Xindu Geng

Northwest University, Ch’ang-an, Shaanxi, China

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Publications (50)78.64 Total impact

  • Ruijuan Niu, Yi Min, Xindu Geng
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    ABSTRACT: A novel method for the fast separation of native proteins was investigated using sub-2 µm nonporous silica packing inside a chromatographic cake having a diameter much larger than its thickness. Various silica-based particles ranging from 630 nm to 1.2 µm were synthesized and chemically modified with polyethylene glycol 600. The packing material was laterally packed into a series of chromatographic cakes containing the same diameter (10 mm) and different thicknesses, ranging from 2 to 10 mm, and tested by hydrophobic interaction chromatography. The results showed that the sub-2 µm NPS particles in a small chromatographic cake were found to have a high efficiency at a flow rate of 10 mL/min and a backpressure of <20 MPa. The effect of the thickness of the chromatographic cake on the resolution of the proteins was also investigated and it was found that too short a column length could dramatically decrease the protein resolution; the minimum column length was also qualitatively evaluated. The presented method is expected to be useful for routine analysis of native and/or intact proteins in hospitals and as a tool for the fast screening protein drugs and optimization of experimental laboratory conditions. Copyright © 2014 John Wiley & Sons, Ltd.
    Biomedical Chromatography 02/2014; · 1.95 Impact Factor
  • Lili Wang, Xindu Geng
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    ABSTRACT: Protein folding liquid chromatography (PFLC) is a powerful tool for protein refolding with simultaneous purification. We review its recent progress in liquid chromatography and molecular biology, primarily involving the validation of PFLC refolding of proteins containing multiple disulphide bonds, the application of mixed-mode chromatography, PFLC in molecular biology. Representative examples are described.
    Amino Acids 11/2013; · 3.91 Impact Factor
  • Chaozhan Wang, Xindu Geng
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    ABSTRACT: High level expression of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in Escherichia coli (E. coli) usually forms insoluble and inactive aggregates, i.e. inclusion bodies. In the present work, high performance hydrophobic interaction chromatography (HPHIC) was applied to the refolding of rhG-CSF, which was solubilized by 8.0 mol L−1 urea from the inclusion bodies. First a laboratorial scale column (10 mm × 20 mm I.D.) was employed to study the refolding process. Several factors, including concentration of ammonium sulfate, pH of the mobile phase and flow rate, were investigated in details. The results indicated that the rhG-CSF produced by E. coli could be successfully refolded with simultaneous purification by using HPHIC. The refolding process was further scaled up by using a large column (50 mm × 200 mm I.D.). 200 mL of rhG-CSF solution solubilized by 8.0 mol L−1 urea, with a total amount of protein around 1.6 g, could be loaded onto the large column at one time. Under these conditions, the obtained rhG-CSF had a specific activity of 2.3 × 108 IU mg−1 and a purity of 95.4%, the mass recovery during the purification was 36.9%. This work might have great impact on practical production of rhG-CSF, and it also shed a light on protein refolding using liquid chromatography at large scales.
    PROCESS BIOCHEMISTRY 12/2012; 47(12):2262–2266. · 2.41 Impact Factor
  • Fei Wang, Yi Min, Xindu Geng
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    ABSTRACT: Scientists working in many fields require fast separations of intact proteins using liquid chromatography. The fast separations here concern not only the separation step alone but also the complete chromatographic process, including column regeneration, system equilibration, and buffer exchange, in one- and two-dimensional liquid chromatography in addition to fast purification technologies predominantly on the analytical scale with some unique examples on the preparative and industrial scales. This comprehensive review discusses recent developments in methodologies, packing materials, column techniques, and purification technologies in the field of rapid liquid chromatography of intact proteins. Some typical examples are summarized in the tables.
