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Lab Hack: Quickly Reducing GC Inlet Pressure on an Agilent GC Prior to Changing the Septum and/or Inlet Liner

Have you ever been changing your Agilent GC inlet liner (you do change that routinely, right?), particularly a single taper with wool type, and pulled it out to see the wool at the top of the liner (Figure 1) when it should be at the bottom of the liner (Figure 2), the proper placement where injected sample can hit it and be transferred to the GC column?  How does this occur?  Sudden depressurization of the GC inlet can cause the wool to pop to the top of the liner.  It probably doesn’t matter if this happens when you’re changing the liner, since you’re going to throw the old liner away, but it can also happen when you’re changing the GC inlet septum if you haven’t properly depressurized the inlet, and then unknowingly you’re left with the liner wool at the top of the inlet, where it’s not effective for its purposes of keeping the sample from hitting the bottom seal (where compounds like DDT can degrade), heat transfer, and preventing nonvolatile “dirt” from collecting on the head of the more expensive GC column.

In the following sets of figures, I’ll show you how I depressurize my Agilent 6890 GC inlet to prevent the inlet liner wool from jumping up.  As always, it is much better to completely cool all heated zones before inlet maintenance procedures.

Go to the Agilent GC Keyboard, and press “Col 1” (Figure 3), depending on the Column and Inlet in use.  You’ll see the Display showing similarly to Figure 4.  If necessary, use the “Up” or “Down” symbol key on the Keyboard to move the Display arrow to “Pressure” (it is on “Flow” in the Figure 4 example).   When the Display arrow is on “Pressure”, use “0.1 Enter” (or even “0 Enter”) from the Keyboard (Figure 5), which should cause the Pressure to drop down relatively quickly, but not so quickly that it causes the inlet liner wool to move (the pressure does not drop all the way to zero, typically, Figure 6).  [Note:  On newer Agilent GCs you may hear the “rattlesnake” as the pressure drops.]  Use the “Up” symbol key to move the Display back to “Pressure” and then press “Off” on the Keyboard (Figure 7).  Again, the “Pressure” may not completely zero out, but the GC inlet is now ready for a septum and/or liner change.  Why don’t we just use “Off” immediately instead of “0.1”.  When you use “Off” the pressure bleeds off very, very slowly through the GC column and if you open the inlet too soon the liner wool can move in the liner.

Don’t forget to restore pressure/flow before you heat the GC inlet and column up, and it’s always good to leak check after inlet maintenance.

Fig 1x


Fig 2x




Fig 3


Fig 4


 


Fig 5xx


Fig 6



Fig 7


Fig 8

Need help finding the correct ferrule to install your GC column? Part 1: Agilent GC ferrules

You’re ready to install a column into your GC and realize you do not have ferrules to do the job. But which ferrule do you choose?  The parameters to consider when choosing the correct ferrule are instrument make and model, nut size and type, ferrule material, and column ID size. A visit to our ferrules home page will provide a general overview of variety of ferrules offered by Restek.  By navigating the page, you can narrow the selection by choosing these parameters. This is the first in a series of posts that will hopefully make this selection process easier. We begin with Agilent systems because they have more choices available.

Capillary Column Ferrules

For this section, I will assume the GC is already configured to install capillary columns with 1/16 inch fittings. Restek offers two options on nuts and ferrules for Agilent capillary column installation. There are compact ferrules and standard ferrules, each with their own style of nut. The compact ferrule and nut are considered the Agilent-style. The broad taper on the compact ferrules was designed to match the taper on the bottom of the inlet seal. Standard ferrule and nut can also be used effectively on an Agilent system.

