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How (Not) To Cut Your Capillary Column

There are numerous ways to cut a capillary column. Restek has several tools to accomplish this job. There are sapphire scribes, ceramic scoring wafers, wafers with a handle, and diamond blade column cutters. There are also some tools manufactured by other companies that will cut a capillary column. The choice is typically personal preference. No matter what you choose, a square clean cut is extremely important for good chromatography.

A ceramic scoring wafer is used by the majority of chromatographers, so that will be the focus of my post. Restek’s version is pictured below. Ceramic scoring wafers are readily available from most all chromatography companies, are inexpensive, and are fairly easy to use.

The flat straight edge of the wafer is used to lightly score the fused silica tubing. After scoring the fused silica, slight pressure from one’s finger will break the tubing. If done properly, a nice clean square cute is obtained. Here are some good Restek resources, that contain pictures and guidance, for properly cutting columns:

Ceramic Scoring Wafer Instruction Sheet

Column Cutting, for making the Optimal Coupling: Do you use your column-Scoring-Wafer the Right Way?

(S)light pressure and lightly tapping to break the fused silica is stressed in any column cutting instruction guide. If too much pressure is used, you’ll get cuts like the ones in the photos below. These poor cuts have shards of fused silica inside the column and fissures on the side of the column. The shards will create active sites in the column and cause all types of issues. The fissures can perpetuate the break downwards and make the column end brittle. The column on the far right is so badly damaged that it appears to have the pressure from a pair of scissors used to make the cut.

 

If you ever do make a poor cut, make a new cut about 8-12 inches from the end. Cracks can run upwards to 6 inches.  This will help ensure that your new (and good) cut removes any shards and the potential for brittleness caused by a fissure.

As a note, over time a ceramic scoring wafer will dull. As the edge dulls, more pressure will be required to break the fused silica. That excessive pressure will first lead to non-square cuts and eventually to the issues pictured above. When all sides of the wafer start to show signs of being dull, discard and replace it. The same holds true with a sapphire scribe. If you prefer the diamond blade cutter, replace the diamond cutting wheel when you observe poor cuts.

I would like to thank Wendy Henninger, one of our QS Engineers, who provided the detailed photos of the poorly cut columns.

 

 

SPME Arrow Tip = Less Septa Piercing Force

As we indicated in our response to Hailey, the arrow-shaped tip on a SPME Arrow facilitates the smooth penetration of vial and GC septa. We even demonstrated how well GC septa stand up to repeated Arrow injections. However, the following video clearly demonstrates how the Arrow tip enables end users to pierce a vial septum with less force. This means less force spread over a greater area (recall that the SPME Arrow has a beefier shaft than a traditional SPME fiber). All of this contributes to the robustness we have already covered. Without further ado, please enjoy the following video made by Beat Schilling:

Extractable Petroleum Hydrocarbon (EPH) – Extractable Background of Resprep EPH Cartridges

Restek’s Resprep EPH Fractionation SPE Cartridges (cat # 25859) provide method-specific performance for EPH analysis of soil and water samples through complete separation of aliphatic and aromatic compounds into distinct fractions, while providing extractable background levels guaranteed to fall under the strict reporting limits of Massachusetts and New Jersey EPH methods.

 

The cleanliness of silica gel cartridges is critical for EPH analysis. Like many methods, this method is subject to false positives and false negatives when reporting target PAH analytes. Non-targeted hydrocarbons can elute or co-elute within the targeted retention time window, which may lead to falsely identifying or quantifying target analytes. Also, the ability to identify or quantify low concentration target analytes may be inhibited if large, unresolved complex matrices are present beyond the cleanup capacity of the method.

 

In addition to matrix concerns, plasticizers and other compounds can leach from commercially available silica gel cartridges and should be monitored during each analytical batch analysis. The MA EPH and NJ EPH methods specify a procedure involving dichloromethane during the conditioning step followed by an abundant amount of hexane to eliminate or minimize leaching contamination. However, by using this procedure, the amount of solvent used is significantly increased and there is a concern of dichloromethane remaining in the silica gel bed during the aliphatic elution, which could lead to breakthrough issues. Laboratories must report the presence of any contamination leaching from the cartridges within the C11 – C22 aromatics range.