    Journal of Separation Science 10/2012; · 2.59 Impact Factor
  • Yun Yang, Xindu Geng
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    ABSTRACT: Mixed-mode chromatography is a type of chromatography in which a chromatographic stationary phase interacts with solutes through more than one interaction mode. This technique has been growing rapidly because of its advantages over conventional chromatography, such as its high resolution, high selectivity, high sample loading, high speed, and the ability to replace two conventionally corresponding columns in certain circumstances. In this work, some aspects of the development of mixed-mode chromatography are reviewed, such as stationary phase preparation, combinations of various separation modes, separation mechanisms, typical applications to biopolymers and peptides, and future prospects.
    Journal of Chromatography A 12/2011; 1218(49):8813-25. · 4.61 Impact Factor
  • Lili Wang, Chaozhan Wang, Xindu Geng
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    ABSTRACT: A method was developed to increase the recovery of recombinant human stem cell factor (rhSCF) from inclusion bodies using high performance hydrophobic interaction chromatography (HPHIC). The target protein was first solubilized in 8.0 mol/L urea solution, and was purified and refolded simultaneously by HPHIC with different chromatographic cakes. Experimental conditions, such as the ligand structures of stationary phase and the composition of mobile phase, were optimized. Under the optimal conditions, high mass recoveries and specific activities of rhSCF were acquired, the purities of rhSCF were above 95.5%, and the mass recoveries of rhSCF were above 49.6%. The final product was also verified as monomer by size exclusion chromatography and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). These results provided further evidence that HPHIC is an effective tool in the refolding and purification of recombinant proteins.
    Se pu = Chinese journal of chromatography / Zhongguo hua xue hui 01/2011; 29(1):36-41.
  • Quan Bai, Xindu Geng
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    ABSTRACT: A method for carrying out protein folding with simultaneous separation by protein folding liquid chromatography (PFLC) is described herein. Furthermore, a two-dimensional chromatographic column, termed a 2D column, which can be independently employed for accomplishing PFLC in either weak cation exchange mode or hydrophobic interaction chromatography mode is reported. The content of this chapter describes the most commonly employed methods and operations of PFLC, such as the use of urea or guanidine hydrochloride as a denaturant with the protein in either the reduced or oxidized state and solving problems caused by the formation of the precipitates during protein folding. The PFLC can be performed using conventional chromatographic columns and a new chromatographic cake. A protocol for fast renaturation with simultaneous purification of inclusion body protein of the recombinant human interferon-gamma to obtain purity ≥95% and high specific bioactivity in a single step and in 1 h is introduced.
    Methods in molecular biology (Clifton, N.J.) 01/2011; 705:69-85. · 1.29 Impact Factor
  • Yi Min, Gang Chen, Xindu Geng
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    ABSTRACT: Based on the fact that the resolution of intact protein separation is almost independent of column length, the effect on the resolution for intact protein separation causing from dynamic factors in hydrophobic interaction chromatography (HIC) was investigated. A concept of "conditional plate height" (H) for protein separation is firstly suggested for characterizing this effect for protein separation under linear gradient elution. Standard proteins were separated with conventional chromatographic column and chromatographic cake, and the plot of the H vs the linear velocity of mobile phase (u) was made, respectively. It was found that the obtained plot is similar to the conventional van Deemter Plot but has some differences. The optimized u corresponding to the minimum H was determined to be approximate 0.2 mm/s for the chromatographic cake and 1-3 mm/s for the conventional column. Furthermore, in comparison with the latter, optimized u value for the former has much broader range. Based on this fact, the resolutions and speeds for standard protein separation between the chromatographic cake packed with silica-base HIC material and the conventional column packed with soft HIC media were compared. The chromatographic cake (10 mm x 20 mm i.d.) was found to perform a complete separation of seven standard proteins in 10 min, while with the latter (55 mm x 12 mm i.d.) only five standard proteins can be completely separated in 140 min, even though the sample load for the former having bed volume of 3.14 mL, five times of that of the latter. The HIC chromatographic cake was also employed for the renaturation with simultaneous purification of the recombinant human granulocyte colony stimulating factor. The obtained purity was > or = 97%, mass recovery was 39%, specific bioactivity was 1 x 10(8) IU/mg with only one step HIC in 50 min. It would be expected that when a kind of packings having very small particle size is packed into a chromatographic cake with diameter to be greater than its thickness and is employed to separate, and/or renature proteins, a result of high speed and high resolution with simultaneous renaturation under high protein loading ("three H" target) could be obtained.
