separation of flavonoid and alkaloid using collagen fiber adsorbent

Research Article

Separation of flavonoid and alkaloid usingcollagen fiber adsorbent

Flavonoids and alkaloids are two major classes of plant compounds with biological

activities, and they usually coexist in many medicinal herbs. In this study, a novel collagen

fiber adsorbent (CFA) was prepared, and its application for adsorption chromatography

separation of flavonoids and alkaloids was systematically investigated. The typical flavo-

noids, rutin and baicalin, and the typical alkaloids, matrine and caffeine, were selected as

probe molecules for the investigations. The batch adsorption behaviors of these

compounds on CFA in different solvents indicated that hydrogen bond plays a predo-

minant role for the adsorption of flavonoid and alkaloid in pure ethanol, while the

hydrophobic interaction plays a predominant role for the adsorption in water. In column

chromatography separation, flavonoids were completely separated from alkaloids by a

stepwise elution process with pure ethanol followed by aqueous ethanol solution. The two

flavonoids, rutin and baicalin, were also well separated although the two alkaloids,

matrine and caffeine, were washed out together. The optimal loading volume of sample

solution (10 mg/mL) for the separation was determined as 0.66 mL/g CFA. Under these

conditions, flavonoid and alkaloid were effectively separated with a recovery higher than

90% in 8 times repeated applications.

Keywords: Adsorption chromatography separation / Alkaloid / Collagen fiberadsorbent / Flavonoid / Hydrogen bondDOI 10.1002/jssc.200900864

1 Introduction

Flavonoids and alkaloids constitute two major classes of

secondary plant metabolites, which often coexist in plant

extracts [1–3]. Flavonoids are a subgroup of the polyphenols

containing two or more aromatic rings, each with at least

one aromatic hydroxyl [4]. They generally occur in plants as

glycosylated derivatives and could be hydrolyzed into

flavonoids under acidic condition [5]. Alkaloids represent a

highly diverse group of compounds that are related only by

the occurrence of a nitrogen atom in the heterocyclic ring,

and are found in approximately 20% of plant species [6].

Both flavonoids and alkaloids have a long history of use in

traditional Chinese medicine [7]. Recent medical studies

show that flavonoids and alkaloids have different biological

activities and pharmacological effects. Flavonoids have

antioxidant, anti-thrombotic, anti-inflammatory, anti-hyper-

tensive and anti-microbial activities [8]. In recent years,

some flavonoids have been marketed as dietary supplement

products, due to their functions of preventing disease

without side effects [9]. As for alkaloids, some of them have

been found to possess anti-psychotic, anxiolytic, anticancer

and anti-HIV activities [10, 11]. However, some alkaloids,

such as pyrrolizidine alkaloids and taxine alkaloids, have

been demonstrated to be poisonous to human beings

[12, 13]. In addition, flavonoids or alkaloids are usually

used as a whole without further separation in industrial

practices. For instance, many make-ups and skin-care

creams contain the mixture of flavonoids [14], and Sophoraflavescens alkaloids have been used in the pharmacy [15].

Consequently, it is necessary for us to develop an efficient

and simple method for the separation of flavonoids and

alkaloids for the purposes of pharmacological researches

and more effective applications of natural products in drugs.

Some methods for the separation of flavonoids and

alkaloids from plant extracts have been developed, such as

preparative high performance liquid chromatography [16],

and high-speed counter-current chromatography [17, 18].

Due to the high cost and relatively low production efficiency,

these methods are not suitable for large-scale application.

Porous silica, the most common chromatographic separa-

tion material, is also not suitable for the preparative-scale

separation of flavonoids and alkaloids because of low stabi-

lity at high pH, high price and high operating back pressure

[19]. Recently, adsorption separation has been proven to be

an alternative approach for the large-scale separation of

Juan Li1,2

Xuepin Liao1

Guanhao Liao1

Qiang He1

Wenhua Zhang2

Bi Shi2

1Department of BiomassChemistry and Engineering,Sichuan University, Chengdu,P. R. China

2National Engineering Laboratoryfor Clean Technology of LeatherManufacture, SichuanUniversity, Chengdu, P. R. China

Received December 22, 2009Revised May 10, 2010Accepted May 11, 2010

Abbreviations: CFA, collagen fiber adsorbent; BV, bedvolume

Correspondence: Dr. Bi Shi, National Engineering Laboratory forClean Technology of Leather Manufacture, Sichuan University,24, South Section 1, Yihuan Road, Chengdu, Sichuan, 610065,P. R. ChinaE-mail: [email protected]: 186-28-85400356.