 

Restek guarantees a background level in each fraction, aliphatic and aromatic, of less than half the strict reporting limits of MA EPH and NJ EPH methods. The figure below shows the extractable background from C9 – C18 and C19 – C36 in the aliphatic fraction and C9 – C16 and C16 – C36 of the aromatic fraction. The extracted background in region 2 of both fractions has the area of the surrogate peaks removed.

 

Extractable Petroleum Hydrocarbons (EPH) – Lot to Lot Reproducibility of Resprep EPH Cartridges

As previously discussed in my last blog, “Extractable Petroleum Hydrocarbons (EPH) Method – Why it is important?”, Restek’s Resprep EPH Fractionation SPE Cartridges (cat # 25859) provide method-specific performance for EPH analysis of soil and water samples through complete separation of aliphatic and aromatic compounds into distinct fractions, while providing extractable background levels guaranteed to fall under the strict reporting limits of Massachusetts and New Jersey EPH methods. Restek’s newly optimized silica gel cartridges have superior lot-to- lot reproducibility and storage stability ensured by rigorous QC testing and moisture-resistant packaging.

The fractionation step of the silica gel cleanup is a sensitive and critical procedure. Small changes in the elution volumes, analytical equipment, or analytical techniques can impact the proportion of hydrocarbons separated into their respective fractions. According to Massachusetts EPA, “it is recommended that a Fractionation Check Solution be analyzed for each new lot of silica gel/cartridge, to reestablish the optimum volume of hexane elution.” [1] For lab, this can be very time consuming, as well as, a very tedious process. Resprep EPH SPE cartridges offer consistent and complete separation between aliphatic and aromatic compounds from one lot to the next. Figure 1 shows a representative subset of compounds (40 + compounds were analyzed in total) to clearly illustrate the separation between fractions and lack of breakthrough in every lot produced. Restek manufactures quality into each EPH cartridge by closely monitoring the silica gel used and tightly controlling cartridge weight to ensure consistent performance with each cartridge through a lot, as well as, between lots.

 

[1] Massachusetts Department of Environmental Protection, Method For The Determinations of Extractable Petroleum Hydrocarbons (EPH). May 2004. Revision 1.1

 

SPME Arrow Sensitivity = Speed

We have demonstrated that the SPME Arrow is more durable and sensitive than a traditional SPME fiber. What more could you ask for? Well, truth be told that some analysts are content with the durability of their traditional SPME fibers. It is just like any other unhealthy relationship, where they have no clue how good things should be. I know, I digress… The other sensitivity component may not move the needle either, because clearly, they have the sensitivity they need with a traditional SPME fiber. So really a SPME Arrow does little to make their eyes stray from their beloved traditional SPME fiber (yes, relationship reference again). BUT… when you say speed, then everyone listens. Now all of a sudden, the 20- to 25-year-old relationship with SPME is met with the equivalent mid-life crisis solution of the shiny new sports car in the driveway. Enter the SPME Arrow speed!

On the heels of the sensitivity results we showed you last time, we thought that we could take advantage of the Arrow’s increased sensitivity and parlay that into speed. So, Colton and I took a 100 µm PDMS SPME Arrow and a 100 µm PDMS traditional SPME fiber. Again, we sampled headspace (HS) volatile organic compounds (VOCs) which had been spiked in drinking water at 2.5 ppb, as per method ISO 17943. Everything was equivalent (i.e., equilibration times, desorption temperatures, etc…), except for the fibers and the extraction times. We evaluated 15, 30, 60, 120, 240, 480, 960, and 1920 seconds of extraction time for each fiber (n=3 for each fiber and each extraction time). Here is what the results look like:

HS extraction of ISO 17943 VOCs on 100 µm PDMS SPME Arrow and 100 µm PDMS traditional SPME fiber

Here are the following take-away messages we want you to have from the above graph:

  1. There should be no surprise in the increased response associated with the SPME Arrow, as we already demonstrated this last time.
  2. Both fibers equilibrated at ~120 seconds. This makes sense, because both fibers were 100 µm PDMS. Since the phase thickness is the same, equilibrium times are as well.
  3. Pay attention to the response of the SPME Arrow at 15 seconds vs the traditional SPME fiber at 120 seconds (see below).