Here are the visual differences between the standard and compact ferrules:

Standard                                                                         Compact

standferrule                                                             Compferrules          

Capillary Ferrules                                   Compact Ferrules (Fit ≤ 0.28mm ID Fused Silica Columns)

                                                                Compact Ferrules (Fit 0.32mm ID Fused Silica Columns)

                                                                Compact Ferrules (Fit 0.45/0.53mm ID Fused Silica Columns)

 

Here are the catalog numbers of the corresponding applicable nuts:

FTnut                    capnut                 hotswapFT2nut

 

Finger-Tight Capillary Column Nuts Capillary Column Nuts Hot Swap Capillary Column Nuts Looped Finger-Tight Capillary Nuts
Brass Stainless Steel
Standard Ferrules 21041 21879 20883 22347 21312
Compact Ferrules 21040 21878 21884 22348 21311

 

This is a cross-cut look at the insides of the standard capillary nut and shows the length of the ferrule and the depth of the nut hole. This helps to show how necessary it is to match the ferrule with the proper nut; otherwise there will not be an adequate seal. If you are unsure which of these nuts you have on hand to install your columns, take note the Agilent style nut has a notch on the hex-nut portion, the standard does not.

 

comp                                     stand

 

“Compact”  Agilent-style Ferrule and nut                       Standard  ferrules and nut

 

Once you’ve matched the nut type to the correct ferrule type, now we need to match the capillary column ID to the ferrule ID. The table on sizes below gives an overview of how to match fused silica and MXT capillaries.

 20141121105433156

The next step in determining the correct ferrule is the material. The chart below explains the differences between the material choices. For installation into the inlet and most detectors, either graphite or the softer 60/40 vespel graphite should work fine. For GC/MS, it is recommended to use the standard, long ferrule vespel/graphite installed with an MSD source nut.  This material is a good fit for MS because it will not fragment or allow oxygen to permeate into the system under vacuum applications.

Agilent has suggested that 100% vespel ferrules be only used for isothermal analysis.

MSD Source Nut

20643_ph_co_msd

20141118133453588-21

A note on the Agilent TCD, it has an 1/8 inch compression fitting port. In order to use our Agilent style or standard design nuts and ferrules you would need to have the Agilent supplied adaptor fitting. Restek does not have that fitting. Alternatively, Agilent does supply a two piece ferrule to install capillary column with a 1/8 inch compression nut. Restek does not sell the two piece ferrule and we do not recommend using a one piece graphite reducing ferrule.

 

Packed Column Ferrules

Typical Agilent packed column inlets are sized to accommodate ¼ inch OD packed columns; verify the fitting size of your inlet and detector. Agilent systems generally use standard compression style nuts and ferrules. If you doing on column injections, installing a 3/16 or 1/8 inch OD column will require inlet adaptors to center the column inside the ¼ inch injection port. If your instrument is equipped with an adaptor with a glass insert, on column injection will not be necessary, therefore a nut and ferrule would be all that is required to connect the column. Again you will need to verify the size nut and ferrule required. If you are installing a packed column into a capillary column inlet and detector we suggest a pig-tail configuration.

Notes on Agilent detectors- verify the size of the fitting required to connect the column. Agilent TCD’s for example are 1/8 inch compression fittings. If you want to install a 3/16 packed column, make sure your detector is adapted to accommodate ¼ inch packed column so you can use a reducing ferrule and ¼ inch nut.

Packed Column Inlet Adaptor

inletadaptorkit_ph_co_pak

In summary, the key points to remember are:

  • Match the ferrule with the proper nut
  • Know the ID of your column to match with the correct ferrule size.
  • Know system requirements for the ferrule material.
  • For packed columns: Verify the fitting sizes of you inlets and detectors.

 

Look for future posts on nuts & ferrules for other instrument manufacturers. Thanks for reading.

I would like to thank my Colleague, Erin Herrold for her assistance and insight with this blog post.

Residual Solvents in Cannabis…and Terpenes with Simon & Garfunkel

In my last blog post, I wrote about our ongoing method development for residual solvents in cannabis. We’ve been really busy since then, and now we have a complete application note published on this subject. Because we can’t legally get our hands on real cannabis concentrates here in Pennsylvania, the application note proves our concept with some supporting data from our collaborators at CAL Green Solutions. You can check out the full application note here.