    Se pu = Chinese journal of chromatography / Zhongguo hua xue hui 09/2009; 27(5):717-23.
  • Peng Liu, Haiya Yang, Xindu Geng
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    ABSTRACT: Using four commercial weak anion-exchange chromatography (WAX) columns and 11 kinds of different proteins, we experimentally examined the involvement of hydrophobic interaction chromatography (HIC) mechanism in protein retention on the WAX columns. The HIC mechanism was found to operate in all four WAX columns, and each of these columns had a better resolution in the HIC mode than in the corresponding WAX mode. Detailed analysis of the molecular interactions in a chromatographic system indicated that it is impossible to completely eliminate hydrophobic interactions from a WAX column. Based on these results, it may be possible to employ a single WAX column for protein separation by exploiting mixed modes (WAX and HIC) of retention. The stoichiometric displacement theory and two linear plots were used to show that mechanism of the mixed modes of retention in the system was a combination of two kinds of interactions, i.e., nonselective interactions in the HIC mode and selective interactions in the IEC mode. The obtained U-shaped elution curve of proteins could be distinguished into four different ranges of salt concentration, which also represent four retention regions.
    Journal of Chromatography A 08/2009; 1216(44):7497-504. · 4.61 Impact Factor
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    ABSTRACT: A new approach for characterizing the intermediate of urea-denatured alpha-chymotrypsin (alpha-Chy) by hydrophobic interaction chromatography (HIC) is presented. The contact surface region (Z, S), affinity (logI), and the character of interaction force (j) of the alpha-Chy to the stationary phase of HIC (STHIC) between the intermediate (M) and native (N) states were found to be quite different as urea concentration (C(urea)) changes. With the changes in C(urea), a linear relationship between logI and Z was found to exist only for its N state, not for M state, indicating the interaction force between alpha-Chy in N state to the STHIC to be non-selective, but selective one for its M state. Also, the measured magnitude of both logI and Z in M state is only a fifth of that in N state. All three parameters were employed to distinguish protein in the N state from that in the M state. It would be expected that this result could be employed to distinguish any kind of non-functional protein having correct three-, or four-dimensional molecular structure from their stable M state of any kinds of proteins, and/or other proteins in proteome investigation, separation process of protein, and intensively understanding the intrinsic rule of protein folding in molecular biology.
    International Journal of Molecular Sciences 03/2009; 10(2):616-28. · 2.46 Impact Factor
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    ABSTRACT: This paper reports the on-line separation of native (N) proteins by two-dimensional liquid chromatography (2D-LC) using a single column with one phase (called 2D column). The 2D column exhibits excellent resolution, selectivity, and retention of proteins in the N state and functions in two retention modes--hydrophobic interaction chromatography (HIC) and weak-cation exchange chromatography (WCX). We describe a new approach to on-line buffer exchange and collection of fractions from the first retention mode and their quantitative re-injection into the same column, followed by re-separation in the second retention mode. Thus, liquid chromatography in a closed system and in an on-line manner could be successfully carried out. This method was termed on-line protein separation by 2D-LC using only a single column (on-line 2D-LC-1C). The applicability of this method was experimentally demonstrated using standard proteins and a human serum sample. The total hypothetical maximum possible peak capacity n(c,total) and total sample peak capacity n(c,total)(*) of the 2D column were 329 and 199, respectively. By comparison against several popular commercially available columns, it was found that the 2D column had not only comparable resolution and better selectivity but also some unique characteristics. This 2D-LC-1C method could be applied to the fast purification of intact proteins in the N state, such protein drugs from natural products, and recombinant proteins and also for the fast pre-fractionation of intact proteins in the "top-down" MS strategy in proteomics.
