Recycle Chromatography Systems

The first reports on recycle chromatography occurred 70 years ago due to the fact that the particle sizes (30–100 µm) of the stationary phases during the middle of the past century could not overcome the resolution of some complex matrices to avoid the high cost and backdrop pressure requirements of long columns packed with non-uniform stationary phases (Gritti 2021); therefore, the recycling technique was developed with the purpose of increasing the column resolution. For recycling chromatography, two essential systems have been developed: single-column closed loop and alternate two-column switch recycling (Fig. S1). In 1959, the first and simplest recycling system was developed as a closed-loop (Fig. S1a), where the sample is recycled through the same chromatographic column by passing through the pump and the detector with the support of a recycling valve, simulating the use of a longer column (Porter and Johnson 1959). Bailly and Tondeur (1984) recommended different applications of recycling by separation of binary mixtures by ion-exchange chromatography and showed its potential advantages since extensions of these procedures to other chromatographic modes are straightforward (Baily and Tondeur 1982, 1984).

In order to overcome the significant band broadening caused by the pump, Biesenberger and collaborators (Biensenberg et al. 1971) described an alternate pumping recycle chromatography where two identical columns are connected through a multiport valve, and the solute bands are circulated in a closed-loop of the two columns by switching the valve without flowing through the pump (Gritti et al. 2017a, b; Wei et al. 2019). If further purification is required, the analyte can be passed back from the second column to the first column by means of the valve before the pure sample be collected. This process could be repeated as many times to complete the sample purification (Kimata et al. 2019). In this alternate column recycling, the analyte only passes in one occasion through the pump (Fig. S1b). As a result, the separation will be constantly enhanced as the number of cycle increases. In the next section, a brief historical background for the technique optimizations of the recycle chromatography systems is discussed with special reference to the purification of natural products.

Technique OptimizationsSingle-Column Closed-Loop Recycling

This is the simplest version that was developed, where the sample passes through the column, the pump, and the detector, while the sample constituents are separated and purified as they are recycled through the chromatographic column by an additional multiport valve that is included between the detector outlet and pump inlet (Fig. 1). Any preparative HPLC system could be adapted by connecting a recycle valve (available from different suppliers) in which an eluted selected unresolved peak is dispensed back into the same column repeatedly until the desired separation is accomplished. The mobile phase could also be directed into waste or could be recirculated (Fig. 1). Thus, the chromatographic separation takes place in the enclosed volume. The pressure developed for the mobile phase is almost constant and the dead volume in the recycle lines causes band spreading. It is important to mention that no fresh solvent is required in the conventional recycling mode (Fig. 2) and the sample is diluted in each volume of solvent, which each time generates the broadening of the band (Fig. 3). The selection for an adequate polarity of the solvent systems is not important to achieve the separation since the resolution by recycling chromatography depends only on the increment in theoretical plates (number of cycles). However, it is essential to select a mobile phase that provides a retention time of 10–20 min for the selected unresolved peak originally injected to avoid a time-consuming recycling process. In the chromatogram, each cycle would be identified by the injected unresolved mixture to be split into its constituents, or totally resolved, according to the number of passes in the column but maintaining the same retention time throughout the process (Fig. 3).

Fig. 3

Separation of two low-resolution peaks (a and b) in a mixture of closely related natural products (5:1) by column overloading and recycling prepartaive HPLC. Column overloading is useful for the separation of minor components by application of peak saving and heart-cutting methodologies. A cut of the overloaded sample zone gives an enriched fraction in major compound a. After the first recycle of this fraction, the separation of the two constituents is satisfactory to collect minor peak b. When the eluate is returned to the column, peak a can then be isolated in pure state by achieving resolution with a sequence of separation cycles to eliminate a trace impurity (asterisk)

A closed-loop system can be operated in conventional elution or recycling mode, using the peak shaving technique or multiple feed injection followed by peak shaving (Fig. 3). If two peaks are involved, and there is some broadening, the techniques of peak shaving and peak cutting for recycling could be applied to avoid overlapping of the peaks, where the leading and tail ends of two merged peaks are collected as pure components, and the center merged portion is passed through the column to be recycled as many times as needed to be separated and purified by elimination of trace impurities. The sample is collected in the last cycle corresponding to a peak of higher purity with a Gaussian behavior (Fig. 3).