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

J. Sep. Sci. 2010, 33, 2230–22392230

bioactive compounds from plant extracts [20]. The adsorp-

tion separation method based on macroporous resins is

attracting increasing attention in practical applications, due

to its relatively low cost, high stability and easy regeneration

[21]. However, the toxic organic residues introduced from

the synthesis process of macroporous resins, including non-

crosslinked monomer, porogenic agent and dispersant,

may lead to potential pollution of the products separated.

Therefore, new separation materials with good stability and

separation efficiency need to be developed. More recently,

many biopolymers, such as cellulose [22], chitosan [23],

agarose [24], and silk fibroin [25] have been used for

adsorption separation of bioactive compounds from plant

extracts in consideration of their non-toxicity and biocom-

patibility.

Collagen is an abundant protein coming from the skin

of domestic animals, and it is composed of three polypep-

tide chains with triple helical structure. Naturally, collagen

molecules are aggregated through hydrogen bond and

covalent bond to form water-insoluble collagen fiber [26].

Collagen fiber contains an abundance of functional groups,

including –OH, –NH2, and –COOH, and therefore, it has

potential to be prepared into collagen fiber-based functional

materials through chemical modifications [27]. Our previous

study indicated that an adsorbent made by collagen fiber

exhibits excellent adsorption selectivity to tannins in the

plant extracts due to the fact that tannins can associate with

collagen fiber through multi-point hydrogen bonds [28]. The

fact implies that the bioactive compounds in plant extracts

may be separated by collagen fiber adsorbent if they have

different hydrogen binding ability with collagen fiber. As

mentioned above, alkaloids contain at least one nitrogen

atom in a heterocyclic ring, whilst flavonoids contain one or

more aromatic hydroxyls on the aromatic rings. According

to the viewpoint of hydrogen bond, the nitrogen atom in

alkaloids only acts as hydrogen bond acceptor, while the

hydroxyl group of flavonoids serves as both acceptor and

donor of hydrogen bond. The difference in molecular

structures between alkaloids and flavonoids may affect their

capacities of taking hydrogen bond reaction and, therefore,

lead to different affinities with collagen fiber. Hence, it is

reasonable to deduce that collagen fiber could be considered

as a potential adsorbent for the separation of flavonoids and

alkaloids. However, natural collagen fiber is easily attacked

by chemicals and bacteria and has a limited hydrothermal

stability. In order to improve the mechanical strength and

chemical stability, a proper modification of collagen fiber

should be carried out. It is well known that the functional

groups (-OH, -NH2, and -COOH) of collagen fibers can form

interhelical cross-links with crosslinking agent, either

within or between fibrils. These cross-links can increase the

mechanical strength and physical properties of collagen

fiber [29]. Glutaraldehyde is the most widely used cross-

linking agent due to its high efficiency and low toxicity. The

crosslinking reaction of glutaraldehyde mainly takes place at

amino groups of lysine and hydroxylysine amino acid resi-

dues of polypeptide chains [30].

On the basis of chemical structures, flavonoids are divided

into nine categories. Rutin (1) and baicalin (2) (as shown in

Fig. 1) are typical flavonol and flavone, respectively, and are

widely presented in many kinds of plants [7]. These two

flavonoids are attracting more and more attention due to their

various biological activities [31, 32]. As for alkaloids, approxi-

mately 12,000 compounds have been identified, which can be

divided into groups based on their carbon skeletal structures.

Matrine (3) and caffeine (4) (as shown in Fig. 1) are the

representative compounds of quinolizidine alkaloid and purine

alkaloid, respectively, and they both contain several nitrogen

atoms in heterocyclic rings [7]. Matrine and caffeine are two

common alkaloids existing in plants, and a great amount of

researches has focused on them due to their wide pharmaco-

logical activities [33, 34]. In addition, these two alkaloids

usually coexist with flavonoids in plants [35, 36]. Therefore, in

the present investigation, rutin, baicalin, matrine, and caffeine

were selected as probe molecules to simulate the plant extracts

containing both flavonoids and alkaloids. Batch adsorption

experiments were carried out to investigate the fundamental

adsorption mechanism and adsorption selectivity of collagen

fiber adsorbent for flavonoids and alkaloids in different

solvents. The column chromatography separation of the

mixture of flavonoids and alkaloids was performed by a step-

wise elution with ethanol and aqueous ethanol solution.

Furthermore, the sample loading volume and the reusability of

collagen fiber adsorbent for the separation of flavonoids and

alkaloids were evaluated.

2 Experimental

2.1 Reagents

Rutin (1), baicalin (2), matrine (3) and caffeine (4) were

bought from Hui Ke Botanical Research and Development

Corp. Ltd, China. The purity of these compounds was

Z98.0%. Double distilled water was used in HPLC analyses.

