Table 3.

Separation methods commonly used to isolate GAG mixtures

Separation method	Pros	Cons
Size-exclusion chromatography (SEC)	• Separate chain lengths by increments of dp2 • Simple and robust	• Multiple species with different degree of sulfation can elute at same time • Ion suppression is often needed
Strong anion exchange (SAX)	• Separates based on charge • Can isolate different degree of sulfation	• High number of inorganic salts resulting in contamination
Reverse-phase ion pairing (RPIP)	• Can separate similar GAGs	• Ion pairing reagent is needed • Unable to separate highly polar and ionic compounds
Hydrophilic interaction liquid chromatography (HILIC)	• Polar stationary phase • Separates based on polarity	• Longer equilibration time than reversed-phase LC • Mobile-phase pH shift can affect retention times
Ion mobility spectrometry (IMS)	• Fast separation • Occurs after ionization • Can be used with direct infusion	• Instrument specific
High field asymmetric waveform ion mobility spectrometry (FAIMS)	• Separates spatially and by differential mobility • Easily coupled with slow MS acquisition	• More time needed for ion accumulation
Capillary zone electrophoresis (CZE)	• Separates based on size, shape, and charge • Can distinguish isomers, including C-5 stereochemistry	• Sensitive to salt • Requires interface for pairing with MS