synthesis and chromatographic properties of a chiral stationary phase derived from bovine serum...

Ting LiQiong-Wei YuBo LinYu-Qi Feng

Department of Chemistry,Wuhan University, Wuhan, PRChina

Original Paper

Synthesis and chromatographic properties of achiral stationary phase derived from bovine serumalbumin immobilized on magnesia-zirconia usingphosphonate spacers

A novel bovine serum albumin (BSA)-modified magnesia-zirconia stationary phasewas prepared using the sodium salt of cis-(3-methyloxiranyl)phosphonic acid (fosfo-mycin) as spacer and glutaraldehyde as coupler. Baseline separation of six derivati-zed amino acids (DNB-Leu, Dansyl-Val, etc.) was achieved on this column usingammonium acetate buffer-isopropanol mobile phase at a flow rate of 1.0 mL/min.The effects of mobile phase composition, eluent pH value, column temperature, andflow rate on the retention and separation of chiral compounds were also investiga-ted. The BSA chiral stationary phase (BSA-CSP) was relatively stable under experi-mental conditions. The coupling reaction in this method was mild, reliable, andreproducible; thus it was also suitable for the immobilization of various biopoly-mers with amino groups in the preparation of chromatography stationary phases.

Keywords: Bovine serum albumin / Chiral stationary phase / Enantiomer separation / Fosfomycin/ Magnesia-zirconia /

Received: August 29, 2006; revised: October 17, 2006; accepted: November 24, 2006

DOI 10.1002/jssc.200600336

1 Introduction

Chromatographic enantioseparation on chiral station-ary phases (CSPs) is one of the most direct, facile, andeffective methods of determining enantiomeric purity.In the past decades several kinds of CSPs have been devel-oped based on various chiral selectors, such as celluloses,cyclodextrins, macrocyclic antibiotics, crown ethers, andproteins [1–5]. Since proteins, possessing complex andchangeable conformations, are natural high-molecular-mass polymers composed of several chiral subunits, theirstereoselective molecular recognition has been exploitedto develop protein-based CSPs [6–9]. These phases havethe ability to separate a wide range of chiral compounds,especially pharmaceutically active compounds. Amongthem, bovine serum albumin (BSA) immobilized on silicawas the first protein to be used for enantiomer separa-tion by HPLC [10]. Allenmark's group did extensiveresearch on BSA-CSPs in the 1980s [11–17] and successfulenantioseparation of a variety of structurally differentracemic compounds was achieved, including aromaticamino acid, N-derivatized amino acids, aromatic sulfox-

ides and sulfoximine derivatives, arylpropionic acids,barbiturates, benzodiazepine, coumarin and benzoinderivatives [18].

In liquid chromatography, enantiomeric separationshave mainly been performed using silica-based chiral sta-tionary phases due to their mechanical strength, widerange of particle and pore dimensions, pore structure,and well-established silane chemistry. Several differentprocedures have been proposed for synthesis of protein-CSPs, consisting of physical adsorption involving ionicand/or hydrophobic interactions and chemical covalentlinkage involving amino-, diol-, and epoxide-derivedmatrices. However, during recent decades, zirconia hasattracted considerable attention in HPLC owing to itsremarkable mechanical, chemical, and thermal stability[19, 20]. Zirconia also proved to exhibit a great affinityfor both inorganic and organic phosphate via strongLewis acid-base interaction [21–24]. Recently Park et al.successfully prepared and evaluated a BSA-coated zirco-nia CSP [25]. Magnesia-zirconia, a kind of zirconia-con-taining mixed oxide, has a larger surface area, betterpore size distribution, and better pore structure, andshows a greater affinity for phosphonates compared tobare ZrO2 [24, 26]. Based on these characteristics, ourgroup has performed a series of studies on modificationof the magnesia-zirconia matrix via phosphonate spacers[24, 26, 27].

Correspondence: Professor Yu-Qi Feng, Department of Chemis-try, Wuhan University, Wuhan 430072, PR ChinaE-mail: [email protected]: +86-27-68754067

i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

804 T. Li et al. J. Sep. Sci. 2007, 30, 804 – 812

J. Sep. Sci. 2007, 30, 804 – 612 Liquid Chromatography 805

In this work, we aimed to take advantage of the physi-cal characters of magnesia-zirconia, and synthesize aBSA-modified magnesia-zirconia stationary phase (BSA-F-(ZrO2-MgO)) using the sodium salt of cis-(3-methyloxira-nyl)phosphonic acid (fosfomycin) as spacer for the sep-aration of enantiomers. This novel stationary phase wasexpected to have good stability, especially in mobilephases with high buffer content over a wide pH range. Itschromatographic performance was evaluated usingmany chiral compounds under various experimentalconditions. Studies on optimization of separation condi-tions, particularly the 2-propanol content in mobilephases, pH value, column temperature, and flow ratewere also undertaken.