Chemical studies began to apply conventional recycling HPLC and its variant since the early 1980s for isolation of natural products. Closed-loop recycling has been the most widely used approach for preparative purifications with its modification of external recycle chromatography that includes the reinjection of unevaporated impure eluted fractions, as a single-volume overload injection, back onto the original column to enhance the total recovery and purity of sample components from a separation of adjacent overlapping peaks (Crary et al. 1989). Recycling in a single column means that only certain parts of the elution profile including the target components may be repeatedly recycled until the required separation is reached. Consequently, the separation efficiency is limited to samples containing a limited number of constituents. For the purification of complex mixtures, they must be prepared by the multiple pre-procedures mentioned above to inject each target fraction independently.

Early application of recycling HPLC in plant chemistry used normal phase chromatography with silica cartridges. Mixtures of isomeric labdadienes and labdatrienes were resolved by extensive use of the peak shaving-recycling (i.e., collect-recycle-collect) technique even though the HPLC chromatogram showed only one peak for all isomers in each case. There was a 98% recovery of starting material and, since hexane was used as the only solvent system, it was also recycled after each recycling routine (Mohanraj and Herz 1981). Tylolupenols A and B, pentacyclic triterpenoids, from the Chinese Vincetoxicum kerrii (Craib) A.Kidyoo, Apocynaceae, were purified by recycling HPLC in silica gel (mPorasil) with hexane saturated with H2O-i-propylether-i-propanol (97.75:2.00:0.25) (Kawanishi et al. 1985). The sesquiterpene isomeric mixture of curcumanolides A and B from the stem-distilled volatile oils of the roots from Curcuma heyneana Valeton & Zijp, Zingiberaceae, was resolved in a silica gel column with CHCl3 after a recycle sequence of 95 times (Firman et al. 1988). Likewise, guaiane-type sesquiterpenes characterized by the presence of a hydroxyperoxy function, were isolated from the wood of Viburnum awabuki K.Koch, Viburnaceae, and purified by recycling HPLC in silica with CHCl3 (Fukuyama et al. 1996).

What started in the early 1990s, close loop recycling in a reversed-phase chromatography with a C-18 column (ODS type), in which octadecyl groups are bonded to a silica base to deliver wide-ranging separation applicability, has become the most extensive system used for the purification of small natural products (see sections “Reversed-Phase HPLC” and “Applications in Natural Products”). The following chemical studies constitute examples of the initial applications of reversed-phase recycling chromatography: 4-quinolinone alkaloids from Esenbeckia leiocarpa Engl., Rutaceae, with antifeedant activities against the pink bollworm, Pectinophora gossypiella, were purified by recycling in a C-18 column with MeOH-H2O-MeCN (50:25:10) (Nakatsu et al. 1990). Purification of glycosphingolipids from the marine annelid Neanthes diversicolor was performed by means of recycling on a preparative C-18 column using MeOH-CHCl3 (10:1) (Naoki et al. 1993). Methyl 4-hydroxy-3-(3′-methyl-2′-butenyl)benzoate, the major insecticidal principle from Piper guanacostense C.DC., Piperaceae, was purified from its EtOH transesterification artifact by recycling through a semipreparative C-18 column with CH3CN-H2O (7:3) (Pereda-Miranda et al. 1997). The hepatotoxic mixtures of hydroxystilbene tetramers, vitisin A and cis-vitisin A, from Vitis coignetiae Pulliat ex Planch., Vitaceae, were resolved by recycling HPLC on a C-8 (octyl) column with MeOH-H2O (3:2) (Ito et al. 1998).

Finally, polyvinyl alcohol columns were also used for size exclusion chromatography to achieve the purification of phytoecdysteroids (Zhang et al. 1992) and anthraquinone glycosides (Kubo et al. 1992). Two polystyrene columns in tandem with CHCl3 (Fig. S2A) were unsatisfactory to separate the closely related macrolide alkaloids cathedulines E3, E4, and ES with insect growth inhibitory activity from the MeOH extract of the aerial parts of Catha edulis (Vahl) Endl., Celastraceae. However, recycling chromatography on polyvinyl alcohol resin column and methanol as an eluent was appropriate to provide the necessary resolution to achieve total purity of individual alkaloids. As illustrated in Fig. S2B, a principal peak with two shoulders was detected during the first pass through the polyvinyl alcohol column. However, after recycling two times, three individual peaks were observed which were resolved by applying the peak shaving-recycling technique. A single injection (95 mg) of the alkaloidal fraction was resolved within 5 h. Catheduline E5 (15 mg) was isolated after two cycles. Cathedulines E3 (43 mg) and E4 (22 mg) were purified after a third cycle (Kubo et al. 1987). An alternative closed-loop recycling technique with a periodic intra-profile is a binary chromatographic separation where the sample is injected into the interior of the circulating chromatographic profile and fractionation is achieved by collecting the leading and trailing edges during each cycle (Grill 1998). Closed-loop recycling is the most frequently used technique for natural product isolation (Sidana and Joshi 2013; Rohaity et al. 2017; Chen et al. 2019; He et al. 2020; Li et al. 2021).