HS extraction of ISO 17943 VOCs on 100 µm PDMS SPME Arrow and 100 µm PDMS traditional SPME fiber

Right there lies your speed!!! You will notice the SPME Arrow produced 2x the response @ 15 seconds of extraction when compared to a traditional SPME fiber @ 120 seconds. That is twice the response in 1/8th the time. Admittedly, for the current example the rate limiting step would be a GC run time short enough to really capitalize on the 15 second extraction. Not exactly reasonable for the 90 some VOCs we looked at for ISO17943. However, if you were looking at less compounds and/or running a fast screening run, then you could really take advantage of this. It is also important to note that when using a CTC PAL, we were able to achieve ~5 RSDs on replicate runs using a 15 second extraction, which is about what the RSDs were for 120 seconds of extraction. It is important to note that if you are doing manual injections, a 1 second shift in extraction time could have a more significant impact on response with a target extraction time of 15 seconds compared to 120 seconds. So, you better keep a close eye on your watch!

Now picture this: you could potentially take your current 40-minute extraction time down to 5 minutes and walk away with two times the response. Time is money, so I know that I am now speaking in a language almost every analyst can understand.

SPME Arrow Size = Sensitivity

By now you should know what a SPME Arrow is and that you should be using a SPME Arrow instead of a traditional SPME fiber, because the Arrow is more durable. The same physical dimensions, which make an Arrow more mechanically robust, also afford an increase in sensitivity. Last time, you saw that the phase volumes could increase by approximately 4 to 6x greater on an Arrow. This time, we see how that correlates to an increase in response.

Colton and I took a 100 µm PDMS traditional SPME fiber and a 100 µm PDMS SPME Arrow. We sampled headspace (HS) volatile organic compounds (VOCs) which had been spiked in drinking water at 2.5 ppb, as per method ISO 17943. Everything was equivalent (i.e., equilibration times, extraction times, desorption temperatures, etc…), except for the fibers.

Here is what the respective c-grams look like:

2 minute HS extraction of ISO 17943 VOCs on 100 µm PDMS SPME Arrow and 100 µm PDMS traditional SPME fiber

 

The following are three take-away messages I would like you to have:

  1. Obviously, the SPME Arrow’s response is higher than a traditional fiber, which should be of no surprise. It all makes sense that if you increase your phase volume, you increase your volume of target analyte collected, and thereby increase your analytical response.
  2. It is hard to accurately quantify this by the chromatogram overlay; however, you will have to trust me that the SPME Arrow demonstrated a ~4x increase in response over traditional SPME fibers for most compounds. Low and behold it is right on target with the phase area/volume increase we discussed last time.
  3. I say “on average” in the previous point, because it is important to note that the increase in response for very volatile compounds like vinyl chloride is ~10x on the SPME Arrow vs the traditional SPME fiber. Whereas, a semi-volatile compound like naphthalene only sees a 2x increase in sensitivity on the SPME Arrow compared to the traditional SPME fiber. This has more to do with partial pressures (i.e., Henry’s law constants) of these compounds and therefore their availability in the headspace being the rate-limiting step. If you have not already done so, take a look back at the chromatogram and you will see what I am talking about.

So now you have the option of a more durable and more sensitive SPME product. What more could you want!? Well, stay tuned for next time when I talk about how you can trade in your Arrow’s increase in sensitivity for speed…

Upcoming Restek Cannabis Testing Seminar

10/9 UPDATE- This seminar has been cancelled. Stay tuned for future dates. 

New to chromatography? Looking to brush up on your current analytical skills? Interested in what different types of cannabis testing currently exist?

The analytical market for cannabis is a rapidly growing, burgeoning field. As more and more states legalize cannabis, an increased number of analytical labs and scope of services will be necessary.

If you are involved with a lab that analyzes cannabis by chromatography, then this seminar is tailor-made for you.

Restek will be holding this one-day seminar on Friday, October 13 in Manchester, NH. The registration fee is only $50 for this event.