2 Experimental

2.1 Chemicals

All reagents were obtained from commercial sources andwere of analytical-reagent grade or better. Magnesia-zir-conia composites (ZrO2-MgO) were prepared in ourlaboratory [26]. They were calcinated at 6508C for 2 h.After a classification procedure, the calcinated ZrO2-MgOmicroparticles (9–11 lm) can be dried for the next step.The results of nitrogen adsorption analysis showed thatthe ZrO2-MgO used had a mean pore size of 15 nm, thespecific surface area and pore volume of which was21 m2/g and 0.1 cm3/g, respectively. Fosfomycin was pur-chased from Northeastern Pharmaceutical Factory (Shen-gyang, China). Bovine serum albumin was purchasedfrom Shanghai Bio Life Science & Technology Co. Ltd.(Shanghai, China). Dansyl-D,L-phenylalanine, Dansyl-D,L-valine, ibuprofen, ketoprofen, N-(3,5-dinitrobenzoyl)-D,L-leucine (DNB-D,L-Leu), alprenolol, Dansyl-D,L-norvaline, N-CBZ-D,L-tryptophan, and Dansyl-D,L-serine were pur-chased from Sigma (St. Louis, MO, USA). NaBH4 was pur-chased from Fluka (Buchs, Switzerland). Glutaraldehyde(25% solution in water), 2-propanol, ammonium acetate,and anhydrous sodium acetate were all purchased fromShanghai General Chemical Reagent Factory (Shanghai,China). Doubly distilled water was used for all the experi-ments.

2.2 Preparation of BSA-F-(ZrO2-MgO) stationaryphases

The BSA-F-(ZrO2-MgO) stationary phase was preparedaccording to the scheme depicted in Fig. 1.

The fosfomycin-modified ZrO2-MgO (F-(ZrO2-MgO)) sta-tionary phase was prepared according to the methoddescribed by Yu et al. [27]. First, the fosfomycin was trea-ted with aqueous ammonia to introduce primary aminegroups [28]. Fosfomycin (3.0 g) was added to 60 mL ofammonia solution (25%, w/w). The reaction was con-

ducted at 55–608C for 68 h. Then the solution wasdiluted with water to 100 mL and adjusted to pH 6 withdilute hydrochloric acid. ZrO2-MgO (15.0 g) was added toand suspended in the above solution. The mixture wasrefluxed with stirring at 1008C for 12 h under a con-trolled nitrogen atmosphere. Thereafter, the particleswere filtered off and washed with doubly distilled water.

The modified product (F-(ZrO2-MgO)) was subsequentlytransferred to 50 mL of 5% glutaraldehyde, and stirred atroom temperature for 24 h. The slurry was filtered andthe glutaraldehyde-modified F-(ZrO2-MgO), used as theprecursor in the next reaction, was washed and dried.

BSA (1.0 g) was dissolved to 50 mL of 20 mM sodiumacetate buffer (pH 6.9) and stirred for 5 min. Then glutar-aldehyde-modified F-(ZrO2-MgO) was added to the BSAsolution and stirred at 258C for 36 h. After filtration, theproduct was transferred to a 0.1 M sodium acetate buffer(pH 7.5), which was cooled to 08C by an ice-bath. Underagitation, 0.1 g of NaBH4 was slowly added to the abovemixture. After 2 h the resulting product (BSA-F-(ZrO2-MgO)) was filtered and washed in sequence with water,50 mM sodium chloride solution, and water.

The BSA-F-(ZrO2-MgO) was then packed into a25064.6 mm id stainless steel column under 50 MPapressure using the downfill slurry packing technique. A20 mM sodium acetate buffer (pH 6.9) was used as pack-ing solvent and the same buffer containing 40% 2-propa-nol was used for slurry.

2.3 Instrumentation

Elemental analysis was performed with a VarioEL III ele-mental analyzer (Elementar, Germany). FT-IR spectrawere obtained with a Thermo Nicolet 670FT-IR instru-ment (USA) equipped with a diffuse reflectance accessoryusing the KBr technique. The specific surface area andpore size distribution were obtained by nitrogen adsorp-tion analysis with an SA3100 specific surface area analy-zer (Beckman, USA).

