Recycle HPLC: A Powerful Tool for the Purification of Natural Products
Natural compounds occur as various isomeric or closely related structures in biological matrices. These compounds are difficult to separate from the complex mixtures, and hence, the need for effective and innovative separation techniques arises. Recycle HPLC allows the recycling of sample, in part or full, and increases the separation efficiency of the process while keeping the peak dispersion to a minimum. Recycling in an HPLC system has been used in the isolation and purification of different types of natural products including enantiomers, diastereomers, epimers, positional isomers, and structurally related or unrelated compounds having similar retention characteristics. The present paper overviews the development of instrumentation and setup of recycle HPLC and its applications in the separation of natural products.
The ever increasing needs for the isolation and purification of natural products have resulted in the constant improvements and advancements in separation techniques and instrumentation. The efficiency of a separation may be increased in a number of ways. A few include (1) increasing the column length; (2) using two columns in series, or (3) recycling the sample (in part or full) through the column. The first option, that is, to increase the column length, may not be feasible on an industrial scale as the columns used are of fixed dimensions. Sample recycling in an HPLC system came into picture in the middle of the twentieth century. Two key studies highlighted the instrumentation and applications of recycle HPLC [1, 2].
The aim of recycling is to increase the separation efficiency of the process while keeping the peak dispersion to a minimum. This aim is achieved by incorporating a recycle valve in the HPLC (preparative or semipreparative scale) system to recirculate the unresolved peaks into the column. Another added advantage of this recycling is that no fresh solvent is required in the recycling period.
Currently, several recycling setups, based on mathematical models, have been devised, and the operating policies of these setups have been experimentally verified to be capable of improving product yields at laboratory, pilot, and industrial scales [1, 2]. These techniques have been utilized in the isolation and purification of a plethora of natural and synthetic molecules. As many as 23 recycles in the chromatographic system have been reported for the separation of structurally related compounds .
If a comparison is made between the conventional separations of isomeric/structurally related compounds and separation by recycle HPLC, the latter offers higher resolution in maximum cases. However, in the separation of triacylglycerol positional isomers, recycle HPLC on a polymeric ODS column met with mixed results. Triacylglycerol positional isomeric pairs consisting of two C8 and one C10 fatty acid chains could not be separated even after several recycles. Similar tendency was observed with other triacylglycerol isomers .
It is important to mention here that triacylglycerol isomeric pair with two docosahexaenoic acids and one oleic acid was not separated, but the pair comprising two docosahexaenoic acids and one elaidic acid could be separated using a recycle system. The difference between oleic acid and elaidic is the type of the double bond present (cis in case of former and trans in case of latter at the same position in the structure). The cis configuration of the double bond in oleic acid makes it bulky and therefore does not allow sufficient interaction with the ODS groups of stationery phase . Hence, it can be concluded that the type of stationery phase and its interaction with the analyte(s) play a significant role in the separation by recycling. This review is an account of instrumentation and applications of recycle HPLC in separation of different types of compounds.
2. Instrumentation and Setup
2.1. Closed Loop Recycling
Closed loop recycling is perhaps the oldest version of recycle HPLC. The peaks of interest in the chromatogram, after passing through the column and detector, are directed to the suction side of the pump through a recycle valve. Owing to the increase in the number of theoretical plates, the resolution of the components in the recycled peak(s) increases. Figure 1 presents the block diagram of a closed loop recycling HPLC system.
Teoh and coworkers  have commented on different operating policies of closed loop recycling. Conventional recycling involves the reinjection(s) of the sample (in part or full) on to the HPLC column and collection of the fractions when the appropriate separation is achieved. In this case, recycling can be done till the sample is spread over the length of the column. Further recycling will possibly lead to the mixing of the trailing part of one peak with the leading part of the next peak and hence decreasing the resolution achieved. To avoid this, the trailing and leading edges of the peaks are eluted, and the rest of the sample is recycled into the column. The phenomenon called as peak shaving.
In case of recycling with peak shaving, sufficiently purified portions (generally the trailing and the leading edges) of the overlapping peaks are eluted, and the rest of the fraction is recycled. This technique is known to give better recovery as compared to the conventional recycling.
Another mode of recycling is multiple feed injection(s) followed by peak shaving. Because of the peak shaving, the sample quantity reinjected to the column after each recycle decreases. This can be made up by sample injection(s) onto the column at a particular point in the chromatographic procedure .
Bailly and Tondeur  suggested mixing of the unresolved portion of the chromatogram with fresh feed and then reinjection into the column. This type of recycling can be of use in case of binary separations wherein a partial separation of two components of the mixture is achieved. The nonseparated part of the effluent is recycled with the fresh sample . However, this technique of recycling could not attract much attention as the partial resolution of the sample was destroyed by mixing with fresh feed .