Column Switch Closed-Loop Recycling

A variant of closed-loop recycling is the column switch recycling methodology but with the use of two coupled columns, where the sample passes from one column to the other through a recycling valve (Fig. S1b). The sample does not pass through the pump, and this can be used with isocratic or gradient mode, for the latter two valves are used (Wei et al. 2021). The impurities are removed using one two-position multi-port valve during the initial gradient step and the baseline resolution is guaranteed using a second two-position multi-port recycling valve (Gritti 2021). This technique has also allowed the separation of proteins in a wide molecular weight range by closed-loop recycling in a size exclusion chromatographic system (Yuan et al. 2009). Ten anthocyanins from red cabbage were isolated by recycling preparative HPLC using two twin Waters Sunfire preparative columns, which were used and manually switched by a 10 port-2 position valve. So, the unwanted low retention constituents were excluded, and the compounds of interest were redirected to column 2, until sufficient separation achieved (Chen et al. 2018).

On-column stopped-flow bidimensional recycling was designed for enantiomeric separation of chiral drugs as a variant of column switch recycling. In a chiral column, the two enantiomers are initially separated, and the pharmacologically active isomer is collected; then, the mobile phase flow in the chiral column is stopped and the other inactive isomer is trapped in the achiral column by switching the second valve, which was filled with an appropriate solvent where the sample undergoes acid catalyzed racemization. Then, the enantiomeric mixture is pushed back by the first valve to the chiral column at appropriate time where separation occurs. This process is repeated to obtain an enantiomeric enrichment to obtain about 95% of the pure pharmacologically active isomer. The procedure could be applied both in reverse-phase mode and in normal-phase mode (Cannazza et al. 2009, 2011). Until now, there are no reports on the separation of natural products by this technique.

Reversed-Phase HPLC

RP-HPLC is a technique using surface-modified silica, R(CH3)2OSi-OSi(OH)(CH3)2, where R is a covalently bonded alkyl group, such as C-4 (-C4H9), C-8 (-C8H17), C-18 (-C18H37), phenyl-hexyl, cyano (CN(CH2)3-), or amino (NH2(CH2)3-), to the surface of silica particles or closely related materials used as the stationary phase in order to create a non-polar hydrophobic stationary phase. The mobile phase is very polar and composed with water or mixtures of water with water-miscible polar organic solvents, such as methanol, acetonitrile, isopropanol, or tetrahydrofuran (their elution strength increases in this order). Isocratic elution with a fixed proportion of solvents is the most commonly used method at the semipreparative and preparative levels. Column characterization and selection systems in RP-HPLC have been recently reviewed (Žuvela et al. 2019). The analyte retention time increases with the increment of the (i) hydrophobicity of the solute, (ii) hydrophobicity of the stationary-phase surface, and (iii) polarity of the mobile phase. The interaction of the analyte and the stationary phase results in a reduction of the total hydrophobic area exposed to the mobile phase which represents the driving force for the reversed-phase retention (for a recent example, see Moreno-Velasco et al. (2024)).

Two physicochemical phenomena are involved in the course of separation, i.e., the partitioning process when the analyte molecules are totally immerse into the bonded phase and/or adsorption phenomenon at the bonded-phase/solvent interface. In addition, the logP value, the pKa value of the analyte, chromatographic parameters like mobile phase pH, and organic modifiers also influence the retention and selectivity of the analyte. It was found that hydrophobic, electrostatic, and hydrogen bonding and other specific interactions between the stationary phase and the solutes, along with the hydrophobicity of a molecule (logP), modify the retention behavior of the analytes. While the overall retention mechanism remains the same, subtle differences in the surface chemistries of the different stationary phases will lead to changes in selectivity. The mechanisms of retention and various factors that influence the retention behavior of analytes were recently revised with suitable examples by Ganesh et al. (2022). In addition, the basic principles of semi- and prep-HPLC (Surve et al. 2023), as well as several applications for the isolation of natural products from plants, bacteria, fungi, and marine organisms, were recently reviewed (Niculau et al. 2022; Queiroz et al. 2024).