Figure 1. UV spectra and molecular structures of rutin (1),baicalin (2), matrine (3) and caffeine (4).

J. Sep. Sci. 2010, 33, 2230–2239 Liquid Chromatography 2231


Methanol used for HPLC was of chromatographic grade

(Tedia, USA). All other reagents were of analytical grade and

obtained from Chengdu Kelong Chemical Reagent Co Ltd,

China. All solutions prepared for HPLC analysis were

filtered using 0.45 mm cellulose membranes before use.

2.2 Equipment

HPLC 1100 (Agilent Technologies, USA), equipped with

Archrom Bond-AQ C18 reversed-phase column

(150 mm� 4.6 mm, I.D., 5 mm) and G1315B diode array

detector, was used to analyze the concentration of compo-

nents in solutions. UV/VIS Spectrometer (Perkin-Elmer

Lambda 25, German) was used to perform the UV

absorbance measurement. Differential scanning calorimetry

(2000PC, NETZSCH, Germary) was used to detect the

thermal denaturation temperature of collagen fiber adsor-

bent. The morphologies of collagen fiber and CFA were

studied using a JSM-5900LV scanning electron microscope

(JEOL Co. Ltd., Japan). The specific surface areas of collagen

fiber and CFA were measured by a surface area and porosity

analyzer (TriStar3000, Micrometitics, USA), and calculated

by BET equation.

2.3 Preparation of collagen fiber

Collagen fiber was prepared according to the procedures in our

previous work [28]. Briefly, bovine skin was cleaned, limed,

split and delimed according to the procedures of leather

manufacture to remove the non-collagen components. Then

the skin was treated with aqueous acetic acid solution

(concentration 16.0 g/L) for three times to remove mineral

substances. The pH was then adjusted to 4.8–5.0 by acetic acid-

sodium acetate buffer solution and the skin was subsequently

dehydrated with absolute ethyl alcohol, dried in vacuum to

moisture content r10.0%, ground and sieved. Then the

collagen fiber was obtained with particle size of 0.1–0.25 mm,

moisture r12.0%, ash content r0.3% and pH 5.0–5.5.

2.4 Preparation of collagen fiber adsorbent

The preparation of collagen fiber adsorbent (CFA) was

described in detail in our previous work [28]. Briefly, 15.0 g

collagen fiber was soaked in 300 mL distilled water at room

temperature for 24 h. Then, 0.75 g glutaraldehyde was added

and reacted at 301C with constant stirring for 2 h. A sufficient

amount of NaHCO3 solution (15% w/w) was gradually added

within 2 h in order to increase the pH of the solution to 7.0

and then continuously reacted at 401C for another 4 h. When

the reaction was completed, the product was collected by

filtration and fully washed with distilled water and dried in

vacuum at 501C, and finally the collagen fiber adsorbent

(CFA) was obtained. The crosslinking reaction between

collagen fiber and glutaraldehyde is shown in Fig. 2 [37].

2.5 Establishment of working calibration curve

HPLC with a DAD detector was used for the quantification

in order to accurately determine the concentrations of rutin,

baicalin, matrine and caffeine in the elution fractions. The

standards of rutin (1), baicalin (2), matrine (3) and caffeine

(4) were dissolved in methanol to prepare standard

solutions. The working calibration curves of the four

components were established by using 6 different concen-

trations of standards. Gradient elution was used in HPLC

runs. The gradient was formed by varying the proportion of

solvent (A) 0.5% (w/w) aqueous phosphoric acid and solvent

(B) methanol. That is, the volume of solvent B increased

from 20% to 50% during 0–5 min, from 50 to 70% during

5–8 min, and kept at 70% during 8–13 min. According to

the spectra shown in Fig. 1, the detection wavelengths were

280 nm for baicalin and caffeine, 220 nm for matrine, and

360 nm for rutin. The flow rate was 0.8 mL min�1, and the

injection volume was 20 mL. The column temperature was

maintained at 301C. The retention times of rutin, baicalin,

matrine and caffeine were found to be 9.2 min, 10.2 min,

3.6 min and 6.8 min, respectively. The working calibration

curve based on rutin, baicalin, matrine and caffeine

standard solutions showed good linearity over the range of

10–160 mg/mL. The regression lines of rutin, baicalin,

matrine and caffeine were y 5 33.452x�12.804 (R2 5 1,

n 5 6), y 5 47.371x164.838 (R2 5 0.9998, n 5 6), y 5 15.868x13.3 (R2 5 0.9999, n 5 6), y 5 55.985x1135.13 (R2 5 0.9995,

n 5 6), respectively.

