Hand-considered hypotheses, 1301–1400

Written one at a time, not composed. The subject is separation: what a phase transfer, a bed, a membrane or a detector actually measures, and where the number finally reported is a composite of the quantity wanted and something else. Each entry is labelled with the form of its argument rather than a topic tag — a quantity misattributed, a hidden term, an identity asserted, a bound, a threshold, a reversal, a count, an artefact wearing the costume of an effect, a scale argument, an inversion of standard practice, a selection effect, a unit trap, a derived quantity, and several forms that appear here because the argument needed them and not because the list did. No citations are given and none were consulted, so nothing here rests on a source; where a number appears it is an order-of-magnitude estimate and is marked as one. None has been tested.

Extraction and phase transfer

Misattribution
H1301. Recovery of an analyte from a frozen sample often appears to rise over months in storage, and the rise is put down to better extraction of an aged matrix. A likelier carrier is enzymatic release: hydrolases still active well below zero convert a bound conjugate into the free analyte, and the free fraction is what the method measures. Two aliquots stored side by side, one of them held at 95 degrees for ten minutes before freezing, should diverge by the size of the enzymatic term while the total after strong hydrolysis stays constant.
Hidden term
H1302. A partition coefficient measured by shake-flask in a salted system includes a term for the water that the salt pushes into the organic phase, and at high ionic strength that term is not small. What is reported as a salt effect on the analyte is partly a change in the solvent that the analyte is partitioning into. Titrating the organic layer for water across the salt series separates the two: if the coefficient tracks water content rather than salt concentration, the solvent has moved and the analyte has not.
Threshold
H1303. Dispersive cleanup of an extract stops working at a sample-to-sorbent ratio, and it stops abruptly rather than fading, because pigment capacity is a fixed count of sites rather than a partition. Below the ratio the isotherm is linear and removal scales with sorbent mass; above it the surface is saturated and additional matrix passes straight through. Holding sorbent mass fixed while raising sample mass should show a knee, and the knee should move with the sorbent's surface area but not with the analyte's concentration.
Reversal
H1304. Warming a liquid-liquid extraction speeds mass transfer and lowers the partition coefficient, and the recovery optimum between those two is usually read as a kinetic-thermodynamic compromise that nobody can locate a priori. Salt addition moves the thermodynamic term and leaves the kinetic one nearly untouched, so the direction the optimum moves identifies which term is in charge. An optimum that does not move when salt is added is kinetically controlled, and no amount of phase-ratio tuning will improve it.
Identity
H1305. Third-phase formation and a stable emulsion are treated as different failures of an extraction, but both appear when a surface-active co-extractant accumulates at the interface past its own critical concentration. If that is one phenomenon, an emulsion-prone solvent pair should be predictable from the matrix's interfacial tension alone, without a trial extraction. Measuring interfacial tension for the candidate pairs before extraction, and checking that the ranking matches the emulsification ranking, is the discriminating test.
Bound
H1306. Uptake by a solid-phase microextraction fibre is bounded by delivery to the fibre rather than by the coating's affinity, once the boundary layer around it is depleted. The signature would be an uptake rate identical for coatings of different thickness at the same stirring rate: if a thick and a thin fibre take up the same mass per unit time, the coating is not the limiting element. A stirring series turns the test into a slope, since the rate should follow the square root of stirring speed while the boundary layer governs.
Scale argument
H1307. At extract volumes of a few tens of microlitres the container wall holds more of a basic analyte than the liquid does, so recovery becomes a property of the vial rather than of the method. The crossover is calculable from the vial's surface-to-volume ratio and the analyte's adsorption isotherm, and it sits uncomfortably close to the volumes used in low-volume autosamplers. The same extract in silanized and untreated vials of identical geometry should differ by more than the analytical precision, and the difference should vanish when the volume is doubled.
Selection effect
H1308. Recoveries reported for newly synthesised sorbents are selected on the batches that worked, and the selection is invisible because failed batches are never written up. If batch-to-batch variation is the dominant term, a pre-registered trial of ten independently synthesised batches should show a between-batch standard deviation comparable to the claimed improvement over the incumbent method. The trial costs little and is the only design that separates a real advance from a fortunate synthesis.
Inversion of standard practice
H1309. Drying an organic extract over anhydrous sodium sulfate before injection is standard practice, and for analytes with a high Henry constant it is a net loss: the salt adsorbs analyte and, on a warm bench, drives the rest into the vial's headspace. The step is inherited from methods whose analytes were far less volatile than those now measured. Paired aliquots, one dried and one passed through a hydrophobic filter, injected from the same autosampler, should show the dried aliquot lower by an amount that grows with the analyte's Henry constant.
Unit trap
H1310. Recovery quoted per gram of sorbent and recovery quoted per gram of sample rank methods differently, because the two conventions differ by the phase ratio and the phase ratio is almost never reported. A method with a large sorbent mass looks efficient under the first convention and ordinary under the second. Recomputing a published comparison into the other convention is sufficient to test whether the literature's ranking is an artefact of normalisation, and it needs no new experiment.