    Journal of Chromatography A 02/2009; 1216(16):3553-62. · 4.61 Impact Factor
  • Chaozhan Wang, Lili Wang, Xindu Geng
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    ABSTRACT: Immobilized metal ion affinity chromatography (IMAC) is a new technique for protein refolding, but it is limited to the refolding of fusion proteins with histidine affinity tags. In the present work, a non-fusion recombinant protein, recombinant human granulocyte colony-stimulating factor (rhG-CSF) expressed in Escheriachia coli (E. coli) in the form of inclusion bodies was successfully refolded with simultaneous purification by IMAC. rhG-CSF inclusion bodies solubilized in 8.0 mol/L urea was injected into a Cu(II)-iminodiacetic acid (IDA)-IMAC column, the soluble and active form of rhG-CSF in aqueous solution was obtained after desorbed from the column by linear increase of imidazole concentration. Several factors in the refolding process, including urea concentration and pH of mobile phases, type of buffer, glycerol concentration and loading sample volume, were investigated, respectively. When 200 μL of denatured/reduced rhG-CSF solution at a total protein concentration of 2.8 mg/mL was loaded on the IMAC column, rhG-CSF with a specific activity of 2.3 × 108 IU/mg and a mass recovery of 39% was obtained after IMAC refolding, and rhG-CSF was also purified during this chromatographic process, its purity was determined to be 97%.
    Biochemical Engineering Journal. 01/2009;
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    Xindu Geng, Lili Wang
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    ABSTRACT: Many recombinant proteins (rPRTs) have a high bioactivity and some of them may eventually be classified as drugs beneficial to human health, recombinant human protein drugs (rPDs). rPDs are a high-technology product with all the associated economic benefits, therefore the liquid chromatography (LC) of rPRT is different from that of proteins isolated in laboratory scale for purely research purposes. The design of a purification scheme for an rPRT depends on the intended function of the purified rPRT, as a pure sample for research in small scale, or as a product for industrial production. This review paper mainly deals with the latter instance, producing rPD at a large scale. Pharmaceutical economics is considered not only for each step of purification, but also the whole production process. This strategy restricts the content of this review paper to the factors affecting the optimization source, the character of rPRT in up-stream technology and the purification of the rPRT in down-stream production. In the latter instance, the purification step is required to be as efficient as possible and LC is the core of the refined purification method, which is either a single LC method or combination of LC methods, sometimes, it may be a combination of LC and other non-LC separation methods comprising an optimized purification technology. Here some typical examples of rPRT purification at the large scale, recent developments, such as protein folding liquid chromatography, short column chromatography, and new packing material and column techniques are introduced.
    Journal of Chromatography B 05/2008; 866(1-2):133-53. · 2.49 Impact Factor
  • Dan Wu, Dong Gao, Quan Bai, Xindu Geng
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    ABSTRACT: The renaturation with simultaneous purification of recombinant human interferon-gamma (rhIFN-gamma) expressed as inclusion bodies in Escherichia coli (E. coli) was accomplished by the stationary phase of hydrophobic interaction chromatography (STHIC) with the end group of poly(ethylene glycol) (PEG)(PEG200) packed in a chromatographic column and a chromatographic pie by nonlinear gradient, separately. In order to provide more selections for the chromatographic separation of rhIFN-gamma from different sources, the chromatographic behavior of rhIFN-gamma in reversed-phase liquid chromatography, ion-exchange chromatography and immobilized-nickel affinity chromatography were also studied. The fraction of the renatured and purified rhIFN-gamma from HIC was desalted by the size exclusion chromatography, subsequently freeze-dried to powder. With matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), monomeric and dimeric rhIFN-gamma were found in the powder due to the freeze-dried process and their relative molecular masses were 17 184.0 and 34 204.4, respectively. With the bioactivity assay by cytopathic effect inhibition (CPEI), the specific bioactivity of rhIFN-gamma was 9.5 x 10(8) IU/mg, which was higher than that of the required criteria in the phar macopoeia of China, because the presence of dimeric rhIFN-gamma which has much higher specific bioactivity than its monomer in the powder. The obtained mass recovery, purity, specific bioactivity of the purified monomeric rhIFN-y were 93.7%, > 95%, and 4.3 x 10(7) IU/mg, respectively. The results showed that the renaturation with simultaneous purification of rhIFN-gamma by PEG200-STHIC is a kind of efficient method.