Closed loop recycling in a size exclusion chromatographic system has been used in the separation of macromolecules, but its use remains limited due to several limitations. In a study involving the separation of β-lactoglobulin, myoglobin, ribonuclease, and cytochrome c, the use of serially coupled columns increased the resolution in comparison to the use of a single column. However, baseline separation was not achieved. Myoglobin and ribonuclease (having a molecular weight difference of 2500 Da) could be separated after seven recycles by closed loop method. Similar conditions were applied for the separation of a mixture of five proteins (molecular weight range 10 to 80 kDa), but the resolution was compromised because of the wide molecular weight distribution. After three recycles, the early eluted components caught up with the late eluted ones resulting in poor overall resolution. These results demonstrated that recycle size exclusion chromatography with coupled columns is applicable only for the analysis of simple samples with narrow molecular weight distribution.
Column switch recycling size exclusion chromatography (csrSEC) is another variant of closed loop recycling used in the separation of protein mixtures. It allows the separation of proteins in a wide molecular weight range and offers improved separation window and resolution. In this method, the proteins were separated by two serially coupled columns (second column being much longer than the first). The parts of the protein mixture having similar molecular weights were recycled to first column (after passing through the second column) by a recycle valve and repeatedly separated in the first column till satisfactory resolution is achieved .
2.2. Closed Loop Recycling Technique with Periodic Intraprofile Injection
Grill has described another closed loop recycling technique with periodic intraprofile injection (CLRPIPI), also called closed loop steady state recycling (SSR) . This binary chromatographic technique is similar to simulated moving bed (SMB) chromatography as the fresh sample is injected at a precise location in the circulating chromatographic profile. The chromatographic profile is governed by the size of fractions collected and the point of injection of fresh sample. Grill and Miller have compared the separation efficiency, product purities and recoveries, and costs involved in the separation of enantiomers by different methods such as closed loop recycling, SSR, and SMB [9, 10].
2.3. On-Column Stopped Flow Bidimensional Recycling HPLC
Cannazza et al.  have developed a recycling HPLC method for separation of enantiomers from racemic mixtures. In this method, the two enantiomers of a compound are separated on a chiral column, the isomer of interest (usually pharmacologically active) is collected, and the other isomer is directed to an achiral column (connected serially to the chiral column) wherein racemization takes place. This racemic mixture is again pumped to the chiral column, and the process is repeated until the enantiomer of interest is completely resolved.
For racemization to take place, appropriate solvent conditions, pH, temperature, and so forth require strict control. A second pump is installed into the HPLC setup for this purpose. This pump pushes the trapped undesired enantiomer into the achiral column where it racemizes in stopped flow conditions. The racemized mixture is again taken further to the chiral column with the mobile phase pumped by the first pump, and the cycle of racemization-enantioseparation continues .
This technique allows the purification of single enantiomer from racemic mixtures without expensive enantio-purification processes. The application of this method will be limited to the cases where racemization of undesired isomer takes place rapidly on-column under the conditions compatible with HPLC method used for enantio-separation. The second limitation of this method is that it requires customization of the HPLC instrument.
3. Recycle HPLC for Purification of Natural Products: Examples
Recycling in an HPLC system has been used in the isolation and purification of different types of natural products including enantiomers, diastereomers, epimers, positional isomers, and structurally related or unrelated compounds having similar retention characteristics (Figure 2) [12–18]. In the case of isolation of natural products from complex mixtures, the most commonly used variant of recycle HPLC is closed loop recycling technique, wherein the mixture is first subjected to isocratic or gradient HPLC separation, and preliminary fractionation is carried out. The fractions are then recycled in an isocratic mode to achieve the desired separation of components. For example, euglobal Bl-1 (1) and 1b (2) have been purified from a crude euglobal-rich fraction (ERF) prepared from the leaves of Eucalyptus loxophleba. Initially, the ERF was fractionated into six individual peaks separated on an ODS column when eluted with methanol-water-acetic acid (100 : 5 : 3) isocratically. One of these separated peaks was subjected to recycling over ODS using acetonitrile as mobile phase. The stereoisomers 1 and 2 were completely separated after eight recycles (Figure 3) .
Both normal and reverse phase silica supports have been used in recycle HPLC. Polyvinyl alcohol (PVA) columns have been used for the separation of phytoecdysteroids  and anthraquinone glycosides . The principle of separation in PVA columns is size exclusion, but owing to its hydrophilicity, it also acts as ODS . Amino columns have been used for the separation of glycosides and glycolipids. Along with the nature of stationary phase used, the length and diameter of the columns are of prime importance in any HPLC analysis. When the principle of separation is size exclusion, longer columns (typically 50 cm long) with narrow internal diameter (0.76 to 2.0 cm) are used .
