HILIC for separation of co-eluted flavonoids under RP-HPLC mode

J. Sep. Sci. 2008, 31, 1623 –1627 H. Zhang et al. 1623

Hui ZhangZhimou GuoFeifang ZhangQing XuXinmiao Liang

Dalian Institute of ChemicalPhysics, Chinese Academy ofSciences Dalian, China

Short Communication

HILIC for separation of co-eluted flavonoids underRP-HPLC mode

Hydrophilic interaction chromatography (HILIC) was employed to separate the co-eluted flavonoids from licorice extract under RP-HPLC mode. HILIC separations werecarried out with the Atalantis HILIC Silica column and the CD-based column. Theco-eluted flavonoids were well retained and separated on the two HILIC columnsunder HILIC mode. Similar results were obtained in the separation of another isofla-vones sample, from kudzu extract under HILIC mode.

Keywords: HILIC / RP-HPLC / Stationary phase / Separation selectivity /

Received: December 13, 2007; revised: January 23, 2008; accepted: January 24, 2008

DOI 10.1002/jssc.200700656

1 Introduction

RP-HPLC is commonly used for the analysis of complexsamples, such as environmental, biological and naturalproduct samples. The choice of employing RP-HPLC foranalysis is mostly due to features like the wide range ofapplicability, robustness and convenience. Recently,remarkable advances have been made in column andinstrument technology [1–4]. However there are stillmany problems in the application of RP-HPLC. For exam-ple, the limited separation selectivity. Some componentsin the complex samples prefer co-eluting due to theirsimilar retention behaviors under RP-HPLC. In pharma-ceutical analysis, the impurities or minor componentsmay be overlooked due to their being co-eluted and over-lapped by major components, which is important forearly-phase drug development. The selectivity of RP-HPLCis not sufficient for the separation of the co-eluted com-ponents even with higher efficiency columns or longerelution time, etc. Therefore, it is necessary to develop analternative method to resolve the co-eluted compoundsunder RP-HPLC.

Hydrophilic interaction chromatography (HILIC) is aneffective technique in the separation of polar com-pounds. It was first proposed by Alpert in 1990 [5] andpromoted by Strege [6–8]. HILIC employs a polar station-ary phase eluted with aqueous/organic mobile phases.HILIC offers different separation selectivity from RP-HPLC because of their different retention mechanisms

and the retention orders. Despite the progress inresearch of HILIC over the last ten years [9], it is primarilyused for separating polar compounds or compounds thatare not sufficiently retained under RP-HPLC, such as pep-tides [10, 11], amino acids [5, 12], oligonucleotides [13,14], polar compounds in natural products and drug sub-stances [6, 8, 15–17]. HILIC has been rarely used for theseparation of the well-retained compounds on RP col-umns. However, HILIC might be an attractive alternativeto RP-HPLC when the selectivity of RP-HPLC is not suffi-cient to resolve the well-retained but co-eluted com-pounds.

In this work, HILIC was employed to separate the co-eluted flavonoids under RP-HPLC. The HILIC methodshould be useful for the separation of the co-eluted flavo-noids which were sufficiently retained but difficult to bewell separated under RP-HPLC.

2 Experimental

2.1 Chemicals

HPLC grade ACN was obtained from Fisher (USA). Formicacid of HPLC grade was purchased from Acros (USA).Water was prepared by a MilliQ purification system(USA).

2.2 Equipment

The chromatographic system consisted of a Waters 2695HPLC pump, equipped with an autosampler and a Waters2996 diode-array detector (Waters, USA). The followingcolumns were used under RP-HPLC mode: Tigerkin C18column (25064.6 mm id, 5 lm, Sipore, Dalian, China),ZORBAX Eclipse XDB-C8 column (15064.6 mm id, 5 lm,Agilent, USA), Inertsil CN-3 column (25064.6 mm id,

Correspondence: Dr. Xinmiao Liang, Dalian Institute of Chemi-cal Physics, Chinese Academy of Sciences, No. 457 ZhongshanRoad, Dalian 116023, ChinaE-mail: [email protected]: +86-411-8437-9539

Abbreviations: HILIC, hydrophilic liquid chromatography

i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

1624 H. Zhang et al. J. Sep. Sci. 2008, 31, 1623 – 1627

5 lm, DIKMA, USA). The separations under HILIC modewere performed on two columns, Atalantis HILIC Silicacolumn (25064.6 mm id, 5 lm, Waters, USA) and CD-based column (15064.6 mm id, 5 lm, made by ourselves[18]). The column temperature was maintained at 308C.Mobile phase A (water with 0.1% formic acid) and mobilephase B (ACN with 0.1% formic acid) were prepared bythe addition of formic acid to water or ACN.

