Topics in GC & HPLC

Sometimes we get customer reaction that porous polymer PLOT columns bleed a lot. They are used to the low bleed values using Rxi-series of columns and wonder why this is not the case with porous polymers. Porous polymer columns have a huge amount of stationary phase to generate sufficient loadability and retention. Typically porous layers are upto 20 um in thickness, That means for a 30m column, a porous polymer PLOT has approx. 200 x more stationary phase compared with a 30/0.25/0.25 type Rxi column.

Therefore much higher bleed values can be expected for the porous polymer type columns. If we aim for same bleed as Rxi, we must realize, the upper temperature should be listed 100C lower.

The max temp. listed for porous polymer columns is kept high, to be able to bake-out impurities that may accumulate on the column. The new generation Rt-Q-BOND columns do offer a lower bleed while films are thicker, reducing background but with higher capacity so technology is improving.

Lower elution temperature will make a component elute at a more stable baseline, which can be especially of interest if there is a lot background developed. Many methods are inherited from time where column inertness was not controlled as well as we do now. Often thicker films were chosen to get sufficient symmetrical peaks. Thick films will give high elution temperatures and they will bleed a lot.

Today’s GC Columns (implementing Rxi-technology) have MUCH higher inertness, so you do not have to use a thick-film to get a good peak.. Using a 2x thinner film reduces bleed about 50% AND reduces elution temperature by approx 16 degrees... Lowering elution temperature by 16 degrees will also reduce the background a additional factor 2.

In total, the background signal will be lowered a factor 4 providing highest signal/noise, a faster analysis and you can use the same analysis conditions (easy to try!) Check Restek's GC program if your phase has an Rxi-equivalent with thinner film

Instead of trying to bribe shy readers with autographed photos like some of my other colleagues, I'm going to ask for help on an issue that's been bugging me for the past couple of days. Anybody who has any feedback will get 10 wizard dollars. That's enough for a t-shirt. Those of you who aren't interested in Restek apparel should still read on, because this is an interesting phenomenon, and I'd like some help explaining it. While performing a splitless injection of amphetamines in methanol, I see a large peak that doesn't show up in split injections. It looks like a separate compound forming from my primary amine in the injection port, as there is a common mass difference of 12m/z for elements of the two spectra. This peak disappears when the sample solvent is changed to ethanol. For a full explanation, check out the figure. I wouldn't recommend methanol for amphetamines analysis anyway, so it's kind of a moot point, but it would be nice to have an explanation for what's happening.

Everybody knows that column cutting is important. For some injection techniques, its more important then for others. With a split-injection we usually get away easy. With on-column and direct injection the challenge is bigger. Also, If we want to make coupling, we need an ideal cut. Fused silica can be cut with different devices, of which the “wafer” is the most simple and the cheapest..

Cutting metal (MXT) columns is another story. When using a split injection one may use the wafer:most of the time MXT columns are used with Valves, on-column injection or a PTV type injection

To reduce dead volumes in these systems, one needs the best column cut. A device to help us, has become available. Its called a column “scorer”. By scoring the MXT tubing on the outside, trimming becomes easy and a perfect end cut is created, allowing:

- no issues with on-column injection

- the best possible “seal” using direct injection liners (PTV)

- minimal dead volume in Valve connections

Stationary phase (i.e. column type) is a key contributor to selectivity in LC. But what about the mobile phase? A significant advantage, and sometimes challenge, in LC is that both the stationary and mobile phase chemistries can be modified to optimize our method. This contrasts with GC, where the main selectivity option is the stationary phase. LC mobile phase chemistry can be altered by changing the pH, adding ion pair reagents or other modifiers, or changing the type of organic solvent used. For example, phenyl-based stationary phases can offer significantly different selectivity for aromatic, nitro-, and sulfone-containing analytes when using methanol vs. acetonitrile. This is demonstrated for a series of NSAIDs – with acetonitrile, the selectivity of a Biphenyl phase is very similar to a C18. With methanol, however, the Biphenyl vs. C18 selectivity is radically different (see figure). Mobile phase chemistry – another useful tool in LC method development!