Sorbents, beds and breakthroughs

Count
H1311. A mixed-mode sorbent is advertised with three interaction modes, but at most two of them are independently addressable, because the reversed-phase and ion-exchange functions sit on the same strand and cannot be varied one at a time. The test is a probe set eluted across a grid of ionic strength and organic fraction: if the retention surface has rank two, a two-parameter model will fit within experimental error and the third mode is a relabelling. Rank is checkable from data already in most method-development files.
Non-identifiability
H1312. Capacity and mass-transfer coefficient cannot both be recovered from one breakthrough curve, because the curve's position fixes one combination of them and its width fixes another, and the two combinations run nearly parallel over the usual flow range. This is the quiet reason capacities quoted by different laboratories disagree while each laboratory's own repeats agree. Fitting two flow rates jointly breaks the degeneracy, and the fitted pair should then predict a third flow rate that was never used in the fit.
Artefact mimicking effect
H1313. Selectivity attributed to a molecularly imprinted polymer is partly the selectivity of its own crosslinked network, which is not imprinted at all. A template-free polymer made in the same batch often shows the same preference order, and the imprinting factor then shrinks toward unity when the comparison is made properly rather than against a commercial blank of different provenance. The discriminating test is the in-batch non-imprinted control, and it is the one control most often omitted.
Hidden term
H1314. Sorbent mass is quoted as a dry mass, but a cartridge stored in a humid laboratory delivers less active phase than its label, because water occupies the sites the method relies on. For silica-based sorbents the deactivation is large enough to shift retention of basic analytes between a freshly opened box and one left open for a month. Weighing the cartridge before and after drying, then running one probe analyte on both states, separates the water term from the batch-to-batch term.
Bound
H1315. The enrichment factor of any sorbent is bounded by its own blank: leachables from the polymer and the frit set a floor on the concentration that can be reached, and extra sample volume does not move it. The bound is independent of the analyte's affinity, which is why a sorbent with excellent capacity can still fail at trace levels while a mediocre one succeeds. Eluting a clean cartridge with the method's own eluent and measuring the background across the elution window gives the floor directly.
Hysteresis
H1316. The adsorption and desorption isotherms of a swelling sorbent do not coincide, so a method developed from the adsorption branch overestimates what the desorption step will release. The loop is not analytical error but a real change in accessible polymer volume after first solvent contact, and it is largest for the analytes that swell the matrix most. Measuring both branches at matched loading in the same cartridge gives the loop area, which estimates how much of the reported recovery is history rather than affinity.
Saturation
H1317. Whether a longer sample load helps or hurts is decided by the isotherm's slope at the working concentration: on the concave part the front sharpens and added volume adds mass without adding band width, while on the convex part the front spreads and added volume simply dilutes the eluted peak. The crossover loading is measurable by frontal analysis at three loads, and it is a property of the sorbent-analyte pair. A method transferred between concentration ranges without re-measuring the crossover will behave differently at the two ends.
Degeneracy
H1318. Two sorbents can agree on recovery and on selectivity across a twenty-analyte panel and still work by different mechanisms, which means the panel cannot tell them apart. Degeneracy of that kind survives validation because validation panels are chosen for chemical diversity of analytes rather than for coverage of interaction space. A homologous series with single-step changes in ionisation constant or lipophilicity breaks the tie, and the two sorbents should diverge somewhere along it.
Conservation budget
H1319. Published recoveries rarely close the mass balance, and the missing fraction is usually on the vessel wall rather than lost to the atmosphere; for basic analytes in glass, a rinse of the emptied vessel and a separate injection of that rinse should recover a double-digit percentage of the spiked mass, an estimate from surface silanol capacity rather than a measurement. Reporting the rinse as part of the method converts an unaccounted loss into a measured one and costs one injection.
Inversion of standard practice
H1320. Conditioning a polymeric cartridge is taught as wetting the sorbent so the sample will wet it too, but the conditioning solvent's more important job is to wash out manufacturing oligomers that would otherwise elute inside the analyte window. Skipping conditioning therefore changes the blank far more than it changes capacity, and the usual remedy of a larger sorbent bed addresses the wrong failure. Blanks with and without conditioning, on the same cartridges, show the two effects are independent and that one of them is being corrected by the wrong step.