Chromatographic Foundations for Cannabis Testing Seminar

Join Restek for this three-hour overview of the fundamentals of chromatography for cannabis-testing laboratories. This event will be valuable to any scientist or technician working directly with the chromatographic instruments used for the wide variety of testing performed on cannabis samples. A brief historical example will serve to highlight the foundations common to all chromatographic techniques. Next, this seminar will explore the major differences between the principal types of chromatography — gas chromatography and liquid chromatography — using examples from the cannabis industry to illustrate the advantages of each type. Finally, a survey of sample introduction and detection techniques, including headspace sampling and mass spectrometry, will introduce the audience to the variety of options available to supplement the separations performed by the chromatograph.

Schedule
8:30–9:00 a.m. — Registration & breakfast
9:00 a.m.–12:00 noon — Chromatographic Foundations for Cannabis Testing 
12:00–1:00 p.m. — Lunch

Cost
$50 (includes breakfast, lunch, and course materials)

Outline
— Universal chromatographic foundations
— GC-specific foundations
— LC-specific foundations
— Special topics: Sample introduction & detection
— Questions and answers

Complete details, along with lodging information and registration instructions, can be found here.

We look forward to seeing you!

Cassini-Huygens: 20-Year Mission Accomplished

Copyright: ESA/NASA/JPL/University of Arizona

Cassini’s Probe Huygens Decent to the Surface of Saturn’s Moon Titan. Courtesy: ESA / NASA / JPL / University of Arizona (5)

On October 15th, 1997, the $3 billion spacecraft went on a seven year, two-billion-mile journey to study the planet Saturn along with its moons and rings. After arriving at the Saturnian system, Cassini deployed the 700-pound Huygens probe to its largest moon, Titan. At 100 miles above the surface the aerosol collector pyrolizer (ACP) and gas chromatograph – mass spectrometer (GC-MS) identified the components in the atmosphere (1). Initial observations after touchdown revealed methane rain drenching the hills and staining the ground with streaks left from higher molecular weight hydrocarbons. The probe was only expected to survive for several minutes, however, the battery had enough power to operate on the surface for nearly 70 minutes. While several different manufacturers were evaluated for capillary columns, two of Restek’s MXT columns were chosen; MXT-1 & MXT-1701 in the 0.18mm internal diameter format (2,3).  The most abundant organic compounds on the surface of the planet were evaporating gases such as; ethane, acetylene, cyanogen and carbon dioxide (4). Most interesting was the discovery that the probe’s landing had allowed for an increase of methane indicating the ground contains high amounts of the liquid. Further investigation by the Cassini craft orbiting Titan discovered an ocean of liquid methane 200 miles under the crust. Today Cassini ends its 13-year orbit around Saturn and NASA has steered the craft into the atmosphere. The gravitational forces of the planet accelerated the spacecraft to 75,000 miles per hour, effectively vaporizing it. We are proud to have been able to contribute our columns to such a monumental mission.

Further Reading: Sitting Down with a Chromatography Icon: Dr. Robert Sternberg

http://www.restek.com/Technical-Resources/Technical-Library/Editorial/general_gen_0030

 

  1. DiGregorio, B. E. GC-MS Analysis on Titan Mission. May 2005. Spectroscopy. http://www.spectroscopyonline.com/gc-ms-analysis-titan-mission?id=&sk=&date=&pageID=3

2. Navale, V., Harpold, D. and Vertes, A. Development and Characterization of Gas Chromatographic Columns for the Analysis of Prebiologic Molecules in Titan’s Atmosphere. Anal. Chem. 1998, 70, 689-697. http://pubs.acs.org/doi/abs/10.1021/ac9708598

3. Szopa C., Sternberg R., Rodier C., Coscia D., and Raulin F. Development and Analytical Aspects of Gas Chromatography for Space Exploration. February 2001. LCGC Europe. http://alfresco.ubm-us.net/alfresco_images/pharma/2014/08/22/5eca4f66-0659-4f22-afdb-5a63cb4a2095/article-7464.pdf

4. Niemann, H.B., Atreya, S.K., Bauer S.J., Biemann K., et. al. The Gas Chromatograph Mass Spectrometer for the Huygens Probe. Space Science Reviews. 2002. 104: 553-591. https://link.springer.com/article/10.1023/A:1023680305259

5. Image provided Courtesy of ESA / NASA / JPL / University of Arizona http://www.esa.int/spaceinimages/Images/2017/09/Descent_to_Titan

 

Which LC column should I use for my method?