By now, a lot of people in the industry know that headspace-gas chromatography (HS-GC) is the ideal analytical technique for analysis of residual solvents in cannabis concentrates, but wouldn’t it be nice to be able to perform another pertinent cannabis analysis on our fancy headspace instrument? It turns out that we can. The beauty of HS-GC is its ability to separate volatile analytes from difficult sample matrices with very little sample preparation. While terpenes are less volatile than residual solvents, all cannabis terpenes (with the exception of phytol) are volatile enough to analyze using HS-GC. With that in mind, we developed a preliminary method for HS-GC analysis of terpenes in cannabis.

In order to keep things as simple as possible, we developed our terpenes method on the Rxi-624Sil MS column, which is the same column that is recommended for residual solvent analysis. Luckily, Jack Cochran had already established that the column does a really good job at resolving terpenes. This means that you can run two different analyses on the same instrumental setup, getting more use out of your instrument investment – hooray! Here’s what the chromatography looks like for a comprehensive set of terpenes:

 

 

Beautiful Terpene Chromatogram

The Rxi-624Sil MS nicely separates a comprehensive set of terpenes.

 

To test out the method sans cannabis, we decided to determine terpene profiles for some of the following common herbs: parsley, sage, rosemary, and thyme (now the title makes sense!):

 

Terpene profiles of common herbs using the Rxi-624Sil MS

Terpene profiles of common herbs using the Rxi-624Sil MS

 

As a disclaimer, this data should be considered semi-quantitative, and the method is preliminary, but our overall profiles matched up well with what we could find about terpene profiles of these herbs. We also profiled hops, which are a close cousin to cannabis. This work can be found in this application note which describes the analytical procedure and our results in detail.

I will be discussing this and other work at The Emerald Conference in San Francisco on January 23rd. The conference agenda includes some good speakers from the cannabis industry, and it looks like it will be a valuable conference for anyone who can attend. 

How Dirty Are You? Part 3…Gloves…The Answers

Read The Question first…

Let’s review…we soaked small pieces of different gloves in acetonitrile for 30 minutes. The samples were tested using GC-TOFMS. For relative intensity, a dashed line at the peak height of a 2 ppm PAH standard is drawn on the chromatograms.

Which gloves produced the worst background? GREEN GLOVES

gloves1

Bright green, yellow latex and royal blue gloves had similar background levels even though the chromatograms look different. Baby blue gloves and green gloves had higher background. Green gloves had the highest intensity background peaks. Let’s look at the data…

brightgreen cgram

 

yellow glove

 

 

 

royalblue

 

 

 

babyblue

 

green2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

I have to admit that I was a bit surprised at the answer to the next question. Several people emailed me answers and UV stabilizers was the most popular choice. However, we saw several antioxidant compounds in the glove samples. A couple of compounds are shown below. Check out the signal from antioxidant “Nocrack NS 6″ in the baby blue gloves!

Antioxidants are added to rubber to prevent oxidative damage that degrade the physical and mechanical properties of rubber materials.

q2 gloves

 

glove antiox 1 glove antiox 2

 

 

 

Philae: Goodnight but Not Goodbye. Unlocking the Origins of Life: Comet 67P/Churyumov-Gerasimenko

Rosetta's Philae on the surface of 67P/C-G. Photo Courtesy of ESA (European Space Agency).

Rosetta’s Philae on the surface of 67P/C-G. Photo Courtesy of the European Space Agency.

On September 28th, 1969 a bright fireball exploded, shaking houses as it lit up the daytime sky outside of Murchison, Australia. Over the next forty-five years scientists have studied the Murchison Meteorite and found 14,000 compounds to include 92 different amino acids. Of the amino acids discovered, only 19 are found on earth. This rock, and evidence from other encounters with space objects, reveals that life-forming amino acids may have originated deep in space even before the formation of the sun.  In addition amino acids have been found on Saturn’s moon Titan, comets and newly forming stars (Large Molecule Heimat, Sagittarius B2).