2.6 Batch adsorption experiments

The mixture solutions of rutin, baicalin, matrine and

caffeine were prepared by dissolving them in 100 mL

of aqueous ethanol solution. The ethanol concentrations

(%, v/v) of the solutions were 0%, 30%, 50%, 70%, 80%,

85%, 90%, 95% and 100%, respectively, and the content of

each component in the solutions was 40 mg/mL.

For each experiment, 0.15 g (dry weight) CFA and

15 mL sample solution were introduced into a flask. Then,

the adsorption was conducted with constant shaking at 251C

for 12 h. In our preliminary experiments, the concentration

of each component in solution was analyzed by HPLC every

2 h, and it was found that 12 h is sufficient for the adsorp-

Figure 2. The crosslinking reaction between collagen fiber andglutaraldehyde.

J. Sep. Sci. 2010, 33, 2230–22392232 J. Li et al.


tion equilibrium of each component. The batch adsorption

experiments in the presence of urea or n-propanol were also

carried out, in order to investigate the adsorption mechan-

ism. The adsorption extent of each component was calcu-

lated by Equation (1).

E ¼ C0 � Ce

C0� 100% ð1Þ

where E is the adsorption extent (%); C0 and Ce are the

initial and equilibrium concentrations of each component in

the solutions, respectively (mg/mL).

2.7 Column chromatography separation

The two-component mixture solutions of rutin–matrine,

rutin–caffeine, baicalin-matrine and baicalin-caffeine were

prepared by dissolving them in 50% aqueous ethanol

solution. The content of each component in the solutions

was 5 mg/mL. Similarly, the four-component mixture solu-

tion of rutin–baicalin–matrine–caffeine was prepared by

dissolving them in 50% aqueous ethanol solution, and the

content of each component in the solutions was 5 mg/mL.

6.0 g of CFA was soaked in distilled water for 12 h and

then packed into a glass column (16 mm� 40 cm). Similar

as packing the silica column, CFA can be firmly packed into

the column without other supporting materials due to its

good mechanical strength. The bed volume (BV) was 40 mL,

the packed height of CFA bed was 20 cm and the void

volume was 10.8 mL. The column was equilibrated with

pure ethanol (the first eluent) or water for control experi-

ment. Then, 1 mL of sample solution was loaded on the top

of column followed by stepwise elution at a constant flow

rate of 0.3 BV/h controlled by a constant flow pump, and

0.15 BV of effluent solution was collected every 30 min by

using an automatic collector. The separation was monitored

by HPLC analysis of the effluent solution at an interval of

0.15�0.9 BV, depending on the concentrations of the four

components. The eluent was changed when no component

was detected in the effluent solution.

2.8 Effect of sample loading volume

The mixture solution of baicalin and matrine was prepared by

dissolving them in 50% aqueous ethanol solution. The content

of each component in the solution was 5 mg/mL. 1 mL, 2 mL,

4 mL and 6 mL of baicalin-matrine mixture solution were

respectively used to test the influence of sample loading

volume on column separation performance. Pure ethanol and

50% aqueous ethanol solution were used as the eluent

solution. The separation was performed at a constant flow

rate of 0.3 BV/h, and 0.15 BV of effluent solution was collected

every 30 min. The concentrations of baicalin and matrine in

the effluent solution were analyzed by HPLC at an interval of

0.15�0.3 BV, depending on the concentrations of the two

components in solution. The fractions of baicalin and matrine

were collected separately, and the concentrations of baicalin

and matrine were determined by HPLC. Equation (2) was used

to calculate recoveries of baicalin and matrine.

R ¼ m

M� 100% ð2Þ

where R is the recovery (%), M is the total mass of baicalin or

matrine in loading sample solution, m is the mass of baicalin

or matrine purified.

2.9 Reusability of CFA

The mixture solution of baicalin and matrine prepared as in

Section 2.8 was used as loading sample, and the concentra-

tion of each component in the solution was 5 mg/mL. The

sample loading volume was 4.0 mL. The column separation

was performed under optimized operation conditions for 8

times to test the reusability of CFA. The stepwise elution

was conducted with 2 BVs of pure ethanol followed by 4 BVs

of 50% aqueous ethanol solution. The effluent solutions of

the stepwise elution were collected separately, and the

recoveries of baicalin and matrine were calculated using

Equation (2). The CFA column was washed at the flow rate

of 0.3 BV/h with 2 BVs of 70% aqueous ethanol solution and

4 BVs of pure ethanol between the repetitive runs.

3 Results and discussion

3.1 Characterization of collagen fiber adsorbent

(CFA)

Both collagen fiber and CFA are in a fibrous state with visual

inspection. The surface morphology of collagen fiber and

CFA were observed by scanning electron microscopy (SEM).