We understand that with so many products on the market, choosing a column to get started can be difficult. Of course, column selection depends on what type of method you are following and what kind of LC system you have in your lab. Let’s begin by looking at our choices for the more traditional, fully porous particle LC columns.

 

 

If your method is a USP or other compendial method, the preferred column would be one of our Roc HPLC columns. The ROC phase selections we offer encompass most of what you would need for these methods. The Roc columns are very appropriate for any highly regulated work environment where a rugged and long-lasting column is desired and lot to lot reproducibility is critical. Most regulations also allow for slight modifications in column dimensions, as well as other parameters for example, as indicated here in this document from the FDA: https://www.fda.gov/downloads/ScienceResearch/FieldScience/LaboratoryManual/UCM173085.pdf

 

 

If your method is not a USP method, but is very well established for a 3 or 5 µm particle column, simple in design, and requires very high reproducibility, the Roc HPLC columns are still preferred. If you find that the phase or dimensions you need are not offered as a Roc column, the next place to look would be within our Ultra columns. Ultra columns are also available in preparative sizes if you are doing purification work with large sample sizes.

 

 

 

 

 

If you have a challenging method that is written for UHPLC (<2.0 µm fully porous particles) or one that you would like to eventually convert to a UHPLC method, our new Force LC columns are the ideal product. We are excited to offer these columns made with the highest quality silica in a 1.8 µm particle size, as well as 3 and 5 µm for easy method transfer between HPLC and UHPLC systems. Force columns are available in C18, Biphenyl, and Fluorophenyl phases.

 

 

 

 

 

If your method requires an SPP (superficially porous particle) column or you are looking for superior separation and a fast analysis, but you do not have a UHPLC system, the Raptor columns are ideal. Raptor 2.7 µm columns are preferred for large analyte panels such as pesticides, steroids, and drugs, and are typically used with LC-MS/MS.

Raptor 5.0 µm columns won’t be able to separate quite as many analytes, but separation is still better than you would get with a fully porous 5.0 µm column. Often when using LC-MS/MS, less chromatographic separation is acceptable because the MS can identify closely eluting and even coeluting compounds, unless they are isobaric, so a 5.0 µm column is often still useful. Although it depends on the exact dimensions, generally speaking, the 2.7 µm columns are intended for use with LC systems that have a 600 bar (8700 psi) pressure limit, whereas the 5.0 µm columns are intended for those with a 400 bar (5800 psi) limit. Please see the blog post “Should I use a 2.7 or 5 µm Raptor column?” for more discussion of this.

If you need help selecting a guard cartridge and/or holder for any of these, please see the earlier blog post “Which guard cartridges and holders go with which LC analytical columns?”.

To find a listing of all LC columns we offer, you might also find our LC Columns Physical Characteristics Chart useful.

I hope you have found this post helpful. Thank you for reading.

 

Should I use LC or GC for my analysis?

Although most analysts already know which approach they need to use for analysis, this is occasionally a topic of discussion.  While this blog post is not meant to give an absolute answer for each specific application, I hope to provide some tips to steer you in the general direction towards a solution, if this is your dilemma. I will focus here on characteristics of the analyte compound, which should be the primary concern. Other factors, such as cost and detection methods will not be discussed in this blog post.