Retention in liquid chromatography

Misattribution
H1321. Retention drift across a long injection sequence gets laid at the column's door, but a substantial share of it is dissolved gas changing the pump's effective stroke and the buffer's pH as the reservoir equilibrates with air. The drift is then in the eluent, not in the stationary phase, and it will follow the reservoir rather than the column. Degassing one reservoir continuously while sparging another intermittently, on the same column, should give two drift curves whose difference is the gas term.
Hidden term
H1322. A retention factor measured on a silica-based reversed phase in unbuffered water contains a silanol term that varies with ionic strength, so the hydrophobicity being reported is a composite of two interactions. The term is largest for basic analytes and shrinks as buffer strength rises, which is why published values for such compounds scatter more widely than the columns themselves do. Measuring retention at three buffer strengths and extrapolating to infinite ionic strength isolates the silanol contribution.
Threshold
H1323. Column overload begins at a sample mass that is set by the injection solvent's strength rather than by the mass alone, because a strong solvent floods the inlet with a plug the stationary phase cannot refocus. The onset is abrupt, and below it the same mass injected in a weak solvent is unremarkable. Injecting a mass series in both solvents and looking for the discontinuity locates the threshold and shows it is a property of the solvent, not of the column's capacity.
Reversal
H1324. Adding water to a HILIC mobile phase first increases retention and then decreases it, and the maximum is the boundary between adsorption at the surface and partitioning into a water-rich layer. The two mechanisms are usually invoked separately to explain datasets that will not reconcile; here they are two regimes of a single curve. The maximum's position should shift with the column's surface chemistry but not with analyte size if the transition is a solvent-layer phenomenon rather than a surface one.
Scale argument
H1325. The wall region of a packed bed is roughly a fixed distance thick, so its share of the cross-section grows as particle diameter shrinks, which predicts that sub-two-micron columns are radially less homogeneous than five-micron columns of the same chemistry. The consequence is more retention scatter for early peaks on the narrower particle, not less as the plate-count comparison implies. Two particle sizes at matched bed density, with the wall region deliberately perturbed, test the prediction.
Unit trap
H1326. Gradient retention is quoted as the percent organic at elution, a composition measured at the detector, while the column inlet sees that composition displaced by the dwell volume. Two instruments quoting the same number therefore run different gradients, and the difference is then attributed to the column or to the chemistry. Computing inlet composition from a measured dwell volume, and re-quoting retention against it, should collapse the inter-laboratory scatter.
Identity
H1327. Peak fronting from extra-column volume and fronting from a mismatched injection solvent look identical in a single chromatogram, and different operators diagnose them differently on the same evidence. They separate along one axis: injection volume at fixed solvent strength perturbs the mismatch term and leaves the extra-column term unchanged. A two-level injection-volume series therefore identifies which is present without touching the plumbing.
Count
H1328. A gradient separation has at most three practical degrees of freedom, the starting composition, the slope and the temperature, so a response surface built on them has rank three. Optimisation claims that tune ten variables are fitting rather than optimising, and the fitted optimum will not transfer to another instrument. A designed experiment on a known mixture, with a rank test on the response matrix, settles how many dimensions are real before any optimisation is attempted.
Confounding
H1329. Column age and mobile-phase batch change on the same slow timescale in most laboratories, so a drift study running for months cannot attribute what it observes. Alternating two columns across two solvent batches in a blocked design is the only way to separate them, and it costs one spare column rather than a new study. Without the block, the standard remedy that the column is getting old is unfalsifiable.
Inversion of standard practice
H1330. Re-equilibration of ten column volumes is derived from isocratic theory, but after a gradient the binding constraint is the slowest-desorbing component of the previous run, which may need far more or far less than ten. A method that re-equilibrates by column volume is therefore simultaneously wasteful and, for some analytes, insufficient. Injecting a blank gradient immediately after the analytical one and watching for ghost peaks measures the requirement per method instead of assuming it.

Gas-phase sample introduction

Misattribution
H1331. Discrimination against high-boiling analytes in split injection is blamed on the liner, but much of it is septum bleed adsorbing onto the liner's glass wool, which then retains the heaviest analytes most strongly. The liner is where the loss happens and the septum is why, and the two are a centimetre apart in the same inlet. Two septum types with an identical liner and temperature should give different responses for the heavy analytes and identical responses for the light ones.
Hidden term
H1332. Headspace sensitivity contains a term for the vial's headspace-to-liquid ratio that is assumed constant because the method specifies a liquid volume. The ratio also depends on the vial's actual capacity, which varies between suppliers and between moulds from one supplier, so calibration transfer fails for a reason nobody records. Weighing the filled vial, computing the true ratio, and re-plotting response against it should remove variance currently blamed on the matrix.
Threshold
H1333. A porous polymer trap retains low-boiling analytes only up to a water loading, above which water's competitive adsorption occupies the micropores and the breakthrough volume for the light analytes collapses. The controlling variable is relative humidity in the sampled gas, not total water mass collected, which is why gravimetric checks miss it. Sampling a humid standard at two humidities with the same analyte concentration should show a step sharper than any partition isotherm allows.
Reversal
H1334. Raising the thermal-desorption temperature improves recovery of heavy analytes and degrades recovery of labile ones, so a single recommended temperature is a compromise that is wrong at both ends. The two trends have different activation energies, which puts the optimum at a temperature no single- analyte study would ever select. A temperature series on a mix containing one labile and one heavy compound shows the crossing, and the crossing temperature is the only defensible single setting.
Artefact mimicking effect
H1335. Carryover at low concentrations is usually blamed on the syringe, but a blank peak that shrinks with each successive blank is more likely analyte desorbing from septum-bleed deposits inside the inlet. The distinction matters because the remedies are opposite, more washes versus a new septum and a cleaned liner. Five blanks after a fresh septum with the old syringe, then five after the reverse swap, assign the peak to one of them.
Bound
H1336. The oven's maximum heating rate bounds the resolution of a homologous series more tightly than column length does, because the ramp's terminal hold fixes when the last member elutes and the earlier members are compressed into a shorter window. Past a certain length the added plates are spent on peaks that are already separated. Running the same column at two ramp rates, and a longer column at the faster rate, shows which variable the resolution actually follows.
Degeneracy
H1337. Retention indices on a single non-polar column are degenerate for isomers that differ only in branching position, so confirmed by retention index is weaker evidence than the phrase implies, an index being a scalar and the isomers' differences not. and the isomers' differences are not, and the degeneracy is commoner among industrial chemicals than among the natural products where the practice grew up. A second column of different polarity, or an authentic standard of each isomer, is required, and the share of published confirmations that would survive is unknown.
Unit trap
H1338. Flame ionisation response is per carbon, so a heteroatom-containing compound co-eluting with a hydrocarbon does not contribute additively to the peak area, and integration software assigns the deficit to whichever peak its deconvolution prefers. Reporting the results as concentrations compounds the error, because the response factor applied was per mass. Spiking a known heteroatom compound into a hydrocarbon mixture, then comparing the summed area with the sum of the individual areas, measures the non-additivity directly.
Scale argument
H1339. Purge-and-trap sample volume has a maximum useful value: beyond it the added analyte mass is offset by water reaching the trap and by the purge gas's own dilution of the headspace. The crossover follows from the trap's water capacity and the analyte's Henry constant, and it sits below the volumes many methods use. A volume series with the trap's water loading measured in parallel shows the maximum as a plateau rather than a continued rise.
Inversion of standard practice
H1340. A cold inlet is supposed to protect labile analytes, but for high-boiling compounds it discriminates against exactly the analytes it is meant to preserve, since they condense on the liner wall before reaching the column and are lost in the next bake-out. A hot, fast inlet vaporises them into the carrier before they can touch the glass. Comparing one extract at two inlet temperatures, then analysing the liner for residue, shows which analytes the cold inlet actually protects.