The enantiomers of hapten derivative of propranolol [d and l forms of 1-(2-methoxycarbonylethyl)amino-3-(1-naphthoxy)-2-propanol] (3) have been separated on a cyanopropyl silica column using dichloromethane-hexane-acetonitrile (79 : 20 : 1), containing 5 mM d-10-camphorsulfonic acid and 2.5 mM tert-butylamine as mobile phase. The column was equilibrated with the mobile phase for 6 h to completely saturate the stationary phase with the ion pairs of tert-butylamine and d-10-camphorsulfonic acid. The enantiomers could be separated after four recycles in the HPLC system. The mechanism of separation of the two enantiomers involved the competition of tert-butylamine with the protonated hapten derivatives of propranolol for interaction with d-10-camphorsulfonic acid .
This technique has also been used in separation and purification of components from sugar mixtures. Turanose (4) is a noncariogenic and low-calorigenic structural isomer of sucrose. Due to lack of the low cost and efficient processes for the production of turanose, industrial application of turanose has been limited. Recently, 4 has been separated after nine recycles from other maltooligosaccharides and monosaccharides by elution with HPLC grade water. The total time taken to complete one cycle was 10 h, and no organic solvent was used in the process. The product purity and recovery yield were estimated to be 94.7% and 97.5%, respectively .
Two pentacyclic triterpene alcohols, namely, tylolupenols A (5) and B (6), have been isolated from the roots of Tylophora carrii. From a mixture of 5 and 6, individual components were purified by recycle NP-HPLC using n-hexane saturated with water-diisopropyl ether-isopropyl alcohol (97.75 : 2.00 : 0.25) . Phytoecdysteroids 20-hydroxyecdysone-20,22-monoacetonide (7), and ajugasterone C-20,22-monoacetonide (8) have been separated on a poly vinyl alcohol (PVA) column using methanol isocratically . Galphimines A (9) and H (10) along with other nor-secofriedelanes have been isolated from Galphimia glauca. Recycle HPLC of crude mixture of galphimines was carried out on an ODS column (19 × 300 mm) using acetonitrile-water (45 : 55) at a flow rate of 10 mL/min. Detection was done at 232 nm .
Two quinolinone alkaloids, named leiokinine A (11) and B (12), have been isolated from the biocidal fractions of the leaves of Esenbeckia leiocarpa after recycle RP-HPLC using methanol-water-acetonitrile (50 : 25 : 10) as mobile phase .
Glycosphingolipids have been purified from enriched fractions by recycle HPLC over C18 column (20 × 250 mm, 10 μ) using chloroform-methanol (1 : 10) at a flow rate of 4.0 mL/min. Compound 13 was purified, from a fraction prepared by RP-HPLC, after as many as fifteen recycles accomplished in a period of 540 min . Structurally similar compounds have been purified under similar HPLC conditions . A synthetic mixture of ten benzo(a)pyrone phenols has been separated by recycle HPLC over normal phase HPLC column run isocratically with hexane-dioxane-formic acid (9 : 1 : 0.02) .
Two new anthrone C-glycosides, named picramnioside D (14) and E (15), have been isolated from the ethyl acetate extract of the bark of Picramnia teapensis. The anthraquinone-rich fractions were chromatographed over a polymeric packing (Asahipak GS-310 P, 21.5 × 50.0 cm) using methanol at a flow rate of 8 mL/min. The separation was monitored at 254 nm. Three recycles in a duration of 60 min. afforded 14 and 15 along with other related compounds . Similar HPLC conditions have been used for the purification of other anthraquinone glycosides .
Recycle HPLC has also been used for the isolation of compounds from complex mixtures of unrelated components. 2,4-bis-(p-Hydroxyphenyl)-2-butenal (16) has been isolated from fructose-tyrosine Maillard reaction products. The bio-assay guided fractionation of Maillard reaction products for antiproliferative activity led to an active fraction that was purified by RP-HPLC followed by recycling on a JAIGEL-GS310 column (20 × 500 mm) using methanol as eluent at a flow rate of 3 mL/min. The components were detected at 206 nm .
Glycolipids of the genus Ipomoea have been isolated from partially purified fractions of the resins by recycle HPLC [17, 32–38]. Several of these compounds have been purified by recycle chromatography over ODS. Purginosides I (17) and II (18) are the pentasaccharides reported from I. purga . For structures 1–18, see Figure 4.
4. Conclusion and Future Prospects
Recycling, in an HPLC system, for the purification of natural products, has not been utilized to its full. Closed loop recycling is the most popular technique used in the isolation of small molecules. Several custom-made instrument designs for recycle HPLC have been reported to suit specific industrial requirements. The potential of recycle HPLC is evidenced by the purification of both chiral and achiral natural molecules using diverse stationery phases. This technique needs to be popularized amongst the isolation chemists, so that the difficulties involved in the separation of natural products may be minimized.
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