Comets are of specific interest because they are the oldest objects in the Solar System, preserving four-billion-year-old organic molecules. Comet 67P/Churyumov-Gerasimenko is a short period comet (Jupiter Family) with an orbital period of 6.5 years. These objects are believed to originate from the Kuiper belt, a region of icy rocks just outside of Neptune’s orbit. A collision with another object can cause these rocks to fall into orbit around the sun.

Philae’s COSAC (Cometary Sampling and Composition) experiment was able to analyze samples taken from below the surface of the comet before the batteries died from lack of sunlight. ESA (European Space Agency) reported the lander bounced and is located under a cliff where it only receives 90 minutes of sunlight every 12 hours. When Philae receives enough sunlight it will again power up and send a signal to Rosetta. The last transmission was received on Friday, November 15th but ground control confirmed that COSAC sent back analytical data.

Striking images of Philae's harrowing journey to 67P/C-G. Courtesy of European Space Agency.

Striking images of Philae’s harrowing journey to 67P/C-G. Courtesy of European Space Agency.

Restek in Space: http://www.restek.com/news/view/?p=851

Comet Impacts May Have Jump-Started Life on Earth

http://www.space.com/20102-comets-impact-earth-life.html

Amino Acid Detected in Space

http://physicsworld.com/cws/article/news/2003/aug/11/amino-acid-detected-in-space

New Organic Molecule in Space

http://www.mpg.de/567495/pressRelease20080325

 

Comet 67P/Churyumov-Gerasimenko: Data from Rosina.

Mosaic of 4 images combined. Gas and dust from comet can clearly be seen from the center. Courtesy: European Space Agency (ESA).

Mosaic of 4 images, gas and dust from comet can clearly be seen from the center. Courtesy: European Space Agency (ESA).

While anxiously waiting for data from the COSAC (Cometary Sampling and Composition Experiment) aboard the Philae lander, we turn to some of the information that has already been sent by the Rosetta’s Rosina (Rosetta Orbiter Spectrometer for Ion and Neutral Analysis) instrument.  While sampling the comet’s coma they have found carbon dioxide in nearly the same proportions as water. Rosina has the ability to measure the ratio of hydrogen to deuterium in water.  Deuterium is believed to have been created in the Big Bang 13.8 billion years ago.  Since deuterium accounts for approximately 0.0156% of all hydrogen found on earth, this data may shed light on the origin of our water.

So far the following compounds have been detected by Rosina:

Water, Carbon monoxide, Carbon dioxide, Ammonia, Methane, Methanol, Formaldehyde, Hydrogen sulfide, Hydrogen cyanide, Sulphur dioxide, Carbon disulfide and ethanol.

 

The ‘Perfume’ of 67P/C-G

http://blogs.esa.int/rosetta/2014/10/23/the-perfume-of-67pc-g/

Restek in Space

http://www.restek.com/news/view/?p=851

 

Catching a Comet Traveling at 40,000 Miles per Hour

Comet 67P/Churyumov–Gerasimenko at less than 10 km from its surface. Photo Courtesy of ESA

Comet 67P/Churyumov–Gerasimenko at less than 10 km from its surface. Photo Courtesy of ESA

In 1993 the International Rosetta Mission was approved and over the next 21 years an estimated 1 billion Euros was invested in an audacious plan to catch a comet. On March 3rd, 2004 a European Ariane 5 rocket propelled the Rosetta on a ten year mission to orbit a comet.  While there have been 6 brief intercepts with comets in the past this will be the first time a spacecraft will fly alongside and land on a comet as it heads back into our solar system.  The craft has 21 instruments to include 8 parallel gas chromatographic columns on board, each equipped with a thermal conductivity detector (TCD). Using thermally activated switches effluent from these TCDs can be directed to a single Time-of-Flight Mass Spectrometer.  Restek provides four of the columns to include the MXT-UPLOT, MXT-1701, MXT-20 and MXT-1.