    Se pu = Chinese journal of chromatography / Zhongguo hua xue hui 03/2008; 26(2):206-11.
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    ABSTRACT: Recombinant human stem cell factor (rhSCF) was solubilized and renatured from inclusion bodies expressed in Escherichia coli. The effect of both pH and urea on the solubilization of rhSCF inclusion bodies was investigated; the results indicate that the solubilization of rhSCF inclusion bodies was significantly influenced by the pH of the solution employed, and low concentration of urea can drastically improve the solubilization of rhSCF when solubilized by high pH solution. The solubilized rhSCF can be easily refolded with simultaneous purification by ion exchange chromatography (IEC), with a specific activity of 7.8 x 10(5) IU x mg(-1), a purity of 96.3%, and a mass recovery of 43.0%. The presented experimental results show that rhSCF solubilized by high pH solution containing low concentration of urea is easier to be renatured than that solubilized by high concentration of urea, and the IEC refolding method was more efficient than dilution refolding and dialysis refolding for rhSCF. It may have a great potential for large-scale production of rhSCF.
    Applied Biochemistry and Biotechnology 03/2008; 144(2):181-9. · 1.89 Impact Factor
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    Chaozhan Wang, Lili Wang, Xindu Geng
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    ABSTRACT: Protein folding liquid chromatography (PFLC) is a powerful tool for simultaneous refolding and purification of recombinant proteins in inclusion bodies. Urea gradient size exclusion chromatography (SEC) is a recently developed protein refolding method based on the SEC refolding principle. In the presented work, recombinant human granulocyte colony-stimulating factor (rhG-CSF) expressed in Escheriachia coli (E. coli) in the form of inclusion bodies was refolded with high yields by this method. Denatured/reduced rhG-CSF in 8.0 mol.L(-1) urea was directly injected into a Superdex 75 column, and with the running of the linear urea concentration program, urea concentration in the mobile phase and around the denatured rhG-CSF molecules was decreased linearly, and the denatured rhG-CSF was gradually refolded into its native state. Aggregates were greatly suppressed and rhG-CSF was also partially purified during the refolding process. Effects of the length and the final urea concentration of the urea gradient on the refolding yield of rhG-CSF by using urea gradient SEC were investigated respectively. Compared with dilution refolding and normal SEC with a fixed urea concentration in the mobile phase, urea gradient SEC was more efficient for rhG-CSF refolding--in terms of specific bioactivity and mass recovery, the denatured rhG-CSF could be refolded at a larger loading volume, and the aggregates could be suppressed more efficiently. When 500 microL of solubilized and denatured rhG-CSF in 8.0 mol.L(-1) urea solution with a total protein concentration of 2.3 mg.mL(-1) was loaded onto the SEC column, rhG-CSF with a specific bioactivity of 1.0 x 10(8) IU.mg(-1) was obtained, and the mass recovery was 46.1%.