Recycling Prep-HPLC

Scaling up the selected analytical conditions to prep-HPLC can be a relatively easy task if the only variables to be enlarged are the diameter and/or the length of the column. A direct linear scale-up can be achieved using the following equation:

$$\mathrm\;\mathrm\; \mathrm\;\mathrm=\left[L__\right]/\left[L__\right]$$

where L is the length and Ac is the cross-sectional area of the (P) preparative and (A) analytical columns.

The direct scale factor allows the calculation of the scaled up flow rate and an estimation of the amount of sample that can be injected into the preparative column. Changes between the analytical and preparative systems require some modifications to be made at the preparative stage to achieve the optimal separation and avoid the development of high column backpressure. This situation could require a reduction of the flow rate and/or a change in the composition for the eluation mixture to maintain backpressure down. It is important to consider that in order to execute the calculations for the scale-up factors correctly, the analytical and preparative columns must contain the same stationary phases with equal particle sizes and preferably manufactured by the same company. The composition of the mobile phases must also remain the same. Concentration overload is normally used to inject a greater amount of sample (Niculau et al. 2022). Column overloading increases the capacity for peak detection and preparative-scale recycling work. If low-resolution peaks are sufficiently close together with minimal peak broadening and the preparative system is fitted with a recycle valve, the peaks can be passed through the column again and further separated as a consequence of the increment in the number of theoretical plates. Recently, state-of-the-art trends in preparative chromatography, which included gradient transfer from analytical to preparative scale HPLC, for the purification of natural products were reviewed (Queiroz et al. 2024).

Recycling prep-HPLC is used in the isocratic elution mode and low solvent flows are generally applied. In addition, recycling chromatography cannot proceed beyond the point where the sample is spread over the whole column. Further recycling from the optimum number of cycles would lead to a reduction in resolution by the overlapping of successive cycles and peak broadening. Application of peak shaving may further improve the performance of recycling chromatography since the overlap between the peaks stemming from two consecutive cycles can be avoided or delayed and, consequently, the purification can be enhanced in a cost-effective way (Teoh et al. 2003). Solvent gradients have been developed to solve purification problems encountered in the pharmaceutical industry for reinforcing the isolation of minor impurities in drugs (Gritti 2021; Wei et al. 2021). Recycling prep-HPLC is widely used in organic synthesis of therapeutical substances to separate impurities from target compounds and to collect large volumes of active ingredients in pharmaceutical developments (Gritti et al. 2018; Lamotte et al. 2017). Recycling prep-HPLC has also been applied during the isolation of natural products (Sidana and Joshi 2013).

Scale-up to Recycling Prep-HPLC

Some protocols for scaling up instrumental conditions to prep-HPLC in natural product isolation have been reviewed (Majors 2004; Latif and Sarker 2012). An isocratic scale-up method for recycling prep RP-HPLC to isolate resin glycosides (acylsugars) from Ipomoea purga Hayne, Convolvulaceae (Mexican jalap root), with laxative activity is shown in Fig. S3 to illustrate this process.

Reversed-phase chromatography with C-4, C-8, and C-18 has a stronger affinity for the hydrophobic portions in these amphipathic acylsugars, such as the aglycone (mono- and dihydroxylated C14-C18 fatty acids) and esterifying residues of the saccharide cores, which is evident from the time that the analyte spends in the column as a function of the hydrophobicity of each constituent of the resin glycoside matrix. Therefore, the longer retention time only depends on the adsorption via hydrophobic interactions. The stronger the interaction, the higher the resulting retention time (Moreno-Velasco et al. 2022, 2024).

