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# The Ultimate Technique for Complex Mixture Separation: FUPLC-NMR-CE6-GC-IR-ICP-MS-MS-MS-MS

Posted by Günther Schlonk
February 11, 2022February 11, 2022
Posted in Articles, Journal of Immaterial Science
Tags: Chemistry, Research, Satire, Science

Günther Schlonk,A Günther Schlonk,A Günther Schlonk,A and Günther SchlonkB*

Abstract: Nothing we can write is funnier than a journal called Anal. Chem.

Specific: We have observed the trend of combining analytical instruments in series, and extrapolated it to the max.

The march of analytical chemistry is relentless. In the century-and-a-bit since the invention of chromatography, it has made great strides, from tubes full of sand to the million-dollar instruments of today. We have seen LC become HPLC, then UPLC, and MS become MS-MS, then MS-MS-MS. At this rate, the acronyms will take over by 2050, and chemistry will become just as incomprehensible as molecular biology.

The Schlonk lab is infamous for creating mixtures of hellish complexity, as a result of spectacularly failed reactions.1,2 Consequently, we have a need for extremely powerful analytical techniques to help us figure out what on earth happened to our reactants. Conventional techniques such as UPLC and MS3 are incommensurate with our needs, so we have been driven to develop our own instrumentation and methodology. We have constructed a device capable of isolating and fully characterising a new molecule from a complex mixture. This was accomplished by stringing together every analytical instrument we had in series. The result is the FUPLC-NMR-CE6-GC-IR-ICP-MS-MS-MS-MS. (Figure 1).

Figure 1: Two operators at the controls of the FUPLC-NMR-CE6-GC-IR-ICP-MS-MS-MS-MS.

The instrument is about the same size as a semi-trailer, and currently occupies a small building adjacent to the chemistry department. Power consumption is equivalent to a medium sized city block, and the instrument requires copious amounts of argon, hydrogen, xenon, solvent and Taxol buffer solution. Thus it may be some time before a portable version becomes available. Figure 2 shows a diagrammatic representation of the instrument.

Figure 2: Diagrammatic representation of the instrument.

FUPLC-NMR

The Instrument is designed to accept liquid samples, which are placed on a stone plinth recessed into one side. Appropriate offerings and propitiations are made before the injectionator descends. Thanks to a fruitful collaboration with Flagellant Technologies, a range of commercially available columns have been adapted to function with our instrument (table 1).

Abnormal Phase | Perverse Phase
Silica | C10 Silica
Alumina | C18 Silica
PtO/Pentazole MILF | Sand + Butter
Powdered Mentos | Styrofoam Packing Beads
Icing Sugar | Golf Balls
Coffee Grounds | Macerated Tyres
Smythe’s Column (LiOH slurry) | Oceanic Microplastic3

Table 1: Compatiblestationary phases for FUPLC.

Of particular note is the platinum oxide/pentazole metal-inorganically-linked-framework (PtO/N5 MILF) developed by Kimmel and Klein,4 which contains enough theoretical plates for an entire Christmas dinner. Conjectural crockery aside, coffee grounds have recently been proposed as a sustainable and biodegradable column packing material. This may be the case, but running a blank on a new column is essential. We ignored this step, which led to our erroneous claim that Antarctic sea-ice contains caffeine at 2000 ppm.5 The Smythe type column is ideal for the separation of basic molecules such as amines. It is most effective when freshly prepared by filling an empty column with n-BuLi and waiting a while.

Perverse phase columns are available for the separation of analytes at the far ends of the polarity spectrum. Conventional C18 columns are effective but expensive. A cheaper alternative is to treat sand with butter, which yields a passable imitation of C18 silica (though much less durable). Golf balls are also cheap and have a non-polar surface, but large void spaces reduce the efficiency of such columns. Styrofoam pellets have a much larger surface area, but careful selection of eluent is crucial to prevent the stationary phase dissolving. Oceanic microplastics are an ideal column packing: cheap, readily available (to excess, some might say), robust and with adjustable particle sizes.

A range of eluents can be used the instrument, in either gradient or autocratic solvent programs (table 2).

Non-Polar Eluent | Polar Eluent
Silicon Oil | DMSO
Vacuum Grease | Spryte (5% carbonic acid)
Des-methyl Toluene | CDCl3
Perfluorohexane | Magic Acid (1% aq.)
Crude Oil | Brine

Table 2: Solvent systems for autocratic elution.

Extensive optimisation is required for most samples, but as a general rule a gradient from 0–100% silicon oil/DMSO is sufficient to elute most analytes from a perverse-phase column. Some time ago, we won a 420 MHz NMR spectrometer as the main prize at VariBrük’s annual quiz night, (for those interested, the crucial question was “what’s the difference between a duck?”).6 The spectrometer contains a new generation of Rh/Sr/Ho superconductors capable of operating at liquid H2 temperatures, and is technically a bench-top instrument (provided you have an extremely large bench). Using the latest advances in NMR-flow technology, we have run the FUPLC-column through the guts of the spectrometer. This modification allows us to collect spectral information on our analytes in real time (sort of). Flow-NMR has some significant challenges associated with it, the first of which is the solvent. Several workarounds exist to prevent analyte peaks being swamped by solvent signals. The simplest, though most expensive, is to use deuterated solvents in the FUPLC. This is manageable when chloroform is a suitable eluent, but cost can escalate rapidly when THF is involved. Aprotic solvents like perfluorohexane and diphosgene also circumvent these issues, albeit with some added toxicity. Finally, a harsh program of solvent-repression can be conducted, to supress unwanted peaks.