As shown in Fig. 3, the outer diameters of both collagen fiber

and CFA are approximately 5–10 mm. It is obvious that the

fibrous structure of collagen fiber was not changed after

crosslinking reaction of glutaraldehyde. It has been reported

that the adsorbents with fibrous structure usually have a high

mass transfer rate and a low back pressure, in comparison

with the conventional porous materials, such as porous silica

gel [19]. Therefore, the fibrous structure of CFA is beneficial

to large-scale industrial separation.

The specific surface areas of collagen fiber (2.13 m2/g) and

CFA (2.05 m2/g) are in the same level with those of other

Figure 3. Scanning electron microscopy images of collagenfiber (A) and CFA (B) (1500�).



polymer phases [38]. There are no remarkable differences in

the BET surface area between collagen fiber and CFA, which is

consistent with the observation of SEM. The denaturation

temperature of collagen fiber is 60–651C. As for CFA, it is

increased to 80–861C. The crystalline structure of collagen

molecules collapses at the denaturation temperature [29].

Thereby, the denaturation temperature of collagen fiber is

remarkably improved after crosslinking by glutaraldehyde,

indicating higher hydrothermal stability in the applications.

3.2 Influence of solvent on the adsorption of flavo-

noids and alkaloids in batch experiments

Considering the ecological and dietary restraints, water and

ethanol were used for all the separation processes. The

ethanol concentration (%, v/v) of solution was 0%, 30%,

50%, 70%, 80%, 85%, 90%, 95% and 100%, respectively. All

of these experiments were conducted at 251C, and the CFA/

liquid ratio was 0.15:15 (g/mL). The initial concentration of

each component in the solution was 40 mg/mL. Fig. 4 shows

the influence of ethanol concentration on the adsorption

extents of flavonoids and alkaloids in batch experiments. It

was found that the adsorption extents of both rutin and

baicalin on CFA were low when the concentration of ethanol

was below 70%, while they significantly increased when the

concentration of ethanol was higher than 80%. However,

the adsorption extent of matrine and caffeine were all

limited when the concentration of ethanol varied from 0%

to 100%. The adsorption extents of rutin and baicalin were

much higher than those of matrine and caffeine when the

ethanol concentration was higher than 80%. The experi-

mental results indicate that the adsorption selectivity of CFA

towards flavonoids (rutin, baicalin) is enhanced in pure

ethanol compared with that in aqueous solution. On the

other hand, it implies that most of rutin and baicalin

adsorbed on CFA could be eluted by an aqueous ethanol

solution of 50%–70%. Therefore, it is reasonable to suggest

that rutin and baicalin can be separated from matrine and

caffeine by stepwise elution with varying concentration of

the aqueous ethanol solution.

3.3 Adsorption mechanism

The hydrogen bond and hydrophobic interaction may play

important roles for the adsorption of flavonoids and

alkaloids on CFA. According to our previous study [39,

40], the adsorption mechanism of flavonoids and alkaloids

on CFA should be a synergetic effect of hydrogen bond and

hydrophobic interaction. In aqueous solution, water mole-

cules are good hydrogen bond donors and acceptors [41], so

they can form hydrogen bonds not only with CFA, but also

with flavonoids and alkaloids, which may reduce the

probability of the formation of hydrogen bonds between

CFA and flavonoids or alkaloids. As a result, although

hydrogen bonds still have an effect on the adsorption, the

dominant adsorption force of rutin, baicalin, matrine and

caffeine on CFA at lower ethanol concentration (o30%) is

hydrophobic interaction between the hydrophobic domain

of adsorbates and polypeptide chains of CFA. It is known

that hydrophobic interaction is a non-specific interaction

[42, 43], because this interaction is the tendency of any

hydrocarbons or lipophilic hydrocarbon-like groups in

solutes to form intermolecular aggregates in an aqueous

medium [44]. Therefore, CFA exhibits a lower adsorption

selectivity at lower ethanol concentration, as shown in

Fig. 4.

According to the definition of hydrophobic interaction

mentioned above [44], the hydrophobic interaction is more

strongly suppressed in the solution with less water percen-

tage, while the hydrogen bonds are easier to be formed due

to the lower dielectric constant of ethanol compared with

that of water. Therefore, the hydrogen bonds play a predo-

minant role for the adsorption in pure ethanol. As shown in

Fig. 1, the main hydrogen bond sites of rutin and baicalin

are hydroxyls, while they are carbonyl oxygen and tertiary

amine nitrogen for matrine and caffeine. The hydroxyls of

rutin and baicalin are both hydrogen bond donor and

acceptor, whereas the carbonyl oxygen atoms and tertiary

amine nitrogen atoms of matrine and caffeine only have the

role as hydrogen bond acceptor. On the other hand, the

electronegativity of oxygen atoms in rutin and baicalin is

higher than that of nitrogen atoms in matrine and caffeine.