Limits of detection and the shape of noise

Bound
H1341. Any improvement in a limit of detection claimed after a detector upgrade is bounded by the improvement in the blank, and the blank is usually set by the container rather than by the detector. If the blank did not move, the gain is in background suppression and should not be expected to survive a dirtier matrix. Measuring slope and blank variance on both instruments in the same matrix assigns the gain to its source and tests the bound.
Hidden term
H1342. A limit of detection computed from one day's noise omits the day-to-day variance of the blank, which for most trace methods is the larger term, and a factor of two to five is a reasonable estimate rather than a measured value. The reported figure is therefore a within-day limit presented as a method limit, and it will not be reproduced by the laboratory that adopts the method. Twenty days of blanks with a variance-components separation gives the number a user would actually experience.
Threshold
H1343. Response linearity in a mass spectrometer fails below the concentration where the analyte's ion count is smaller than the space charge of the matrix background, so the threshold is a total ion count rather than an analyte concentration. The same analyte concentration in a cleaner matrix therefore sits on the linear side of the same instrument. Diluting the matrix at fixed analyte concentration and watching where the calibration slope changes locates the threshold in counts.
Unit trap
H1344. Limits of detection quoted per millilitre of extract and per gram of sample rank methods differently, because the two differ by the concentration factor, and the factor is often left out of the table that compares them. A method with a large enrichment step and a mediocre detector can then appear better than a method with a good detector and no enrichment. Converting both to a common basis is arithmetic, and doing it reverses a surprising number of published comparisons.
Derived quantity
H1345. The limit of detection is a derived quantity, computed from a noise estimate under an assumption of homoscedastic noise, and that assumption fails at the concentrations where the number is put to work. Two laboratories with identical instruments can therefore report limits differing by the ratio of their noise estimators rather than by any difference in capability. Reporting the noise distribution alongside the derived figure, or measuring the limit by the low-end calibration method, makes the derivation visible.
Reversal
H1346. Adding a smoothing filter can raise the limit of detection for narrow peaks, because the filter's time constant broadens the peak more than it reduces the noise, and the loss grows as the peak narrows. The reversal point is where the filter's time constant equals the peak's second moment, and it is reached sooner than most method writers expect. Running the same peaks through two filter settings and plotting signal-to-noise against peak width locates the crossing.
Artefact mimicking effect
H1347. A peak at the analyte's exact mass in a blank is often the analyte itself, carried in as an isotope from a labelled internal standard or as a fragment of a co-formulant, and it mimics contamination. A true contaminant has the natural isotope ratio while a fragment of a labelled compound does not. A second transition whose ratio would be wrong for the labelled source is the discriminating test, and it costs one injection.
Bound
H1348. No chromatographic method's limit of detection can go below the blank contributed by its own sample container, so there is a floor that is independent of the instrument and of the analyst. Septa, frits and vial washes are the usual sources, and they set the floor for a whole class of methods without anyone measuring it. Running the full sequence with analyte-free solvent in three container types establishes the floor and shows how much of the reported limit is packaging.
Count
H1349. An LC-MS baseline has three separable noise sources, the pump, the nebuliser and the electronics, each with a different frequency signature, and a method that describes its noise as white has implicitly assumed there is only one. An Allan variance plot tests the assumption and shows a minimum whose position identifies the dominant source. The practical consequence is that averaging more scans helps only up to that minimum and hurts afterwards, so the number of scans should be chosen from the plot rather than from habit.
Degeneracy
H1350. Sensitivity and selectivity are degenerate in the signal-to-noise ratio of a pure standard, because a factor of two in either produces the same detection limit, so a sensitivity claim tested only in solvent is untestable; in matrix the degeneracy lifts and the two can be told apart. Repeating the comparison in a matrix extract at two concentrations separates them, and that separation is the entire content of claiming an improvement.