2.3 Sample preparation

The aqueous licorice extract was separated using prepa-rative RPLC system on the Tigerkin C18 preparative col-umn (360680 mm id, 10–20 lm). The mobile phase wascomposed of water (A) and methanol (B) with gradientelution at a flow rate of 200 mL/min. The gradient was asfollows: 20–85% B from 0 to 40 min, 85 –100% B from 40to 42 min, and 100% B from 42 to 50 min. The eluatefrom 14 to 19 min was collected. According to the UVspectra the components of this sample were identified asflavonoids. After concentration, the flavonoids samplewas re-dissolved in ACN and filtered through a 0.22 lm

pore filter for subsequent experiments. In order to easilycompare results, labeling of the peaks in the separationswas used. The labeling of the peaks was to show howmany peaks were obtained in every separation.

3 Results and discussion

In this study RP-HPLC and HILIC were employed to sepa-rate the flavonoids sample. Separations under RP-HPLCmode were carried out with the XDB-C8 column and theInertsil CN-3 column. The Atalantis HILIC Silica columnand the CD-based column were used under HILIC mode.Chromatographic conditions for the separations of thepresent work were summarized in Table 1.

The flavonoids fraction from preparative RPLC wasanalyzed with the Tigerkin C18 analytical column(25064.6 mm id, 5 lm). The chromatogram is shown inFig 1. The components of this sample were found to beco-eluted within a very short time due to their similarretention behaviors. The retention times ranged from 33to 36 min. Four main peaks were observed in the chroma-togram and the peak shapes were broad and overlapped.


Table 1. Chromatographic conditions for the separations of the present work

Method name Columna) LC mode Gradient(%B)b)

Method A 25064.6 mm id, 5 lm Tigerkin C18 column RP-HPLC 5/25/40/95/95% B in 0/40/45/50/60 minMethod B 15064.6 mm id, 5 lm Zorbax XDB-C8 column RP-HPLC 5/5/20/20% B in 0/2/62/65 minMethod C 25064.6 mm id, 5 lm Inertsil CN-3 column RP-HPLC 5/5/25/25% B in 0/2/62/65 minMethod D 25064.6 mm id, 5 lm HILIC Silica column HILIC 98/95/95% B in 0/30/35 minMethod E 15064.6 mm id, 5 lm CD-based column HILIC 98/90/90% B in 0/30/35 minMethod F 25064.6 mm id, 5 lm Tigerkin C18 column RP-HPLC 10/50/95/95% B in 0/60/62/65 min

a) Flow rate was set at 1.0 mL/min for all the columns, and temperature of the columns was maintained at 308C.b) B represents the mobile phase B, 0.1% formic acid in ACN. Mobile phase A used in this study was 0.1% formic acid aqueous

solution.

Figure 1. Chromatogram for the co-eluted flavonoids with the Tigerkin C18 column (25064.6 mm id, 5 lm) under RP-HPLCmode. Method A in Table 1 was employed. Injection volume, 5.0 lL; UV detection, 330 nm.

J. Sep. Sci. 2008, 31, 1623 –1627 Liquid Chromatography 1625

There might be minor components overlapped by majorcomponents. It was very difficult to separate the co-eluted flavonoids with the C18 column, even wheneluted within a longer gradient time, or a column withhigher efficiency was employed.

Separations of the co-eluted flavonoids were also car-ried out with the XDB-C8 column and the Inertsil CN-3column under the conditions shown in Table 1. Threemain peaks were observed in the chromatogram of theXDB-C8 column, as shown in Fig. 2. The retention timesof the flavonoids were from 32 to 42 min. The possibility

to improve the separation of the co-eluted flavonoids wasquite limited owing to the correlated selectivity betweenC8 and C18 caused by the highly correlated retentionmechanisms [19]. The separation selectivity wasimproved to some extent by using the Inertsil CN-3 col-umn. Five peaks were observed in the chromatogramand the retention times of the flavonoids were rangedfrom 20 to 30 min. Furthermore, the broad and over-lapped doublet (peak 1 and peak 2) was observed in thechromatogram. Therefore, the separation selectivity pro-vided by the Inertsil CN-3 column was still not sufficient


Figure 2. Chromatograms for theco-eluted flavonoids under RP-HPLC mode with (a) the XDB-C8column (15064.6 mm id, 5 lm),Method B in Table 1 wasemployed; and (b) the IntersilCN-3 column (25064.6 mm id,5 lm), Method C in Table 1 wasemployed. Injection volume,5.0 lL; UV detection, 330 nm.

