our linearity looks beautiful for all of the cannabinoids, indicating the derivatization does indeed go to completion.
In addition to verifying that the derivatization reaction goes to completion in the presence of plant matrix, we also verified the procedure using several different samples which were generated at the same time as the sample shown in the figure above. Our preliminary work is still looking good, which is exciting, but what about all of the other matrices cannabis chemists have to work with? Well, we’re planning on moving the work forward into edible matrices next, so stay tuned for an update!
Using the QuEChERS extraction and cleanup method for high fat commodities such as, nuts, dairy, fish, and avocado can be difficult to analyze for pesticide residues. Using the dispersive solid phase (dSPE) cleanup sometimes is just not enough to adequately remove the fatty acid co-extractives. Check out this technical article on how to quickly yet effectively remove more of those fatty acid interferences!
Wildfires in the Western United States: The smoke can be as dangerous as the flames, especially for firefighters.
The drought continues and wildfires are raging across the Western United States. In Washington State alone, the wildfires have burned over 1400 square miles (that’s 3600 km2). Brave men and women from across the United States are coming to the aid and trying to contain these historical fires. Unfortunately lives of the firefighters are in danger from the moment they approach the scene. While the fire itself poses a huge risk to the firefighters, neighboring communities, and wildlife, the even larger area of smoke coverage is another health concern. Smoke contains many known hazards in the form of particulate matter and chemicals produced from the incomplete combustion of wood and other organic materials. Carbon monoxide, aldehydes, benzenes, polycyclic aromatic hydrocarbons (PAHs) are just a few of the chemicals that are found in smoke that can cause health problems if inhaled. Residents in the affected areas are cautioned to limit their exposure by staying indoors and keep windows and doors closed.
At the 4th Multidimensional Workshop in 2013, the late Brian McCarry presented on the Identification of new Markers of Wood Smoke Exposures in Firefighters using GCxGC-TOFMS. He used a 60 m x 0.25 mm x 0.25 µm Rxi-5Sil MS in the first dimension and a 1 m x 0.1 mm x 0.1 µm Rxi-17Sil MS in the second dimension on the LECO Pegasus 4D instrument. He analyzed firefighter’s urine pre and post-smoke exposure. After he evaluated chemicals found in wood smoke and compared that to the compounds detected in post-smoke urine samples, he found several potential marker compounds for wood smoke exposure. Some of the marker compounds identified were methoxyphenols (guaiacols and syringols), resin acids and PAHs. A quantitative method was then developed using GC-MS/MS for wood smoke markers in air, on skin and in urine. By monitoring these markers, the true exposure to firefighters can be better understood and therefore, better health risk assessments can be made to hopefully limit their exposure.
Just like in my original post, Technical Service “Red Flags” – GC, this post contains several additional Red Flags we look (email) and listen (phone) for in technical service.
Trying to set the carrier gas flow rate for a packed column without using a flow meter. You may want to review this previous post.
When you have poor or no compound response for only the early eluting or later eluting compounds, but response for the other compounds are normal.
Purchasing a compressed gas cylinder/tank regulator without knowing which CGA fitting is needed. Need some help in choosing the correct gas regulator?
Trying to cut capillary fused-silica or metal (MXT) tubing with scissors or wire cutters (or some similar household cutting tool).
Using a splitless liner for split injections. While you may obtain acceptable results for sensitivity and reproducibility, obtaining a linear calibration curve may be difficult.
Trying to analyze Polywax® standards without using an on-column injection port, the proper simulated distillation GC column, and FID.
I hope you have found this second installment of “Red Flags” interesting and informative. Thanks for reading.
A dip tube (also known as an outage tube) provides vapor space above liquefied gases in a sampling cylinder so that if expansion occurs with an increase in temperature, the pressure is not significantly increased. Basically, the length of the dip tube is used to determine the filling capacity of the cylinder.
Outage is expressed as a percent of the total cylinder volume, based on the ratio of the vapor space to the total length of the cylinder with a maximum available outage of 50%. The dip tube is welded directly to the male inlet of the valve and is cut to a length ranging from one inch to 5 ¼ inches (available in ~1/8 inch increments).
• Dip Tube Length = Cylinder Length x Percent Outage (as a decimal)
Typically, the ideal dip tube length will provide a ~20% void within the cylinder based on the cylinder length and the physical (expansion) properties of the sample. To calculate for this length, determine the overall length of the cylinder and multiply by 0.20 to obtain the desired dip tube length in inches. Note that this is an approximate calculation (+/-20%) since several cylinder design variables will influence the actual volume.
Restek offers 10, 20, 30, 40, and 50% outage options. A 10% outage would translate to a 90% fill capacity, 20% would equate to 80% fill, 30% would be a 70% fill, etc.
The following chart provides suggested dip tube lengths for a ~20% outage (~80% fill) based on Restek supplied sample cylinder dimensions.
To calculate a ~10% outage, multiply the cylinder length by 0.10. For example, Restek catalog #22925 (1000cc High Pressure Cylinder) has a length of 10.9 inches. 10.9 inches x 0.10 = 1.09 inches (or about 1 1/8 inch). Multiply by 0.30, 0.40, or 0.50 respectively for 30, 40, or 50% outage values. Again, keep in mind that all these calculations and the associated dip tube lengths are approximate.
We offer two types of dip tube materials: stainless steel and Sulfinert-treated stainless steel. Stainless steel is recommended for general purpose applications, while Sulfinert is superior for the collection and analysis of low-level sulfurs in the ppb range. Make sure to match the dip tube (stainless steel or Sulfinert) to the sample cylinder material.
Placing an order for a Restek supplied dip tube is easy. Simply contact our US Customer Service Team at email@example.com or your local Restek representative. Specify dip tube length in inches or percent outage when ordering. Note that when ordering a treated dip tube, the end of the part will not be Sulfinert treated after we cut the tube to length.
To learn more about dip tubes, refer to ASTM D3700 (Standard Practice for Obtaining LPG Samples Using a Floating Piston) and ASTM 1265-05 (Standard Practice for Sampling Liquefied Petroleum (LP) Gases Manual Method) as well as the link below.
In summary, appropriate dip tube selection is a critical part of a successful liquefied petroleum gas sampling. Review your method specifications, select the dip tube length based on cylinder volume and sample composition, and determine the best material based on sample inertness requirements to ensure successful sampling.