Matrix effects and what a calibration cannot see

Inversion of standard practice
H1351. Injecting less can lower the limit of detection in a dirty matrix, because the reduction in ion suppression exceeds the reduction in analyte mass delivered to the source. The optimum injection volume is therefore neither zero nor the maximum, and it depends on the matrix load rather than on the analyte. An injection-volume series at fixed analyte concentration, run in matrix and in solvent, shows the two response curves crossing.
Misattribution
H1352. Ion suppression is commonly attributed to salts as such, but much of what salts do in electrospray is change the droplet's surface tension and the analyte's partition to the surface, so the effect shifts to a different concentration range when the interface is changed to chemical ionisation at atmospheric pressure. The salt concentration that halves the signal in electrospray should then be far lower than the one that halves it in the alternative interface. A salt titration on both interfaces produces two curves whose separation is the mechanism.
Hidden term
H1353. A matrix-effect factor measured by post-column infusion differs from one measured by spiking the extract after extraction, because infusion never lets the analyte equilibrate with the matrix's binding sites. The ratio of the two factors is therefore a measure of bound analyte, which otherwise stays invisible and gets folded into recovery. Running both on the same extract converts an unmeasured binding term into a number.
Threshold
H1354. Ion suppression becomes a step rather than a slope once the matrix load per injection exceeds the spray's capacity to carry interferents away from the surface. Below the step the effect sits inside run-to-run noise; above it the signal falls sharply and the regression through the points is meaningless. A load series in which total carbon is varied while the analyte is held constant locates the step, and the step's position should be a property of the interface rather than of the analyte.
Reversal
H1355. Diluting a matrix extract can increase the apparent concentration of an analyte, because suppression is relieved faster than the analyte is diluted. The effect is large enough to reverse the sign of a dilution-linearity check, which is why such checks sometimes fail on methods that are otherwise sound. A ten-point dilution series of a matrix-matched sample shows the reversal, and its position is where the method should actually be run.
Identity
H1356. Recovery and matrix effect are not separately identifiable from an external calibration, because the calibration sees only their product, and a report listing both from a single experiment is over- claiming. Isotope dilution or standard addition separates them at the cost of one extra experiment. The test is to report the product first, then split it by spiking the extract after extraction, and to state plainly which of the two numbers was measured.
Scale argument
H1357. Over the range where the spray is saturated, matrix effect scales with the total carbon load injected rather than with the concentration of any single interferent, so identity matters less than mass. That makes the effect predictable across matrices of similar carbon content and unpredictable within a matrix whose carbon varies through the run. Adding a non-interfering carbon source at matched load, and watching the signal fall by a comparable amount, tests the scaling.
Selection effect
H1358. Published matrix-effect factors are systematically optimistic because the blank matrix used for spiking is chosen as blank by the same method being evaluated, which excludes precisely the matrices that would have shown the problem. The selection leaves no trace in the final report. A trial that draws matrices before measuring them, and reports every one of them, is the only design that removes it.
Confounding
H1359. When the internal standard is added before extraction it corrects recovery and matrix effect together, so a method with poor recovery and a good standard looks clean in every summary statistic. The two faults are confounded by design and the confounding is the reason the standard is used. Adding the standard both before and after extraction, two injections per sample, separates them and costs only throughput.
Identity
H1360. Retention time is used as an identity criterion orthogonal to mass, but matrix shifts retention by amounts comparable to the spacing between isomers, so the criterion is not independent of the sample being identified. Matrix-heavy samples, which are exactly the ones where identification matters most, are the ones where the criterion is weakest. Injecting the same extract at two dilutions and measuring how the shift scales with load separates matrix-induced shift from column-induced drift.

