Table 2.

Comparison of direct and indirect methods for confirming and quantifying REG modification.

Method	Sample preparation	Detection	Pros/Cons	Ref.
AFM	drying on surface	microscopy	− requires introduction of bulky functionality + direct confirmation of reaction	[105]
TEM	staining & drying on TEM grids or setting in polymer gel for cutting (Cryo‐EM: freezing in holey TEM grids)	microscopy	− requires introduction of bulky functionality, with sufficient contrast + direct confirmation of reaction + can be combined with electron diffraction	[22, 54, 88, 103]
solution‐state NMR	dissolution into [P4444][OAc]/[D6]DMSO	NMR	+ high‐resolution quantitative information on chemical regioselectivity is possible − interpretation can be complicated and may require model compound synthesis	[75, 176, 177]
BCA	formation of CuI bicinchoninate complex after CuII oxidation	photometric absorption at 560 nm	+ simple method for REG quantification − further validation studies required for CNCs	[56, 104]
AA	reductive amination with anthranilic acid	fluorescence emission at 330 & 425 nm	+ simple method for REG quantification − further validation studies required for CNCs	[56]
hydroxylamine	formation of oxime and HCl by reaction with hydroxylamine hydrochloride	pH titration with NaOH	+ simple method for REG quantification − further validation studies required for CNCs	[110]
chlorite oxidation	NaClO2 oxidation to carboxylates at pH 3.5 and acidification to carboxylic acids	conductivity titration with NaOH	+ simple method for REG quantification as carboxylates − further validation studies required for CNCs	[110, 184]
Purpald®	reaction with Purpald® reagent under oxidative conditions	photometric absorption at 570 nm	+ simple method for REG quantification − further validation studies required for CNCs	[183]
CCOA	reaction with CCOA alkoxyamine to form oxime, under heterogeneous (aqueous) or homogeneous (LiCl/DMA) conditions	GPC‐MALLS with fluorescence and RI detection	+ well‐validated method for REG determination + may potentially offer more information than simple REG quantification‐based methods − time‐consuming	[73, 74, 75]
FDAM	reaction of carboxylic acids with FDAM diazo compound to form ester (REGs first require chlorite oxidation & acidification)	GPC‐MALLS with fluorescence and RI detection	+ well‐validated method for carboxylic acid determination − time‐consuming + may potentially offer more information than simple REG quantification‐based methods	[184]