Previous encounters with comets and ground-based observations indicate that comets contain complex organic molecules containing carbon, hydrogen, oxygen, nitrogen and sulfur and may hold the key to understanding the origins of life on our planet.  Today at 11:00 AM EST the Philae lander will touch down on the comet and the European Space Agency (ESA) will webcast live coverage from mission control in Darmstadt, Germany.

 

 

The pneumatic hardware for the 8 capillary columns. Photo Courtesy of Max Plank Institute for Solar Resarch, Katlenburg-Lindau, Germany.

The pneumatic hardware for the 8 capillary columns on board the Philae lander. Photo Max Plank Institute for Solar Resarch, Katlenburg-Lindau, Germany.

 

Check out our press release: http://www.restek.com/news/view/?p=851

Live Webcast of from Mission Control: http://new.livestream.com/esa/cometlanding

Track Rosetta’s Location: http://sci.esa.int/where_is_rosetta/

COSAC – Cometary Sampling and Composition Experiment: http://www.mps.mpg.de/1979406/COSAC

 

 

 

 

 

How to decrease run time for reversed phase LC

There are several ways to accomplish this, although it boils down to roughly 4 possibilities.

  1. Use a shorter column length.

This may be appropriate if you have plenty of retention for your analyte(s), if you only have one or a few analytes AND if you do not have interference peaks in your chromatogram. It does, however, require purchasing a new column and shortening the length (L) does reduce the number of theoretical plates (N) in your column. The good news is that the end result might be better than you would expect. Shortening your column length by a factor of 2 does not reduce resolution (R) by a factor of 2, but only reduces it by a factor of 1.4, since R is proportional to the square root of N.

R for hpclN vs dp

  1. Optimize conditions for your existing column.

Look at the following and see what you might be able to tweak:

Flow rate- First of all, if you are running at less than the optimum flow rate, you should definitely increase it as long as your system can handle a little more pressure. Secondly, you may be able to go slightly above what is recommended as the optimum flow rate if you don’t need to have perfectly symmetric peaks and/or separation from other peaks. If you’re not sure what the optimal flow rate for your column should be, there are some listed in the blog post for Building up Pressure.

Mobile phase (s)- If you have not investigated this yet, try increasing the organic content of your mobile phase. You can also try using a different organic solvent. There are some cases when methanol will provide better selectivity, such as with biphenyl phases. Otherwise, acetonitrile may provide shorter retention times because it is more nonpolar. Solvent choice should be made on a case by case basis, though, as sometimes it may surprise you what works better. (Please keep in mind that this is only the case for reversed phase LC.)

Gradient- Try starting with higher organic and/or ramping up to more organic faster and see if that helps.

Temperature – Try increasing temperature. This usually speeds things up and often increases resolution also. Just make sure you don’t go beyond what is recommended for the column you have. Restek HPLC columns generally have a maximum temperature limit of 80°C. A good article to read on this topic is HPLC solutions #53: Temperature and Retention by John Dolan, published by Separation Science.

 

  1. Use a smaller particle size column

This will increase column efficiency and retain the efficiency even at higher flow rates. This is because efficiency is inversely proportional to the particle size, as shown in the two equations above. The following graph demonstrates the effect of linear velocity on H, the height equivalent of a theoretical plate. Keep in mind that a lower H value indicates greater efficiency, more plates (N) per unit length (L).

 

H vs dp and u

Since you will have more plates per unit length, you can either keep the same length and get increased separation or you can shorten the length accordingly to get the same separation with a shortened analysis time. The relationship between particle size and length is illustrated by the following equation.