Gas chromatography can be used widely, but when components get thermolabile we have to be careful. When a component decomposes while it is traveling through the column, the peak shape will tell us. The component will decompose most of the time into a more volatile component that elutes faster from the column. We observe a typical peak shape as shown in the figure: the peak shows a strong “lead”.

The example shown here is a flame retardant. There components are designed to decompose but we have to keep them in shape to quantify them. This can be done by reducing the elution temperature. If BDE elutes below 295C, the peak will be near symmetrical.

Ways to reduce decomposition in the column:

• Use higher flow rate, a flow program or a pressure program.
• Use a slower temperature program
• One can also use hydrogen as the carrier gas instead of helium.
• Use columns with thinner films: Use a capillary with a 0.32mm internal diameter
• Use shorter columns

I hate to think of myself as a cynic but when the request came from a leading lab in May of 2000 for ppb levels of alcohols by P&T I “proved” it could not be done (Search website GC_EV00401). This c-gram showed ethanol at 10ppm by purge and trap and my advice at the time was, “You can get 500ppb using optimized flash injection,” & that echoed conventional wisdom at the time. Trent Sprenkle & Laura Chambers disagreed and produced an outstanding application note (O.I.Analytical #25370206) which evaluated sample temp. & trap temp. (during purge) and were able to prove that it could be done even for ethanol at single ppb levels. I revisited this application for a customer sample in 2004 and found that it was quite straightforward (see figure). I ran 100ppb and had enough sensitivity on an older GC/MS to see 10ppb without any effort. For the complete answer to oxygenates and alcohols for the analysis of gasoline take a look at both of these apps notes & remember nothing is impossible.

Testing for alcohol metabolites such as ethyl glucuronide (EtG) and ethyl sulfate (EtS) is growing in popularity because these metabolites can be used to indicate alcohol consumption up to 80 hours after ingestion. Current methods for analysis of EtG and EtS generally use RP-HPLC, but since both compounds are polar, they are difficult to retain on reversed phase columns. Enter the ion pairing reagent. Gasp! I know, ion pairing (IP) reagents shouldn't be used on LCMS instruments, right? Well, there are certainly several IP reagents that should exclusively be used for UV analyses, but there are some volatile IP reagents that are suitable for LCMS analyses. One such reagent was used to generate this chromatogram, and my LCMS is still chugging along as I type this post. When compared to other methods, retention is much improved, while run time is reduced due to the shorter equilibration times needed for shorter columns. Feel free to contact me for more information. I want to hear from you!

This will be a short post without informative and useful data or exciting offers to win autographed photos of Restek celebrities (for the former, see my colleagues’ posts below, for the latter, see mine). Instead, I just want to call your attention to the new Comments feature added to the blog. It makes providing your valuable comments, questions, or concerns a little easier than before. The key to science is discourse (and often times disagreement), and we are very eager to hear your questions, experiences, or concerns about the topics posted here. So don’t be shy! Wing your comments our way and let’s start talking. Oh, and if you want that picture, be sure to follow the link below (shameless, I know).

Analysis of thermal labile compounds is challenging. The component can decompose in injector and/or column. When decomposition happens in the injector we usually see smaller peaks and we may also see the degradation product as a peak. Example in the figure are carbamates which are decomposing in a hot injector during splitless injection forming phenolic degradation products.

On column injection would be the best, but we may also consider:

- use lowest possible injection port temp.

- use highest possible flow (use 0.32mm ID fs)

- reduce splitless time by using a pressure pulse

- use inert liners (siltek or specially deactivated)

- avoid glass wool packings as these may initiate decomposition

Alternate injection technique to consider is the PTV. Here the sample is introduced in a cold liner, and flash evaporated when the injector is heated. It’s not as good as the cold-on-column, but better than the splitless technique. And then we have also the option of LC..