NMR experiments must be conducted rapidly to account for the constant flow of analyte through the sample chamber. Conventional 1D-NMR spectra can be acquired, with some minor peak broadening (Figure 3).

Figure 3: 1H NMR of ethyl acetate under flow conditions.

Two-dimensional techniques such as the 1H-1H HASTY (Highly Accelerated Spectroscopy) experiment can provide more comprehensible spectra (Figure 4).

Figure 4: 1H-1H HASTY spectrum of serotonin.

CE6

One of my students helpfully pointed out that this grey box I was using as a coffee table was actually a capillary-electrophoresis mass-spectrometer. We dusted it off and installed a new six-barrelled cartridge designed by Smythe and Wesson, allowing six fractions to be separated simultaneously. Chiral separations can be achieved with phthalidomide-coated capillaries and 1 mM Taxol background electrolyte. The voltage is supplied by a lightning rod on the roof, and detection is by Calamitously Capricious Contactless Conductivity Detection(C4D).

GC-IR

Output from CE is split into two streams, one of which is nebulised, volatilized, and disorganised in an Aspirationalizer (patent rejected). Volatile components enter a GC column, propelled by xenon as carrier gas. While in the gas phase, the analyte molecules are irradiated with IR light, and absorbance spectra are obtained. Of particular interest are the C-H twerking, C-C bopping and C-O thrusting frequencies. Output from the GC is split between inductively coupled plasma (ICP) and MS4 systems.

ICP

Analyte molecules are obliterated in a whirling vortex of argon ions at 10000 oC. This serves no practical purpose but is immensely satisfying to watch (Figure 5).

Figure 5: Some scientists just want to watch the world burn. (Photo credit: Wblanchard CC BY-SA 3.0 via Wikimedia Commons)

MS-MS-MS-MS

The final stage of analysis combines the feeds from both the CE6 and GC-IR and infuses them into a veritable mountain of mass spectrometers. Electrosquirt Idolisation (ESI) is used to produce the sample ions, which pass through an ion trapdoor into the vacuum chamber. A pressure of 10-10 Torr is maintained within the chamber by a vacuous pump, which scares gas molecules out of the chamber by playing Keeping Up With the Kardashians at high frequency. Ions are accelerated from 0 to 60 keV in 7 ms by charged plates designed by Ferrari.7

The first quadrupole is a traditional mass filter, while the second is a derision cell, in which ions are bombarded with insults to weed out those lacking commitment. The truly motivated ions make it to the third quadrupole, which can be tuned for specific analytes. For most analyses, tuning the MS to C major is sufficient, and causes the ions to oscillate rhythmically (a phenomenon known as quadrupoledancing). The final element is a dipolar political polarisation filter (DPPF), which separates the ions by their views on universal healthcare. Mass analysers are positioned to the left and far right, which provide the final signal output. The entire MS array is contained in a black box, both literally and metaphorically.

Data Processing

We have found that quantum computing is the only technique with sufficient power to process the complex data generated by the instrument. However, the instrument is only compatible with Windows XP, so good luck with that.

Conclusions

The FUPLC-NMR-CE6-GC-IR-ICP-MS-MS-MS-MS should prove a powerful addition to any analytical laboratory. The instrument costs slightly less than the Iraq war, or about the same as the loose change down the back of Elon Musk’s couch. Running costs equate to one student’s kidney’s per fortnight. With a full team of eight operators, a method can be developed and optimised in as little as six months, and a single sample can be analysed in <48 hours. The instrument is capable of performing the full characterisation of a novel molecule in one go. The only thing it can’t do is X-ray crystallomancy, but it can make a coffee while you wait.

Acknowledgments

Günther Schlonk thanks Günther Schlonk for his diligent work on this project. Günther Schlonk acknowledges Günther Schlonk, it was a pleasure to work with him. Günther Schlonk, however, thinks that Günther Schlonk didn’t pull his weight on this one, and that Günther Schlonk should be first author instead of Günther Schlonk.

About the Authors

Demeritus Professor Günther Schlonk seized power in a palace coup in mid 2021, and appointed himself Chemistry Tzar of the University of West Failure. His other titles include Satrap of Satire, Toastmaster General and Provost of the Plebs. He is the Imperial Editor in Perpetuity of The Journal of Immaterial Science.

Conflicts of Disinterest

Günther Schlonk is not an analytical chemist, and this article has made that abundantly clear.

Notes and references

1 G. Schlonk et al. J. Immat. Sci. 2021, 1, 1–35.

2 G. Schlonk et al. J. Immat. Sci. 2021, 1, 35–69.

3 C. Klein, J. Kimmel, T. Kapputke, O. Yogi, R. A. Freud, J. Immat. Sci. 2021, 1, 43.

4 Microplastic was responsibly sourced from the stomachs of deceased sea turtles.

5 G. Schlonk, Sci. Part. Envi, 2020, 4, 568–567 (retracted).

6 Answer: one of its legs is both the same!

7 R. Hammond, J. May, J. Clarkson, A. Twat, Top J. Chem. 2011, 69, S1E1–

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