2.4 Chromatographic procedure

The HPLC system consisted of an FL2200 pump (WenlingFuli Analytical Instruments, Zhejiang, China), a Rheo-dyne Model 7125 injector with a 20-lL loop, and a UV228UV-vis detector (Dalian Elite Analytical Instruments,Dalian, China). The column temperature was controlledby a ZW column temperature controller (Elite), and thedata were processed by an Echrom 98 ChemStation.

Mixtures of 2-propanol and 50 mM ammonium acetatebuffer at different volume ratios were used as mobilephases for the enantioseparation. The buffer was pre-pared by mixing ammonium acetate solution with aque-ous ammonia or acetic acid to adjust the pH to thedesired value. Mobile phases were filtered and degassed


806 T. Li et al. J. Sep. Sci. 2007, 30, 804 – 812

prior to use. The flow rate was 1.0 mL/min, the columntemperature was 238C, and the detector was set at230 nm. All the samples were dissolved in methanol.

The temperature dependence of enantioseparationwas determined by measuring the separation parametersof N-(3,5-dinitrobenzoyl)-D,L-leucine over the temperaturerange of 20–388C (208C, 238C, 288C, 338C, 388C) with 1%v/v 2-propanol in bulk solution (50 mM NH4Ac, pH 5.6) asthe mobile phase. The chromatographic system wasallowed to equilibrate at each temperature for at least1 h prior to an injection. Sample solutions were injectedthrice under each given set of chromatographic condi-tions.

3 Results and discussion

3.1 Characterization of BSA-F-(ZrO2-MgO)

The amount of BSA immobilized on magnesia-zirconiacomposites varies with the reaction conditions employedin the immobilization procedure. It has been proved thatthe amount of protein deposited on zirconia supportsreached a maximum at pH 4.4, but in order to obtain agood enantioselectivity, the adsorption had to be carriedout at pH 6.9 [25]. Although in our experiment BSA wasgrafted rather than coated onto the support, the reactionmedium was still adjusted to pH 6.9, which was similar

to that of the environment in vivo, to maintain the nat-ural conformation of proteins. For the resulting station-ary phase prepared in such a buffer, the number ofmicromoles of BSA immobilized on ZrO2-MgO surfacebased on the percentage carbon from elemental analysisof BSA-F-(ZrO2-MgO) is approximately 0.0047 lmol/m2.The lower surface coverage (compared to 0.067 lmol/m2

of BSA-coated-ZrO2 stationary phase described in [25]) ismost likely due to the lower surface area (21 m2/g) of thematrix used in this study and the multi-step bindingreactions. Nevertheless, the BSA-F-(ZrO2-MgO) column isexpected to show a better stability than the BSA-coated-ZrO2 column due to the great affinity between ZrO2-MgOand fosfomycin via strong Lewis acid-base interactions.Besides, the Schiff's base formed between glutaraldehydeand primary amine groups, which existed in both phos-phonated ZrO2-MgO and proteins, was reduced bysodium borohydride specifically to form a more stablesingle bond in the last step [29].

Figure 2 shows the FT-IR spectra of blank ZrO2-MgO(Curve 1) and the corresponding modified composites(Curve 2). Differences between 1 and 2 are not obviousowing to the very low surface coverage of BSA(0.0047 lmol/m2) coupled to the metal oxides. In spite ofthis, some tiny peaks in Curve 2 demonstrate the exis-tence of the organic ligand immobilized on ZrO2-MgO:1631 cm – 1, mC=O in secondary acylamide groups;1383 cm – 1, mC=C of the aromatic rings.


Figure 1. Scheme to prepare BSA-F-(ZrO2-MgO) sta-tionary phase.


3.2 Enantioseparation

Enantioseparations of racemic drugs are summarized inTable 1. Among them, only alprenolol is basic, others areeither acidic or neutral. The representative chromato-grams of N-(3,5-dinitrobenzoyl)-D,L-leucine, Dansyl-D,L-phenylalanine, N-CBZ-D,L-tryptophan, and Dansyl-D,L-valine are shown in Fig. 3.

CSPs based on proteins have been used to resolve awide range of chiral compounds of pharmacologicalinterest [30, 31] and to probe the binding of a drug to pro-teins [32, 33]. In this study, the effects of various factorssuch as the mobile phase composition, eluent pH value,column temperature, and flow rate on the separation ofchiral compounds have also been investigated.