L vs dp

Another option for smaller particles may be to increase the flow rate, since the column remains efficient at higher flow rates versus a comparable column with larger particle size, with all other dimensions remaining the same. For reference, please see the graph above of H vs. Linear Velocity, µ. Also keep in mind that any change in the inner diameter results in a change of linear velocity. A reduction in ID actually requires decreasing the flow rate to maintain the same linear velocity, so that must be considered as well. Generally, decreasing the ID is more useful in terms of saving solvent, not in shortening the analysis time.

Please note that decreasing particle size also results in a higher backpressure, so the instrument hardware must be considered when determining what particle size to use. The use of our 1.9 µm particle Pinnacle DB columns does require a UHPLC system (equipped for 1000 bar or higher max pressure).

 

  1. Use a superficially porous particle (SPP) column

In terms of Restek products, these are the Raptor™ columns. Otherwise known as fused core, particles in this column result in greater efficiency, similar to using a smaller particle, but without the backpressure that is normally associated with that particle size. A simple explanation of these benefits can be found at the blog post, What is SPP and when should I use a Raptor™ column?. Using SPP columns often produces a marked improvement in sharpness of peaks, resulting in greater overall sensitivity for the analysis. Often data can be produced that is UHPLC quality on an LC that is not technically a UHPLC system. If you need help determining whether to use a 2.7 µm of 5 µm column, please see the blog post Should I use a 2.7 or 5 µm Raptor™ column?.

 

Suggested links for reading:

Easy Transfer of HPLC Methods to UHPLC

Explaining the Small Particle Advantage

The Effects of LC Particle Choice on Column Performance: Switching from 3 and 5 µm Fully Porous articles (FPP) to 5 µm Superficially Porous Particles (SPP)

The Essential CHROMacademy Guide, Mobile Phase Optimization Strategies for Reversed Phase HPLC

 

I hope that you find this useful. Thank you for reading.

 

Which GC instrument should I use for the analysis of pesticides in herbal tea? GC-MS/MS or GCxGC-TOFMS?

You may remember a while ago I posted a blog about the analysis of herbal tea using QuEChERS and GCxGC-TOFMS.   The LECO Pegasus GCxGC-TOFMS is a great instrument for analyzing complex samples because of the comprehensive chromatographic separation achieved (two columns with different selectivities).  The GCxGC-TOFMS also excels at non-target screening of compounds.  Lucky for us, we also have a Thermo TSQ 8000 GC-MS/MS in our lab to use.  The GC-MS/MS is also a great instrument for analyzing complex samples, like herbal tea, because of the selectivity afforded by the Selected Reaction Monitoring (SRM) transitions.  While the GC-MS/MS cannot do non-target screening like the TOFMS can, it is significantly more sensitive for targeted analysis.   My colleague Julie Kowalski and I set out to do a head to head comparison of the two instruments for the analysis of pesticides in herbal tea.  Check out the data and analysis conditions in my presentation that I gave at the recent EPA Region 6 Quality Assurance Conference in Dallas, TX.

Overall the instruments performed similarly, with only a few notable differences.  One of the major differences was that we could accurately quantify at the 10 ppb fortification level (5 pg on-column) on the GC-MS/MS where we could not get that sensitivity on the GCxGC-TOFMS system.

So which GC instrument should I choose for the TARGETED analysis of pesticides in herbal tea – the GC-MS/MS.  However, I couldn’t do this with the GC-MS/MS though.  So, in the end I am happy to have both instruments in the lab! Please let me know if you have any questions or want more info on this analysis.

GC capillary columns for the European Pharmacopoeia methods

Have you ever viewed a European Pharmacopoeia method and tried to figure out which Restek GC capillary column you should use? To make the selection a little easier for our customers, I decided to match the nomenclature listed in the European Pharmacopoeia “Reagents” chapter to the appropriate Restek columns.  I hope you find the table below helpful.  If you have any questions, simply send them to support@restek.com or contact your local distributor.  Thank you.

 

column

EU Capillary 5