Reversed-phase semiprep-scale up recycling HPLC afforded retention times that reflected the interaction of each analyte with the C-18 chains attached to the surface of silica. The initial optimized analytical conditions used to achieve the resolution of the resin glycosides from the jalap root were as follows (Fig. S3A): a Symmetry C-18 Waters column (4.6 × 250 mm, 5 µm), a mobile phase of MeOH at a flow rate of 0.4 ml/min with a refractive index detector and a sample injection volume of 1 ml (sample concentration, 0.1 mg/ml). Once these analytical conditions were found, they were extrapolated at the semipreparative level by the calculation of the scale flow rate and an estimation of the amount of sample to be injected without losing the appropriate resolution for the chromatogram to achieve the isolation and purification of the major individual constituents in each of the collected peaks. Scale-up was performed on a semiprep Symmetry C-18 Waters column (19 × 300 mm, 7 µm); mobile phase, MeOH; flow rate, 8.0 ml/min; and the sample injection volume of 500 ml (sample concentration, 0.1 mg/ml) (Fig. S3B). The following retention times for the hydrophobic purginoside series, pentaglycosidic macrolactones with the same oligosaccharide core, operculinic acid A, and their differences occur in the acylating residues at the positions C2, C3, and/or C4 of the last external rhamnose unit with units of (2S)-methylbutyric (mba), n-hexanoic (hexa), and trans-cinnamic acids (Cna), were recorded as follows: tR 22.0 min for purginoside I (1) and IV (4), 23.0 min for purginoside II (2) and 26.6 min for purginoside III (3), the retention properties of these analytes were detected as a linear function of the number of acylating substituents and the number of methylene groups in the fatty acid residues (Fig. S3B). Recycling of the selected unresolved peak (tR 20 min) for over 18 cycles assured 100% purity for purginoside IV (4) with a retention time of 345 min after the whole recycling process, while purginoside I (1) corresponded to the collected minor peak at 150 min by heart-cutting (Fig. 4). The purgin series (5–7) eluated first due to an increase in the hydrophilic area as a consequence of their ester-type dimeric structure with tR 16.6 min for purgin II (6), 18.3 min for purgin III (27), and 19.9 min for purgin I (5). Consequently, the longer the esterification groups, i.e., n-dodecanoic (dodeca) vs. n-decanoic (deca), the deeper the penetration into the ligand layer of the reversed phase.

Fig. 4

Recycling semiprep-HPLC for the resolution of a fraction containing two related resin glycosides from the Mexican jalap root, Ipomoea purga. The unresolved peak (tR 20 min) was recycled to purify a major component by heart-cutting and elimination of impurities (asterisk) with a Waters C-18 Symmetry column (19 × 300 mm, 7 µm), a mobile phase of MeOH-CH3CN (9:1), and a flow rate of 8.0 ml/min for over 15 cycles to assure 100% purity for purginoside IV (4) with a retention time of 345 min after the whole recycling process. Purginoside I (1) corresponded to the collected minor peak at 150 min by heart-cutting. The purification process continued by recycling the remaining peak and the use of peak saving as indicated by the vertical longdashed lines, where the leading and tail ends were directed to waste, while the center portion was recycled

Structural elucidation of metabolites is always performed off-line following recycling prep-HPLC purification (Pereda-Miranda et al. 2010). Once the purification process is accomplished to yield single chemical entities, their structural elucidation and absolute configuration determinations are accomplished by the combination of NMR (Breton and Reynolds 2013), MS (Demarque et al. 2016), and chiroptical spectroscopy (Pereda-Miranda et al. 2023). Then, pure isolated compounds could be submitted to biological evaluations. For example, purgin II (2) enhanced vinblastine activity > 2140-fold when incorporated at 25 μg/ml against resistant human breast carcinoma cells (MCF-7) overexpressing glycoprotein (Pg-p) (Castañeda-Gómez et al. 2013).

Applications in Natural ProductsGlycans

Glycoconjugates comprise complex carbohydrates, or glycans, linked to a protein, lipid, peptide, and secondary metabolites. They are the most profuse and multipurpose biopolymers mostly used for energy production and as structural materials by plants. Glycans are built by monosaccharides linked through glycosidic linkages. Significant biological roles of complex carbohydrates, carbohydrate polymers, and glycoconjugates, and their interactions with other biomolecules, such as carbohydrate-protein and carbohydrate-carbohydrate interactions, have been comprehensively documented (Bucior et al. 2009; Zeng et al. 2012). These interactions control and modulate a variety of cell processes, such as differentiation, proliferation and adhesion, inflammation, as well as the immune response (Townsend 2023) and as a main force to initiate cell-cell recognition (Bucior and Burger