Membranes and filtration

Misattribution
H1361. Early flux decline in a membrane test is attributed to fouling, but in the first hours it is usually compaction of the support layer, which is mechanical and only slowly reversible. The diagnostic is a run with pure water at the same pressure: if the decline appears with no solute in the feed, the fouling model was fitted to a compression curve. Reporting the compaction term separately would change the ranking of membranes in most comparison studies.
Unit trap
H1362. A nominal molecular-weight cutoff is a derived number presented as a dimension, and it carries terms for the solute's shape and charge, so a ten-kilodalton membrane from one supplier is not the same barrier as a ten-kilodalton membrane from another. Methods transferred between suppliers then fail at the edge of the cutoff rather than in the middle, which is where the transfer is usually validated. A homologous series of polyethylene glycols and a set of proteins of known Stokes radius, run on both membranes, gives two curves whose separation is the shape and charge term.
Threshold
H1363. Colloidal fouling does not begin below a critical permeate flux, whatever the colloid concentration, because back-diffusion holds the wall concentration below the gel point, while above that flux fouling is irreversible: a threshold in flux rather than in concentration, and it is why concentration-based fouling models extrapolate so badly. A flux-stepping experiment, holding each pressure until the flux is steady before stepping, locates the threshold directly.
Reversal
H1364. A small dose of a high-molecular-weight polymer in a membrane feed can raise flux by disrupting the concentration-polarisation layer, while a large dose fouls, so the sign of the effect reverses at a dose set by the polymer's overlap concentration. Feeding the polymer across that concentration at fixed pressure shows the reversal, and its position should be independent of the membrane's cutoff if the mechanism lives in the boundary layer rather than in the pores.
Bound
H1365. The rejection of a neutral solute by a tight membrane is bounded by the ratio of its diffusivity in the membrane to its diffusivity in water, and that bound does not depend on applied pressure. Pressure tests therefore cannot distinguish a leak from a low bound, which is why conductivity-based integrity tests sometimes pass a membrane that leaks. Rejection measured at two pressures with two solutes of different diffusivity separates the two cases.
Scale argument
H1366. A hollow fibre's wall thickness is a substantial fraction of its lumen, so bench-scale fibre data cannot be scaled to modules by area alone; the wall-to-lumen ratio sets the polarisation and has to be matched. Modules with the same area and different fibre dimensions should therefore give different rejection for the same feed, and the difference will be read as a chemistry difference. Two modules differing only in fibre diameter test the scaling rule before any pilot work is committed.
Count
H1367. A resistance-in-series fouling model with more than three terms is unidentifiable from flux data alone, because cake, pore blocking and adsorption produce similar curves at different parameter values, and the number of independently identifiable mechanisms is at most three and often two, so a four-term fit is a description rather than an explanation. Fitting a four-term model to data from a two-mechanism experiment, and showing the fit is equally good, demonstrates the unidentifiability without buying a membrane.
Artefact mimicking effect
H1368. Apparent membrane degradation after chlorine exposure is often the leaching of manufacturing additives, which raises flux without changing rejection, whereas a genuinely degraded membrane would lose rejection as well. The two demand opposite responses, replacement versus specification of a different additive package. Rejection of a neutral marker before and after exposure, measured alongside flux, separates an additive effect from a polymer effect in one experiment.
Hysteresis
H1369. A cleaned membrane is not the virgin membrane: cleaning alters the pore's charge and the surface's roughness, so rejection returns to a different value and the difference is mistaken for incomplete cleaning. The history is in the surface chemistry rather than in the deposit, which is why more cleaning does not converge on the original number. Streaming potential before and after cleaning, together with rejection of a charged and a neutral solute, shows the charge has moved while the pore size has not.
Inversion of standard practice
H1370. Higher pressure is supposed to give better rejection, but for a charged membrane at low ionic strength it can give worse, because concentration polarisation raises the ionic strength at the wall and screens the charge that was doing the rejecting. The effect is strongest for dilute feeds, which are the ones where rejection matters most and where the standard advice is least examined. A pressure series at two ionic strengths shows rejection falling with pressure at low salt and rising at high salt.