Thus, the multi-hydrogen bonds between flavonoids and

CFA are much stronger than that between alkaloids and

CFA, resulting in considerably higher adsorption selectivity

of CFA towards flavonoids in pure ethanol, as shown in

Fig. 4. In addition, the adsorption extent of rutin is lower

than that of baicalin, which could be ascribed to the steric

hindrance of the hydrophilic 6-deoxy-a-L-mannopyranosyl

and b-D-glucopyranosyl to C-3 position of rutin molecule.

When the ethanol concentration is in the range of 50%

to 70%, both hydrophobic interaction and hydrogen bond

between adsorbates and CFA are inhibited. As a result, the

minimum adsorption capacity of CFA to rutin, baicalin,

matrine and caffeine is observed, as shown in Fig. 4.

From elucidations above, it can be concluded that the

hydrophobic interaction should be the main mechanism in

water and in low ethanol concentration solution for the

Figure 4. Adsorption extents of flavonoids and alkaloids on CFAin different aqueous ethanol solutions.

J. Sep. Sci. 2010, 33, 2230–22392234 J. Li et al.


adsorption of rutin, baicalin, matrine and caffeine on CFA,

while the hydrogen bond is predominant in pure ethanol

and in high ethanol concentration solution.

In order to further confirm the adsorption mechanism

of CFA to flavonoids and alkaloids, batch adsorption

experiments were carried out in the presence of urea and

n-propanol, respectively. Urea was used as the hydrogen

bonds breaking agent [45], while n-propanol was used as the

hydrophobic interaction breaking agent [46]. The solvent

was pure ethanol and water, respectively. All the experi-

ments were run at 251C, and the CFA/liquid ratio was

0.15:15. The initial concentration of each component in the

solution was 40 mg/mL.

Figs. 5 and 6 exhibit the adsorption extents of rutin,

baicalin, matrine and caffeine on CFA in water or ethanol in

the presence of urea. As can be seen from Fig. 5, there is

only a slight decrease in adsorption extents of the four

components in water when the urea concentration increased

from 0 to 1.0 M, suggesting a limited effect of hydrogen

bond on adsorption of flavonoids and alkaloids. When the

urea concentration increased to 2.0 M, the adsorption

extents of the four components are all obviously decreased

due to the fact that a part of intermolecular hydrogen bonds

of collagen fiber are broken down by urea [47]. On the

contrary, the adsorption extents of the four components are

dramatically decreased with the increase of urea concen-

tration in ethanol, as shown in Fig. 6. When the urea

concentration is 0.75 M, nearly no adsorption was observed

for rutin, matrine and caffeine, and the adsorption extent of

baicalin is lower than 20%. These facts confirm that the

hydrogen bond plays a predominant role for the adsorption

of flavonoids and alkaloids on CFA in pure ethanol or in

high ethanol concentration solution.

Adsorption extents of rutin, baicalin, matrine and

caffeine on CFA in water or ethanol in the presence of

n-propanol are shown in Figs. 7 and 8, respectively. It can be

found that the adsorption extents of the four components

are greatly decreased in water when the concentration of

n-propanol increased from 10% to 30%. However, there is

no significant decrease for the adsorption extent in ethanol

when the concentration of n-propanol increased from 0 to

40%. The comparison of these results suggests that the

hydrophobic interaction plays an important role for the

adsorption of flavonoids and alkaloids on CFA in water or in

low ethanol concentration solution.

3.4 Column chromatography separation

3.4.1 Column chromatography separation of two-

component mixture

Column chromatography separation experiment of each two-

component mixture of flavonoid and alkaloid was conducted

Figure 5. Adsorption extents of flavonoids and alkaloids on CFAin water in the presence of urea.

Figure 6. Adsorption extents of flavonoids and alkaloids on CFAin ethanol in the presence of urea.

Figure 7. Adsorption extents of flavonoids and alkaloids on CFAin water in the presence of n-propanol.