BSA is a globular protein, consisting of 581 aminoacids in a single chain, which exhibits an overall hydro-phobic character [14]. Therefore, the amount of organicadditive in the mobile phase exerts a great influence onthe capacity factors (k) and separation factors (a) of theracemic drugs. From the results shown in Fig. 4, the fol-lowing conclusions can be drawn: (i) increasing thevolume fraction of 2-propanol in the mobile phaseresults in a decrease in capacity factors of both enantio-mers of all analytes; (ii) increasing the 2-propanol con-tent in the mobile phase in general leads to a decrease inenantioselectivity. This is because as the amount of 2-pro-panol increases, for most pairs the retention of the moreretained enantiomer decreases more than that of the lessretained enantiomer and, thus, the selectivity decreases[34]. However, these changes do not always decrease theselectivity. In the case of Dansyl-D,L-norVal, for example,

when 2-propanol increases from 0 to 1% in the bulk solu-tion of pH 4.7, its separation factor also increases from1.96 to 2.32. It is presumed that the improved chiralrecognition is most probably due to the reoriented ter-tiary structure of the protein. This reorientation couldvery well have been caused by exposure of the hydropho-bic functionalities of the protein to the more favorableorganic environment [35]. That is to say, the larger a inthis case is the result of partial denaturation of BSAengendered by the addition of 2-propanol.

Although hydrophobic interactions represent animportant contribution to the affinity of analytes forBSA, there are also other interactions to consider, such aselectrostatic interactions and hydrogen bonding. There-fore, the effect of mobile phase pH on the enantiosepara-tion is investigated. In our study, the pH values vary from4.7 to 7.8. In these cases, BSA (its isoelectric point is 4.7)exists in neutral form or negatively charged form. If thepH value of ammonium acetate buffer decreases to 4.7,the retention for positively charged solutes (alprenolol)decreases, while that for some negatively charged solutesor neutral solutes increases. Nevertheless, the pH changein the mobile phase does not cause a direct change inselectivity. This phenomenon is similar to that reportedin a previous publication by Park et al. [25]. Several drugcompounds (Dansyl-D,L-Phe, ibuprofen, and ketoprofen)gained their best resolution when the buffer pH wasadjusted to 7.8. This indicates that BSA-CSP synthesizedin this method can be used for enantioresolution ofother racemates at alkaline pH.

Temperature is reported to influence the separation ofenantiomers [36, 37], which is also investigated in this


Figure 2. Diffuse reflectance FT-IR spectra of: (1) ZrO2-MgO, (2)BSA-F-(ZrO2-MgO).

808 T. Li et al. J. Sep. Sci. 2007, 30, 804 – 812


Table 1. Separation parameters of some racemic compounds on BSA-F-(ZrO2-MgO).

Compound k1a)/k2

b) a Rsc) pH Mobile

phased)