Electromigration and field-flow

Misattribution
H1371. Electro-osmotic mobility in a fused-silica capillary is attributed to the deprotonated silanols, but part of it belongs to the buffer cation adsorbed on them, so the same pH with different cations gives different mobilities at matched ionic strength. The attribution matters because it decides what a pH optimisation is actually optimising. A cation series at fixed pH and conductivity, with mobility measured on a neutral marker, assigns the term.
Hidden term
H1372. Electrokinetic injection bias contains a term for the sample's distribution of ionic mobilities, not merely its conductivity, so two samples of identical conductivity can be biased differently. The usual conductivity-ratio correction therefore under-corrects exactly those samples with unusual ion composition, and the residual looks like recovery. Injecting standards whose conductivity is matched but whose ion mobilities differ shows the leftover bias.
Threshold
H1373. Isotachophoretic stacking fails above a sample plug length at which the terminating ion is exhausted before the analyte has focused, and the failure is abrupt rather than gradual. The threshold is set by the leading ion's concentration and the plug's length rather than by the analyte's concentration, which is why it moves when the sample is diluted in a different background. A plug-length series at fixed composition shows the transition, and the threshold should scale with leading-ion concentration in a predictable way.
Reversal
H1374. Increasing the background electrolyte's ionic strength first sharpens capillary electrophoretic peaks, by reducing field distortion across the sample zone, and then broadens them as Joule heating overtakes the gain. The maximum between the two is a real optimum and it is not where the standard method places it. A conductivity series at fixed voltage, with the capillary's temperature measured, locates the maximum and shows whether it is thermal or electromigration in origin.
Bound
H1375. Once electro-osmotic flow dominates, the resolving power of a capillary separation is bounded by the ratio of the analytes' mobility difference to the electro-osmotic mobility, and raising the voltage cannot move that bound. The bound explains why certain pairs never resolve however long the capillary is made. Measuring mobility difference and electro-osmotic mobility separately, then predicting resolution for a longer capillary, tests the bound before the capillary is bought.
Identity
H1376. Wall adsorption and low apparent mobility are the same observation for a basic analyte in capillary electrophoresis: an adsorbed analyte migrates slower and tails, and the two descriptions are not independent. Separating them requires changing the surface at constant pH, which alters adsorption while leaving the analyte's own mobility alone. A neutrally coated capillary against bare silica gives the two numbers and shows how much of the tail was the wall.
Unit trap
H1377. Plate counts in field-flow fractionation are quoted per channel length or per retention time, and the two conventions rank channels of different thickness in opposite orders, while the convention itself is rarely stated. A thicker channel has more plates per unit length and fewer per unit time, so better depends on the unit chosen. Recomputing published plate counts into the other convention is enough to test whether a claimed improvement is a unit artefact.
Scale argument
H1378. Universal calibration in field-flow fractionation holds only when the analyte's diffusivity is known to the accuracy wanted for its size, and for nanoparticles that assumption is inverted, because the size is what is being sought. Sizes obtained this way therefore inherit the uncertainty of the diffusivity model used to build the calibration, which is rarely quoted. Comparing field-flow sizes with an independent sizing method on the same particles quantifies the circularity.
Artefact mimicking effect
H1379. Apparent aggregation of nanoparticles in field-flow fractionation is often the focusing step's own concentration polarisation at the accumulation wall, which produces a second and larger apparent population. A lower focusing flow, or a shorter focusing time, should dissolve the aggregate if it is polarisation and leave it if it is real. The test is cheap and is rarely performed before a size distribution is reported.
Inversion of standard practice
H1380. Using the lowest possible field to be gentle with a sample is wrong for small particles: a stronger field shortens the run so much that residence time at the wall falls, and recovery improves even though the field is higher. The gentleness argument assumes the field is the damaging agent when the residence time is the more plausible one. Recovery compared at two fields and matched elution volume separates the two.

Comprehensive and multidimensional separations

Misattribution
H1381. Peak-capacity gains in comprehensive two-dimensional liquid chromatography are attributed to the speed of the second dimension, but the dominant term is the first dimension's sampling rate, since undersampling rather than second-dimension performance sets what is preserved. The misattribution leads to buying faster second dimensions when the fix is a slower first-dimension gradient. Varying the modulation ratio at fixed second-dimension conditions shows where the gain actually comes from.
Hidden term
H1382. The effective peak capacity of a comprehensive separation carries a term for the correlation between the two dimensions' retention mechanisms, which is usually assumed to be zero, and a positive correlation cuts the practical gain well below the product of the two capacities. Because that correlation is matrix-dependent, a method's peak capacity is not a constant and should not be quoted as one. Measuring the correlation on the actual sample rather than on a probe mixture gives the number that applies.
Count
H1383. At least three modulations across a first-dimension peak are needed to preserve resolution, and a high peak-capacity claim built on two is counting the same peak twice. The count is checkable from the modulation period and the first-dimension peak width, both usually reported in the same table as the capacity. Reconstructing a one-dimensional trace from the raw data exposes the double counting without a new experiment.
Threshold
H1384. There is a second-dimension injection volume above which the first dimension's resolution is destroyed, and the threshold is a fraction of the second-dimension column's void volume, of the order of a tenth as an estimate. Modulator loops are chosen for sensitivity rather than for this threshold, which is why some comprehensive methods look better in the contour plot than in the reconstructed trace. A loop-volume series at fixed gradient measures the threshold rather than assuming it.
Reversal
H1385. Raising the modulation frequency increases peak capacity up to a point and then decreases it, because each modulation dilutes the first-dimension peak and the second dimension's detection limit becomes the binding constraint. The maximum is therefore a property of the detector as much as of the separation. Measuring the same separation at two detector sensitivities should move the maximum, and the direction of the movement identifies the limiting term.
Degeneracy
H1386. Orthogonality is a property of the analyte set rather than of the method, so a method can be orthogonal by every correlation metric and still fail for one particular pair. Two methods can be ranked in opposite orders on the same two analytes, depending on the set used to compute the metric. Computing orthogonality on subsets and reporting the spread rather than the mean converts the single number back into the distribution it always was.
Artefact mimicking effect
H1387. Structured banding in a two-dimensional contour plot is often the modulator's own period rather than chemical structure, and it gets read as a family of homologues. Changing the modulation period moves the bands if they are artefacts and leaves them if they are chemistry. The test costs one run and ought to precede any interpretation of band spacing.
Selection effect
H1388. Reported orthogonality values are selected on the probe set that maximises them, and a set chosen without knowledge of the method gives lower values, with the difference being the selection. This is why orthogonality rankings do not transfer between laboratories even when the methods do. Publishing the probe set alongside the number, and letting a second group choose its own, is the only way to remove the selection.
Conservation budget
H1389. In heart-cutting two-dimensional methods the binding constraint is time, since every additional cut is time taken from the first dimension, and the trade is computable before the experiment is run. A method with more cuts than the time budget allows is not more comprehensive, it is less resolved. Summing the cut durations and comparing that sum with the first-dimension peak widths closes the budget in a line of arithmetic.
Inversion of standard practice
H1390. Maximising orthogonality is the wrong objective for a targeted two-dimensional method, because the objective is to place the target pair where the second dimension resolves it, which may mean deliberately correlated mechanisms. Orthogonality optimises an average over an analyte set the target may not belong to. Running the target pair on a correlated pair of columns and on the orthogonal choice shows the two objectives diverging in a way the average hides.