 

To use GC for analysis:

First of all, the analyte must be volatile.  This is because it will need to exist in a vapor state in order to partition between the carrier gas stream (mobile phase) and the stationary phase inside the column.  While it depends somewhat on the choice in column, generally the compound should have a boiling below about 400°-500°C (at atmospheric pressure of 760 mm Hg).  For this reason, most GC analytes are smaller compounds with a molecular weight of less than 1000.  An example of a compound that works well for GC analysis is naphthalene, which has a boiling point of 217.9°C.  Although we have many examples of analyses that include naphthalene, here is a chromatogram that represents one of the most common applications (EPA method 8270):

http://www.restek.com/images/cgram/gc_ev1418.pdf

In order for partition to occur in the vapor state, the molecule must also remain intact. Ideally, it should not decompose upon heating.  In other words, it must be thermally stable (not thermally labile).  For example, riboflavin decomposes between 278-282°C and is generally not analyzed using GC.  Often in cases like this, GC analysis can be done if the compounds are derivatized.

 

Molecules that can be analyzed by GC or LC:

There are some compounds that could be analyzed equally well by GC or LC.  Bisphenol A is a good example of this.  Here is a link to example chromatograms for both LC and GC analyses of this:

http://www.restek.com/chromatogram/search?s=bisphenol%20A

Some other good examples include compounds like nitrobenzene:

http://www.restek.com/chromatogram/search?s=nitrobenzene

Sometimes analytes that need to be derivatized for GC analysis do not need derivatization for LC.  Chlorophenoxy acid herbicides are a good example of this. Here is an example of GC analysis for these herbicides (derivatized to methyl ester form):

http://www.restek.com/images/cgram/gc_ev00091.pdf

And an example of LC analysis (underivatized):

http://www.restek.com/images/cgram/lc_ev0355.pdf

Although there are many examples of compounds that can be done either way, LC is considered more universal and generally does not require derivatization as often.

 

To use LC for analysis:

Analytes for LC need to be soluble in a suitable mobile phase, but they do not need to be volatile at all. As a result, compounds range from small to very large.  As is the case for GC, most applications for LC are for organic molecules. Although it may be possible under certain circumstances for some inorganic compounds to be analyzed by LC, this would be beyond the scope of what Restek products can accomplish and will not be discussed here.

LC analysis is usually more difficult for the smallest of molecules, particularly if they coincide with the solvents in the mobile phase, for example methanol, acetonitrile or water.  Also, compounds that exist as gases at room temperature cannot be analyzed by LC, or at least it would not be practical.

In LC, to allow partitioning between the liquid mobile phase and the stationary phase inside the column, a compound must be reasonably soluble in the mobile phase and it must have some affinity toward the stationary phase. The phrase “like dissolves like” is very applicable when considering solubility. A compound’s chemical interaction toward a stationary phase could occur in several different ways.  If interested in reading more on this topic, I suggest reading USLC Column Selection and Mobile Phase Adjustment Guide. As discussed in the guide, four of the primary mechanisms are dispersion, polarizability, hydrogen-bonding and cation exchange.

As in GC, an analyte for LC also must remain intact and not decompose. Fortunately, thermal stability is not a concern for LC, since analysis can usually be performed at or near room temperature.  I mentioned earlier that riboflavin does decompose upon heating.  We find that riboflavin is analyzed fairly easily by LC, though, as shown in the following chromatogram:

http://www.restek.com/images/cgram/lc_ff0558.pdf

While decomposition is not common with LC analyses, ionization in aqueous solution occurs quite often. Consequently, such analytes are affected dramatically by the pH of the mobile phase.  To control these affects, most analysts will use buffer in the mobile phase. If interested in reading more on this topic, please refer to the following:

When should you use a buffer for HPLC, how does it work and which one to use?

New Advice on an Old Topic: Buffers in Reversed-Phase HPLC

 

Resources available:

A good tool to use that is at your disposal is Restek Searchable Chromatogram Library.  To look for example analyses for compound(s) of interest, simply type their name or CAS number in the search box. You may see examples for the analysis done by LC or GC, or perhaps both.  If the compounds of interest tend to decompose, become reactive or are difficult to detect, you may see examples of the analysis done by derivatization.

If interested in reading more on this topic, here are some articles that may be helpful:

http://bitesizebio.com/29109/run-fly-comparison-hplc-gc/

http://www.news-medical.net/life-sciences/Liquid-Chromatography-versus-Gas-Chromatography.aspx

http://lab-training.com/2014/04/02/what-are-the-differences-between-gc-and-hplc/

 

I hope you find this helpful. Thank you for reading.