    Biotechnology Progress 02/2008; 24(1):209-13. · 1.85 Impact Factor
  • Chaozhan Wang, Lili Wang, Xindu Geng
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    ABSTRACT: Protein refolding is a key step for the production of recombinant proteins, especially at large scales, and usually their yields are very low. Application of liquid chromatography to protein refolding is an exciting step forward for this field. In this work, recombinant human granulocyte colony-stimulating factor (rhG-CSF) expressed in Escherichia coli was renatured with simultaneous purification by ion exchange chromatography (IEC) with a Q Sepharose FF column. Several chromatographic parameters affecting the refolding yield of the denatured/reduced rhG-CSF, such as the urea concentration, pH value, concentration and ratio of reduced/oxidized glutathione in the mobile phase, as well as the flow rate of the mobile phase, were investigated in detail and indicated that the urea concentration and the pH value were of great importance. At the optimal conditions, the renatured and purified rhG-CSF was found to have a specific bioactivity of 3.0 x 10(8) IU/mg, a purity of 96%, and a mass recovery of 49%. Compared with the usual dilution method, the IEC method developed here is more effective for rhG-CSF refolding in terms of specific bioactivity and mass recovery.
    Biomedical Chromatography 01/2008; 21(12):1291-6. · 1.95 Impact Factor
  • Chaozhan Wang, Lili Wang, Xindu Geng
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    ABSTRACT: Recombinant human granulocyte colony-stimulating factor (rhG-CSF) in the form of inclusion bodies expressed in Escherichia coli (E. coli) was simultaneously refolded and purified using protein folding liquid chromatography (PFLC). Cu2+ -iminodiacetic acid (IDA) Sepharose was selected as the stationary phase for immobilized metal ion affinity chromatography. rhG-CSF was purified and the aggregates were diminished under a linear gradient elution of imidazole in the presence of a suitable concentration of urea. Using only one PFLC run, the refolded rhG-CSF had a specific bioactivity of 1.8 x 10(8) IU/mg and a purity of 97%, with the mass recovery of 32%.
    Se pu = Chinese journal of chromatography / Zhongguo hua xue hui 08/2007; 25(4):514-7.
  • Xindu Geng, Chaozhan Wang
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    ABSTRACT: The ultimate goal of proteomics is to identify biologically active proteins and to produce them using biotechnology tools such as bacterial hosts. However, proteins produced by Escherichia coli must be refolded to their native state. Protein folding liquid chromatography (PFLC) is a new method developed in recent years, and it is widely used in molecular biology and biotechnology. In this paper, the new method, PFLC is introduced and its recent development is reviewed. In addition the paper includes definitions, advantages, principles, applications for both laboratory and large scales, apparatus, and effecting factors of PFLC. In addition, the role of this method in the future is examined.
    Journal of Chromatography B 05/2007; 849(1-2):69-80. · 2.49 Impact Factor
  • Dan Wu, Chaozhan Wang, Xindu Geng
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    ABSTRACT: Protein refolding is a key process for the production of recombinant proteins from E. coli. One of the effective techniques for protein refolding is through liquid chromatography named as protein folding liquid chromatography (PFLC). Mobile phase composition is one of the most important factors affecting the efficiency of protein renaturation in PFLC. Optimization of mobile phase composition in PFLC for protein renaturation with simultaneous purification is thus much more important than that in usual liquid chromatography. This study presents an approach with regard to the increase of protein production through optimizing the mobile phase composition of PFLC. Recombinant human interferon-gamma (rhIFN-gamma) was purified with simultaneous renaturation by high performance hydrophobic interaction chromatography (HPH-IC) using a stationary phase with an end group of PEG-200. Effects of mobile phase composition, gradient elution mode, and flow rate on the recoveries of mass and bioactivity of rhIFN-gamma were investigated. It was found that these factors were very important for refolding with simultaneous purification of rhIFN-gamma by PFLC. This study highlights the importance of optimizing the mobile phase composition in PFLC for increasing the recovery of protein. Taking 3.0 mol/L (NH4 )2 SO4 + 0. 05 mol/L KH2 PO4 (pH 7. 0) and 0. 05 mol/L KH2 PO4 (pH 7. 0) as mobile phases A and B, respectively, the highest mass recovery was obtained under a nonlinear gradient elution for 35 min.
    Se pu = Chinese journal of chromatography / Zhongguo hua xue hui 04/2007; 25(2):197-202.