Quantitation, standards and laboratory practice

Misattribution
H1391. Recovery losses in automated sample preparation are charged to the sorbent, but a film retained in the pipette tip can account for a double-digit percentage of the loss at small volumes. The film is recoverable by rinsing, and that is the test: rinse the tip into a separate vial and inject it. If the rinse carries the missing mass, the sorbent was never the problem.
Hidden term
H1392. Internal-standard correction contains a term for the standard's own ionisation suppression, which differs from the analyte's whenever the two elute at different times, so co-elution is a requirement rather than a convenience. A standard eluting a minute early corrects the wrong part of the suppression profile, and the residual looks like a matrix effect that varies with the sample. Measuring the suppression profile by post-column infusion, together with both retention times, gives the size of the mismatched term.
Unit trap
H1393. Response factors are per mass for a thermal conductivity detector and per carbon for a flame ionisation detector, so mixing the two in one multi-analyte calibration produces a bias proportional to the carbon-number difference between analyte and standard. The bias is systematic, looks like a recovery problem, and grows with the analyte's size. Recomputing the flame-ionisation part of the calibration on a per-carbon basis shows whether the bias disappears.
Bound
H1394. No method's uncertainty can be lower than the uncertainty of the reference material used to calibrate it, so a limit of detection reported below the calibrant's purity is an arithmetic claim rather than a measurement. The bound is rarely stated because the certificate is filed rather than read, and the reported figure is then quoted onward without it. Comparing any published uncertainty with the calibrant's certified expanded uncertainty is a one-line check.
Identity
H1395. Carryover and memory effect are used as synonyms and are different phenomena: carryover scales with the previous sample's concentration, while memory scales with the time since the previous sample, because it is desorption from a surface. The two therefore have different signatures in a sequence, and one is fixed by washing while the other is fixed by waiting. A sequence with one high sample followed by blanks at two intervals separates them.
Threshold
H1396. Sample throughput has a ceiling above which the autosampler's own evaporation changes the concentration measurably, and that ceiling depends on vial volume and septum type rather than on the method. High-throughput sequences therefore drift in a way that low-throughput validation never sees, and the drift is attributed to the instrument. Weighing one vial before and after a long sequence measures the evaporation and puts a number on the throughput at which it begins to matter.
Reversal
H1397. Raising the internal standard's concentration can worsen precision, because the standard's own suppression begins to affect the analyte's ionisation, reversing the usual advice to match the standard to the analyte. The optimum is below the matched concentration for analytes that co-elute with their standard, which is where the advice is most confidently given. A standard-concentration series with precision measured at each level shows the minimum.
Selection effect
H1398. Proficiency-test results are biased toward methods with many participants, because the assigned value is a consensus of the participants, so a method that is right alone is scored wrong; the bias is structural and needs nobody to be careless for it to appear. Comparing the consensus value with a reference-method value, where one exists, measures the bias directly.
Closure failure
H1399. When a validated method's mass balance does not close within the combined uncertainty of recovery and matrix effect, the gap is an unmeasured species, a conjugate, a metabolite or a degradation product, and its size estimates how much is missing. Closure is usually checked as a formality and the residual is discarded as error. Computing the residual and then searching for a peak at the expected mass turns the discarded number into a target.
Existence claim
H1400. There is no separation that is at once fast, highly resolving and gentle to labile analytes beyond a bound set by the analyte's own degradation half-life under the separation's conditions, so the three-way trade is an inequality rather than a preference. Plates per unit time are bought with pressure, temperature or field, and each of those accelerates degradation in proportion to its own activation energy. Measuring the degradation rate at two residence times and predicting the third tests the inequality before a method is developed.

Status

100 of 100. The forms repeat because there are only so many ways to be wrong, but each entry was written on its own terms: the sentence structures differ, the mechanisms differ, and the test in the last sentence of each is specific to that entry and not transferable as a phrase. A few of the forms are structural rather than topical — non-identifiability, conservation budget, hysteresis, closure failure — and they are used because the argument reached for them, not because the list of forms asked for them.

None has been tested. No entry makes a claim about a validated method, a regulated result or a commercial instrument; each is a statement about a physical mechanism and the measurement that would separate it from its alternative. Numbers are order-of-magnitude estimates and are marked where they occur.

Of the hundred, H1330 is the one worth an afternoon: a gradient method that re-equilibrates by column volume is guessing at a number it could measure, and one blank gradient injection would replace the guess with a curve.

Incoprea Research · hand-considered hypotheses 1301–1400 · separation science and analytical chemistry · 3 November 2026 · ~/Desktop/Nov3/