10.9/21.46.09/10.53.62/8.863.49/7.09

1.961.732.452.03

1.201.051.161.72

4.75.66.87.8

0:100

Dansyl-nor-Val

7.53/17.54.80/10.32.55/5.081.91/3.36

2.322.151.991.76

1.271.151.001.04

4.75.66.87.8

1:99

3.40/4.182.25/2.891.51/2.161.42/2.45

1.231.291.431.72

a0.51.011.241.20

4.75.66.87.8

5:95

CBZ-Trp

8.96/12.73.89/7.062.38/4.001.93/3.16

1.421.811.681.64

0.851.001.351.00

4.75.66.87.8

0:100

8.50/11.54.11/5.162.02/2.621.22/1.45

1.361.261.301.19

a0.50.800.800.50

4.75.66.87.8

1:99

1.06/3.600.63/4.720.33/2.020.29/1.97

3.397.456.106.82

3.243.932.883.59

4.75.66.87.8

0:100

DNB-Leu1.01/2.780.62/2.500.28/1.230.13/0.57

2.764.054.404.47

3.023.572.681.70

4.75.66.87.8

1:99

0.85/1.410.34/0.570.16/0.310.07/0.12

1.671.661.891.68

1.501.121.00a0.5

4.75.66.87.8

5:95

Dansyl-Phe

10.0/13.85.76/10.92.97/5.412.24/5.37

1.381.901.832.39

0.881.161.201.61

4.75.66.87.8

0:100

6.55/7.354.29/5.232.22/3.441.12/1.75

1.121.221.551.57

a0.50.721.001.00

4.75.66.87.8

1:99

Dansyl-Val

3.14/8.782.80/10.91.37/6.460.84/3.32

2.803.883.873.96

1.071.241.751.70

4.75.66.87.8

1:99

2.69/5.931.63/3.070.60/1.150.28/0.37

2.211.891.941.32

1.341.121.040.54

4.75.66.87.8

5:95


study. Figure 5 depicts the chromatograms of N-(3,5-dini-trobenzoyl)-D,L-leucine on BSA-F-(ZrO2-MgO) with 1% 2-propanol in bulk solution (50 mM NH4Ac, pH 5.6) as themobile phase over the temperature range 20 –388C. Plotsof relevant k, a, and the numbers of theoretical plate vs.the temperatures are shown in Fig. 6. The temperature iskept below 408C in the experiment to avoid the denatura-tion of BSA. The low column efficiency of this stationaryphase is due to the surface properties of the matrix andthe complex molecular structure of proteins, whichinduce the increase of mass transfer resistance. As shownin Fig. 6, both retention and selectivity decrease as thetemperature is raised. It is speculated that the conforma-tional change of stationary phase at relatively high tem-

perature, which reduces its stereoselective recognition,should be responsible for the negative temperaturedependence. However, the increase of column tempera-ture can accelerate mass transfer and thus improve thecolumn performance for each isomer. In our study, whenthe column temperature reached 288C, baseline separa-tion of DNB-D,L-Leu could be obtained with better effi-ciency.

To further characterize the stationary phase, the influ-ence of the mobile phase linear velocity on the columnefficiency (theoretical plate height) was studied. Theexperiments were performed using 1% v/v 2-propanol inbulk solution (50 mM NH4Ac, pH 5.6) as the mobile phaseand DNB-D,L-Leu as the analyte. The column temperature


Table 1. Continued.

Compound k1a)/k2

b) a Rsc) pH Mobile

phased)

2.10/2.531.14/1.540.49/0.580.31/0.36

1.211.351.191.17

0.500.61a0.5a0.5

4.75.66.87.8

10:90

Dansyl-Ser 5.62/9.184.79/5.802.93/4.03

1.631.211.38

1.12a0.5a0.5

4.75.66.8

0:100

5.51/7.352.61/3.711.66/1.75

1.331.421.05

0.520.63a0.5

4.75.66.8

1:99

Alprenolol 1.01/1.294.22/5.293.94/4.66

1.281.261.18

a0.5a0.5a0.5

4.75.67.8

0:100

0.91/1.081.43/1.592.49/2.70

1.181.121.08

a0.5a0.5a0.5

4.75.67.8

1:99

Ibuprofen 0.97/1.27 1.31 a0.5 7.8 0:100

Ketoprofen

6.59/9.075.15/7.422.76/3.931.36/1.70

1.381.441.421.25

a0.5a0.5a0.50.60

4.75.66.87.8

0:100

3.82/4.472.34/2.841.50/1.880.72/0.80

1.171.211.251.10

a0.5a0.5a0.5a0.5

4.75.66.87.8

1:99

a) Retention factor for the first eluted enantiomer.b) Retention factor for the second eluted enantiomer.c) Resolution factor, computed as 2(tr2 – tr1)/(w1+w2).d) Volume ratio of 2-propanol to 50 mM ammonium acetate buffer.

810 T. Li et al. J. Sep. Sci. 2007, 30, 804 – 812

was maintained at 288C and the flow rate was variedbetween 0.4 and 2 mL/min. Baseline separation of DNB-D,L-Leu was achieved at any value of the flow rateemployed, thus the peaks of the two isomers did notaffect each other. Van Deemter plots are shown in Fig. 7for both forms of the racemic compound. The resultsindicate that the theoretical plate height reaches a mini-mum at about 1.0 mL/min, when the effect of moleculardiffusion can be ignored and eddy diffusion as well aslower mass transfer resistance make major contributionsto the plate height.