Figure 8. Adsorption extents of flavonoids and alkaloids on CFAin ethanol in the presence of n-propanol.



as described in Section 2.7. The sample loading volume was

1.0 mL, and the initial concentration of each component in

the loading sample solution was 5 mg/mL. According to the

results of batch adsorption, pure ethanol and 70% aqueous

ethanol solution were used as eluent solutions for the

separation of rutin-matrine and rutin-caffeine mixtures, and

pure ethanol and 50% aqueous ethanol solution were used as

eluent solutions for baicalin-matrine and baicalin-caffeine

mixtures. The separation was performed at the flow rate of

0.3 BV/h, and the effluent solution was analyzed by HPLC at

an interval of 0.15�0.9 BV.

The results of the two-stepwise elution of the two-

component mixtures are shown in Fig. 9. It can be observed

that for all of the two-component mixtures, matrine or

caffeine is firstly eluted from the CFA column with 80 mL

(2 BVs) of pure ethanol, and rutin or baicalin cannot be

detected by HPLC in the effluent. Then rutin or baicalin is

washed out from the CFA column by 160 mL (4 BVs) of 70%

or 50% aqueous ethanol solution, and no matrine or

caffeine is detected by HPLC in the effluent. The elution

order follows the adsorption mechanism described in

Section 3.3. When eluted by pure ethanol at first, the

hydrogen bond between CFA and matrine or caffeine is very

weak, while that between CFA and rutin or baicalin is

relatively stronger. Therefore, matrine and caffeine can be

eluted by pure ethanol, while rutin and baicalin are retained

on the CFA column. When aqueous ethanol solution is used

as eluent solution, the hydrogen bond between CFA and

flavonoids is strongly suppressed, thus rutin and baicalin

can be eluted from the CFA column.

Therefore, the retention of flavonoids and alkaloids on

the CFA column can be controlled by adjusting the

composition of the eluent, and then flavonoids and alkaloids

can be easily separated on the CFA column by two-stepwise

elution. However, it is usual that a variety of flavonoids and

alkaloids coexist in plant extracts. So it is necessary to

explore the separation ability of CFA to the mixture

containing more components of flavonoids and alkaloids.

3.4.2 Column chromatography separation of four-

component mixture

Column chromatography separation experiments of the

four-component mixture of flavonoids and alkaloids were

performed as described in Section 2.7. The sample loading

volume was 1.0 mL, and the initial concentration of each

component in the loading sample solution was 5 mg/mL.

The stepwise elution was carried out with 100%, 90%, 80%,

70% and 50% aqueous ethanol solution in turn. As a

contrast, the column separation was also carried out by

stepwise elution with water, 10%, 30% and 50% aqueous

ethanol solution in turn. The effluent solution was analyzed

by HPLC at an interval of 0.15�0.9 BV.

The chromatograms of stepwise elution of mixture of

rutin, baicalin, matrine and caffeine on CFA column are

presented in Fig. 10. It can be seen in Fig. 10A, rutin and

baicalin are separated from matrine and caffeine by the

stepwise elution of 100%, 90%, 80%, 70% and 50% aqueous

ethanol solution, which indicates that the CFA column has

excellent separation ability to the mixture of flavonoids and

alkaloids. The two alkaloids, matrine and caffeine, were

eluted simultaneously, which implies that they have no

remarkable difference in hydrogen bonding with CFA. It

should be noted that rutin and baicalin are completely

separated on CFA column by using 80% and 70% aqueous

ethanol solution as eluent solutions, which implies that the

Figure 9. Chromatograms of two-component mixture of flavonoid andalkaloid on CFA column (flow rate0.3 BV/h, sample loading volume1.0 mL). (A,B) Stepwise elution withpure ethanol and 70% aqueous etha-nol solution; (C,D) stepwise elutionwith pure ethanol and 50% aqueousethanol solution.

J. Sep. Sci. 2010, 33, 2230–22392236 J. Li et al.


CFA column could be also used for separation of mixture of

flavonoids. In fact, the hydrogen bond between the CFA and

flavonoids depends on the number and position of hydroxyl

groups as well as the aglycone skeleton and the number of

saccharide groups in flavonoid molecules [48, 49]. There-

fore, it is reasonably expected that the separation of the

mixture of flavonoids can be achieved on the CFA column

by stepwise elution with appropriate solvents.

In the case of stepwise elution with water, 10%, 30%

and 50% aqueous ethanol solution in sequence, the four

components are eluted by water simultaneously, as shown

in Fig. 10B. In batch adsorption experiments, it has been

observed that the adsorption extents of rutin, baicalin,

matrine and caffeine in water were very low compared with

that in ethanol (Fig. 4). Therefore, the adsorption-desorption

process of the four components on the CFA column cannot

create enough difference to separate the mixture into indi-

vidual components.