Figure 3. Chromatograms for theco-eluted flavonoids under HILICmode with (a) the Atalantis HILICSilica column (25064.6 mm id,5 lm), Method D in Table 1 wasemployed; and (b) the CD-basedcolumn (15064.6 mm id, 5 lm),Method E in Table 1 wasemployed. Injection volume,10.0 lL; UV detection, 330 nm.

1626 H. Zhang et al. J. Sep. Sci. 2008, 31, 1623 – 1627

to resolve the co-eluted flavonoids. According to theabove results, it was suggested that the separation modewith different retention mechanism from that of RP-HPLC might be an alternative to resolve the co-eluted fla-vonoids.

HILIC offers different separation selectivity from RP-HPLC because of their different retention mechanisms.As an alternative, the separations of the co-eluted flavo-noids under HILIC mode were performed on two differ-ent columns, the Atalantis HILIC Silica column and theCD-based column [18]. The separations are illustrated inFig 3. Surprisingly, the components of the co-eluted fla-vonoids were well retained under HILIC mode. Morepeaks were separated by the two HILIC columns than bythe RP columns. The separation selectivity of the co-eluted flavonoids was greatly improved on the two HILICcolumns because of the different retention mechanismsbetween HILIC and RP-HPLC mode. The flavonoids werewell retained on the Atalantis HILIC Silica column and atotal of 12 peaks were observed in the chromatogram.The retention times of the flavonoids were ranged from 3to 16 min. Most of the components were well separatedon the HILIC Silica column. Besides, the flavonoids werealso well retained on the CD-based column and 14 peakswere observed as shown in Fig. 3. Baseline separationcould be achieved for most of the components in thissample. The peaks were evenly distributed over theentire chromatogram. The retention times of these com-ponents on the CD-based column ranged from 2 to24 min, while that on the Atalantis HILIC Silica columnwere from 3 to 16 min. It seemed that these flavonoidshad better retention on the CD-based column than onthe Atalantis HILIC Silica column, which may be a resultof the molecular recognition ability of the CD-based sta-

tionary phase. Furthermore, according to the UV and thechromatographic profiles, the selectivities of the two col-umns were complementary to each other.

The isoflavones sample from kudzu extract was alsoseparated under HILIC mode with the two HILIC col-umns, the Atalantis HILIC Silica column and the CD-based column. As shown in Fig. 4, at least nine peakswith retention times from 24 to 32 min were observedon the Tigerkin C18 column, which was difficult to sepa-rate well under RP-HPLC. Separations performed on thetwo HILIC columns are illustrated in Fig. 5. The isofla-vones were well retained on the two columns underHILIC mode. Ten peaks on the HILIC Silica column and 14peaks on the CD-based column were observed in the chro-matograms, respectively. The peaks were evenly distrib-uted over the entire chromatogram. According to thechromatographic profiles, the selectivities of the two col-umns were also complementary to each other. Theresults were similar to that of the flavonoids samplefrom licorice extract under HILIC mode.

4 Concluding remarks

The co-eluted flavonoids on C18 were well retained onthe two HILIC columns under HILIC mode. HILIC has alsoproven to be a powerful tool for the separation of the fla-vonoids, despite their polar character, which were wellretained, but co-eluted under RP-HPLC mode. The resultssuggest that HILIC can also be used for the separation ofwell-retained compounds on RP columns. Combinationof HILIC and RP-HPLC has the potential in 2-D methodsdevelopment for the research of complex samples.Related research, such as retention behaviors and prepa-


Figure 4. Chromatogram for the isoflavones sample from kudzu extract under RP-HPLC mode with the Tigerkin C18 column(25064.6 mm id, 5 lm). Method F in Table 1 was employed. Injection volume, 2.0 lL; UV detection, 300 nm.

J. Sep. Sci. 2008, 31, 1623 –1627 Liquid Chromatography 1627

rative HPLC under HILIC mode, are underway in our labo-ratory. We believe that HILIC, together with the MS detec-tions will become more useful in the research of naturalproducts.

We acknowledge the grant of the project supported by the Knowl-edge Innovation Program of DICP, CAS (K2006A3).

The authors declared no conflict of interest.

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Figure 5. Chromatograms for theisoflavones sample from kudzuextract under HILIC mode with(a) the Atalantis HILIC Silica col-umn (25064.6 mm id, 5 lm),Method D in Table 1 wasemployed; and (b) the CD-basedcolumn (15064.6 mm id, 5 lm),Method E in Table 1 wasemployed. Injection volume,2.0 lL, UV detection 300 nm.

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HILIC for separation of co-eluted flavonoids under RP-HPLC mode