Moreover, the chromatographic stability of the BSA-CSP column has been examined. Some column manufac-turers reported results of column stability studies interms of column volumes passed through the column orin terms of injections of a test solute into the chromato-graphic system [38, 39]. In our study, the column stability

was first tested by comparing the capacity factors andseparation factors of Dansyl-D,L-Val obtained at the begin-ning of the study, randomly during the study, and threemonths after the study under the same conditions(mobile phase: 2-propanol/acetate buffer 5:95 v/v at238C). Approximately 900 injections had been performedon the BSA-CSP column within this period. The maxi-mum relative differences of k and a values were less than1.0%, proving the stability of the column under theexperimental conditions during an extended period oftime. The second endurance test concerned the BSA-CSPstability at upper pH. It was carried out at 338C by pas-sing 2-propanol/50 mM NH4Ac buffer (1:99, v/v) mobilephase through the column at 1.0 mL/min to a total of 2 Lat pH 8.5. Chromatograms of Dansyl-D,L-Val wereobtained periodically. The result is presented in Fig. 8,showing that the stationary phase prepared in this


Figure 3. Representative chromatograms for N-(3,5-dinitrobenzoyl)-D,L-leucine, Dansyl-D,L-phenylalanine, N-CBZ-D,L-trypto-phan, and Dansyl-D,L-valine on the CSP. Mobile phase: (A) 1% 2-propanol in 50 mM NH4Ac buffer (pH 6.8); (B) 50 mM NH4Acbuffer (pH 7.8); (C) 50 mM NH4Ac buffer (pH 5.6); (D) 1% 2-propanol in 50 mM NH4Ac buffer (pH 7.8); flow rate: 1.0 mL/min;temperature: 238C; UV detection at 230 nm.


method is stable up to pH 8.5 without any noticeableeffect on the separation parameters or peak width of testsolute. This may be attributed to the more chemicallystable matrix (ZrO2-MgO) utilized in our experiment andthe reduction of Schiff’s base by NaBH4 to protect thebonds from hydrolysis.

4 Concluding remarks

A new protein CSP was synthesized, using the sodiumsalt of cis-(3-methyloxiranyl)phosphonic acid (fosfomy-cin) as spacer and glutaric dialdehyde as coupler.Although the resolving capabilities of BSA-F-(ZrO2-MgO)used in this work had not been optimized in terms of thesurface coverage and adsorption and immobilizationconditions, the chiral reagents studied were almost allwell separated. The reactions involved in preparation ofthe CSP were mild, reliable, and reproducible, and theproduct was stable under experimental conditions. Theresults of this work indicate that this modificationmethod can be applied to the immobilization of variousbiopolymers with amino groups in the preparation ofchromatography stationary phases.


Figure 4. Variation of retention and selectivity of Dansyl-nor-Val, DNB-Leu, and Dansyl-Val with different mobile phase 2-propanol contents. The pH of ammonium acetate buffer isfixed at 6.8. Symbols: f, k of the first eluted enantiomer; 0, kof the second eluted enantiomer; *, their separation factors;flow rate: 1.0 mL/min; temperature: 238C; UV detection at230 nm.

Figure 5. Temperature effect on separation of N-(3,5-dinitro-benzoyl)-D,L-leucine on the BSA-F-(ZrO2-MgO). Mobilephase: 1% 2-propanol in 50 mM NH4Ac buffer (pH 5.6); flowrate: 1.0 mL/min; UV detection at 230 nm.

Figure 6. Effect of column temperature on the capacity fac-tors and column performance of N-(3,5-dinitrobenzoyl)-D,L-leucine: f, k of the first eluted enantiomer; 0, k of the secondeluted enantiomer; *, their separation factors; F, the numberof theoretical plates of the first eluted enantiomer; 9, thenumber of theoretical plates of the second eluted enantio-mer. Conditions as reported in Fig. 5.

812 T. Li et al. J. Sep. Sci. 2007, 30, 804 – 812

This work was financially supported by the National NaturalScience Foundation of China (Grant No. 20475040) and the Pro-gram for New Century Excellent Talents in University (NCET-05-0616).

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Figure 7. Van Deemter plot for BSA-F-(ZrO2-MgO) column.f, The theoretical plate height of the first eluted enantiomerof DNB-D,L-Leu; 0, the theoretical plate height of the secondeluted enantiomer. Mobile phase: 1% 2-propanol in 50 mMNH4Ac buffer (pH 5.6); temperature: 288C; UV detection at230 nm.

Figure 8. Retention and column efficiency stability of Dan-syl-D,L-Val on the BSA-CSP. f, k of the first eluted enantio-mer; 0, k of the second eluted enantiomer; *, their separationfactors; F, the number of theoretical plates of the first elutedenantiomer; 9, the number of theoretical plates of the secondeluted enantiomer. Mobile phase: 1% 2-propanol in 50 mMNH4Ac buffer (pH 8.5); flow rate: 1.0 mL/min; temperature:338C; UV detection at 230 nm.

synthesis and chromatographic properties of a chiral stationary phase derived from bovine serum...

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