3.5 Effect of sample loading volume

1 mL, 2 mL, 4 mL and 6 mL of baicalin-matrine mixture

solution were tested, respectively, and the initial concentra-

tion of each component in the mixture solution was

5 mg/mL. Pure ethanol and 50% aqueous ethanol

solution were used as the eluent solutions, and the effluent

solution was monitored at an interval of 0.15�0.3 BV. The

results of separation are shown in Fig. 11. The recoveries of

baicalin and matrine for each sample loading are listed in

Table 1.

As can be seen in Fig. 11 and Table 1, in the loading

volume range of 1.0 mL to 4.0 mL, baicalin and matrine can

be completely separated by eluting with 2 BVs of pure

ethanol and 4 BVs of 50% aqueous ethanol solution, and the

recovery of both baicalin and matrine is greater than 92%.

When the sample loading volume is 6.0 mL, baicalin and

matrine still can be separated by the stepwise elution, but

the recovery of baicalin is only 79.6%, and the band broad-

ening is more obvious compared with that in 4.0 mL. The

worse recovery of baicalin in 6.0 mL of sample loading

solution should be due to the peak tailing caused by over-

loading. In fact, a small amount of baicalin can be detected

by additional elution using 2 BVs of 50% aqueous ethanol

solution. Therefore, sample loading volume is an important

factor for the adsorption chromatography separation of

baicalin and matrine.

3.6 Reusability of the CFA

It is economically required that an adsorbent should be

reused for a large-scale separation process. Therefore, it is

necessary to investigate the reusability of the CFA for the

separation of flavonoids and alkaloids under the optimal

separation conditions. For each repeated application, 4 mL

baicalin-matrine mixture solution (5 mg/mL) was loaded, and

eluted by 2 BVs of pure ethanol and 4 BVs of 50% aqueous

ethanol solution respectively at the flow rate of 0.3 BV/h. The

reusability of the CFA was characterized based on the

recoveries of baicalin and matrine. The results of 8 times

repeated applications are listed in Table 2. The mixture of

baicalin and matrine are completely separated in the 8

repeated applications. The recoveries of both baicalin and

matrine are all greater than 90%, indicating that

the CFA possess excellent reusability in the separation

of flavonoids and alkaloids. The consistent recoveries of

baicalin and matrine in the same elution volume (4 BVs of

50% aqueous ethanol solution for baicalin, 2 BVs of pure

Figure 10. Chromatograms of four-component mixture offlavonoids and alkaloids on CFA column (flow rate 0.3 BV/h,sample loading volume 1.0 mL). (A) Stepwise elution with 100%,90%, 80%, 70% and 50% aqueous ethanol solution; (B) stepwiseelution with water, 10%, 30% and 50% aqueous ethanol solution.

Figure 11. Chromatograms of baicalin-matrine mixture onCFA column with different sample loading volume (flow rate0.3 BV/h). The eluent solutions of the two-stepwise elution arepure ethanol and 50% aqueous ethanol solution.



ethanol for matrine) indicate the good reproducibility of the

CFA column. In addition, after 25 times of successive cycles,

the color at the top of the column turned to brown yellow due

to column fouling. After washed with 30% (w/w) acetic acid

[50, 51], the separation efficiency of CFA column was restored

to the original state. After all the separation cycles, the packed

height of CFA bed was 19.8 cm. The height change rate is

only 1%, indicating that the CFA is well settled during the

separation process. Therefore, it is proven that CFA is a

promising separation material for the plant extracts.

4 Concluding remarks

In this study, we explored the potential use of collagen

fiber adsorbent (CFA) for the adsorption chromatography

separation of flavonoids and alkaloids by using four

typical flavonoids and alkaloids as probe molecules. The

batch adsorption behaviors of these compounds on CFA

indicate that hydrogen bond plays a predominant role for

the adsorption of flavonoids and alkaloids in pure ethanol,

while hydrophobic interaction plays a predominant role for

the adsorption in water. Under these optimal conditions, the

flavonoids and alkaloids in two- and four-component

mixture solutions can be separated on the CFA column by

the stepwise elution process with high recovery. The

recycling application tests demonstrate that CFA possesses

excellent reusability for the separation of flavonoids and

alkaloids. Compared with other separation materials, such

as C18 silica and macroporous resins, CFA is much cheaper

and easily obtained material (no more than $50/kg).

Therefore, CFA would be a cost-efficient material for large

scale separation of flavonoids and alkaloids.

The authors gratefully acknowledge the financial support byNational Natural Science Foundation of China (20976111),Key Program of National Science Fund of China (20536030),and National Technologies R&D Program (2006BAC02A09).

The authors have declared no conflict of interest.

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separation of flavonoid and alkaloid using collagen fiber adsorbent

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